Mapping cellular Fe-S cluster uptake and exchange reactions - divergent pathways for iron-sulfur cluster delivery to human ferredoxins.

PubMed

Fidai, Insiya; Wachnowsky, Christine; Cowan, J A

2016-12-07

Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe-2S] or [4Fe-4S] clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe-2S] cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular cluster trafficking. This is the first step toward building a network map for all of the possible iron-sulfur cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

Pyruvate:Ferredoxin Oxidoreductase Is Coupled to Light-independent Hydrogen Production in Chlamydomonas reinhardtii*

PubMed Central

Noth, Jens; Krawietz, Danuta; Hemschemeier, Anja; Happe, Thomas

2013-01-01

In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H2) as one of several fermentation products. H2 is generated mostly by the [Fe-Fe]-hydrogenase HYDA1, which uses plant type ferredoxin PETF/FDX1 (PETF) as an electron donor. Dark fermentation of the alga is mainly of the mixed acid type, because formate, ethanol, and acetate are generated by a pyruvate:formate lyase pathway similar to Escherichia coli. However, C. reinhardtii also possesses the pyruvate:ferredoxin oxidoreductase PFR1, which, like pyruvate:formate lyase and HYDA1, is localized in the chloroplast. PFR1 has long been suggested to be responsible for the low but significant H2 accumulation in the dark because the catalytic mechanism of pyruvate:ferredoxin oxidoreductase involves the reduction of ferredoxin. With the aim of proving the biochemical feasibility of the postulated reaction, we have heterologously expressed the PFR1 gene in E. coli. Purified recombinant PFR1 is able to transfer electrons from pyruvate to HYDA1, using the ferredoxins PETF and FDX2 as electron carriers. The high reactivity of PFR1 toward oxaloacetate indicates that in vivo, fermentation might also be coupled to an anaerobically active glyoxylate cycle. Our results suggest that C. reinhardtii employs a clostridial type H2 production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H2 evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA. PMID:23258532

Ferredoxin:NAD + oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation [Identification of a ferredoxin:NAD + oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation

DOE PAGES

Tian, Liang; Lo, Jonathan; Shao, Xiongjun; ...

2016-09-30

Ferredoxin:NAD + oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD +. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705more » resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. As a result, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.« less

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy.

PubMed

Bowen, Alice M; Johnson, Eachan O D; Mercuri, Francesco; Hoskins, Nicola J; Qiao, Ruihong; McCullagh, James S O; Lovett, Janet E; Bell, Stephen G; Zhou, Weihong; Timmel, Christiane R; Wong, Luet Lok; Harmer, Jeffrey R

2018-02-21

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe 2 S 2 ] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe 2 S 2 ] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

An Electron-bifurcating Caffeyl-CoA Reductase*

PubMed Central

Bertsch, Johannes; Parthasarathy, Anutthaman; Buckel, Wolfgang; Müller, Volker

2013-01-01

A low potential electron carrier ferredoxin (E0′ ≈ −500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD+ oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA. PMID:23479729

Energetic Selection of Topology in Ferredoxins

PubMed Central

Kim, J. Dongun; Rodriguez-Granillo, Agustina; Case, David A.; Nanda, Vikas; Falkowski, Paul G.

2012-01-01

Models of early protein evolution posit the existence of short peptides that bound metals and ions and served as transporters, membranes or catalysts. The Cys-X-X-Cys-X-X-Cys heptapeptide located within bacterial ferredoxins, enclosing an Fe4S4 metal center, is an attractive candidate for such an early peptide. Ferredoxins are ancient proteins and the simple α+β fold is found alone or as a domain in larger proteins throughout all three kingdoms of life. Previous analyses of the heptapeptide conformation in experimentally determined ferredoxin structures revealed a pervasive right-handed topology, despite the fact that the Fe4S4 cluster is achiral. Conformational enumeration of a model CGGCGGC heptapeptide bound to a cubane iron-sulfur cluster indicates both left-handed and right-handed folds could exist and have comparable stabilities. However, only the natural ferredoxin topology provides a significant network of backbone-to-cluster hydrogen bonds that would stabilize the metal-peptide complex. The optimal peptide configuration (alternating αL,αR) is that of an α-sheet, providing an additional mechanism where oligomerization could stabilize the peptide and facilitate iron-sulfur cluster binding. PMID:22496635

Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring H nuclear magnetic resonances in clostridial-type ferredoxins. [Clostridium acidi-urici, C. pasteurianum, C. perfringens, Peptococcus aerogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Packer, E.L.; Sweeney, W.V.; Rabinowitz, J.C.

1977-04-10

We have directly assigned the /sup 1/H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the /sup 1/H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and /sup 13/C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppM (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the /sup 1/H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The /sup 1/H NMR spectramore » of the ..beta.. protons of the cysteinyl residues of these ferredoxins differ, however, from the /sup 1/H NMR spectra of equivalent ..beta.. protons of the methylene carbon atoms bonded via a sulfur atom to (4Fe-4S) clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the ..beta.. protons appear as a single resonance shifted 10 ppM from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl ..beta.. protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppM, relative to the free amino acid resonance position.« less

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

PubMed Central

Yoon, K S; Ishii, M; Igarashi, Y; Kodama, T

1996-01-01

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively. PMID:8655524

Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

PubMed

Kurdrid, Pavinee; Subudhi, Sanjukta; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Hongsthong, Apiradee

2007-12-01

When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro

PubMed Central

Yacoby, Iftach; Pochekailov, Sergii; Toporik, Hila; Ghirardi, Maria L.; King, Paul W.; Zhang, Shuguang

2011-01-01

Photosynthetic water splitting, coupled to hydrogenase-catalyzed hydrogen production, is considered a promising clean, renewable source of energy. It is widely accepted that the oxygen sensitivity of hydrogen production, combined with competition between hydrogenases and NADPH-dependent carbon dioxide fixation are the main limitations for its commercialization. Here we provide evidence that, under the anaerobic conditions that support hydrogen production, there is a significant loss of photosynthetic electrons toward NADPH production in vitro. To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP+-oxidoreductase (FNR), and NADP+. Replacing the hydrogenase with a ferredoxin-hydrogenase fusion switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction. These results suggest a new direction for improvement of biohydrogen production and a means to further resolve the mechanisms that control partitioning of photosynthetic electron transport. PMID:21606330

Alternative Pathway of Metronidazole Activation in Trichomonas vaginalis Hydrogenosomes

PubMed Central

Hrdý, Ivan; Cammack, Richard; Stopka, Pavel; Kulda, Jaroslav; Tachezy, Jan

2005-01-01

Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes. PMID:16304169

The structurally unique photosynthetic Chlorella variabilis NC64A hydrogenase does not interact with plant-type ferredoxins.

PubMed

Engelbrecht, Vera; Rodríguez-Maciá, Patricia; Esselborn, Julian; Sawyer, Anne; Hemschemeier, Anja; Rüdiger, Olaf; Lubitz, Wolfgang; Winkler, Martin; Happe, Thomas

2017-09-01

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H 2 ) production and H 2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H 2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity). Copyright © 2017 Elsevier B.V. All rights reserved.

Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

PubMed

Plaga, W; Lottspeich, F; Oesterhelt, D

1992-04-01

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

DOE PAGES

Lo, Jonathan; Zheng, Tianyong; Olson, Daniel G.; ...

2015-06-29

NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP +. The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. In this paper, activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H 2more » formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity. Importance: Thermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P) +. The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP + and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. Finally, we report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes.« less

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

USDA-ARS?s Scientific Manuscript database

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium perfringens

USDA-ARS?s Scientific Manuscript database

Clostridium related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

Formate Dehydrogenase from Clostridium acidiurici

PubMed Central

Kearny, James J.; Sagers, Richard D.

1972-01-01

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO2 was not accomplished. PMID:4333376

Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuzawa, Jun; Aikawa, Hiroki; Umeda, Takashi

2014-09-25

A crystal was obtained of the complex between reduced terminal oxygenase and oxidized ferredoxin components of carbazole 1,9a-dioxygenase. The crystal belonged to space group P2{sub 1} and diffracted to 2.25 Å resolution. The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Å and belonged to space group P2{submore » 1}, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 Å, α = γ = 90, β = 100.1°. The V{sub M} value is 2.85 Å{sup 3} Da{sup −1}, indicating a solvent content of 56.8%.« less

Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus.

PubMed

Nguyen, Diep M N; Schut, Gerrit J; Zadvornyy, Oleg A; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L; Adams, Leslie A; Dinsmore, Jessica T; Nixon, William J; Boyd, Eric S; Bothner, Brian; Peters, John W; Adams, Michael W W

2017-09-01

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP + oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Thioredoxins in evolutionarily primitive organisms

NASA Technical Reports Server (NTRS)

Buchanan, B. B.

1986-01-01

Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction

NASA Astrophysics Data System (ADS)

Diakonova, A. N.; Khrushchev, S. S.; Kovalenko, I. B.; Riznichenko, G. Yu; Rubin, A. B.

2016-10-01

Ferredoxin (Fd) protein transfers electrons from photosystem I (PSI) to ferredoxin:NADP+-reductase (FNR) in the photosynthetic electron transport chain, as well as other metabolic pathways. In some photosynthetic organisms including cyanobacteria and green unicellular algae under anaerobic conditions Fd transfers electrons not only to FNR but also to hydrogenase—an enzyme which catalyzes reduction of atomic hydrogen to H2. One of the questions posed by this competitive relationship between proteins is which characteristics of thylakoid stroma media allow switching of the electron flow between the linear path PSI-Fd-FNR-NADP+ and the path PSI-Fd-hydrogenase-H2. The study was conducted using direct multiparticle simulation approach. In this method protein molecules are considered as individual objects that experience Brownian motion and electrostatic interaction with the surrounding media and each other. Using the model we studied the effects of pH and ionic strength (I) upon complex formation between ferredoxin and FNR and ferredoxin and hydrogenase. We showed that the rate constant of Fd-FNR complex formation is constant in a wide range of physiologically significant pH values. Therefore it can be argued that regulation of FNR activity doesn’t involve pH changes in stroma. On the other hand, in the model rate constant of Fd-hydrogenase interaction dramatically depends upon pH: in the range 7-9 it increases threefold. It may seem that because hydrogenase reduces protons it should be more active when pH is acidic. Apparently, regulation of hydrogenase’s affinity to both her reaction partners (H+ and Fd) is carried out by changes in its electrostatic properties. In the dark, the protein is inactive and in the light it is activated and starts to interact with both Fd and H+. Therefore, we can conclude that in chloroplasts the rate of hydrogen production is regulated by pH through the changes in the affinity between hydrogenase and ferredoxin.

Structural Investigations of the Ferredoxin and Terminal Oxygenase Components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferraro,D.; Brown, E.; Yu, C.

The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-F{sub B1}). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-O{sub B1}), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo(a)pyrene. Results: In this study, crystal structures of BPDO-O{sub B1} in both native and biphenylmore » bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-F{sub B1} has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted. Conclusion: This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.« less

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle.

PubMed

Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R; Nguyen, Diep M N; Schut, Gerrit J; Adams, Michael W W; Peters, John W; Boyd, Eric S; Bothner, Brian

2018-01-01

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP + oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron‑sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units. Copyright © 2017 Elsevier B.V. All rights reserved.

Electron spin resonance studies of the ovary of the rat

NASA Astrophysics Data System (ADS)

Andersen, Roy S.; Curtis, Joseph C.

1988-11-01

Electron spin resonance spectra of rat ovaries, isolated ovarian compartments, and ovarian subcellular fractions were compared with spectra of rat adrenals. Rat ovaries were found to exhibit ESR signals similar to those previously described in studies of mammalian adrenal and testis. Observations were made at 113 K in an anaerobic environment. ESR signals of the low-spin ferric cytochrome P-450, the non-heme protein ferredoxin, and the non-heme glycoprotein transferrin were consistently observed in whole ovaries. The first two signals were detected in mitochondrial fractions isolated from ovaries, while only cytochrome P-450 was detected in microsomal fractions. Signals from ferredoxin and cytochrome P-450 were also consistently observed in both whole adrenals and adrenal mitochondrial fractions. However, in the microsomal fraction only cytochrome P-450 was present. The g values for the cytochrome P-450 and ferredoxin signals found in this study of ovaries were identical to those previously reported and also found in this study in spectra of rat adrenals. The concentration of ferredoxin per milligram wet mass in rat ovaries appears to be only one-sixth of that in the rat adrenal. The concentration of cytochrome P-450 appears to be only one-ninth of that in the adrenal. Signals from ferredoxin were detected in all ovarian compartments except granulosa cells isolated from Graafian follicles. The third signal, that of transferrin, while often observed in the spectra of whole ovaries, has been attributed to residual blood in the tissues examined. The effects of oxygen on these spectra has been found to be considerable and is discussed.

The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

PubMed

Szekeres, M; Droux, M; Buchanan, B B

1991-03-01

The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form.

A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway.

PubMed

Bahar, M; de Majnik, J; Wexler, M; Fry, J; Poole, P S; Murphy, P J

1998-11-01

Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.

Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashikawa, Yuji; Uchimura, Hiromasa; Fujimoto, Zui

2007-06-01

The NAD(P)H:ferredoxin oxidoreductase in carbazole 1,9a-dioxygenase from Janthinobacterium sp. J3 was crystallized and diffraction data were collected to 2.60 Å resolution. Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 Å and belonged to space group P4{sub 2}2{sub 1}2, with unit-cell parameters amore » = b = 158.7, c = 81.4 Å. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 Å resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 Å.« less

Increasing the electron-transfer ability of Cyanidioschyzon merolae ferredoxin by a one-point mutation – A high resolution and Fe-SAD phasing crystal structure analysis of the Asp58Asn mutant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ueno, Yuko; Matsumoto, Takashi; Yamano, Akihito

2013-07-12

Highlights: •A single amino acid change on the ferredoxin surface affects electron transfer. •Precise positions of amide atoms were located utilizing no prior structural data. •Ultra high resolution and SAD phasing may be used for bias-free model building. -- Abstract: Cyanidioschyzon merolae (Cm) is a single cell red algae that grows in rather thermophilic (40–50 °C) and acidic (pH 1–3) conditions. Ferredoxin (Fd) was purified from this algae and characterized as a plant-type [2Fe–2S] Fd by physicochemical techniques. A high resolution (0.97 Å) three-dimensional structure of the CmFd D58N mutant molecule has been determined using the Fe-SAD phasing method tomore » clarify the precise position of the Asn58 amide, as this substitution increases the electron-transfer ability relative to wild-type CmFd by a factor of 1.5. The crystal structure reveals an electro-positive surface surrounding Asn58 that may interact with ferredoxin NADP{sup +} reductase or cytochrome c.« less

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.

PubMed

Webert, Holger; Freibert, Sven-Andreas; Gallo, Angelo; Heidenreich, Torsten; Linne, Uwe; Amlacher, Stefan; Hurt, Ed; Mühlenhoff, Ulrich; Banci, Lucia; Lill, Roland

2014-10-31

Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis.

Atomic Resolution Modeling of the Ferredoxin:[FeFe] Hydrogenase Complex from Chlamydomonas reinhardtii

PubMed Central

Chang, Christopher H.; King, Paul W.; Ghirardi, Maria L.; Kim, Kwiseon

2007-01-01

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H2 from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H2 evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2. PMID:17660315

Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii.

PubMed

Chang, Christopher H; King, Paul W; Ghirardi, Maria L; Kim, Kwiseon

2007-11-01

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Ying; Xu, Feng, E-mail: xuf@xtal.tsinghua.edu.cn; Bell, Stephen G.

Palustrisredoxin reductase (RPA3782, PuR), a flavin-dependent ferredoxin reductase, is an essential component of the Class I cytochrome P450 systems in Rhodopseudomonas palustris CGA009. Crystals of PuR that diffract to 2.2 Å resolution have been obtained. Palustrisredoxin reductase from Rhodopseudomonas palustris CGA009, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (ONFR) family, catalyzes electron transfer from NADH to ferredoxins. It is an essential component of the cytochrome P450 systems in R. palustris CGA009, a model organism with diverse metabolic pathways. Here, the crystallization of palustrisredoxin reductase is reported. The crystals belong to the trigonal space group P3{sub 2}21, with unit-cell parametersmore » a = 107.5, b = 107.5, c = 69.9 Å, and diffract to 2.2 Å resolution on a synchrotron source.« less

The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

PubMed Central

Szekeres, M; Droux, M; Buchanan, B B

1991-01-01

The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form. Images PMID:1705544

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: Involvement of electrostatic interactions and cofactors

PubMed Central

Moczygemba, Charmaine; Guidry, Jesse; Jones, Kathryn L.; Gomes, Cláudio M.; Teixeira, Miguel; Wittung-Stafshede, Pernilla

2001-01-01

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (Tm) of 122°C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (ΔH[Tm]) and Tm from which an upper limit for the heat capacity of unfolding (ΔCP) was determined to be 3.15 ± 0.1 kJ/(mole • K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20°C) observed ([GuSCN]1/2 = 3.1 M; ΔGU[H2O] = 79 ± 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, Tm is 64 ± 1°C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (ΔGU[H2O] = 20 ± 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron–sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron–sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation. PMID:11468351

Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii.

PubMed

Chowdhury, Nilanjan P; Kahnt, Jörg; Buckel, Wolfgang

2015-08-01

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air. © 2015 FEBS.

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

PubMed Central

Negi, Surendra S.; Carol, Andrew A.; Pandya, Shivangi; Braun, Werner; Anderson, Louise E.

2008-01-01

In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex. PMID:17945509

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species.

PubMed

Droux, M; Jacquot, J P; Miginac-Maslow, M; Gadal, P; Huet, J C; Crawford, N A; Yee, B C; Buchanan, B B

1987-02-01

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20

PubMed Central

Price, Morgan N.; Ray, Jayashree; Wetmore, Kelly M.; Kuehl, Jennifer V.; Bauer, Stefan; Deutschbauer, Adam M.; Arkin, Adam P.

2014-01-01

Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13) is sometimes important alongside Rnf and may perform an electron bifurcation to generate more reduced ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1) is important and may generate reduced ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions, although we cannot rule out genetic redundancy. PMID:25400629

The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

NASA Technical Reports Server (NTRS)

Huppe, H. C.; de Lamotte-Guery, F.; Buchanan, B. B.

1990-01-01

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

PubMed Central

Wittkopp, Tyler M.; Warakanont, Jaruswan; Dubini, Alexandra; Catalanotti, Claudia; Kim, Rick G.; Nowack, Eva C. M.; Mackinder, Luke C. M.; Aksoy, Munevver; Page, Mark Dudley; D’Adamo, Sarah; Saroussi, Shai; Heinnickel, Mark; Johnson, Xenie; Richaud, Pierre; Alric, Jean; Boehm, Marko; Jonikas, Martin C.; Benning, Christoph; Merchant, Sabeeha S.; Posewitz, Matthew C.; Grossman, Arthur R.

2015-01-01

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions. PMID:26627249

An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy.

PubMed Central

Geary, P J; Saboowalla, F; Patil, D; Cammack, R

1984-01-01

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis. PMID:6324743

Molybdenum and vanadium do not replace tungsten in the catalytically active forms of the three tungstoenzymes in the hyperthermophilic archaeon Pyrococcus furiosus.

PubMed Central

Mukund, S; Adams, M W

1996-01-01

Three different types of tungsten-containing enzyme have been previously purified from Pyrococcus furiosus (optimum growth temperature, 100 degrees C): aldehyde ferredoxin oxidoreductase (AOR), formaldehyde ferredoxin oxidoreductase (FOR), and glyceraldehyde-3-phosphate oxidoreductase (GAPOR). In this study, the organism was grown in media containing added molybdenum (but not tungsten or vanadium) or added vanadium (but not molybdenum or tungsten). In both cell types, there were no dramatic changes compared with cells grown with tungsten, in the specific activities of hydrogenase, ferredoxin:NADP oxidoreductase, or the 2-keto acid ferredoxin oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Compared with tungsten-grown cells, the specific activities of AOR, FOR, and GAPOR were 40, 74, and 1%, respectively, in molybdenum-grown cells, and 7, 0, and 0%, respectively, in vanadium-grown cells. AOR purified from vanadium-grown cells lacked detectable vanadium, and its tungsten content and specific activity were both ca. 10% of the values for AOR purified from tungsten-grown cells. AOR and FOR purified from molybdenum-grown cells contained no detectable molybdenum, and their tungsten contents and specific activities were > 70% of the values for the enzymes purified from tungsten-grown cells. These results indicate that P. furiosus uses exclusively tungsten to synthesize the catalytically active forms of AOR, FOR, and GAPOR, and active molybdenum- or vanadium-containing isoenzymes are not expressed when the cells are grown in the presence of these other metals. PMID:8550411

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity.

PubMed

Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

2017-03-21

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20

DOE PAGES

Price, Morgan N.; Ray, Jayashree; Wetmore, Kelly M.; ...

2014-10-31

Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13) is sometimes important alongside Rnf and may perform an electron bifurcation to generate more reduced ferredoxin from NADH to allow further ion pumping. Similarly,more » during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1) is important and may generate reduced ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce ferredoxin, thus providing a substrate for Rnf. We found that during hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions, although we cannot rule out genetic redundancy.« less

Ferredoxin is involved in secretion of cytotoxic necrotizing factor 1 across the cytoplasmic membrane in Escherichia coli K1.

PubMed

Yu, Hao; Kim, Kwang Sik

2010-02-01

We previously showed that cytotoxic necrotizing factor 1 (CNF1) contributes to Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) and interacts with the receptor on the surface of HBMEC. CNF1 is the cytoplasmic protein, and it remains incompletely understood how CNF1 is secreted across the inner and outer membranes in E. coli K1. In order to investigate the genetic determinants for secretion of CNF1 in E. coli K1, we performed Tn5 mutagenesis screening by applying beta-lactamase as a reporter to monitor secretion of CNF1. We identified a Tn5 mutant that exhibited no beta-lactamase activity in the culture supernatant and in which the mutated gene encodes a ferredoxin gene (fdx). In the fdx deletion mutant, there was no evidence of translocation of CNF1 into HBMEC. Western blot analysis of the fdx deletion mutant revealed that ferredoxin is involved in translocation of CNF1 across the cytoplasmic membrane. The fdx mutant exhibited significantly decreased invasion of HBMEC, similar to the decreased HBMEC invasion observed with the CNF1 mutant. The failures to secrete CNF1 and invade HBMEC of the fdx mutant were restored to the levels of the parent strain by complementation with fdx. These findings demonstrate for the first time that ferredoxin is involved in secretion of CNF1 across the inner membrane in meningitis-causing E. coli K1.

Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

PubMed Central

Roy, Roopali; Mukund, Swarnalatha; Schut, Gerrit J.; Dunn, Dianne M.; Weiss, Robert; Adams, Michael W. W.

1999-01-01

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS−) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit. PMID:9973343

Trichomonads, hydrogenosomes and drug resistance.

PubMed

Kulda, J

1999-02-01

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.

A Novel Phenanthrene Dioxygenase from Nocardioides sp. Strain KP7: Expression in Escherichia coli

PubMed Central

Saito, Atsushi; Iwabuchi, Tokuro; Harayama, Shigeaki

2000-01-01

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases. PMID:10735855

Identification and functional analysis of cytochrome P450 complement in Streptomyces virginiae IBL14

PubMed Central

2013-01-01

Background As well known, both natural and synthetic steroidal compounds are powerful endocrine disrupting compounds (EDCs) which can cause reproductive toxicity and affect cellular development in mammals and thus are generally regarded as serious contributors to water pollution. Streptomyces virginiae IBL14 is an effective degradative strain for many steroidal compounds and can also catalyze the C25 hydroxylation of diosgenin, the first-ever biotransformation found on the F-ring of diosgenin. Results To completely elucidate the hydroxylation function of cytochrome P450 genes (CYPs) found during biotransformation of steroids by S. virginiae IBL14, the whole genome sequencing of this strain was carried out via 454 Sequencing Systems. The analytical results of BLASTP showed that the strain IBL14 contains 33 CYPs, 7 ferredoxins and 3 ferredoxin reductases in its 8.0 Mb linear chromosome. CYPs from S. virginiae IBL14 are phylogenetically closed to those of Streptomyces sp. Mg1 and Streptomyces sp. C. One new subfamily was found as per the fact that the CYP Svu001 in S. virginiae IBL14 shares 66% identity only to that (ZP_05001937, protein identifer) from Streptomyces sp. Mg1. Further analysis showed that among all of the 33 CYPs in S. virginiae IBL14, three CYPs are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three CYPs with ATP/GTP binding proteins, four CYPs arranged with transcriptional regulatory genes and one CYP located on the upstream of an ATP-binding protein and transcriptional regulators as well as four CYPs associated with other functional genes involved in secondary metabolism and degradation. Conclusions These characteristics found in CYPs from S. virginiae IBL14 show that the EXXR motif in the K-helix is not absolutely conserved in CYP157 family and I-helix not absolutely essential for the CYP structure, too. Experimental results showed that both CYP Svh01 and CYP Svu022 are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively. PMID:23442312

Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.

PubMed

Chowdhury, Nilanjan Pal; Mowafy, Amr M; Demmer, Julius K; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

2014-02-21

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans*

PubMed Central

Chowdhury, Nilanjan Pal; Mowafy, Amr M.; Demmer, Julius K.; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

2014-01-01

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD+ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH− is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH− by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD⨪, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH− that converts crotonyl-CoA to butyryl-CoA. PMID:24379410

Towards a mechanistic and physiological understanding of a ferredoxin disulfide reductase from the domains Archaea and Bacteria.

PubMed

Prakash, Divya; Walters, Karim A; Martinie, Ryan J; McCarver, Addison C; Kumar, Adepu K; Lessner, Daniel J; Krebs, Carsten; Golbeck, John H; Ferry, James G

2018-05-02

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster.  UV-Vis, EPR and Mӧssbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active-site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR advancing the physiological understanding of FDRs role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show FDR homologs are widespread in diverse microbes from the domain Bacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I.

PubMed

Hedderich, Reiner

2004-02-01

[NiFe] hydrogenases are well-characterized enzymes that have a key function in the H2 metabolism of various microorganisms. In the recent years a subfamily of [NiFe] hydrogenases with unique properties has been identified. The members of this family form multisubunit membrane-bound enzyme complexes composed of at least four hydrophilic and two integral membrane proteins. These six conserved subunits, which built the core of these hydrogenases, have closely related counterparts in energy-conserving NADH:quinone oxidoreductases (complex I). However, the reaction catalyzed by these hydrogenases differs significantly from the reaction catalyzed by complex I. For some of these hydrogenases the physiological role is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or poly-ferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase family mainly function to provide the cell with reduced ferredoxin with H2 as electron donor in a reaction driven by reverse electron transport. As complex I these hydrogenases function as ion pumps and have therefore been designated as energy-converting [NiFe] hydrogenases.

Engineering electron metabolism to increase ethanol production in Clostridium thermocellum.

PubMed

Lo, Jonathan; Olson, Daniel G; Murphy, Sean Jean-Loup; Tian, Liang; Hon, Shuen; Lanahan, Anthony; Guss, Adam M; Lynd, Lee R

2017-01-01

The NfnAB (NADH-dependent reduced ferredoxin: NADP + oxidoreductase) and Rnf (ion-translocating reduced ferredoxin: NAD + oxidoreductase) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) + . Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes had little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted. Copyright © 2016 International Metabolic Engineering Society. All rights reserved.

Energy Conservation Associated with Ethanol Formation from H2 and CO2 in Clostridium autoethanogenum Involving Electron Bifurcation

PubMed Central

Mock, Johanna; Zheng, Yanning; Mueller, Alexander P.; Ly, San; Tran, Loan; Segovia, Simon; Nagaraju, Shilpa; Köpke, Michael; Dürre, Peter

2015-01-01

ABSTRACT Most acetogens can reduce CO2 with H2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO2 and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD+ oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2 and H2. IMPORTANCE Ethanol formation from syngas (H2, CO, and CO2) and from H2 and CO2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2 and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase). PMID:26148714

[Brownian dynamics simulations of protein-protein interactions in photosynthetic electron transport chain].

PubMed

Khruschev, S S; Abaturova, A M; Diakonova, A N; Fedorov, V A; Ustinin, D M; Kovalenko, I B; Riznichenko, G Yu; Rubin, A B

2015-01-01

The application of Brownian dynamics for simulation of transient protein-protein interactions is reviewed. The review focuses on theoretical basics of Brownian dynamics method, its particular implementations, advantages and drawbacks of the method. The outlook for future development of Brownian dynamics-based simulation techniques is discussed. Special attention is given to analysis of Brownian dynamics trajectories. The second part of the review is dedicated to the role of Brownian dynamics simulations in studying photosynthetic electron transport. Interactions of mobile electron carriers (plastocyanin, cytochrome c6, and ferredoxin) with their reaction partners (cytochrome b6f complex, photosystem I, ferredoxin:NADP-reductase, and hydrogenase) are considered.

Fat Metabolism in Higher Plants

PubMed Central

Jacobson, Bruce S.; Jaworski, J. G.; Stumpf, P. K.

1974-01-01

Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds. PMID:16658913

Homology modeling and in silico site directed mutagenesis of pyruvate ferredoxin oxidoreductase from Clostridium thermocellum.

PubMed

Saranyah, Kannuchamy; Kalva, Sukesh; Mukund, Nisha; Singh, Sanjeev Kumar; Saleena, Lilly M

2015-01-01

Pyruvate ferredoxin oxidoreductase is the crucial enzyme that involves in bioethanol synthesis pathway of Clostridium thermocellum. It is an ethanologenic organism but has been investigated less on its enzyme structure. The amino acid sequence of Pyruvate ferredoxin oxidoreductase was derived from UNIPROT and the screened crystal structure was taken as the template for homology modeling using MODELLER 9V11. The model was loop refined and was validated using RMSD, ProSA and PROCHECK. The docking and per residue interaction studies were carried out to elucidate the interaction energies of amino acid residues with pyruvate. To enhance the binding of pyruvate with the enzyme, mutation studies were carried out by replacing Thr31 as it had a less interaction energy. Out of 10 mutants, T31N, T31Q and T31G were selected using potential energy and the residual energy calculations. Five nanoseconds explicit MD simulations were run for apo, wild type and mutants T31N, T31Q and T31G using Desmond. RMSD, RMSF, distance plots and H-bonds analysis proved T31G to be a favorable mutant for binding of pyruvate. Thus, modeling PFOR would help in profound understanding of its structural clefts and mutation studies would aid in improving the enzyme efficiency.

A physiological perspective on the origin and evolution of photosynthesis

PubMed Central

Martin, William F; Bryant, Donald A; Beatty, J Thomas

2017-01-01

Abstract The origin and early evolution of photosynthesis are reviewed from an ecophysiological perspective. Earth's first ecosystems were chemotrophic, fueled by geological H2 at hydrothermal vents and, required flavin-based electron bifurcation to reduce ferredoxin for CO2 fixation. Chlorophyll-based phototrophy (chlorophototrophy) allowed autotrophs to generate reduced ferredoxin without electron bifurcation, providing them access to reductants other than H2. Because high-intensity, short-wavelength electromagnetic radiation at Earth's surface would have been damaging for the first chlorophyll (Chl)-containing cells, photosynthesis probably arose at hydrothermal vents under low-intensity, long-wavelength geothermal light. The first photochemically active pigments were possibly Zn-tetrapyrroles. We suggest that (i) after the evolution of red-absorbing Chl-like pigments, the first light-driven electron transport chains reduced ferredoxin via a type-1 reaction center (RC) progenitor with electrons from H2S; (ii) photothioautotrophy, first with one RC and then with two, was the bridge between H2-dependent chemolithoautotrophy and water-splitting photosynthesis; (iii) photothiotrophy sustained primary production in the photic zone of Archean oceans; (iv) photosynthesis arose in an anoxygenic cyanobacterial progenitor; (v) Chl a is the ancestral Chl; and (vi), anoxygenic chlorophototrophic lineages characterized so far acquired, by horizontal gene transfer, RCs and Chl biosynthesis with or without autotrophy, from the architects of chlorophototrophy—the cyanobacterial lineage. PMID:29177446

Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

PubMed Central

Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

2015-01-01

Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC. PMID:26287179

Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria.

PubMed

Sauber, K; Fröhner, C; Rosenberg, G; Eberspächer, J; Lingens, F

1977-03-15

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000,red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.

Amino acid residues in Anabaena ferredoxin crucial to interaction with ferredoxin-NADP+ reductase: site-directed mutagenesis and laser flash photolysis.

PubMed

Hurley, J K; Salamon, Z; Meyer, T E; Fitch, J C; Cusanovich, M A; Markley, J L; Cheng, H; Xia, B; Chae, Y K; Medina, M

1993-09-14

Ferredoxin (Fd) functions in photosynthesis to transfer electrons from photosystem I to ferredoxin-NADP+ reductase (FNR). We have made several site-directed mutants of Anabaena 7120 Fd and have used laser flash photolysis to investigate the effects of these mutations on the kinetics of reduction of oxidized Fd by deazariboflavin semiquinone (dRfH.) and the reduction of oxidized Anabaena FNR by reduced Fd. None of the mutations influenced the second-order rate constant for dRfH. reduction by more than a factor of 2, suggesting that the ability of the [2Fe-2S] cluster to participate in electron transfer was not seriously affected. In contrast, a surface charge reversal mutation, E94K, resulted in a 20,000-fold decrease in the second-order rate constant for electron transfer from Fd to FNR, whereas a similar mutation at an adjacent site, E95K, produced little or no change in reaction rate constant compared to wild-type Fd. Such a dramatic difference between contiguous surface mutations suggests a very precise surface complementarity at the protein-protein interface. Mutations introduced at F65 (F65I and F65A) also decreased the rate constant for the Fd/FNR electron transfer reaction by more than 3 orders of magnitude. Spectroscopic and thermodynamic measurements with both the E94 and F65 mutants indicated that the kinetic differences cannot be ascribed to changes in gross conformation, redox potential, or FNR binding constant but rather reflect the protein-protein interactions that control electron transfer. Several mutations at other sites in the vicinity of E94 and F65 (R42, T48, D68, and D69) resulted in little or no perturbation of the Fd/FNR interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

PubMed Central

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P.; Köpke, Michael

2013-01-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP+ with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0′ = −520 mV). PMID:23893107

Spectroscopic and Redox Studies of Valence-Delocalized [Fe2S2]+ Centers in Thioredoxin-Like Ferredoxins

PubMed Central

Subramanian, Sowmya; Duin, Evert C.; Fawcett, Sarah E. J.; Armstrong, Fraser A.; Meyer, Jacques; Johnson, Michael K.

2015-01-01

Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2]+ clusters, which constitute a fundamental building block of all higher nuclearity Fe-S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2]2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV-visible-NIR absorption, CD and variable-temperature MCD, and protein-film electrochemistry. The results indicate that the [Fe2S2]+ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0, due to protonation of the coordinated serinate residue. However, freezing high-pH samples results in partial or full conversion from valence-delocalized S = 9/2 to valence-localized S = 1/2 [Fe2S2]+ clusters. MCD saturation magnetization data for valence-delocalized S = 9/2 [Fe2S2]+ centers facilitated determination of transition polarizations and thereby assignments of low-energy MCD bands associated with the Fe−Fe interaction. The assignments provide experimental assessment of the double exchange parameter, B, for valence-delocalized [Fe2S2]+ centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized S = 9/2 [Fe2S2]+ fragments in higher nuclearity Fe-S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe-S clusters in general is discussed in light of these results. PMID:25790339

NADP-specific electron-bifurcating [FeFe]-hydrogenase in a functional complex with formate dehydrogenase in Clostridium autoethanogenum grown on CO.

PubMed

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P; Köpke, Michael; Thauer, Rudolf K

2013-10-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0' = -520 mV).

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii: Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site▿

PubMed Central

Imkamp, Frank; Biegel, Eva; Jayamani, Elamparithi; Buckel, Wolfgang; Müller, Volker

2007-01-01

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes H2-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits α (EtfA) and β (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD+ oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na+ pump. These data suggest the following electron transport chain: H2 → ferredoxin → NAD+ → Etf → caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD+ reduction catalyzed by Rnf. PMID:17873051

Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.

PubMed

Seo, Daisuke; Soeta, Takahiro; Sakurai, Hidehiro; Sétif, Pierre; Sakurai, Takeshi

2016-06-01

Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (<10 s(-1)). Mixing BsFd(red) with BsFNR(ox) induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s(-1) at 10°C, which increased as BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs. Copyright © 2016 Elsevier B.V. All rights reserved.

Ab initio single and multideterminant methods used in the determination of reduction potentials and magnetic properties of Rieske ferredoxins

NASA Astrophysics Data System (ADS)

Powers, Nathan Lee

2008-10-01

The [Fe2S2]2+/[Fe2S 2]+ electronic structure of seven Rieske protein active sites (bovine mitochondrial cytochrome bc1 complex, spinach chloroplast cytochrome b6f complex, Rieske-type ferredoxin associated with biphenyl dioxygenase from Burkholderia cepacia, yeast cytochrome bcl complex from Saccharomyces cerevisiae, Rieske subunit of arsenite oxidase from Alcaligenes faecalis, respiratory-type Rieske protein from Thermus thermophilus, and Rieske protein II (soxF) from Sulfolobus acidocaldarius), which lie in a reduction potential range from -150 mV to 375 mV, have been studied by both single and multi-determinant quantum mechanical methods. Calculated reduction potentials and magnetic properties are found comparable to experimental values.

A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase

DOE PAGES

Wei, Yifeng; Li, Bin; Prakash, Divya; ...

2015-11-04

Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRsmore » reflects the diversity of electron carriers used in anaerobic metabolism« less

The Arnon-Buchanan cycle: a retrospective, 1966-2016.

PubMed

Buchanan, Bob B; Sirevåg, Reidun; Fuchs, Georg; Ivanovsky, Ruslan N; Igarashi, Yasuo; Ishii, Masaharu; Tabita, F Robert; Berg, Ivan A

2017-11-01

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO 2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO 2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO 2 , reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO 2 . Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

Insight Into the Radical Mechanism of Phycocyanobilin-Ferredoxin Oxidoreductase (Pcya) Revealed By X-Ray Crystallography And Biochemical Measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu, S.-L.; Rockwell, N.; Lagarias, J.C.

2007-07-13

The X-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) from the cyanobacterium Nostoc sp. PCC7120 has been solved at 2.5 angstrom resolution. A comparative analysis of this structure with those recently reported for substrate-bound and substrate-free forms of PcyA from the cyanobacterium Synechocystis sp. PCC6803 (Hagiwara et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 27-32; Hagiwara et al. (2006) FEBS Lett. 580, 3823-3828) provides a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. On the basis of these structures and the biochemical analysis ofmore » site-directed mutants of Nostoc PcyA, including mutants reported in recent studies (Tu et al. (2006) J. Biol. Chem. 281, 3127-3136) as well as mutants described in this study, a revised mechanism for the PcyA-mediated four-electron reduction of biliverdin IX{alpha} to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates is proposed. The mechanistic insight of these studies, along with homology modeling, have provided new insight into the catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family that are widespread in oxygenic photosynthetic organisms.« less

Expression of Critical Sulfur- and Iron-Oxidation Genes and the Community Dynamics During Bioleaching of Chalcopyrite Concentrate by Moderate Thermophiles.

PubMed

Zhou, Dan; Peng, Tangjian; Zhou, Hongbo; Liu, Xueduan; Gu, Guohua; Chen, Miao; Qiu, Guanzhou; Zeng, Weimin

2015-07-01

Sulfate adenylyltransferase gene and 4Fe-4S ferredoxin gene are the key genes related to sulfur and iron oxidations during bioleaching system, respectively. In order to better understand the bioleaching and microorganism synergistic mechanism in chalcopyrite bioleaching by mixed culture of moderate thermophiles, expressions of the two energy metabolism genes and community dynamics of free and attached microorganisms were investigated. Specific primers were designed for real-time quantitative PCR to study the expression of these genes. Real-time PCR results showed that sulfate adenylyltransferase gene was more highly expressed in Sulfobacillus thermosulfidooxidans than that in Acidithiobacillus caldus, and expression of 4Fe-4S ferredoxin gene was higher in Ferroplasma thermophilum than that in S. thermosulfidooxidans and Leptospirillum ferriphilum. The results indicated that in the bioleaching system of chalcopyrite concentrate, sulfur and iron oxidations were mainly performed by S. thermosulfidooxidans and F. thermophilum, respectively. The community dynamics results revealed that S. thermosulfidooxidans took up the largest proportion during the whole period, followed by F. thermophilum, A. caldus, and L. ferriphilum. The CCA analysis showed that 4Fe-4S ferredoxin gene expression was mainly affected (positively correlated) by high pH and elevated concentration of ferrous ion, while no factor was observed to prominently influence the expression of sulfate adenylyltransferase gene.

Evolution of major metabolic innovations in the Precambrian

NASA Technical Reports Server (NTRS)

Barnabas, J.; Schwartz, R. M.; Dayhoff, M. O.

1982-01-01

A combination of information on the metabolic capabilities of prokaryotes with a composite phylogenetic tree depicting an overview of prokaryote evolution based on the sequences of bacterial ferredoxin, 2Fe-2S ferredoxin, 5S ribosomal RNA, and c-type cytochromes shows three zones of major metabolic innovation in the Precambrian. The middle of these, which reflects the genesis of oxygen-releasing photosynthesis and aerobic respiration, links metabolic innovations of the anaerobic stem on the one hand and, on the other, proliferation of aerobic bacteria and the symbiotic associations leading to the eukaryotes. Those pathways where information on the structure of the enzymes is known are especially considered. Halobacterium and Thermoplasma (archaebacteria) do not belong to a totally independent line on the basis of the composite tree but branch from the eukaryote cytoplasmic line.

Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review

PubMed Central

Buckel, Wolfgang; Thauer, Rudolf K.

2018-01-01

Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0′ = −420 mV) with NADH (−320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (−10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms—in which they have been found—are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered. PMID:29593673

Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH

PubMed Central

Losey, Nathaniel A.; Mus, Florence; Peters, John W.; Le, Huynh M.

2017-01-01

ABSTRACT Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by Syntrophomonas wolfei would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that S. wolfei has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in E. coli, demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms. PMID:28802265

Energetic basis on interactions between ferredoxin and ferredoxin NADP{sup +} reductase at varying physiological conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Misaki; Kim, Ju Yaen; Kume, Satoshi

In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP{sup +} reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.0), NaCl concentrations (0–200 mM), and temperatures (19–28 °C) for mimicking physiological conditions in chloroplasts. Energetically unfavorable endothermic enthalpy changes were accompanied by Fd:FNR complexation at all conditions. This energetic cost was compensated by favorable entropy changes, balanced by conformational and hydrational entropy. Increases inmore » the NaCl concentration and pH weakened interprotein affinity due to the less contribution of favorable entropy change regardless of energetic gains from enthalpy changes, suggesting that entropy drove complexation and modulated affinity. Effects of temperature on binding thermodynamics were much smaller than those of pH and NaCl. NaCl concentration and pH-dependent enthalpy and heat capacity changes provided clues for distinct binding modes. Moreover, decreases in the enthalpy level in the Hammond's postulate-based energy landscape implicated kinetic advantages for FNR activity. All these energetic interplays were comprehensively demonstrated by the driving force plot with the enthalpy-entropy compensation which may serve as an energetic buffer against outer stresses. We propose that high affinity at pH 6.0 may be beneficial for protection from proteolysis of Fd and FNR in rest states, and moderate affinity at pH 8.0 and proper NaCl concentrations with smaller endothermic enthalpy changes may contribute to increase FNR activity. - Highlights: • Energetics of Fd:FNR binding were examined by considering physiological conditions. • NaCl and pH affect energetically Fd:FNR binding with minimal effects of temperature. • Enthalpy and heat capacity may modulate binding kinetics and modes for FNR activity. • Entropy drives complexation by overcoming unfavorable enthalpy and tunes affinity. • Driving force plot reveals condition-dependent energetic interplays for complexation.« less

Identification of aldolase and ferredoxin reductase within the dbt operon of Burkholderia fungorum DBT1.

PubMed

Piccoli, Stefano; Andreolli, Marco; Giorgetti, Alejandro; Zordan, Fabio; Lampis, Silvia; Vallini, Giovanni

2014-05-01

Burkholderia fungorum DBT1, first isolated from settling particulate matter of an oil refinery wastewater, is a bacterial strain which has been shown capable of utilizing several polycyclic aromatic hydrocarbons (PAHs) including dibenzothiophene (DBT). In particular, this microbe is able to efficiently degrade DBT through the Kodama pathway. Previous investigations have lead to the identification of six genes, on a total of eight, required for DBT degradation. In the present study, a combined experimental/computational approach was adopted to identify and in silico characterize the two missing genes, namely a ferredoxin reductase and a hydratase-aldolase. Thus, the finding of all enzymatic components of the Kodama pathway in B. fungorum DBT1 makes this bacterial strain amenable for possible exploitation in soil bioremediation protocols. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei.

PubMed

Worm, Petra; Stams, Alfons J M; Cheng, Xu; Plugge, Caroline M

2011-01-01

Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO(2) to formylmethanofuran. The best candidates for F(420)-dependent N(5),N(10)-methyl-H(4) MPT and N(5),N(10),-methylene-H(4)MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases.

Crystal Structure and Biochemical Characterization of Chlamydomonas FDX2 Reveal Two Residues that, When Mutated, Partially Confer FDX2 the Redox Potential and Catalytic Properties of FDX1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boehm, Marko; Alahuhta, Markus; Mulder, David W.

2015-11-03

The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2more » protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H 2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.« less

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

PubMed

Cho, Min Seok; Lee, Jang Ha; Her, Nam Han; Kim, Changkug; Seol, Young-Joo; Hahn, Jang Ho; Baeg, Ji Hyoun; Kim, Hong Gi; Park, Dong Suk

2012-06-01

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

Role of CadC and CadD in the 2,4-dichlorophenoxyacetic acid oxygenase system of Sphingomonas agrestis 58-1.

PubMed

Kijima, Kumiko; Mita, Hajime; Kawakami, Mitsuyasu; Amada, Kei

2018-02-02

In the present study, we confirm that 2,4-dichlorophenoxyacetic acid (2,4-D) oxygenase from Sphingomonas agrestis 58-1 belongs to the family of Rieske non-heme iron aromatic ring-hydroxylating oxygenases, which comprise a core enzyme (oxygenase), ferredoxin, and oxidoreductase. It has previously been shown that cadAB genes are necessary for the conversion of 2,4-D to 2,4-dichlorophenol; however, the respective roles of ferredoxin and oxidoreductase in the 2,4-D oxygenase system from S. agrestis 58-1 remain unknown. Using nucleotide sequence analysis of the plasmid pCADAB1 from Sphingomonas sp. ERG5, which degrades 4-chloro-2-methylphenoxyacetic acid and 2,4-D, Nielsen et al. identified orf95, upstream of cadA, and orf98, downstream of cadB, which were predicted and designated as cadD (oxidoreductase) and cadC (ferredoxin), respectively (Nielsen et al., PLoS One, 8, 1-9, 2013). These designations were the result of sequence analysis; therefore, we constructed an expression system of CadABC and CadABCD in Escherichia coli and assayed their enzyme activities. Our findings indicate that CadC is essential for the activity of 2,4-D oxygenase and CadD promotes CadABC activity in recombinant E. coli cells. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Ferredoxin:thioredoxin reductase (FTR) links the regulation of oxygenic photosynthesis to deeply rooted bacteria.

PubMed

Balsera, Monica; Uberegui, Estefania; Susanti, Dwi; Schmitz, Ruth A; Mukhopadhyay, Biswarup; Schürmann, Peter; Buchanan, Bob B

2013-02-01

Uncovered in studies on photosynthesis 35 years ago, redox regulation has been extended to all types of living cells. We understand a great deal about the occurrence, function, and mechanism of action of this mode of regulation, but we know little about its origin and its evolution. To help fill this gap, we have taken advantage of available genome sequences that make it possible to trace the phylogenetic roots of members of the system that was originally described for chloroplasts-ferredoxin, ferredoxin:thioredoxin reductase (FTR), and thioredoxin as well as target enzymes. The results suggest that: (1) the catalytic subunit, FTRc, originated in deeply rooted microaerophilic, chemoautotrophic bacteria where it appears to function in regulating CO(2) fixation by the reverse citric acid cycle; (2) FTRc was incorporated into oxygenic photosynthetic organisms without significant structural change except for addition of a variable subunit (FTRv) seemingly to protect the Fe-S cluster against oxygen; (3) new Trxs and target enzymes were systematically added as evolution proceeded from bacteria through the different types of oxygenic photosynthetic organisms; (4) an oxygenic type of regulation preceded classical light-dark regulation in the regulation of enzymes of CO(2) fixation by the Calvin-Benson cycle; (5) FTR is not universally present in oxygenic photosynthetic organisms, and in certain early representatives is seemingly functionally replaced by NADP-thioredoxin reductase; and (6) FTRc underwent structural diversification to meet the ecological needs of a variety of bacteria and archaea.

Method of controlled reduction of nitroaromatics by enzymatic reaction with oxygen sensitive nitroreductase enzymes

DOEpatents

Shah, Manish M.; Campbell, James A.

1998-01-01

A method for the controlled reduction of nitroaromatic compounds such as nitrobenzene and 2,4,6-trinitrotoluene by enzymatic reaction with oxygen sensitive nitroreductase enzymes, such as ferredoxin NADP oxidoreductase.

Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains.

PubMed

Jensen, Kenneth; Johnston, Jonathan B; de Montellano, Paul R Ortiz; Møller, Birger Lindberg

2012-02-01

The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP(+) to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement.

PubMed Central

Martín, A E; Burgess, B K; Stout, C D; Cash, V L; Dean, D R; Jensen, G M; Stephens, P J

1990-01-01

Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure. PMID:2153958

Bundle-sheath thylakoids from NADP-malic enzyme-type C4 plants require an exogenous electron donor for enzyme light activation.

PubMed

Lavergne, D; Droux, M; Jacquot, J P; Miginiac-Maslow, M; Champigny, M L; Gadal, P

1985-10-01

Light activation of either NADP-malate dehydrogenase (EC 1.1.1.82) or fructose-1,6-bisphosphate phosphatase (EC 3.1.3.11) was assayed in a reconstituted chloroplastic, system comprising the isolated proteins of the ferredoxin-thioredoxin light-activation system and thylakoids from either mesophyll or bundle-sheath tissues of different C4 plants. While C4-plant thylakoids functionned almost equally well with C3-or C4-plant proteins, the photosyntem-II-deficient bundle-sheath thylakoids from the NADP-malic enzyme type, were unable to perform enzyme photoactivation unless supplemented with an electron donor to photosystem I. Bundle-sheath thylakoids isolated from plants showing no photosystem-II deficiency did not require such an addition. The results are discussed with respect to a possible requirement for a physiological reductant of ferredoxin for enzyme light activation in bundle-sheath, tissues.

Energy-converting [NiFe] hydrogenases: more than just H2 activation.

PubMed

Hedderich, Reiner; Forzi, Lucia

2005-01-01

The well-characterized [NiFe] hydrogenases have a key function in the H2 metabolism of various microorganisms. A subfamily of the [NiFe] hydrogenases with unique properties has recently been identified. The six conserved subunits that build the core of these membrane-bound hydrogenases share sequence similarity with subunits that form the catalytic core of energy-conserving NADH:quinone oxidoreductases (complex I). The physiological role of some of these hydrogenases is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or polyferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase subfamily mainly function in providing the cell with reduced ferredoxin using H2 as electron donor in a reaction driven by reverse electron transport. These hydrogenases have therefore been designated as energy-converting [NiFe] hydrogenases. Copyright 2005 S. Karger AG, Basel.

An Ancient Pathway Combining Carbon Dioxide Fixation with the Generation and Utilization of a Sodium Ion Gradient for ATP Synthesis

PubMed Central

Poehlein, Anja; Schmidt, Silke; Kaster, Anne-Kristin; Goenrich, Meike; Vollmers, John; Thürmer, Andrea; Bertsch, Johannes; Schuchmann, Kai; Voigt, Birgit; Hecker, Michael; Daniel, Rolf; Thauer, Rudolf K.; Gottschalk, Gerhard; Müller, Volker

2012-01-01

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes. PMID:22479398

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1

PubMed Central

Kounosu, Asako; Hasegawa, Kazuya; Iwasaki, Toshio; Kumasaka, Takashi

2010-01-01

The hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe–2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-­terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 Å resolution and belonged to the tetragonal space group P43212, with unit-cell parameters a = 60.72, c = 83.31 Å. The asymmetric unit contains one protein molecule. PMID:20606288

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1.

PubMed

Kounosu, Asako; Hasegawa, Kazuya; Iwasaki, Toshio; Kumasaka, Takashi

2010-07-01

The hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe-2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 A resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 60.72, c = 83.31 A. The asymmetric unit contains one protein molecule.

The drug ornidazole inhibits photosynthesis in a different mechanism described for protozoa and anaerobic bacteria.

PubMed

Marcus, Yehouda; Tal, Noam; Ronen, Mordechai; Carmieli, Raanan; Gurevitz, Michael

2016-12-01

Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP + reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

PubMed Central

Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

2017-01-01

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the “core” nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin–NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR–ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops. PMID:28193863

The End of the Line: Can Ferredoxin and Ferredoxin NADP(H) Oxidoreductase Determine the Fate of Photosynthetic Electrons?

PubMed Central

Goss, Tatjana; Hanke, Guy

2014-01-01

At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems. PMID:24678667

Electronic, Magnetic, and Redox Properties of [MFe(3)S(4)] Clusters (M = Cd, Cu, Cr) in Pyrococcus furiosus Ferredoxin.

PubMed

Staples, Christopher R.; Dhawan, Ish K.; Finnegan, Michael G.; Dwinell, Derek A.; Zhou, Zhi Hao; Huang, Heshu; Verhagen, Marc F. J. M.; Adams, Michael W. W.; Johnson, Michael K.

1997-12-03

The ground- and excited-state properties of heterometallic [CuFe(3)S(4)](2+,+), [CdFe(3)S(4)](2+,+), and [CrFe(3)S(4)](2+,+) cubane clusters assembled in Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR and variable-temperature/variable-field magnetic circular dichroism (MCD) studies. The results indicate Cd(2+) incorporation into [Fe(3)S(4)](0,-) cluster fragments to yield S = 2 [CdFe(3)S(4)](2+) and S = (5)/(2) [CdFe(3)S(4)](+) clusters and Cu(+) incorporation into [Fe(3)S(4)](+,0) cluster fragments to yield S = (1)/(2) [CuFe(3)S(4)](2+) and S = 2 [CuFe(3)S(4)](+) clusters. This is the first report of the preparation of cubane type [CrFe(3)S(4)](2+,+) clusters, and the combination of EPR and MCD results indicates S = 0 and S = (3)/(2) ground states for the oxidized and reduced forms, respectively. Midpoint potentials for the [CdFe(3)S(4)](2+,+), [CrFe(3)S(4)](2+,+), and [CuFe(3)S(4)](2+,+) couples, E(m) = -470 +/- 15, -440 +/- 10, and +190 +/- 10 mV (vs NHE), respectively, were determined by EPR-monitored redox titrations or direct electrochemistry at a glassy carbon electrode. The trends in redox potential, ground-state spin, and electron delocalization of [MFe(3)S(4)](2+,+) clusters in P. furiosus ferredoxin are discussed as a function of heterometal (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Tl).

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

PubMed Central

Sridhar Prasad, G.; Kresge, N.; Muhlberg, A. B.; Shaw, A.; Jung, Y. S.; Burgess, B. K.; Stout, C. D.

1998-01-01

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258. PMID:9865948

Method of controlled reduction of nitroaromatics by enzymatic reaction with oxygen sensitive nitroreductase enzymes

DOEpatents

Shah, M.M.; Campbell, J.A.

1998-07-07

A method is described for the controlled reduction of nitroaromatic compounds such as nitrobenzene and 2,4,6-trinitrotoluene by enzymatic reaction with oxygen sensitive nitroreductase enzymes, such as ferredoxin NADP oxidoreductase. 6 figs.

Evolution of prokaryote and eukaryote lines inferred from sequence evidence

NASA Technical Reports Server (NTRS)

Hunt, L. T.; George, D. G.; Yeh, L.-S.; Dayhoff, M. O.

1984-01-01

This paper describes the evolution of prokaryotes and early eukaryotes, including their symbiotic relationships, as inferred from phylogenetic trees of bacterial ferredoxin, 5S ribosomal RNA, ribulose-1,5-biphosphate carboxylase large chain, and mitochondrial cytochrome oxidase polypeptide II.

Phylogenetic Evidence for H2 based Electron Bifurcation In Early Life

NASA Astrophysics Data System (ADS)

Adams, M. W.; Boyd, E. S.; Schut, G.; Peters, J.

2012-12-01

Energy conservation is a fundamental underpinning of all life and is accomplished by electron transport phosphorylation and/or substrate level phosphorylation. A third mechanism, known as flavin-based electron bifurcation, has recently been established as a mechanism by which life can conserve energy. In this mechanism, a flavin-containing multienzyme complex catalyzes the thermodynamically unfavorable reduction of low potential ferredoxin using electron donors with higher potentials, such as NAD(P)H or H2. Such endergonic reactions are driven forward through the simultaneous oxidation of the electron donor with higher potential acceptors such as NAD+ or heterodisulfide. Membrane associated energy converting [NiFe]hydrogenases (Ech, Eha) link the oxidation of ferredoxin with the production of H2 and in the process conserve energy in the form of an ion (Na+/H+) gradient. Ech/Eha exhibit a modular composition represented by a Na+/H+ antiporter domain and a [NiFe] hydrogenase domain. In addition, Ech/Eha can be accompanied by a formate dehyrogenase, carbon monoxide dehydrogenase, or an FAD/NAD(P)H module that enables coupling with these substrates. Representatives of Ech/Eha have been identified among anaerobic Archaea and Bacteria, including deeply rooted methanogens, sulfur-reducing Crenarcheota/Euryarchaeota as well as Thermotogae. Ech exhibit extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family (Nuo, Fpo). Metabolically, Ech generally couple the oxidation of carbon monooxide, formate or ferredoxin to the production of H2. In contrast, the Eha complex couples the translocation of Na+ and the oxidation of H2 to the reduction of ferredoxin, which is then available for the reduction of CO2 in methanogens. In the case of Eha, the gradient of Na+/H+ produced through translocation coupled to ferredoxin oxidation can in be used to drive the phosphorylation of ADP via an ATP synthase complex, thereby representing one of the most simple forms of ATP production supporting life. This finding is consistent with phylogenetic analyses which indicate a close phylogenetic relationship between Ech/Eha and Nuo/Fpo, with [NiFe]-hydrogenase typically involved in H2 oxidation forming divergent lineages. This suggests that Ech/Eha are most likely to represent an ancestor of the Complex I family and the [NiFe]-hydrogenase family. A concatenation and phylogenetic analysis of the large and small subunits of Ech and Nuo was performed and and additional modules enabling coupling with CO2 (Eha), CO (Ech-CODH), and formate (Ech-Fdh) through H2-based electron bifurcation were overlaid on this phylogeny. The results suggest an origin for H2-based electron bifurcation via Ech/Eha among CO2 reducing hydrogenotrophic methanogenic Archaea or sulfur-reducing Archaea, with evolution towards coupling with formate and CO. These results provide insight into the evolutionary relationships between electron bifurcation-enabled ionic gradients capable of driving phosphorylation and electron transport-based phosphorylation. Moreover, these observations suggest that electron bifurcation may have been important in overcoming key metabolic bottlenecks and may have enabled life to access small energetic gradients to support metabolism on early Earth.

Properties of Intermediates in the Catalytic Cycle of Oxalate Oxidoreductase and Its Suicide Inactivation by Pyruvate

PubMed Central

2017-01-01

Oxalate:ferredoxin oxidoreductase (OOR) is an unusual member of the thiamine pyrophosphate (TPP)-dependent 2-oxoacid:ferredoxin oxidoreductase (OFOR) family in that it catalyzes the coenzyme A (CoA)-independent conversion of oxalate into 2 equivalents of carbon dioxide. This reaction is surprising because binding of CoA to the acyl-TPP intermediate of other OFORs results in formation of a CoA ester, and in the case of pyruvate:ferredoxin oxidoreductase (PFOR), CoA binding generates the central metabolic intermediate acetyl-CoA and promotes a 105-fold acceleration of the rate of electron transfer. Here we describe kinetic, spectroscopic, and computational results to show that CoA has no effect on catalysis by OOR and describe the chemical rationale for why this cofactor is unnecessary in this enzymatic transformation. Our results demonstrate that, like PFOR, OOR binds pyruvate and catalyzes decarboxylation to form the same hydroxyethylidine–TPP (HE–TPP) intermediate and one-electron transfer to generate the HE–TPP radical. However, in OOR, this intermediate remains stranded at the active site as a covalent inhibitor. These and other results indicate that, like other OFOR family members, OOR generates an oxalate-derived adduct with TPP (oxalyl-TPP) that undergoes decarboxylation and one-electron transfer to form a radical intermediate remaining bound to TPP (dihydroxymethylidene–TPP). However, unlike in PFOR, where CoA binding drives formation of the product, in OOR, proton transfer and a conformational change in the “switch loop” alter the redox potential of the radical intermediate sufficiently to promote the transfer of an electron into the iron–sulfur cluster network, leading directly to a second decarboxylation and completing the catalytic cycle. PMID:28514140

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum.

PubMed

Feng, Xueyang; Tang, Kuo-Hsiang; Blankenship, Robert E; Tang, Yinjie J

2010-12-10

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80∼100 units for mixotrophic cultures grown on acetate and 200∼230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.

Antiprotozoal Constituents from Annona cherimola Miller, a Plant Used in Mexican Traditional Medicine for the Treatment of Diarrhea and Dysentery

PubMed Central

Calzada, Fernando; Correa-Basurto, Jose; Barbosa, Elizabeth; Mendez-Luna, David; Yepez-Mulia, Lilian

2017-01-01

Background: Annona cherimola Miller (Annonaceae) is a medicinal plant frequently recommended in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. Objective: This work was undertaken to obtain information that support the traditional use of A. cherimola, on pharmacological basis using in vitro and computational experiments. Material and Methods: Bioassay-guided fractionation of the ethanol extract of the leaves of A. cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin, and rutin. Results: The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different than metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase (PFOR). Conclusion: Considering that PFOR is a target of metronidazole; kaempferol may be a lead compound for the development of novel antiprotozoal agent. Also, these findings give support to the use of A. cherimola in the traditional medicine from México for the treatment of diarrhea and dysentery. SUMMARY Bioassay-guided fractionation of the ethanol extract of the leaves of Annona cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin and rutin. The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different that metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase. Abbreviations used: PFOR:Pyruvate:ferredoxin oxidoreductase, G: lamblia: Giardia lamblia, E: histolytica: Entamoeba histolytica PMID:28216899

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

PubMed

Crawford, N A; Droux, M; Kosower, N S; Buchanan, B B

1989-05-15

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

The Novel Bacterial N-Demethylase PdmAB Is Responsible for the Initial Step of N,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

PubMed Central

Gu, Tao; Zhou, Chaoyang; Sørensen, Sebastian R.; Zhang, Ji; He, Jian; Yu, Peiwen; Li, Shunpeng

2013-01-01

The environmental fate of phenylurea herbicides has received considerable attention in recent decades. The microbial metabolism of N,N-dimethyl-substituted phenylurea herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. The pdmAB genes in Sphingobium sp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted phenylurea herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genes pdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity of pdmAB. Without its cognate electron transport components, expression of PdmAB in Escherichia coli, Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp. strain RW1 greatly enhanced the catalytic activity of PdmAB in E. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved in O- or N-demethylation reactions, indicating that PdmAB is a distinct bacterial RO N-demethylase. PMID:24123738

Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin.

PubMed

Aono, S; Bentrop, D; Bertini, I; Cosenza, G; Luchinat, C

1998-12-01

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

Allostery in the ferredoxin protein motif does not involve a conformational switch.

PubMed

Nechushtai, Rachel; Lammert, Heiko; Michaeli, Dorit; Eisenberg-Domovich, Yael; Zuris, John A; Luca, Maria A; Capraro, Dominique T; Fish, Alex; Shimshon, Odelia; Roy, Melinda; Schug, Alexander; Whitford, Paul C; Livnah, Oded; Onuchic, José N; Jennings, Patricia A

2011-02-08

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.

Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase

PubMed Central

Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

2013-01-01

The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP+-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation. PMID:23936519

Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

PubMed

Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

2013-01-01

The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP(+)-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

DOE PAGES

Lo, Jonathan; Olson, Daniel G.; Murphy, Sean Jean-Loup; ...

2016-10-28

Here, the NfnAB (NADH-dependent reduced ferredoxin:NADP + oxidoreductase) and Rnf ( Rhodobacter nitrogen fixation) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) +. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes hadmore » little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.« less

Photolysis of water for H2 production with the use of biological and artificial catalysts

NASA Astrophysics Data System (ADS)

Hall, D. O.; Adams, M. W. W.; Morris, P.; Rao, K. K.

1980-02-01

An aqueous mixture of chloroplasts, hydrogenase and electron transfer catalyst on illumination liberates H2, the source of the H atoms being water. The rate and duration of H2 production from such a system depends on the stability of chloroplast and hydrogenase activities in light and oxygen. Both chloroplasts and hydrogenases can be stabilized to a certain degree by immobilization in gels or by incubation in bovine serum albumin. Natural electron carriers of hydrogenases are ferredoxin, cytochrome c3 and NAD. Viologen dyes and synthetic iron-sulphur particles (Jeevanu) can substitute for the biological carriers. Methyl viologen, photoreduced in the presence of chloroplasts, can liberate H2 in combination with Pt (Adam's catalyst). An aqueous solution of proflavine can be photoreduced in the presence of organic electron donors such as EDTA, cysteine, dithiothreitol, etc.; the reduced proflavine can subsequently liberate H2 with MV-Pt, MV-hydrogenase, ferredoxin-hydrogenase or cytochrome-hydrogenase systems.

Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lo, Jonathan; Olson, Daniel G.; Murphy, Sean Jean-Loup

Here, the NfnAB (NADH-dependent reduced ferredoxin:NADP + oxidoreductase) and Rnf ( Rhodobacter nitrogen fixation) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) +. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes hadmore » little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.« less

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

PubMed

Pinske, Constanze

2018-01-01

Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H 2 and CO 2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H 2 -production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H 2 -production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA , but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H 2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Clostridium acidurici electron-bifurcating formate dehydrogenase.

PubMed

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Thauer, Rudolf K

2013-10-01

Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD(+) and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2.

Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

PubMed Central

Molnár, István; Hill, D. Steven; Zirkle, Ross; Hammer, Philip E.; Gross, Frank; Buckel, Thomas G.; Jungmann, Volker; Pachlatko, Johannes Paul; Ligon, James M.

2005-01-01

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. PMID:16269733

A novel plant ferredoxin-like protein and the regulator Hor are quorum-sensing targets in the plant pathogen Erwinia carotovora.

PubMed

Sjöblom, Solveig; Harjunpää, Heidi; Brader, Günter; Palva, E Tapio

2008-07-01

Quorum sensing (QS), a population-density-sensing mechanism, controls the production of the main virulence determinants, the plant cell-wall-degrading enzymes (PCWDEs) of the soft-rot phytopathogen Erwinia carotovora subsp. carotovora. In this study, we used random transposon mutagenesis with a gusA reporter construct to identify two new QS-controlled genes encoding the regulator Hor and a plant ferredoxin-like protein, FerE. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and mediated by the global repressor RsmA. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production. Our results showed that FerE contributes to oxidative stress tolerance and in planta fitness of the bacteria and suggest that QS could be central to control of oxidative stress tolerance. The presence of the FerE protein appears to be rather unique in heterotrophic bacteria and suggests an acquisition of the corresponding gene from plant host by horizontal gene transfer.

Molecular interactions between photosystem I and ferredoxin: an integrated energy frustration and experimental model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cashman, Derek J.; Zhu, Tuo; Simmerman, Richard F.

2014-08-01

The stromal domain (PsaC, PsaD, and PsaE) of photosystem I (PSI) reduces transiently bound ferredoxin (Fd) or flavodoxin. Experimental structures exist for all of these protein partners individually, but no experimental structure of the PSI/Fd or PSI/flavodoxin complexes is presently available. Molecular models of Fd docked onto the stromal domain of the cyanobacterial PSI site are constructed here utilizing X-ray and NMR structures of PSI and Fd, respectively. Moreover, predictions of potential protein-protein interaction regions are based on experimental site-directed mutagenesis and cross-linking studies to guide rigid body docking calculations of Fd into PSI, complemented by energy landscape theory tomore » bring together regions of high energetic frustration on each of the interacting proteins. Results identify two regions of high localized frustration on the surface of Fd that contain negatively charged Asp and Glu residues. Our study predicts that these regions interact predominantly with regions of high localized frustration on the PsaC, PsaD, and PsaE chains of PSI, which include several residues predicted by previous experimental studies.« less

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

PubMed

Song, Hyun-Ouk

2016-02-01

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser

PubMed Central

Coe, Jesse; Kupitz, Christopher; Basu, Shibom; Conrad, Chelsie E.; Roy-Chowdhury, Shatabdi; Fromme, Raimund; Fromme, Petra

2015-01-01

Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP+ reductase that reduces NADP+ to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question. PMID:25950978

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

PubMed Central

Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

2010-01-01

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia

PubMed Central

Berney, Michael; Cook, Gregory M.

2010-01-01

Mycobacteria are a group of obligate aerobes that require oxygen for growth, but paradoxically have the ability to survive and metabolize under hypoxia. The mechanisms responsible for this metabolic plasticity are unknown. Here, we report on the adaptation of Mycobacterium smegmatis to slow growth rate and hypoxia using carbon-limited continuous culture. When M. smegmatis is switched from a 4.6 h to a 69 h doubling time at a constant oxygen saturation of 50%, the cells respond through the down regulation of respiratory chain components and the F1Fo-ATP synthase, consistent with the cells lower demand for energy at a reduced growth rate. This was paralleled by an up regulation of molecular machinery that allowed more efficient energy generation (i.e. Complex I) and the use of alternative electron donors (e.g. hydrogenases and primary dehydrogenases) to maintain the flow of reducing equivalents to the electron transport chain during conditions of severe energy limitation. A hydrogenase mutant showed a 40% reduction in growth yield highlighting the importance of this enzyme in adaptation to low energy supply. Slow growing cells at 50% oxygen saturation subjected to hypoxia (0.6% oxygen saturation) responded by switching on oxygen scavenging cytochrome bd, proton-translocating cytochrome bc1-aa3 supercomplex, another putative hydrogenase, and by substituting NAD+-dependent enzymes with ferredoxin-dependent enzymes thus highlighting a new pattern of mycobacterial adaptation to hypoxia. The expression of ferredoxins and a hydrogenase provides a potential conduit for disposing of and transferring electrons in the absence of exogenous electron acceptors. The use of ferredoxin-dependent enzymes would allow the cell to maintain a high carbon flux through its central carbon metabolism independent of the NAD+/NADH ratio. These data demonstrate the remarkable metabolic plasticity of the mycobacterial cell and provide a new framework for understanding their ability to survive under low energy conditions and hypoxia. PMID:20062806

Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 is Involved in Detoxification of Nitrite in Roots.

PubMed

Hachiya, Takushi; Ueda, Nanae; Kitagawa, Munenori; Hanke, Guy; Suzuki, Akira; Hase, Toshiharu; Sakakibara, Hitoshi

2016-11-01

Ferredoxin:NADP(H) oxidoreductase (FNR) plays a key role in redox metabolism in plastids. Whereas leaf FNR (LFNR) is required for photosynthesis, root FNR (RFNR) is believed to provide electrons to ferredoxin (Fd)-dependent enzymes, including nitrite reductase (NiR) and Fd-glutamine-oxoglutarate aminotransferase (Fd-GOGAT) in non-photosynthetic conditions. In some herbal species, however, most nitrate reductase activity is located in photosynthetic organs, and ammonium in roots is assimilated mainly by Fd-independent NADH-GOGAT. Therefore, RFNR might have a limited impact on N assimilation in roots grown with nitrate or ammonium nitrogen sources. AtRFNR genes are rapidly induced by application of toxic nitrite. Thus, we tested the hypothesis that RFNR could contribute to nitrite reduction in roots by comparing Arabidopsis thaliana seedlings of the wild type with loss-of-function mutants of RFNR2 When these seedlings were grown under nitrate, nitrite or ammonium, only nitrite nutrition caused impaired growth and nitrite accumulation in roots of rfnr2 Supplementation of nitrite with nitrate or ammonium as N sources did not restore the root growth in rfnr2 Also, a scavenger for nitric oxide (NO) could not effectively rescue the growth impairment. Thus, nitrite toxicity, rather than N depletion or nitrite-dependent NO production, probably causes the rfnr2 root growth defect. Our results strongly suggest that RFNR2 has a major role in reduction of toxic nitrite in roots. A specific set of genes related to nitrite reduction and the supply of reducing power responded to nitrite concomitantly, suggesting that the products of these genes act co-operatively with RFNR2 to reduce nitrite in roots. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

PubMed Central

Yan, Zhen

2017-01-01

ABSTRACT Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO2-reducing methanogenic anaerobes (methanogens) from the domain Archaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F420 (F420H2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F420H2 and reduction of ferredoxin with the exergonic oxidation of F420H2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. PMID:28174314

Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

PubMed Central

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

2013-01-01

Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD+ and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2. PMID:23872566

Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase

USDA-ARS?s Scientific Manuscript database

Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterised with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion ha...

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

USDA-ARS?s Scientific Manuscript database

Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a novel plant-type ferredoxin/thioredoxin reductase-like protein from Methanosarcina acetivorans

PubMed Central

Kumar, Adepu K.; Yennawar, Neela H.; Yennawar, Hemant P.; Ferry, James G.

2011-01-01

The genome of Methanosarcina acetivorans contains a gene (ma1659) that is predicted to encode an uncharacterized chimeric protein containing a plant-type ferredoxin/thioredoxin reductase-like catalytic domain in the N-terminal region and a bacterial-like rubredoxin domain in the C-terminal region. To understand the structural and functional properties of the protein, the ma1659 gene was cloned and overexpressed in Escherichia coli. Crystals of the MA1659 protein were grown by the sitting-drop method using 2 M ammonium sulfate, 0.1 M HEPES buffer pH 7.5 and 0.1 M urea. Diffraction data were collected to 2.8 Å resolution using the remote data-collection feature of the Advanced Light Source, Lawrence Berkeley National Laboratory. The crystal belonged to the primitive cubic space group P23 or P213, with unit-cell parameters a = b = c = 92.72 Å. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient (V M) of 3.55 Å3 Da−1, corresponding to a solvent content of 65%. PMID:21795791

WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

PubMed Central

Bevers, Loes E.; Bol, Emile; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

2005-01-01

WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (Vmax = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus. PMID:16199576

The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.

PubMed

Souza, André L F; Invitti, Adriana L; Rego, Fabiane G M; Monteiro, Rose A; Klassen, Giseli; Souza, Emanuel M; Chubatsu, Leda S; Pedrosa, Fábio O; Rigo, Liu U

2010-02-01

The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.

Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET.

PubMed

Mons, Cécile; Ferecatu, Ioana; Riquier, Sylvie; Lescop, Ewen; Bouton, Cécile; Golinelli-Cohen, Marie-Pierre

2017-01-01

MitoNEET is the first identified Fe-S protein anchored to mammalian outer mitochondrial membranes with the vast majority of the protein polypeptide located in the cytosol, including its [2Fe-2S] cluster-binding domain. The coordination of the cluster is unusual and involves three cysteines and one histidine. MitoNEET is capable of transferring its redox-active Fe-S cluster to a bacterial apo-ferredoxin in vitro even under aerobic conditions, unlike other Fe-S transfer proteins such as ISCU. This specificity suggests its possible involvement in Fe-S repair after oxidative and/or nitrosative stress. Recently, we identified cytosolic aconitase/iron regulatory protein 1 (IRP1) as the first physiological protein acceptor of the mitoNEET Fe-S cluster in an Fe-S repair process. This chapter describes methods to study in vitro mitoNEET Fe-S cluster transfer/repair to a bacterial ferredoxin used as a model aporeceptor and in a more comprehensive manner to cytosolic aconitase/IRP1 after a nitrosative stress using in vitro, in cellulo, and in vivo methods. © 2017 Elsevier Inc. All rights reserved.

Neutral complexes as oxidants for the reduced form of parsley (Petroselinum crispum) [2Fe--2S] ferredoxin. Evidence for partial blocking by redox-inactive Cr(III) complexes.

PubMed Central

Adzamli, I K; Kim, H O; Sykes, A G

1982-01-01

The 1 : 1 reactions of three neutral Co(III) oxidants, Co(acac)3, Co(NH3)3(NO2)3 and Co(acac)2(NH3)(NO2), with reduced parsley (Petroselinum crispum) [2Fe--2S] ferredoxin (which carries a substantial negative charge), have been studied at 25 degrees C, pH 8.0 (Tris/HCl), I0.10 (NaCl). Whereas it has previously been demonstrated that with Co(NH3)6+ as oxidant the reaction if completely blocked by redox-inactive Cr(NH3)63+, the neutral oxidants are only partially blocked by this same complex. The effects of three Cr(III) complexes, Cr(NH3)63+%, Cr(en)33+ and (en)2Cr . mu(OH,O2CCH3) . CR(en)24+ have been investigated. Kinetic data for the response of 3+, neutral, as well as 1--oxidants to the presence of 3+ (and 4+) Cr(III) complexes can now be rationalized in terms of a single functional site on the protein for electron transfer. Electrostatics have a significant influence on association at this site. PMID:7115307

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy

PubMed Central

Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

2017-01-01

Abstract Iron–sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe–S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. PMID:29040572

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

PubMed

Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Powis, Zoe; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

2017-12-15

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. © The Author 2017. Published by Oxford University Press.

Sunlight mediated synthesis of silver nanoparticles using redox phytoprotein and their application in catalysis and colorimetric mercury sensing.

PubMed

Ahmed, Khan Behlol Ayaz; Senthilnathan, Rajendran; Megarajan, Sengan; Anbazhagan, Veerappan

2015-10-01

Owing to the benign nature, plant extracts mediated green synthesis of metal nanoparticles (NPs) is rapidly expanding. In this study, we demonstrated the successful green synthesis of silver nanoparticles (AgNPs) by utilizing natural sunlight and redox protein complex composed of ferredoxin-NADP(+) reductase (FNR) and ferredoxin (FD). The capping and stabilization of the AgNPs by the redox protein was confirmed by Fourier transform infrared spectroscopy. Light and redox protein is the prerequisite factor for the formation of AgNPs. The obtained result shows that the photo generated free radicals by the redox protein is responsible for the reduction of Ag(+) to Ag(0). Transmission electron microscopy revealed the formation of spherical AgNPs with size ranging from 10 to 15 nm. As-prepared AgNPs exhibit excellent catalytic activity toward the degradation of hazardous organic dyes, such as methylene blue, methyl orange and methyl red. These bio-inspired AgNPs is highly sensitive and selective in sensing hazardous mercury ions in the water at micromolar concentration. In addition, FNR/FD extract stabilized AgNPs showed good antimicrobial activity against gram positive and gram negative bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Photosynthetic and Heterotrophic Ferredoxin Isoproteins Are Colocalized in Fruit Plastids of Tomato1

PubMed Central

Aoki, Koh; Yamamoto, Miyuki; Wada, Keishiro

1998-01-01

Fruit tissues of tomato (Lycopersicon esculentum Mill.) contain both photosynthetic and heterotrophic ferredoxin (FdA and FdE, respectively) isoproteins, irrespective of their photosynthetic competence, but we did not previously determine whether these proteins were colocalized in the same plastids. In isolated fruit chloroplasts and chromoplasts, both FdA and FdE were detected by immunoblotting. Colocalization of FdA and FdE in the same plastids was demonstrated using double-staining immunofluorescence microscopy. We also found that FdA and FdE were colocalized in fruit chloroplasts and chloroamyloplasts irrespective of sink status of the plastid. Immunoelectron microscopy demonstrated that FdA and FdE were randomly distributed within the plastid stroma. To investigate the significance of the heterotrophic Fd in fruit plastids, Glucose 6-phosphate dehydrogenase (G6PDH) activity was measured in isolated fruit and leaf plastids. Fruit chloroplasts and chromoplasts showed much higher G6PDH activity than did leaf chloroplasts, suggesting that high G6PDH activity is linked with FdE to maintain nonphotosynthetic production of reducing power. This result suggested that, despite their morphological resemblance, fruit chloroplasts are functionally different from their leaf counterparts. PMID:9765529

Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa

PubMed Central

Upcroft, Peter; Upcroft, Jacqueline A.

2001-01-01

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated using laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance. PMID:11148007

Potential of bisbenzimidazole-analogs toward metronidazole-resistant Trichomonas vaginalis isolates.

PubMed

Korosh, Travis; Bujans, Emmanuel; Morada, Mary; Karaalioglu, Canan; Vanden Eynde, Jean Jacques; Mayence, Annie; Huang, Tien L; Yarlett, Nigel

2017-10-01

A bisoxyphenylene-bisbenzimidazole series with increasing aliphatic chain length (CH 2 to C 10 H 20 ) containing a meta- (m) or para (p)-benzimidazole linkage to the phenylene ring was tested for ability to inhibit the growth of metronidazole-susceptible (C1) and metronidazole-refractory (085) Trichomonas vaginalis isolates under aerobic and anaerobic conditions. Compound 3m, 2,2'-[α,ω-propanediylbis(oxy-1,3-phenylene)]bis-1H-benzimidazole, displayed a 5.5-fold lower minimum inhibitory concentration (MIC) toward T. vaginalis isolate 085 than metronidazole under aerobic growth conditions, (26 μm compared to 145 μm). A dose of 25 mg/kg per day for four days of compound 3m cured a subcutaneous mouse model infection using T. vaginalis isolates 286 (metronidazole susceptible) and 085 (metronidazole refractory). Compound 3m was weakly reduced by pyruvate:ferredoxin oxidoreductase, but unlike metronidazole was not dependent upon added ferredoxin. It is concluded from structure-activity relationships that there was no obvious trend based on the length of the central aliphatic chain, or the steric position of the bisbenzimidazole enabling prediction of biological activity. The compounds generally fulfill Lipinski's rile of five, indicating their potential as drug leads. © 2017 John Wiley & Sons A/S.

The effect of iron on metronidazole activity against Trichomonas vaginalis in vitro.

PubMed

Elwakil, Hala Salah; Tawfik, Rania Ayman; Alam-Eldin, Yosra Hussein; Nassar, Doaa Ashraf

2017-11-01

Metronidazole is administered in an inactive form then activated to its cytotoxic form within the hydrogenosome of trichomonads. Two hydrogenosomal proteins, pyruvate ferredoxin oxidoreductase (PFOR) and ferredoxin, play a critical role in the reductive activation of metronidazole. The expression of these proteins and other hydrogenosomal proteins are likewise positively regulated by iron. In the present study, the effect of iron on minimal lethal concentration (MLC) of metronidazole on in vitro cultured Trichomonas vaginalis(T. vaginalis) isolates was investigated. Interestingly, Addition of Ferrous ammonium sulphate (FAS) to T. vaginalis culture led to decrease in the MLC of metronidazole. On using aerobic assay, MLC of metronidazole on untreated T. vaginalis of both isolates was 12.5 μg/ml that decreased to 0.38 μg/ml on FAS treated trichomonads. Also anaerobic assay revealed that MLC on untreated parasites was 3.12 μg/ml that decreased to 0.097 μg/ml and 0.19 μg/ml for isolate 1 and isolate 2 respectively after iron addition. It was concluded that, addition of iron to in vitro cultured T. vaginalis decreases metronidazole MLC that was detected by both aerobic and anaerobic assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids.

PubMed

Kozuleva, Marina A; Ivanov, Boris N

2010-07-01

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP(+). The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 microM in the absence of NADP(+) increased the proportion of oxygen reduction by Fd from 25-35 to 40-60% in different experiments. This proportion decreased with increasing light intensity. When NADP(+) was added in the presence of 15 microM Fd, which was optimal for the NADP(+) reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP(+) and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.

Photosynthetic activity of cotyledons is critical during post-germinative growth and seedling establishment.

PubMed

Ojeda, Valle; Nájera, Victoria A; González, Maricruz; Pérez-Ruiz, Juan M; Cejudo, Francisco J

2017-09-02

Thioredoxins (Trxs) play a relevant role in thiol-dependent redox regulation, which allows the rapid adaptation of chloroplast metabolism to unpredictable environmental conditions. In chloroplasts, Trxs use reducing equivalents provided by photoreduced ferredoxin (Fdx) via the action of a ferredoxin-thioredoxin reductase (FTR), thus linking redox regulation to light. In addition, these organelles contain an NADPH-thioredoxin reductase, NTRC, with a Trx domain at the C-terminus. NTRC efficiently reduces 2-Cys peroxiredoxins (Prxs), hence having antioxidant function. However, NTRC also participates in the redox regulation of processes, such as starch and chlorophyll biosynthesis, which are known to be regulated by Trxs. Thus, the question arising is whether there is a cross-talk between the 2 redox systems. Arabidopsis mutants simultaneously devoid of NTRC and Trx x or Trxs f show a dramatic growth inhibition phenotype, indicating that NTRC is required for the function of these unrelated Trxs. Remarkably, both the ntrc-trxx double mutant and, to a higher extent, the ntrc-trxf1f2 triple mutant show high mortality at the seedling stage, which is rescued by sucrose. These findings show the relevant role of redox regulation for chloroplast performance and uncover the key function of cotyledons chloroplasts at the transition to autotrophic metabolism during seedling establishment.

Construction and engineering of a thermostable self-sufficient cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp

2009-06-19

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C.more » Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.« less

Military Munitions-Related Compounds Fate and Effects: A Literature Review Relative to Threatened and Endangered Species

DTIC Science & Technology

2005-03-01

x nigra, DN34) was investigated. The fate, transport, and toxicity were determined. HMX was taken up by poplar cuttings from hydroponic solutions ...4): 513-520. Eight species of plants were exposed to nitrobenzene in a hydroponic solution . Four species experienced no depression of either...NADP oxidoreductase from spinach leaves. Biochem Biophys Res Commun. 241(3): 794-796. Nitrobenzene was reduced in a solution containing ferredoxin

Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts.

PubMed Central

Schmidt, H; Heinz, E

1990-01-01

Envelope membranes isolated from chloroplasts of spinach (Spinacia oleracea) desaturate oleoyl groups in monogalactosyl diacylglycerol to linoleoyl groups. The desaturation requires NADPH in combination with ferredoxin and is not restricted to monogalactosyl diacylglycerol, since it is also observed in biosynthetic intermediates as, for example, in phosphatidic acid. This indicates a certain degree of unspecificity of the oleate desaturase in isolated envelope membranes. Lipid desaturation is another important function of chloroplast envelopes. PMID:11607123

Binding of Pseudomonas aeruginosa Apo-Bacterioferritin Associated Ferredoxin to Bacterioferritin B Promotes Heme Mediation of Electron Delivery and Mobilization of Core Mineral Iron†

PubMed Central

Weeratunga, Saroja K.; Gee, Casey E.; Lovell, Scott; Zeng, Yuhong; Woodin, Carrie L.; Rivera, Mario

2009-01-01

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5–10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) Mol. Microbiol. 45, 1277–1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized. PMID:19575528

Role of a Ferredoxin Gene Cotranscribed with the nifHDK Operon in N2 Fixation and Nitrogenase “Switch-Off” of Azoarcus sp. Strain BH72

PubMed Central

Egener, Tanja; Martin, Dietmar E.; Sarkar, Abhijit; Reinhold-Hurek, Barbara

2001-01-01

The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a ς54-type promoter. Shorter transcripts sequentially missing genes of the 3′ part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% ± 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid “switch-off” of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process. PMID:11371540

[4Fe-4S]-cluster-depleted Azotobacter vinelandii ferredoxin I: a new 3Fe iron-sulfur protein.

PubMed Central

Stephens, P J; Morgan, T V; Devlin, F; Penner-Hahn, J E; Hodgson, K O; Scott, R A; Stout, C D; Burgess, B K

1985-01-01

Fe(CN)6(-3) oxidation of the aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I, (7Fe)FdI, is a degradative reaction. Destruction of the [4Fe-4S] cluster occurs first, followed by destruction of the [3Fe-3S] cluster. At a Fe(CN)6(-3)/(7Fe)FdI concentration ratio of 20, the product is a mixture of apoprotein and protein containing only a [3Fe-3S] cluster, (3Fe)FdI. This protein mixture, after partial purification, has been characterized by absorption, CD, magnetic CD, and EPR and Fe x-ray absorption spectroscopies. EPR and magnetic CD spectra provide strong evidence that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in (7Fe)FdI. Analysis of the extended x-ray absorption fine structure (EXAFS) of (3Fe)FdI finds Fe scattering at an average Fe...Fe distance of approximately equal to 2.7 A. The structure of the oxidized [3Fe-3S] cluster in solutions of oxidized (3Fe)FdI, and, by extension, of oxidized (7Fe)FdI, is thus different from that obtained by x-ray crystallography on oxidized (7Fe)FdI. Possible interpretations of this result are discussed. PMID:2994040

Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

PubMed

Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

2016-12-27

The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro

PubMed Central

Dunn, Linda A.; Burgess, Anita G.; Krauer, Kenia G.; Eckmann, Lars; Vanelle, Patrice; Crozet, Maxime D.; Gillin, Frances D.; Upcroft, Peter; Upcroft, Jacqueline A.

2010-01-01

The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardia lamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID90 values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 μM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID90 values around 100 μM (MTR-susceptible isolates typically have an ID90 of 5–12.8 μM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells. PMID:20456926

A novel mode of lactate metabolism in strictly anaerobic bacteria.

PubMed

Weghoff, Marie Charlotte; Bertsch, Johannes; Müller, Volker

2015-03-01

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-)  + 2 NAD(+)  → pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ μ ˜ Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria

PubMed Central

Kennedy, Andrew J.; Bruce, Alexandra M.; Gineste, Catherine; Ballard, T. Eric; Olekhnovich, Igor N.; Macdonald, Timothy L.

2016-01-01

Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of the Epsilonproteobacteria (Campylobacter and Helicobacter). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests against Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori. Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4′-aminopyrimidine of thiamine pyrophosphate (TPP). The ortho-propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity against C. difficile. Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance. PMID:27090174

Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria.

PubMed

Kennedy, Andrew J; Bruce, Alexandra M; Gineste, Catherine; Ballard, T Eric; Olekhnovich, Igor N; Macdonald, Timothy L; Hoffman, Paul S

2016-07-01

Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of the Epsilonproteobacteria (Campylobacter and Helicobacter). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests against Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4'-aminopyrimidine of thiamine pyrophosphate (TPP). The ortho-propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity against C. difficile Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Alternative 2-keto acid oxidoreductase activities in Trichomonas vaginalis.

PubMed

Brown, D M; Upcroft, J A; Dodd, H N; Chen, N; Upcroft, P

1999-01-25

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus▿†

PubMed Central

Gómez, Cristina; Horna, Dina H.; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J.; Braña, Alfredo F.; Méndez, Carmen; Salas, José A.

2011-01-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin. PMID:21665968

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus.

PubMed

Gómez, Cristina; Horna, Dina H; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J; Braña, Alfredo F; Méndez, Carmen; Salas, José A

2011-08-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.

The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

PubMed

Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

2008-05-01

Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

PETO Interacts with Other Effectors of Cyclic Electron Flow in Chlamydomonas.

PubMed

Takahashi, Hiroko; Schmollinger, Stefan; Lee, Jae-Hyeok; Schroda, Michael; Rappaport, Fabrice; Wollman, Francis-André; Vallon, Olivier

2016-04-04

While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow (CEF) around photosystem I (PSI) and cytochrome b6f generates only ATP. CEF is thus essential to balance the supply of ATP and NADPH for carbon fixation; however, it remains unclear how the system tunes the relative levels of linear and cyclic flow. Here, we show that PETO, a transmembrane thylakoid phosphoprotein specific of green algae, contributes to the stimulation of CEF when cells are placed in anoxia. In oxic conditions, PETO co-fractionates with other thylakoid proteins involved in CEF (ANR1, PGRL1, FNR). In PETO-knockdown strains, interactions between these CEF proteins are affected. Anoxia triggers a reorganization of the membrane, so that a subpopulation of PSI and cytochrome b6f now co-fractionates with the CEF effectors in sucrose gradients. The absence of PETO impairs this reorganization. Affinity purification identifies ANR1 as a major interactant of PETO. ANR1 contains two ANR domains, which are also found in the N-terminal region of NdhS, the ferredoxin-binding subunit of the plant ferredoxin-plastoquinone oxidoreductase (NDH). We propose that the ANR domain was co-opted by two unrelated CEF systems (PGR and NDH), possibly as a sensor of the redox state of the membrane. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.

PubMed Central

McCormick, N G; Feeherry, F E; Levinson, H S

1976-01-01

A variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT), were reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 H2 leads to R-NO H2 leads to R-NHOH H2 leads to R-NH2, 3 mol of H2 was utilized per mol of nitro group. The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups depended on other substituents and on the position of the nitro groups relative to these substituents. In the case of the nitrotoluenes, the para-nitro group was the most readily reduced, the 4-nitro position of 2,4-dinitrotulene being reduced first. The pattern of reduction of TNT (disappearance of TNT and reduction products formed) depended on the type of preparation (cell-free extract, resting cells, or growing culture), on the species, and on the atmosphere (air or H2). The "nitro-reductase" activity of V. alkalescens extracts was associated with protein fractions, one having some ferredoxin-like properties and the other possessing hydrogenase activity. Efforts to eliminate hydrogenase from the reaction have thus far been unsuccessful. The question of whether ferredoxin acts as a nonspecific reductase for nitroaromatic compounds remains unresolved. PMID:779650

Informational biopolymer structure in early living forms.

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.; Mclaughlin, P. J.; Barker, W. C.; Hunt, L. T.

1972-01-01

Some studies devoted to the search in various organisms for 'relics' of the biochemical nature of ancient organisms, preserved by the conservative nature of the evolutionary process in all living species, are reviewed. Investigations of five families of informational molecules constituting such 'relics' in very diverse organisms are reported. They include: cytochrome c, ferredoxin, trypsin, transfer ribonucleic acid (RNA), and 5S ribosomal RNA. It is shown that, even from these few informational molecules, some interesting inferences about early living organisms can be drawn.

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation

DOE PAGES

Hoben, John P.; Lubner, Carolyn E.; Ratzloff, Michael W.; ...

2017-06-14

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power) and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to 'bifurcation'. It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that presence of a short-lived anionic flavin semiquinone (ASQ) is notmore » sufficient to infer existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over two orders of magnitude. Capacity for electron transfer among redox cofactors vs. charge recombination with nearby donors can explain the range of ASQ lifetimes we observe. In conclusion, our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I, and can be an indication of capacity for electron bifurcation.« less

Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen-Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies.

PubMed

Yang, Xiaolu; Nian, Jinqiang; Xie, Qingjun; Feng, Jian; Zhang, Fengxia; Jing, Hongwei; Zhang, Jian; Dong, Guojun; Liang, Yan; Peng, Juli; Wang, Guodong; Qian, Qian; Zuo, Jianru

2016-11-07

Plants assimilate inorganic nitrogen absorbed from soil into organic forms as Gln and Glu through the glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (GS/GOGAT) cycle. Whereas GS catalyzes the formation of Gln from Glu and ammonia, GOGAT catalyzes the transfer of an amide group from Gln to 2-oxoglutarate to produce two molecules of Glu. However, the regulatory role of the GS/GOGAT cycle in the carbon-nitrogen balance is not well understood. Here, we report the functional characterization of rice ABNORMAL CYTOKININ RESPONSE 1 (ABC1) gene that encodes a ferredoxin-dependent (Fd)-GOGAT. The weak mutant allele abc1-1 mutant shows a typical nitrogen-deficient syndrome, whereas the T-DNA insertional mutant abc1-2 is seedling lethal. Metabolomics analysis revealed the accumulation of an excessive amount of amino acids with high N/C ratio (Gln and Asn) and several intermediates in the tricarboxylic acid cycle in abc1-1, suggesting that ABC1 plays a critical role in nitrogen assimilation and carbon-nitrogen balance. Five non-synonymous single-nucleotide polymorphisms were identified in the ABC1 coding region and characterized as three distinct haplotypes, which have been highly and specifically differentiated between japonica and indica subspecies. Collectively, these results suggest that ABC1/OsFd-GOGAT is essential for plant growth and development by modulating nitrogen assimilation and the carbon-nitrogen balance. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoben, John P.; Lubner, Carolyn E.; Ratzloff, Michael W.

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power) and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to 'bifurcation'. It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that presence of a short-lived anionic flavin semiquinone (ASQ) is notmore » sufficient to infer existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over two orders of magnitude. Capacity for electron transfer among redox cofactors vs. charge recombination with nearby donors can explain the range of ASQ lifetimes we observe. In conclusion, our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I, and can be an indication of capacity for electron bifurcation.« less

Effect of redox partner binding on CYP101D1 conformational dynamics.

PubMed

Batabyal, Dipanwita; Poulos, Thomas L

2018-06-01

We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDX D38 which forms an important ion pair with P450cam R112 at the center of the interface is Arx L39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with Arx L39 . Copyright © 2018. Published by Elsevier Inc.

Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme.

PubMed

Selvaraj, Brinda; Buckel, Wolfgang; Golding, Bernard T; Ullmann, G Matthias; Martins, Berta M

2016-01-01

4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs. © 2016 S. Karger AG, Basel.

Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

PubMed Central

Moreno-Vivian, C; Hennecke, S; Pühler, A; Klipp, W

1989-01-01

DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit. PMID:2708314

New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis.

PubMed

Bohrer, Anne-Sophie; Massot, Vincent; Innocenti, Gilles; Reichheld, Jean-Philippe; Issakidis-Bourguet, Emmanuelle; Vanacker, Hélène

2012-11-01

In plants, thioredoxins (TRX) constitute a large protein disulphide oxidoreductase family comprising 10 plastidial members in Arabidopsis thaliana and subdivided in five types. The f- and m-types regulate enzymes involved mainly in carbon metabolism whereas the x, y, and z types have an antioxidant function. The reduction of TRXm and f in chloroplasts is performed in the light by ferredoxin:thioredoxin reductase (FTR) that uses photosynthetically reduced ferredoxin (Fd) as a reductant. The reduction system of Arabidopsis TRXx, y, and z has never been demonstrated. Recently, a gene encoding an atypical plastidial NADPH-dependent TRX reductase (NTRC) was found. In the present study, gene expression analysis revealed that both reductases are expressed in all organs of Arabidopsis and could potentially serve as electron donors to plastidial TRX. This ability was tested in vitro either with purified NTRC in presence of NADPH or with a light-driven reconstituted system comprising thylakoids and purified Fd and FTR. The results demonstrate that FTR reduces the x and y TRX isoforms but not the recently identified TRXz. Moreover, the results show that NTRC cannot be an efficient alternative reducing system, neither for TRXz nor for the other plastidial TRX. The data reveal that TRXf, m, x, and y, known as redox regulators in the chloroplast, have also the ability to reduce TRXz in vitro. Overall, the present study points out the unique properties of TRXz among plastidial TRX.

The genetic basis of natural variation for iron homeostasis in the maize IBM population

PubMed Central

2014-01-01

Background Iron (Fe) deficiency symptoms in maize (Zea mays subsp. mays) express as leaf chlorosis, growth retardation, as well as yield reduction and are typically observed when plants grow in calcareous soils at alkaline pH. To improve our understanding of genotypical variability in the tolerance to Fe deficiency-induced chlorosis, the objectives of this study were to (i) determine the natural genetic variation of traits related to Fe homeostasis in the maize intermated B73 × Mo17 (IBM) population, (ii) to identify quantitative trait loci (QTLs) for these traits, and (iii) to analyze expression levels of genes known to be involved in Fe homeostasis as well as of candidate genes obtained from the QTL analysis. Results In hydroponically-grown maize, a total of 47 and 39 QTLs were detected for the traits recorded under limited and adequate supply of Fe, respectively. Conclusions From the QTL results, we were able to identify new putative candidate genes involved in Fe homeostasis under a deficient or adequate Fe nutritional status, like Ferredoxin class gene, putative ferredoxin PETF, metal tolerance protein MTP4, and MTP8. Furthermore, our expression analysis of candidate genes suggested the importance of trans-acting regulation for 2’-deoxymugineic acid synthase 1 (DMAS1), nicotianamine synthase (NAS3, NAS1), formate dehydrogenase 1 (FDH1), methylthioribose-1-phosphate isomerase (IDI2), aspartate/tyrosine/aromatic aminotransferase (IDI4), and methylthioribose kinase (MTK). PMID:24400634

Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer.

PubMed

Klughammer, Christof; Schreiber, Ulrich

2016-05-01

A newly developed compact measuring system for assessment of transmittance changes in the near-infrared spectral region is described; it allows deconvolution of redox changes due to ferredoxin (Fd), P700, and plastocyanin (PC) in intact leaves. In addition, it can also simultaneously measure chlorophyll fluorescence. The major opto-electronic components as well as the principles of data acquisition and signal deconvolution are outlined. Four original pulse-modulated dual-wavelength difference signals are measured (785-840 nm, 810-870 nm, 870-970 nm, and 795-970 nm). Deconvolution is based on specific spectral information presented graphically in the form of 'Differential Model Plots' (DMP) of Fd, P700, and PC that are derived empirically from selective changes of these three components under appropriately chosen physiological conditions. Whereas information on maximal changes of Fd is obtained upon illumination after dark-acclimation, maximal changes of P700 and PC can be readily induced by saturating light pulses in the presence of far-red light. Using the information of DMP and maximal changes, the new measuring system enables on-line deconvolution of Fd, P700, and PC. The performance of the new device is demonstrated by some examples of practical applications, including fast measurements of flash relaxation kinetics and of the Fd, P700, and PC changes paralleling the polyphasic fluorescence rise upon application of a 300-ms pulse of saturating light.

A novel nematode effector suppresses plant immunity by activating host reactive oxygen species-scavenging system.

PubMed

Lin, Borong; Zhuo, Kan; Chen, Shiyan; Hu, Lili; Sun, Longhua; Wang, Xiaohong; Zhang, Lian-Hui; Liao, Jinling

2016-02-01

Evidence is emerging that plant-parasitic nematodes can secrete effectors to interfere with the host immune response, but it remains unknown how these effectors can conquer host immune responses. Here, we depict a novel effector, MjTTL5, that could suppress plant immune response. Immunolocalization and transcriptional analyses showed that MjTTL5 is expressed specifically within the subventral gland of Meloidogyne javanica and up-regulated in the early parasitic stage of the nematode. Transgenic Arabidopsis lines expressing MjTTL5 were significantly more susceptible to M. javanica infection than wild-type plants, and vice versa, in planta silencing of MjTTL5 substantially increased plant resistance to M. javanica. Yeast two-hybrid, coimmunoprecipitation and bimolecular fluorescent complementation assays showed that MjTTL5 interacts specifically with Arabidopsis ferredoxin : thioredoxin reductase catalytic subunit (AtFTRc), a key component of host antioxidant system. The expression of AtFTRc is induced by the infection of M. javanica. Interaction between AtFTRc and MjTTL could drastically increase host reactive oxygen species-scavenging activity, and result in suppression of plant basal defenses and attenuation of host resistance to the nematode infection. Our results demonstrate that the host ferredoxin : thioredoxin system can be exploited cunningly by M. javanica, revealing a novel mechanism utilized by plant-parasitic nematodes to subjugate plant innate immunity and thereby promoting parasitism. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation.

PubMed

Hoben, John P; Lubner, Carolyn E; Ratzloff, Michael W; Schut, Gerrit J; Nguyen, Diep M N; Hempel, Karl W; Adams, Michael W W; King, Paul W; Miller, Anne-Frances

2017-08-25

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential ( i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I and can be an indication of capacity for electron bifurcation.

Measurement of Mitochondrial Cholesterol Import Using a Mitochondria-Targeted CYP11A1 Fusion Construct.

PubMed

Kennedy, Barry E; Charman, Mark; Karten, Barbara

2017-01-01

All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimer's, and Niemann-Pick Type C Disease. The mechanisms of mitochondrial cholesterol import are not fully elucidated yet, and may vary in different cell types and environmental conditions. Measuring cholesterol trafficking to the mitochondrial membranes is technically challenging because of its low abundance; for example, traditional pulse-chase experiments with isotope-labeled cholesterol are not feasible. Here, we describe improvements to a method first developed by the Miller group at the University of California to measure cholesterol trafficking to the inner mitochondrial membrane (IMM) through the conversion of cholesterol to pregnenolone. This method uses a mitochondria-targeted, ectopically expressed fusion construct of CYP11A1, ferredoxin reductase and ferredoxin. Pregnenolone is formed exclusively from cholesterol at the IMM, and can be analyzed with high sensitivity and specificity through ELISA or radioimmunoassay of the medium/buffer to reflect mitochondrial cholesterol import. This assay can be used to investigate the effects of genetic or pharmacological interventions on mitochondrial cholesterol import in cultured cells or isolated mitochondria.

The Mechanisms of Oxygen Reduction in the Terminal Reducing Segment of the Chloroplast Photosynthetic Electron Transport Chain.

PubMed

Kozuleva, Marina A; Ivanov, Boris N

2016-07-01

The review is dedicated to ascertainment of the roles of the electron transfer cofactors of the pigment-protein complex of PSI, ferredoxin (Fd) and ferredoxin-NADP reductase in oxygen reduction in the photosynthetic electron transport chain (PETC) in the light. The data regarding oxygen reduction in other segments of the PETC are briefly analyzed, and it is concluded that their participation in the overall process in the PETC under unstressful conditions should be insignificant. Data concerning the contribution of Fd to the oxygen reduction in the PETC are examined. A set of collateral evidence as well as results of direct measurements of the involvement of Fd in this process in the presence of isolated thylakoids led to the inference that this contribution in vivo is negligible. The increase in oxygen reduction rate in the isolated thylakoids in the presence of either Fd or Fd plus NADP + under increasing light intensity was attributed to the increase in oxygen reduction executed by the membrane-bound oxygen reductants. Data are presented which imply that a main reductant of the O 2 molecule in the terminal reducing segment of the PETC is the electron transfer cofactor of PSI, phylloquinone. The physiological significance of characteristic properties of oxygen reductants in this segment of the PETC is discussed. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The Effects of Cold Stress on Photosynthesis in Hibiscus Plants

PubMed Central

Paredes, Miriam; Quiles, María José

2015-01-01

The present work studies the effects of cold on photosynthesis, as well as the involvement in the chilling stress of chlororespiratory enzymes and ferredoxin-mediated cyclic electron flow, in illuminated plants of Hibiscus rosa-sinensis. Plants were sensitive to cold stress, as indicated by a reduction in the photochemistry efficiency of PSII and in the capacity for electron transport. However, the susceptibility of leaves to cold may be modified by root temperature. When the stem, but not roots, was chilled, the quantum yield of PSII and the relative electron transport rates were much lower than when the whole plant, root and stem, was chilled at 10°C. Additionally, when the whole plant was cooled, both the activity of electron donation by NADPH and ferredoxin to plastoquinone and the amount of PGR5 polypeptide, an essential component of the cyclic electron flow around PSI, increased, suggesting that in these conditions cyclic electron flow helps protect photosystems. However, when the stem, but not the root, was cooled cyclic electron flow did not increase and PSII was damaged as a result of insufficient dissipation of the excess light energy. In contrast, the chlororespiratory enzymes (NDH complex and PTOX) remained similar to control when the whole plant was cooled, but increased when only the stem was cooled, suggesting the involvement of chlororespiration in the response to chilling stress when other pathways, such as cyclic electron flow around PSI, are insufficient to protect PSII. PMID:26360248

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii.

PubMed

Godaux, Damien; Bailleul, Benjamin; Berne, Nicolas; Cardol, Pierre

2015-06-01

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments. © 2015 American Society of Plant Biologists. All Rights Reserved.

A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

PubMed

Hantzis, Laura J; Kroh, Gretchen E; Jahn, Courtney E; Cantrell, Michael; Peers, Graham; Pilon, Marinus; Ravet, Karl

2018-01-01

Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis ( Arabidopsis thaliana ) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome- b 6 f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency. © 2018 American Society of Plant Biologists. All Rights Reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ledbetter, Rhesa N.; Garcia Costas, Amaya M.; Lubner, Carolyn E.

The biological reduction of dinitrogen (N 2) to ammonia (NH 3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled tomore » reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.« less

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

PubMed

Miginiac-Maslow, M; Jacquot, J P; Droux, M

1985-09-01

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.

PubMed

Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J; Geigenberger, Peter

2015-11-01

Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP(+) and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. © 2015 American Society of Plant Biologists. All Rights Reserved.

NADPH Thioredoxin Reductase C and Thioredoxins Act Concertedly in Seedling Development.

PubMed

Ojeda, Valle; Pérez-Ruiz, Juan Manuel; González, Maricruz; Nájera, Victoria A; Sahrawy, Mariam; Serrato, Antonio J; Geigenberger, Peter; Cejudo, Francisco Javier

2017-07-01

Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid acclimation of chloroplast metabolism to ever-fluctuating light availability. This regulatory mechanism relies on ferredoxin reduced by the photosynthetic electron transport chain, which fuels reducing power to thioredoxins (Trxs) via a ferredoxin-dependent Trx reductase. In addition, chloroplasts harbor an NADPH-dependent Trx reductase, which has a joint Trx domain at the carboxyl terminus, termed NTRC. Thus, a relevant issue concerning chloroplast function is to establish the relationship between these two redox systems and its impact on plant development. To address this issue, we generated Arabidopsis ( Arabidopsis thaliana ) mutants combining the deficiency of NTRC with those of Trxs f , which participate in metabolic redox regulation, and that of Trx x , which has antioxidant function. The ntrc-trxf1f2 and, to a lower extent, ntrc-trxx mutants showed severe growth-retarded phenotypes, decreased photosynthesis performance, and almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover, the combined deficiency of both redox systems provokes aberrant chloroplast ultrastructure. Remarkably, both the ntrc-trxf1f2 and ntrc-trxx mutants showed high mortality at the seedling stage, which was overcome by the addition of an exogenous carbon source. Based on these results, we propose that NTRC plays a pivotal role in chloroplast redox regulation, being necessary for the activity of diverse Trxs with unrelated functions. The interaction between the two thiol redox systems is indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is essential for early plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4-Oxoproline Synthase.

PubMed

Semsary, Siamak; Crnovčić, Ivana; Driller, Ronja; Vater, Joachim; Loll, Bernhard; Keller, Ullrich

2018-04-04

X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X 0 ) or 4-oxoproline (Acm X 2 ) in their β-pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we show-using an artificial Acm half analogue (PPL 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4''-oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

DOE PAGES

Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; ...

2015-09-15

We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzymemore » (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.« less

Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

PubMed Central

Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K.; Dancis, Andrew; Pain, Debkumar

2015-01-01

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. PMID:25398879

Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

NASA Technical Reports Server (NTRS)

Huppe, H. C.; Picaud, A.; Buchanan, B. B.; Miginiac-Maslow, M.

1991-01-01

The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates.

PubMed

Hovey, Raymond; Lentes, Sabine; Ehrenreich, Armin; Salmon, Kirsty; Saba, Karla; Gottschalk, Gerhard; Gunsalus, Robert P; Deppenmeier, Uwe

2005-05-01

Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.

Dynamics of the active site architecture in plant-type ferredoxin-NADP(+) reductases catalytic complexes.

PubMed

Sánchez-Azqueta, Ana; Catalano-Dupuy, Daniela L; López-Rivero, Arleth; Tondo, María Laura; Orellano, Elena G; Ceccarelli, Eduardo A; Medina, Milagros

2014-10-01

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. Copyright © 2014 Elsevier B.V. All rights reserved.

Electron paramagnetic resonance g-tensors from state interaction spin-orbit coupling density matrix renormalization group

NASA Astrophysics Data System (ADS)

Sayfutyarova, Elvira R.; Chan, Garnet Kin-Lic

2018-05-01

We present a state interaction spin-orbit coupling method to calculate electron paramagnetic resonance g-tensors from density matrix renormalization group wavefunctions. We apply the technique to compute g-tensors for the TiF3 and CuCl42 - complexes, a [2Fe-2S] model of the active center of ferredoxins, and a Mn4CaO5 model of the S2 state of the oxygen evolving complex. These calculations raise the prospects of determining g-tensors in multireference calculations with a large number of open shells.

Aqueous light driven hydrogen production by a Ru–ferredoxin–Co biohybrid

DOE PAGES

Soltau, Sarah R.; Niklas, Jens; Dahlberg, Peter D.; ...

2015-06-08

In this paper, we report the creation of a novel solar fuel biohybrid for light-driven H 2 production utilizing the native electron transfer protein ferredoxin (Fd) as a scaffold for binding of a ruthenium photosensitizer (PS) and a molecular cobaloxime catalyst (Co). Finally, EPR and transient optical experiments provide direct evidence of a long-lived (>1.5 ms) Ru(III)–Fd–Co(I) charge separated state formed via an electron relay through the Fd [2Fe–2S] cluster, initiating the catalytic cycle for 2H + + 2e - → H 2.

Evolution of proteins.

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.

1971-01-01

The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58

PubMed Central

Zhao, Qiang; Wang, Wei; Huang, Xian-Qing; Zhang, Xue-Hong

2017-01-01

ABSTRACT Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2, were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3, and ferredoxin-encoding gene, pcaA4, and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography–mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation. IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation. PMID:28188209

Environmental selection pressures related to iron utilization are involved in the loss of the flavodoxin gene from the plant genome.

PubMed

Pierella Karlusich, Juan J; Ceccoli, Romina D; Graña, Martín; Romero, Héctor; Carrillo, Néstor

2015-02-16

Oxidative stress and iron limitation represent the grim side of life in an oxygen-rich atmosphere. The versatile electron transfer shuttle ferredoxin, an iron-sulfur protein, is particularly sensitive to these hardships, and its downregulation under adverse conditions severely compromises survival of phototrophs. Replacement of ferredoxin by a stress-resistant isofunctional carrier, flavin-containing flavodoxin, is a widespread strategy employed by photosynthetic microorganisms to overcome environmental adversities. The flavodoxin gene was lost in the course of plant evolution, but its reintroduction in transgenic plants confers increased tolerance to environmental stress and iron starvation, raising the question as to why a genetic asset with obvious adaptive value was not kept by natural selection. Phylogenetic analyses reveal that the evolutionary history of flavodoxin is intricate, with several horizontal gene transfer events between distant organisms, including Eukarya, Bacteria, and Archaea. The flavodoxin gene is unevenly distributed in most algal lineages, with flavodoxin-containing species being overrepresented in iron-limited regions and scarce or absent in iron-rich environments. Evaluation of cyanobacterial genomic and metagenomic data yielded essentially the same results, indicating that there was little selection pressure to retain flavodoxin in iron-rich coastal/freshwater phototrophs. Our results show a highly dynamic evolution pattern of flavodoxin tightly connected to the bioavailability of iron. Evidence presented here also indicates that the high concentration of iron in coastal and freshwater habitats may have facilitated the loss of flavodoxin in the freshwater ancestor of modern plants during the transition of photosynthetic organisms from the open oceans to the firm land. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis.

PubMed

Harada, Hisashi; Shindo, Kazutoshi; Iki, Kanoko; Teraoka, Ayuko; Okamoto, Sho; Yu, Fengnian; Hattan, Jun-ichiro; Utsumi, Ryutaro; Misawa, Norihiko

2011-04-01

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses. © Springer-Verlag 2011

Insulation of a synthetic hydrogen metabolism circuit in bacteria

PubMed Central

2010-01-01

Background The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued. Results Here we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds. Conclusions Through the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled. PMID:20184755

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apo-ferredoxin in an ATP-dependent reaction†

PubMed Central

Chandramouli, Kala; Johnson, Michael K.

2008-01-01

The role of the Azotobacter vinelandii HscA/HscB co-chaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S] IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulated HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB co-chaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU (Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931), and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed. PMID:16964969

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

PubMed

Ledbetter, Rhesa N; Garcia Costas, Amaya M; Lubner, Carolyn E; Mulder, David W; Tokmina-Lukaszewska, Monika; Artz, Jacob H; Patterson, Angela; Magnuson, Timothy S; Jay, Zackary J; Duan, H Diessel; Miller, Jacquelyn; Plunkett, Mary H; Hoben, John P; Barney, Brett M; Carlson, Ross P; Miller, Anne-Frances; Bothner, Brian; King, Paul W; Peters, John W; Seefeldt, Lance C

2017-08-15

The biological reduction of dinitrogen (N 2 ) to ammonia (NH 3 ) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled to reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

PubMed

McTernan, Patrick M; Chandrayan, Sanjeev K; Wu, Chang-Hao; Vaccaro, Brian J; Lancaster, W Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L; Tainer, John A; Adams, Michael W W

2014-07-11

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and function of CYP108D1 from Novosphingobium aromaticivorans DSM12444: an aromatic hydrocarbon-binding P450 enzyme.

PubMed

Bell, Stephen G; Yang, Wen; Yorke, Jake A; Zhou, Weihong; Wang, Hui; Harmer, Jeffrey; Copley, Rachel; Zhang, Aili; Zhou, Ruimin; Bartlam, Mark; Rao, Zihe; Wong, Luet Lok

2012-03-01

CYP108D1 from Novosphingobium aromaticivorans DSM12444 binds a range of aromatic hydrocarbons such as phenanthrene, biphenyl and phenylcyclohexane. Its structure, which is reported here at 2.2 Å resolution, is closely related to that of CYP108A1 (P450terp), an α-terpineol-oxidizing enzyme. The compositions and structures of the active sites of these two enzymes are very similar; the most significant changes are the replacement of Glu77 and Thr103 in CYP108A1 by Thr79 and Val105 in CYP108D1. Other residue differences lead to a larger and more hydrophobic access channel in CYP108D1. These structural features are likely to account for the weaker α-terpineol binding by CYP108D1 and, when combined with the presence of three hydrophobic phenylalanine residues in the active site, promote the binding of aromatic hydrocarbons. The haem-proximal surface of CYP108D1 shows a different charge distribution and topology to those of CYP101D1, CYP101A1 and CYP108A1, including a pronounced kink in the proximal loop of CYP108D1, which may result in poor complementarity with the [2Fe-2S] ferredoxins Arx, putidaredoxin and terpredoxin that are the respective redox partners of these three P450 enzymes. The unexpectedly low reduction potential of phenylcyclohexane-bound CYP108D1 (-401 mV) may also contribute to the low activity observed with these ferredoxins. CYP108D1 appears to function as an aromatic hydrocarbon hydroxylase that requires a different electron-transfer cofactor protein.

Revisiting the Electronic Structure of FeS Monomers Using ab Initio Ligand Field Theory and the Angular Overlap Model.

PubMed

Chilkuri, Vijay Gopal; DeBeer, Serena; Neese, Frank

2017-09-05

Iron-sulfur (FeS) proteins are universally found in nature with actives sites ranging in complexity from simple monomers to multinuclear sites from two up to eight iron atoms. These sites include mononuclear (rubredoxins), dinuclear (ferredoxins and Rieske proteins), trinuclear (e.g., hydrogenases), and tetranuclear (various ferredoxins and high-potential iron-sulfur proteins). The electronic structure of the higher-nuclearity clusters is inherently extremely complex. Hence, it is reasonable to take a bottom-up approach in which clusters of increasing nuclearity are analyzed in terms of the properties of their lower nuclearity constituents. In the present study, the first step is taken by an in-depth analysis of mononuclear FeS systems. Two different FeS molecules with phenylthiolate and methylthiolate as ligands are studied in their oxidized and reduced forms using modern wave function-based ab initio methods. The ab initio electronic spectra and wave function are presented and analyzed in detail. The very intricate electronic structure-geometry relationship in these systems is analyzed using ab initio ligand field theory (AILFT) in conjunction with the angular overlap model (AOM) parametrization scheme. The simple AOM model is used to explain the effect of geometric variations on the electronic structure. Through a comparison of the ab initio computed UV-vis absorption spectra and the available experimental spectra, the low-energy part of the many-particle spectrum is carefully analyzed. We show ab initio calculated magnetic circular dichroism spectra and present a comparison with the experimental spectrum. Finally, AILFT parameters and the ab initio spectra are compared with those obtained experimentally to understand the effect of the increased covalency of the thiolate ligands on the electronic structure of FeS monomers.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

DOE PAGES

Ledbetter, Rhesa N.; Garcia Costas, Amaya M.; Lubner, Carolyn E.; ...

2017-07-13

The biological reduction of dinitrogen (N 2) to ammonia (NH 3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled tomore » reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.« less

Transcriptional regulation of genes related to progesterone production.

PubMed

Mizutani, Tetsuya; Ishikane, Shin; Kawabe, Shinya; Umezawa, Akihiro; Miyamoto, Kaoru

2015-01-01

Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

Molecular Analysis of a Novel Methanesulfonic Acid Monooxygenase from the Methylotroph Methylosulfonomonas methylovora

PubMed Central

de Marco, Paolo; Moradas-Ferreira, Pedro; Higgins, Timothy P.; McDonald, Ian; Kenna, Elizabeth M.; Murrell, J. Colin

1999-01-01

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria. PMID:10094704

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

DOE PAGES

Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

2015-02-27

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

Fe-S cluster biogenesis in isolated mammalian mitochondria: coordinated use of persulfide sulfur and iron and requirements for GTP, NADH, and ATP.

PubMed

Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K; Dancis, Andrew; Pain, Debkumar

2015-01-02

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [(35)S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-(35)S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the (35)S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Teaching the Z-Scheme of electron transport in photosynthesis: a perspective.

PubMed

Mohapatra, Pradipta Kumar; Singh, Nihar Ranjan

2015-01-01

This paper deals with how Govindjee taught the Z-Scheme of electron transport in oxygenic photosynthesis at Ravenshaw University, Cuttack, Odisha, India, in 2014, in a unique and highly effective fashion-using students to act as molecules, representing the entire electron transport chain from water to nicotinamide adenine dinucleotide phosphate (NADP(+)). It culminated in a show by B.Sc. students in the garden of the Department of Botany, Ravenshaw University. The first author (PKM) personally acted as Ferredoxin NADP Reductase (FNR) catalyzing the reduction of NADP(+) to NADPH, taking electrons from reduced ferredoxin at the end of Photosystem I. On the other hand, the Q-cycle was played by M.Sc. students, who acted as molecules running this ingenious cycle that produces extra protons. An interesting event was when a student, acting as a herbicide, who was dressed like a devil (fierce looking, in black clothes with a sword; "Yamaraj: The God of Death", as he called himself), stopped all reactions by throwing out QB, the second plastoquinone molecule of Photosystem II, and that too aggressively, taking its position instead. The second author was the major organizer of the Z-scheme show. We provide here a basic background on the process, a bit on Govindjee's teaching, and some selected pictures from the drama played in March, 2014 at Ravenshaw University. Here, we also recognize the teacher Govindjee for his ingenious and fun-filled teaching methods that touched the hearts and the souls of the students as well as the teachers of Ravenshaw University. He was rated as one of the most-admired teachers of plant biology at our university.

Cofactor engineering for advancing chemical biotechnology.

PubMed

Wang, Yipeng; San, Ka-Yiu; Bennett, George N

2013-12-01

Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

Proteome insights into the symbiotic relationship between a captive colony of Nasutitermes corniger and its hindgut microbiome

PubMed Central

Burnum, Kristin E; Callister, Stephen J; Nicora, Carrie D; Purvine, Samuel O; Hugenholtz, Philip; Warnecke, Falk; Scheffrahn, Rudolf H; Smith, Richard D; Lipton, Mary S

2011-01-01

We analyzed the metaproteome of the bacterial community resident in the hindgut paunch of the wood-feeding ‘higher' termite (Nasutitermes) and identified 886 proteins, 197 of which have known enzymatic function. Using these enzymes, we reconstructed complete metabolic pathways revealing carbohydrate transport and metabolism, nitrogen fixation and assimilation, energy production, amino-acid synthesis and significant pyruvate ferredoxin/flavodoxin oxidoreductase protein redundancy. Our results suggest that the activity associated with these enzymes may have more of a role in the symbiotic relationship between the hindgut microbial community and its termite host than activities related to cellulose degradation. PMID:20613792

Biophotolysis systems for hydrogen production

NASA Astrophysics Data System (ADS)

Rao, K. K.; Adams, M. W. W.; Morris, P.; Hall, D. O.; Gisby, P. E.

Model systems containing natural and sythetic catalysts were constructed for the production of H2 from water using visible solar radiation as the energy source. Chloroplast membranes were used for light absorption and photodecomposition of water, ferredoxin, flavodoxin, cytochrome, viologen dyes, 'Jeevanu' particles or synthetic clusters containing Fe-Mo-S centers were used as electron transfer catalysts, and hydrogenase or PtO2 served as the proton activator. We have also investigated the use of aqueous systems with proflavine as the light activator and artificial electron donors for subsequent production of H2 when coupled to electron mediators and hydrogenase (or Pt). The characteristics, relative merits and defects of these systems are discussed.

A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Zhen; Wang, Mingyu; Ferry, James G.

Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; but, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2-reducing methanogenic anaerobes (methanogens) from the domainArchaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogenMethanosarcina acetivoranswith unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologsmore » of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domainsBacteriaandArchaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes inEscherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420(F 420H 2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420H 2and reduction of ferredoxin with the exergonic oxidation of F 420H 2and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) byM. acetivoransand uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth ofM. acetivoransand proposed to be essential for growth in the environment when acetate is limiting.IMPORTANCEDiscovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2-reducing methanogens to include diverse prokaryotes from the domainsBacteriaandArchaea. Furthermore, the unprecedented coenzyme F 420-dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4-oxidizing pathways. Our results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.« less

A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

DOE PAGES

Yan, Zhen; Wang, Mingyu; Ferry, James G.; ...

2017-02-07

Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; but, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2-reducing methanogenic anaerobes (methanogens) from the domainArchaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogenMethanosarcina acetivoranswith unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologsmore » of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domainsBacteriaandArchaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes inEscherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420(F 420H 2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420H 2and reduction of ferredoxin with the exergonic oxidation of F 420H 2and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) byM. acetivoransand uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth ofM. acetivoransand proposed to be essential for growth in the environment when acetate is limiting.IMPORTANCEDiscovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2-reducing methanogens to include diverse prokaryotes from the domainsBacteriaandArchaea. Furthermore, the unprecedented coenzyme F 420-dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4-oxidizing pathways. Our results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.« less

A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea.

PubMed

Yan, Zhen; Wang, Mingyu; Ferry, James G

2017-02-07

Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2 -reducing methanogenic anaerobes (methanogens) from the domain Archaea Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420 (F 420 H 2 ) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420 H 2 and reduction of ferredoxin with the exergonic oxidation of F 420 H 2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. Discovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2 -reducing methanogens to include diverse prokaryotes from the domains Bacteria and Archaea The unprecedented coenzyme F 420 -dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4 -oxidizing pathways. The results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth's biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation. Copyright © 2017 Yan et al.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Yifeng; Li, Bin; Prakash, Divya

Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRsmore » reflects the diversity of electron carriers used in anaerobic metabolism« less

The evolution of blue-greens and the origins of chloroplasts

NASA Technical Reports Server (NTRS)

Schwartz, R. M.; Dayhoff, M. O.

1981-01-01

All of the available molecular data support the theory that the chloroplasts of eukaryote cells were originally free-living blue-greens. Of great interest is what the relationships are between contemporary types of blue-greens and eukaryote chloroplasts and whether the chloroplasts of the various eukaryotes are the result of one or more than one symbiosis. By combining information from phylogenetic trees based on cytochrome c6 and 2Fe-2S ferredoxin sequences, it is shown that the chloroplasts of a number of eukaryote algae as well as the protist Euglena are polyphyletic; the chloroplasts of green algae and the higher plants may be the result of a single symbiosis.

Evolution of early life inferred from protein and ribonucleic acid sequences

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.; Schwartz, R. M.

1978-01-01

The chemical structures of ferredoxin, 5S ribosomal RNA, and c-type cytochrome sequences have been employed to construct a phylogenetic tree which connects all major photosynthesizing organisms: the three types of bacteria, blue-green algae, and chloroplasts. Anaerobic and aerobic bacteria, eukaryotic cytoplasmic components and mitochondria are also included in the phylogenetic tree. Anaerobic nonphotosynthesizing bacteria similar to Clostridium were the earliest organisms, arising more than 3.2 billion years ago. Bacterial photosynthesis evolved nearly 3.0 billion years ago, while oxygen-evolving photosynthesis, originating in the blue-green algal line, came into being about 2.0 billion years ago. The phylogenetic tree supports the symbiotic theory of the origin of eukaryotes.

Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (Oryza sativa).

PubMed

Chang, Hsiang; Huang, Hsiang-En; Cheng, Chin-Fu; Ho, Mei-Hsuan; Ger, Mang-Jye

2017-04-01

The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by enhancing the capacity of photosynthetic carbon assimilation.

Diversity and Evolutionary History of Iron Metabolism Genes in Diatoms

PubMed Central

Groussman, Ryan D.; Parker, Micaela S.; Armbrust, E. Virginia

2015-01-01

Ferroproteins arose early in Earth’s history, prior to the emergence of oxygenic photosynthesis and the subsequent reduction of bioavailable iron. Today, iron availability limits primary productivity in about 30% of the world’s oceans. Diatoms, responsible for nearly half of oceanic primary production, have evolved molecular strategies for coping with variable iron concentrations. Our understanding of the evolutionary breadth of these strategies has been restricted by the limited number of species for which molecular sequence data is available. To uncover the diversity of strategies marine diatoms employ to meet cellular iron demands, we analyzed 367 newly released marine microbial eukaryotic transcriptomes, which include 47 diatom species. We focused on genes encoding proteins previously identified as having a role in iron management: iron uptake (high-affinity ferric reductase, multi-copper oxidase, and Fe(III) permease); iron storage (ferritin); iron-induced protein substitutions (flavodoxin/ferredoxin, and plastocyanin/cytochrome c6) and defense against reactive oxygen species (superoxide dismutases). Homologs encoding the high-affinity iron uptake system components were detected across the four diatom Classes suggesting an ancient origin for this pathway. Ferritin transcripts were also detected in all Classes, revealing a more widespread utilization of ferritin throughout diatoms than previously recognized. Flavodoxin and plastocyanin transcripts indicate possible alternative redox metal strategies. Predicted localization signals for ferredoxin identify multiple examples of gene transfer from the plastid to the nuclear genome. Transcripts encoding four superoxide dismutase metalloforms were detected, including a putative nickel-coordinating isozyme. Taken together, our results suggest that the majority of iron metabolism genes in diatoms appear to be vertically inherited with functional diversity achieved via possible neofunctionalization of paralogs. This refined view of iron use strategies in diatoms elucidates the history of these adaptations, and provides potential molecular markers for determining the iron nutritional status of different diatom species in environmental samples. PMID:26052941

Electron Bifurcation Involved in the Energy Metabolism of the Acetogenic Bacterium Moorella thermoacetica Growing on Glucose or H2 plus CO2

PubMed Central

Huang, Haiyan; Wang, Shuning; Moll, Johanna

2012-01-01

Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO2 molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fdred2−) molecules. In the reductive part, 2 CO2 molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO2 are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO2 reduction to acetic acid are generated by the reduction of 2 NADP+ molecules with 1 NADH and 1 Fdred2− catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD+ and Fd with 2 H2 molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H2-CO2-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H2-CO2-grown cells additionally contain high specific activities of an NADP+-dependent hydrogenase that catalyzes the reduction of NADP+ with H2. This activity is hardly detectable in glucose-grown cells. PMID:22582275

Varied clinical presentations of seven patients with mutations in CYP11A1 encoding the cholesterol side-chain cleavage enzyme, P450scc.

PubMed

Tee, Meng Kian; Abramsohn, Michal; Loewenthal, Neta; Harris, Mark; Siwach, Sudeep; Kaplinsky, Ana; Markus, Barak; Birk, Ohad; Sheffield, Val C; Parvari, Ruti; Pavari, Ruti; Hershkovitz, Eli; Miller, Walter L

2013-02-01

The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously. We sought to expand clinical and genetic experience with P450scc deficiency. We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel. Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity. Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

Functional Inactivation of Putative Photosynthetic Electron Acceptor Ferredoxin C2 (FdC2) Induces Delayed Heading Date and Decreased Photosynthetic Rate in Rice

PubMed Central

Ruan, Banpu; Kang, Shujing; He, Lei; Zhang, Sen; Dong, Guojun; Hu, Jiang; Zeng, Dali; Zhang, Guangheng; Gao, Zhenyu; Ren, Deyong; Hu, Xingming; Chen, Guang; Guo, Longbiao; Qian, Qian; Zhu, Li

2015-01-01

Ferredoxin (Fd) protein as unique electron acceptor, involved in a variety of fundamental metabolic and signaling processes, which is indispensable for plant growth. The molecular mechanisms of Fd such as regulation of electron partitioning, impact of photosynthetic rate and involvement in the carbon fixing remain elusive in rice. Here we reported a heading date delay and yellowish leaf 1 (hdy1) mutant derived from Japonica rice cultivar “Nipponbare” subjected to EMS treatment. In the paddy field, the hdy1 mutant appeared at a significantly late heading date and had yellow-green leaves during the whole growth stage. Further investigation indicated that the abnormal phenotype of hdy1 was connected with depressed pigment content and photosynthetic rate. Genetic analysis results showed that the hdy1 mutant phenotype was caused by a single recessive nuclear gene mutation. Map-based cloning revealed that OsHDY1 is located on chromosome 3 and encodes an ortholog of the AtFdC2 gene. Complementation and overexpression, transgenic plants exhibited the mutant phenotype including head date, leaf color and the transcription levels of the FdC2 were completely rescued by transformation with OsHDY1. Real-time PCR revealed that the expression product of OsHDY1 was detected in almost all of the organs except root, whereas highest expression levels were observed in seeding new leaves. The lower expression levels of HDY1 and content of iron were detected in hdy1 than WT’s. The FdC2::GFP was detected in the chloroplasts of rice. Real-time PCR results showed that the expression of many photosynthetic electron transfer related genes in hdy1 were higher than WT. Our results suggest that OsFdC2 plays an important role in photosynthetic rate and development of heading date by regulating electron transfer and chlorophyll content in rice. PMID:26598971

Thermodynamic Driving Force of Hydrogen on Rumen Microbial Metabolism: A Theoretical Investigation

PubMed Central

van Lingen, Henk J.; Plugge, Caroline M.; Fadel, James G.; Kebreab, Ermias; Bannink, André; Dijkstra, Jan

2016-01-01

Hydrogen is a key product of rumen fermentation and has been suggested to thermodynamically control the production of the various volatile fatty acids (VFA). Previous studies, however, have not accounted for the fact that only thermodynamic near-equilibrium conditions control the magnitude of reaction rate. Furthermore, the role of NAD, which is affected by hydrogen partial pressure (PH2), has often not been considered. The aim of this study was to quantify the control of PH2 on reaction rates of specific fermentation pathways, methanogenesis and NADH oxidation in rumen microbes. The control of PH2 was quantified using the thermodynamic potential factor (FT), which is a dimensionless factor that corrects a predicted kinetic reaction rate for the thermodynamic control exerted. Unity FT was calculated for all glucose fermentation pathways considered, indicating no inhibition of PH2 on the production of a specific type of VFA (e.g., acetate, propionate and butyrate) in the rumen. For NADH oxidation without ferredoxin oxidation, increasing PH2 within the rumen physiological range decreased FT from unity to zero for different NAD+ to NADH ratios and pH of 6.2 and 7.0, which indicates thermodynamic control of PH2. For NADH oxidation with ferredoxin oxidation, increasing PH2 within the rumen physiological range decreased FT from unity at pH of 7.0 only. For the acetate to propionate conversion, FT increased from 0.65 to unity with increasing PH2, which indicates thermodynamic control. For propionate to acetate and butyrate to acetate conversions, FT decreased to zero below the rumen range of PH2, indicating full thermodynamic suppression. For methanogenesis by archaea without cytochromes, FT differed from unity only below the rumen range of PH2, indicating no thermodynamic control. This theoretical investigation shows that thermodynamic control of PH2 on individual VFA produced and associated yield of hydrogen and methane cannot be explained without considering NADH oxidation. PMID:27783615

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

PubMed

Ge, Deyong; Xue, Yanfen; Ma, Yanhe

2016-05-11

Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene. Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain. IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

Rapid-mix and chemical quench studies of ferredoxin-reduced stearoyl-acyl carrier protein desaturase.

PubMed

Lyle, Karen S; Haas, Jeffrey A; Fox, Brian G

2003-05-20

Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (approximately 95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O(2) and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for approximately 90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over approximately 1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) approximately 1.5) and k(linear) (D)k(linear) approximately 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.

Phylogenetic analysis of proteins associated in the four major energy metabolism systems: photosynthesis, aerobic respiration, denitrification, and sulfur respiration.

PubMed

Tomiki, Takeshi; Saitou, Naruya

2004-08-01

The four electron transfer energy metabolism systems, photosynthesis, aerobic respiration, denitrification, and sulfur respiration, are thought to be evolutionarily related because of the similarity of electron transfer patterns and the existence of some homologous proteins. How these systems have evolved is elusive. We therefore conducted a comprehensive homology search using PSI-BLAST, and phylogenetic analyses were conducted for the three homologous groups (groups 1-3) based on multiple alignments of domains defined in the Pfam database. There are five electron transfer types important for catalytic reaction in group 1, and many proteins bind molybdenum. Deletions of two domains led to loss of the function of binding molybdenum and ferredoxin, and these deletions seem to be critical for the electron transfer pattern changes in group 1. Two types of electron transfer were found in group 2, and all its member proteins bind siroheme and ferredoxin. Insertion of the pyridine nucleotide disulfide oxidoreductase domain seemed to be the critical point for the electron transfer pattern change in this group. The proteins belonging to group 3 are all flavin enzymes, and they bind flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Types of electron transfer in this group are divergent, but there are two common characteristics. NAD(P)H works as an electron donor or acceptor, and FAD or FMN transfers electrons from/to NAD(P)H. Electron transfer functions might be added to these common characteristics by the addition of functional domains through the evolution of group 3 proteins. Based on the phylogenetic analyses in this study and previous studies, we inferred the phylogeny of the energy metabolism systems as follows: photosynthesis (and possibly aerobic respiration) and the sulfur/nitrogen assimilation system first diverged, then the sulfur/nitrogen dissimilation system was produced from the latter system.

Characterization of Putative Iron Responsive Genes as Species-Specific Indicators of Iron Stress in Thalassiosiroid Diatoms

PubMed Central

Whitney, LeAnn P.; Lins, Jeremy J.; Hughes, Margaret P.; Wells, Mark L.; Chappell, P. Dreux; Jenkins, Bethany D.

2011-01-01

Iron (Fe) availability restricts diatom growth and primary production in large areas of the oceans. It is a challenge to assess the bulk Fe nutritional health of natural diatom populations, since species can differ in their physiological and molecular responses to Fe limitation. We assayed expression of selected genes in diatoms from the Thalassiosira genus to assess their potential utility as species-specific molecular markers to indicate Fe status in natural diatom assemblages. In this study, we compared the expression of the photosynthetic genes encoding ferredoxin (a Fe-requiring protein) and flavodoxin (a Fe-free protein) in culture experiments with Fe replete and Fe stressed Thalassiosira pseudonana (CCMP 1335) isolated from coastal waters and Thalassiosira weissflogii (CCMP 1010) isolated from the open ocean. In T. pseudonana, expression of flavodoxin and ferredoxin genes were not sensitive to Fe status but were found to display diel periodicities. In T. weissflogii, expression of flavodoxin was highly responsive to iron levels and was only detectable when cultures were Fe limited. Flavodoxin genes have been duplicated in most diatoms with available genome data and we show that T. pseudonana has lost its copy related to the Fe-responsive copy in T. weissflogii. We also examined the expression of genes for a putative high affinity, copper (Cu)-dependent Fe uptake system in T. pseudonana. Our results indicate that genes encoding putative Cu transporters, a multi-Cu oxidase, and a Fe reductase are not linked to Fe status. The expression of a second putative Fe reductase increased in Fe limited cultures, but this gene was also highly expressed in Fe replete cultures, indicating it may not be a useful marker in the field. Our findings highlight that Fe metabolism may differ among diatoms even within a genus and show a need to validate responses in different species as part of the development pipeline for genetic markers of Fe status in field populations. PMID:22275908

Evolutionary connections of biological kingdoms based on protein and nucleic acid sequence evidence

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.

1983-01-01

Prokaryotic and eukaryotic evolutionary trees are developed from protein and nucleic-acid sequences by the methods of numerical taxonomy. Trees are presented for bacterial ferredoxins, 5S ribosomal RNA, c-type cytochromes , cytochromes c2 and c', and 5.8S ribosomal RNA; the implications for early evolution are discussed; and a composite tree showing the branching of the anaerobes, aerobes, archaebacteria, and eukaryotes is shown. Single lines are found for all oxygen-evolving photosynthetic forms and for the salt-loving and high-temperature forms of archaebacteria. It is argued that the eukaryote mitochondria, chloroplasts, and cytoplasmic host material are descended from free-living prokaryotes that formed symbiotic associations, with more than one symbiotic event involved in the evolution of each organelle.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bell, Stephen G.; Hoskins, Nicola; Xu Feng

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to {approx}2.0 A have been obtained.

Identification of the Binding Region of the [2Fe-2S] Ferredoxin in Stearoyl-Acyl Carrier Protein Desaturase

PubMed Central

Sobrado, Pablo; Lyle, Karen S.; Kaul, Steven P.; Turco, Michelle M.; Arabshahi, Ida; Marwah, Ashok; Fox, Brian G.

2008-01-01

Stearoyl-acyl carrier protein desaturase (Δ9D) catalyzes the O2 and 2e- dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e- are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Δ9D complex by use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches and molecular docking studies. Treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Δ9D contribute to the cross-linking. The single substitutions of K60A, K56A, and K230A on Δ9D decreased the kcat/KM for Fd by 4-, 22- and 2,400-fold, respectively, as compared to wt Δ9D and a K41A substitution. The double substitution K56A/K60A decreased the kcat/KM for Fd by 250-fold, while the triple mutation K56A/K60A/K230A decreased the kcat/KM for Fd by at least 700,000-fold. These results strongly implicate the triad of K56, K60 and K230 of Δ9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd near to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix-7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Δ9D and other protein-protein complexes. PMID:16605252

Electron Transport in a Dioxygenase-Ferredoxin Complex: Long Range Charge Coupling between the Rieske and Non-Heme Iron Center

PubMed Central

Jono, Ryota; Shimizu, Kentaro

2016-01-01

Dioxygenase (dOx) utilizes stereospecific oxidation on aromatic molecules; consequently, dOx has potential applications in bioremediation and stereospecific oxidation synthesis. The reactive components of dOx comprise a Rieske structure Cys2[2Fe-2S]His2 and a non-heme reactive oxygen center (ROC). Between the Rieske structure and the ROC, a universally conserved Asp residue appears to bridge the two structures forming a Rieske-Asp-ROC triad, where the Asp is known to be essential for electron transfer processes. The Rieske and ROC share hydrogen bonds with Asp through their His ligands; suggesting an ideal network for electron transfer via the carboxyl side chain of Asp. Associated with the dOx is an itinerant charge carrying protein Ferredoxin (Fdx). Depending on the specific cognate, Fdx may also possess either the Rieske structure or a related structure known as 4-Cys-[2Fe-2S] (4-Cys). In this study, we extensively explore, at different levels of theory, the behavior of the individual components (Rieske and ROC) and their interaction together via the Asp using a variety of density function methods, basis sets, and a method known as Generalized Ionic Fragment Approach (GIFA) that permits setting up spin configurations manually. We also report results on the 4-Cys structure for comparison. The individual optimized structures are compared with observed spectroscopic data from the Rieske, 4-Cys and ROC structures (where information is available). The separate pieces are then combined together into a large Rieske-Asp-ROC (donor/bridge/acceptor) complex to estimate the overall coupling between individual components, based on changes to the partial charges. The results suggest that the partial charges are significantly altered when Asp bridges the Rieske and the ROC; hence, long range coupling through hydrogen bonding effects via the intercalated Asp bridge can drastically affect the partial charge distributions compared to the individual isolated structures. The results are consistent with a proton coupled electron transfer mechanism. PMID:27656882

Characterization of the putative iron sulfur protein IdiC (ORF5) in Synechococcus elongatus PCC 7942.

PubMed

Pietsch, Daniel; Staiger, Dorothee; Pistorius, Elfriede K; Michel, Klaus-Peter

2007-10-01

The IdiC protein (iron deficiency induced protein C) is encoded by orf5 (now called idiC), which is part of the iron-responsive idiB operon of Synechococcus elongatus PCC 7942. The 20.5 kDa IdiC protein has a putative transmembrane helix and belongs to the thioredoxin (TRX)-like [2Fe-2S] ferredoxin family. IdiC has the highest similarity to the peripheral subunit NuoE of the Escherichia coli NDH-1 complex. IdiC expression increased under iron starvation and also in the late growth phase, representing growth conditions, which favor photosynthetic cyclic and respiratory electron transport over photosynthetic linear electron transport from water to NADP+. Attempts to insertionally inactivate the idiC gene generated merodiploid mutants with a strongly reduced IdiC content (mutant MuD) but no IdiC-free mutant. Thus, IdiC seems to be an essential protein for the viability of S. elongatus under the used experimental conditions. Comparative analyses of S. elongatus wild type (WT) and mutant MuD showed that under iron limitation in WT and MuD the amount of the reaction center proteins PsbA and PsaA/B was highly reduced. MuD had a lower growth rate, chlorophyll content, and photosynthetic O2 evolving activity with bicarbonate as electron acceptor than WT. Immunoblot analyses also showed that in MuD, when grown under iron limitation, the amount of the proteins IdiC and IdiB was greatly reduced as compared to WT. As a consequence of the reduction of the transcription factor IdiB, IdiA and IrpA expression were also decreased. In addition, the IsiA protein concentration was lower in MuD than in WT, although the isiA mRNA was equally high in MuD and WT. Another significant difference was the lower expression of the ferredoxin:NADP+ oxidoreductase in mutant MuD under iron limitation compared to WT. A possible function of the protein IdiC in cyclic electron transport around photosystem I and/or in respiratory electron transport will be discussed.

Antiprotozoal Nitazoxanide Derivatives: Synthesis, Bioassays and QSAR Study Combined with Docking for Mechanistic Insight

PubMed Central

Scior, Thomas; Lozano-Aponte, Jorge; Ajmani, Subhash; Hernández-Montero, Eduardo; Chávez-Silva, Fabiola; Hernández-Núñez, Emanuel; Moo-Puc, Rosa; Fraguela-Collar, Andres; Navarrete-Vázquez, Gabriel

2015-01-01

In view of the serious health problems concerning infectious diseases in heavily populated areas, we followed the strategy of lead compound diversification to evaluate the near-by chemical space for new organic compounds. To this end, twenty derivatives of nitazoxanide (NTZ) were synthesized and tested for activity against Entamoeba histolytica parasites. To ensure drug-likeliness and activity relatedness of the new compounds, the synthetic work was assisted by a quantitative structure-activity relationships study (QSAR). Many of the inherent downsides – well-known to QSAR practitioners – we circumvented thanks to workarounds which we proposed in prior QSAR publication. To gain further mechanistic insight on a molecular level, ligand-enzyme docking simulations were carried out since NTZ is known to inhibit the protozoal pyruvate ferredoxin oxidoreductase (PFOR) enzyme as its biomolecular target. PMID:25872791

Novel Functions of an Iron-Sulfur Flavoprotein from Trichomonas vaginalis Hydrogenosomes

PubMed Central

Smutná, Tamara; Pilarová, Katerina; Tarábek, Ján; Tachezy, Jan

2014-01-01

Iron-sulfur flavoproteins (Isf) are flavin mononucleotide (FMN)- and FeS cluster-containing proteins commonly encountered in anaerobic prokaryotes. However, with the exception of Isf from Methanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide, none of these proteins has been characterized in terms of function. Trichomonas vaginalis, a sexually transmitted eukaryotic parasite of humans, was found to express several iron-sulfur flavoprotein (TvIsf) homologs in its hydrogenosomes. We show here that in addition to having oxygen-reducing activity, the recombinant TvIsf also functions as a detoxifying reductase of metronidazole and chloramphenicol, both of which are antibiotics effective against a variety of anaerobic microbes. TvIsf can utilize both NADH and reduced ferredoxin as electron donors. Given the prevalence of Isf in anaerobic prokaryotes, we propose that these proteins are central to a novel defense mechanism against xenobiotics. PMID:24663020

Enzymology of the Wood–Ljungdahl Pathway of Acetogenesis

PubMed Central

Ragsdale, Stephen W.

2011-01-01

The biochemistry of acetogenesis is reviewed. The microbes that catalyze the reactions that are central to acetogenesis are described and the focus is on the enzymology of the process. These microbes play a key role in the global carbon cycle, producing over 10 trillion kilograms of acetic acid annually. Acetogens have the ability to anaerobically convert carbon dioxide and CO into acetyl-CoA by the Wood–Ljungdahl pathway, which is linked to energy conservation. They also can convert the six carbons of glucose stoichiometrically into 3 mol of acetate using this pathway. Acetogens and other anaerobic microbes (e.g., sulfate reducers and methanogens) use the Wood–Ljungdahl pathway for cell carbon synthesis. Important enzymes in this pathway that are covered in this review are pyruvate ferredoxin oxidoreductase, CO dehydrogenase/acetyl-CoA synthase, a corrinoid iron-sulfur protein, a methyltransferase, and the enzymes involved in the conversion of carbon dioxide to methyl-tetrahydrofolate. PMID:18378591

Ethanol production by engineered thermophiles.

PubMed

Olson, Daniel G; Sparling, Richard; Lynd, Lee R

2015-06-01

We compare a number of different strategies that have been pursued to engineer thermophilic microorganisms for increased ethanol production. Ethanol production from pyruvate can proceed via one of four pathways, which are named by the key pyruvate dissimilating enzyme: pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), pyruvate formate lyase (PFL), and pyruvate ferredoxin oxidoreductase (PFOR). For each of these pathways except PFL, we see examples where ethanol production has been engineered with a yield of >90% of the theoretical maximum. In each of these cases, this engineering was achieved mainly by modulating expression of native genes. We have not found an example where a thermophilic ethanol production pathway has been transferred to a non-ethanol-producing organism to produce ethanol at high yield. A key reason for the lack of transferability of ethanol production pathways is the current lack of understanding of the enzymes involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poust, Sean; Yoon, Isu; Adams, Paul D.

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-likemore » subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.« less

Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

DOE PAGES

Poust, Sean; Yoon, Isu; Adams, Paul D.; ...

2014-10-06

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-likemore » subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.« less

Contrasting effects of copper limitation on the photosynthetic apparatus in two strains of the open ocean diatom Thalassiosira oceanica

PubMed Central

Allen, Andrew E.; Foster, Leonard J.; Green, Beverley R.; Maldonado, Maria T.

2017-01-01

There is an intricate interaction between iron (Fe) and copper (Cu) physiology in diatoms. However, strategies to cope with low Cu are largely unknown. This study unveils the comprehensive restructuring of the photosynthetic apparatus in the diatom Thalassiosira oceanica (CCMP1003) in response to low Cu, at the physiological and proteomic level. The restructuring results in a shift from light harvesting for photochemistry—and ultimately for carbon fixation—to photoprotection, reducing carbon fixation and oxygen evolution. The observed decreases in the physiological parameters Fv/Fm, carbon fixation, and oxygen evolution, concomitant with increases in the antennae absorption cross section (σPSII), non-photochemical quenching (NPQ) and the conversion factor (φe:C/ηPSII) are in agreement with well documented cellular responses to low Fe. However, the underlying proteomic changes due to low Cu are very different from those elicited by low Fe. Low Cu induces a significant four-fold reduction in the Cu-containing photosynthetic electron carrier plastocyanin. The decrease in plastocyanin causes a bottleneck within the photosynthetic electron transport chain (ETC), ultimately leading to substantial stoichiometric changes. Namely, 2-fold reduction in both cytochrome b6f complex (cytb6f) and photosystem II (PSII), no change in the Fe-rich PSI and a 40- and 2-fold increase in proteins potentially involved in detoxification of reactive oxygen species (ferredoxin and ferredoxin:NADP+ reductase, respectively). Furthermore, we identify 48 light harvesting complex (LHC) proteins in the publicly available genome of T. oceanica and provide proteomic evidence for 33 of these. The change in the LHC composition within the antennae in response to low Cu underlines the shift from photochemistry to photoprotection in T. oceanica (CCMP1003). Interestingly, we also reveal very significant intra-specific strain differences. Another strain of T. oceanica (CCMP 1005) requires significantly higher Cu concentrations to sustain both its maximal and minimal growth rate compared to CCMP 1003. Under low Cu, CCMP 1005 decreases its growth rate, cell size, Chla and total protein per cell. We argue that the reduction in protein per cell is the main strategy to decrease its cellular Cu requirement, as none of the other parameters tested are affected. Differences between the two strains, as well as differences between the well documented responses to low Fe and those presented here in response to low Cu are discussed. PMID:28837661

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids.

PubMed

Terpolilli, Jason J; Masakapalli, Shyam K; Karunakaran, Ramakrishnan; Webb, Isabel U C; Green, Rob; Watmough, Nicholas J; Kruger, Nicholas J; Ratcliffe, R George; Poole, Philip S

2016-10-15

Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2 Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [(13)C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2 However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen

PubMed Central

2014-01-01

Background The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway. Results Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate. Conclusions The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen. PMID:24952578

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

PubMed Central

Terpolilli, Jason J.; Masakapalli, Shyam K.; Karunakaran, Ramakrishnan; Webb, Isabel U. C.; Green, Rob; Watmough, Nicholas J.; Kruger, Nicholas J.; Ratcliffe, R. George

2016-01-01

ABSTRACT Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2. Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [13C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. IMPORTANCE Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2. However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. PMID:27501983

Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium.

PubMed

Abdulmughni, Ammar; Jóźwik, Ilona K; Brill, Elisa; Hannemann, Frank; Thunnissen, Andy-Mark W H; Bernhardt, Rita

2017-11-01

Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D 3 (VD 3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD 3 deficiency and other disorders. However, the chemical synthesis of VD 3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD 3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD 3 . Product yield amounted to 54.9 mg·L -1 ·day -1 , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for VD 3 binding as the related VD 3 hydroxylases CYP109E1 from B. megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of VD 3 hydroxylation. The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2). Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1, UniProt ID: D5DF88, Ferredoxin, UniProt ID: D5DFQ0, cytochrome P450 reductase, EC 1.8.1.2, UniProt ID: D5DGX1. © 2017 Federation of European Biochemical Societies.

Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase.

PubMed

Ashikawa, Yuji; Fujimoto, Zui; Usami, Yusuke; Inoue, Kengo; Noguchi, Haruko; Yamane, Hisakazu; Nojiri, Hideaki

2012-06-24

Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex. In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding. The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O-O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography.

PubMed

Unno, Masaki; Ishikawa-Suto, Kumiko; Kusaka, Katsuhiro; Tamada, Taro; Hagiwara, Yoshinori; Sugishima, Masakazu; Wada, Kei; Yamada, Taro; Tomoyori, Katsuaki; Hosoya, Takaaki; Tanaka, Ichiro; Niimura, Nobuo; Kuroki, Ryota; Inaka, Koji; Ishihara, Makiko; Fukuyama, Keiichi

2015-04-29

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O(+). These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif.

PubMed

Zubieta, Chloe; Krishna, S Sri; Kapoor, Mili; Kozbial, Piotr; McMullan, Daniel; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Ambing, Eileen; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Hampton, Eric; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2007-11-01

BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein). (c) 2007 Wiley-Liss, Inc.

Advances in the Function and Regulation of Hydrogenase in the Cyanobacterium Synechocystis PCC6803

PubMed Central

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2014-01-01

In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H2 production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production. PMID:25365180

Flavodiiron Protein from Trichomonas vaginalis Hydrogenosomes: the Terminal Oxygen Reductase▿

PubMed Central

Smutná, Tamara; Gonçalves, Vera L.; Saraiva, Lígia M.; Tachezy, Jan; Teixeira, Miguel; Hrdý, Ivan

2009-01-01

Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen. PMID:19011120

Artificial photosynthesis for production of hydrogen peroxide and its fuel cells.

PubMed

Fukuzumi, Shunichi

2016-05-01

The reducing power released from photosystem I (PSI) via ferredoxin enables the reduction of NADP(+) to NADPH, which is essential in the Calvin-Benson cycle to make sugars in photosynthesis. Alternatively, PSI can reduce O2 to produce hydrogen peroxide as a fuel. This article describes the artificial version of the photocatalytic production of hydrogen peroxide from water and O2 using solar energy. Hydrogen peroxide is used as a fuel in hydrogen peroxide fuel cells to make electricity. The combination of the photocatalytic H2O2 production from water and O2 using solar energy with one-compartment H2O2 fuel cells provides on-site production and usage of H2O2 as a more useful and promising solar fuel than hydrogen. This article is part of a Special Issue entitled Biodesign for Bioenergetics--The design and engineering of electronc transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2016 Elsevier B.V. All rights reserved.

Sunlight-Dependent Hydrogen Production by Photosensitizer/Hydrogenase Systems.

PubMed

Adam, David; Bösche, Lisa; Castañeda-Losada, Leonardo; Winkler, Martin; Apfel, Ulf-Peter; Happe, Thomas

2017-03-09

We report a sustainable in vitro system for enzyme-based photohydrogen production. The [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii was tested for photohydrogen production as a proton-reducing catalyst in combination with eight different photosensitizers. Using the organic dye 5-carboxyeosin as a photosensitizer and plant-type ferredoxin PetF as an electron mediator, HydA1 achieves the highest light-driven turnover number (TON HydA1 ) yet reported for an enzyme-based in vitro system (2.9×10 6  mol(H 2 ) mol(cat) -1 ) and a maximum turnover frequency (TOF HydA1 ) of 550 mol(H 2 ) mol(HydA1) -1  s -1 . The system is fueled very effectively by ambient daylight and can be further simplified by using 5-carboxyeosin and HydA1 as a two-component photosensitizer/biocatalyst system without an additional redox mediator. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biological iron-sulfur storage in a thioferrate-protein nanoparticle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vaccaro, Brian J.; Clarkson, Sonya M.; Holden, James F.

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. We assembled them from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. We have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssAmore » nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing B 6,400 Fe atoms and B 170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro.« less

Biological iron-sulfur storage in a thioferrate-protein nanoparticle

DOE PAGES

Vaccaro, Brian J.; Clarkson, Sonya M.; Holden, James F.; ...

2017-07-20

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. We assembled them from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. We have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssAmore » nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing B 6,400 Fe atoms and B 170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro.« less

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

PubMed

Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

2017-05-01

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP + reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

PubMed Central

Kovářová, Julie; Horáková, Eva; Changmai, Piya; Vancová, Marie

2014-01-01

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites. PMID:24243795

Mitochondrial and nucleolar localization of cysteine desulfurase Nfs and the scaffold protein Isu in Trypanosoma brucei.

PubMed

Kovárová, Julie; Horáková, Eva; Changmai, Piya; Vancová, Marie; Lukeš, Julius

2014-03-01

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

Insight into Environmental Effects on Bonding and Redox Properties of [4Fe-4S] Clusters in Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niu, Shuqiang; Ichiye, Toshiko

The large differences in redox potentials between the HiPIPs and ferredoxins are generally attributed to hydrogen bonds and electrostatic effects from the protein and solvent. Recent ligand K-edge X-ray absorption studies by Solomon and co-workers show that the Fe-S covalencies of [4Fe-4S] clusters in the two proteins differ considerably apparently because of hydrogen bonds from water, indicating electronic effects may be important. However, combined density function theory (DFT) and photoelectron spectroscopy studies by our group and Wang and co-workers indicate that hydrogen bonds tune the potential of [4Fe-4S] clusters by mainly electrostatics. The DFT studies here rationalize both results, namelymore » that the observed change in the Fe-S covalency is due to differences in ligand conformation between the two proteins rather than hydrogen bonds. Moreover, the ligand conformation affects the calculated potentials by 100 mV and, thus, is a heretofore unconsidered means of tuning the potential.« less

The Acid-Base Properties, Hydrolytic Mechanism, and Susceptibility to O2 Oxidation of Fe4S4(SR)4-2 Clusters

PubMed Central

Bruice, Thomas C.; Maskiewicz, Richard; Job, Robert

1975-01-01

The iron-sulfur cluster compounds Fe4S4(SR)4-2 [where —SR = —SCH3, —S—C(CH3)3, and —S— CH2—CH(CH3)2] have been found to represent the base species of weak acids of pKa comparable to that of carboxylic acids. The acid species Fe4S4(SR)4H- is most subject to reaction with O2 and to acid-catalyzed solvolysis, while the base species Fe4S4(SR)4-2 most readily undergoes ligand exchange. The kinetics for hydrolysis of the isobutyl mercaptide cluster salt has been investigated in detail and a mechanism involving the stepwise process [Formula: see text] has been proposed. The importance of the acid-base equilibria in determining the reactivity of the iron-sulfur clusters and its possible importance as a factor in the determination of the potentials of ferredoxins and high potential iron protein are discussed. PMID:16592211

Genetic architecture of artemisinin-resistant Plasmodium falciparum

PubMed Central

Miotto, Olivo; Amato, Roberto; Ashley, Elizabeth A; MacInnis, Bronwyn; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Mead, Daniel; Oyola, Samuel O; Dhorda, Mehul; Imwong, Mallika; Woodrow, Charles; Manske, Magnus; Stalker, Jim; Drury, Eleanor; Campino, Susana; Amenga-Etego, Lucas; Thanh, Thuy-Nhien Nguyen; Tran, Hien Tinh; Ringwald, Pascal; Bethell, Delia; Nosten, Francois; Phyo, Aung Pyae; Pukrittayakamee, Sasithon; Chotivanich, Kesinee; Chuor, Char Meng; Nguon, Chea; Suon, Seila; Sreng, Sokunthea; Newton, Paul N; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Htut, Ye; Han, Kay Thwe; Kyaw, Myat Phone; Faiz, Md Abul; Fanello, Caterina I; Onyamboko, Marie; Mokuolu, Olugbenga A; Jacob, Christopher G; Takala-Harrison, Shannon; Plowe, Christopher V; Day, Nicholas P; Dondorp, Arjen M; Spencer, Chris C A; McVean, Gilean; Fairhurst, Rick M; White, Nicholas J; Kwiatkowski, Dominic P

2015-01-01

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. PMID:25599401

Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

PubMed

Boniecki, Michal T; Freibert, Sven A; Mühlenhoff, Ulrich; Lill, Roland; Cygler, Miroslaw

2017-11-03

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Solution 1H NMR determination of secondary structure for the three-iron form of ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

PubMed

Teng, Q; Zhou, Z H; Smith, E T; Busse, S C; Howard, J B; Adams, M W; La Mar, G N

1994-05-24

Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica.

PubMed

Rosenthal, B; Mai, Z; Caplivski, D; Ghosh, S; de la Vega, H; Graf, T; Samuelson, J

1997-06-01

Entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (POR), ferredoxin (FD), and alcohol dehydrogenase E (ADHE). The goal of this study was to determine whether the genes encoding these cytosolic E. histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for E. histolytica genes encoding heat shock protein 60, nicotinamide nucleotide transhydrogenase, and superoxide dismutase. In this study, the E. histolytica por gene and the adhE gene of a second amitochondriate protozoan parasite, Giardia lamblia, were sequenced, and their phylogenetic positions were estimated in relation to POR, ADHE, and FD cloned from eukaryotic and eubacterial organisms. The E. histolytica por gene encodes a 1,620-amino-acid peptide that contained conserved iron-sulfur- and thiamine pyrophosphate-binding sites. The predicted E. histolytica POR showed fewer positional identities to the POR of G. lamblia (34%) than to the POR of the enterobacterium Klebsiella pneumoniae (49%), the cyanobacterium Anabaena sp. (44%), and the protozoan Trichomonas vaginalis (46%), which targets its POR to anaerobic organelles called hydrogenosomes. Maximum-likelihood, neighbor-joining, and parsimony analyses also suggested as less likely E. histolytica POR sharing more recent common ancestry with G. lamblia POR than with POR of bacteria and the T. vaginalis hydrogenosome. The G. lamblia adhE encodes an 888-amino-acid fusion peptide with an aldehyde dehydrogenase at its amino half and an iron-dependent (class 3) ADH at its carboxy half. The predicted G. lamblia ADHE showed extensive positional identities to ADHE of Escherichia coli (49%), Clostridium acetobutylicum (44%), and E. histolytica (43%) and lesser identities to the class 3 ADH of eubacteria and yeast (19 to 36%). Phylogenetic analyses inferred a closer relationship of the E. histolytica ADHE to bacterial ADHE than to the G. lamblia ADHE. The 6-kDa FD of E. histolytica and G. lamblia were most similar to those of the archaebacterium Methanosarcina barkeri and the delta-purple bacterium Desulfovibrio desulfuricans, respectively, while the 12-kDa FD of the T. vaginalis hydrogenosome was most similar to the 12-kDa FD of gamma-purple bacterium Pseudomonas putida. E. histolytica genes (and probably G. lamblia genes) encoding fermentation enzymes therefore likely derive from bacteria by horizontal transfer, although it is not clear from which bacteria these amebic genes derive. These are the first nonorganellar fermentation enzymes of eukaryotes implicated to have derived from bacteria.

Crystal Structure and Catalytic Mechanism of 7-Hydroxymethyl Chlorophyll a Reductase*

PubMed Central

Wang, Xiao; Liu, Lin

2016-01-01

7-Hydroxymethyl chlorophyll a reductase (HCAR) catalyzes the second half-reaction in chlorophyll b to chlorophyll a conversion. HCAR is required for the degradation of light-harvesting complexes and is necessary for efficient photosynthesis by balancing the chlorophyll a/b ratio. Reduction of the hydroxymethyl group uses redox cofactors [4Fe-4S] cluster and FAD to transfer electrons and is difficult because of the strong carbon-oxygen bond. Here, we report the crystal structure of Arabidopsis HCAR at 2.7-Å resolution and reveal that two [4Fe-4S]clusters and one FAD within a very short distance form a consecutive electron pathway to the substrate pocket. In vitro kinetic analysis confirms the ferredoxin-dependent electron transport chain, thus supporting a proton-activated electron transfer mechanism. HCAR resembles a partial reconstruction of an archaeal F420-reducing [NiFe] hydrogenase, which suggests a common mode of efficient proton-coupled electron transfer through conserved cofactor arrangements. Furthermore, the trimeric form of HCAR provides a biological clue of its interaction with light-harvesting complex II. PMID:27072131

A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

PubMed

Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

2011-07-01

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

Cyanobacterial Hydrogenases and Hydrogen Metabolism Revisited: Recent Progress and Future Prospects

PubMed Central

Khanna, Namita; Lindblad, Peter

2015-01-01

Cyanobacteria have garnered interest as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms can utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical processes. Our limited understanding of the cellular hydrogen production pathway is a primary setback in the potential scale-up of this process. In this regard, the present review discusses the recent insight around ferredoxin/flavodoxin as the likely electron donor to the bidirectional Hox hydrogenase instead of the generally accepted NAD(P)H. This may have far reaching implications in powering solar driven hydrogen production. However, it is evident that a successful hydrogen-producing candidate would likely integrate enzymatic traits from different species. Engineering the [NiFe] hydrogenases for optimal catalytic efficiency or expression of a high turnover [FeFe] hydrogenase in these photo-autotrophs may facilitate the development of strains to reach target levels of biohydrogen production in cyanobacteria. The fundamental advancements achieved in these fields are also summarized in this review. PMID:26006225

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

PubMed Central

Santangelo, J D; Jones, D T; Woods, D R

1991-01-01

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.

PubMed

Liew, Fungmin; Henstra, Anne M; Kӧpke, Michael; Winzer, Klaus; Simpson, Sean D; Minton, Nigel P

2017-03-01

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Metabolic Reconstruction and Modeling Microbial Electrosynthesis.

PubMed

Marshall, Christopher W; Ross, Daniel E; Handley, Kim M; Weisenhorn, Pamela B; Edirisinghe, Janaka N; Henry, Christopher S; Gilbert, Jack A; May, Harold D; Norman, R Sean

2017-08-21

Microbial electrosynthesis is a renewable energy and chemical production platform that relies on microbial cells to capture electrons from a cathode and fix carbon. Yet despite the promise of this technology, the metabolic capacity of the microbes that inhabit the electrode surface and catalyze electron transfer in these systems remains largely unknown. We assembled thirteen draft genomes from a microbial electrosynthesis system producing primarily acetate from carbon dioxide, and their transcriptional activity was mapped to genomes from cells on the electrode surface and in the supernatant. This allowed us to create a metabolic model of the predominant community members belonging to Acetobacterium, Sulfurospirillum, and Desulfovibrio. According to the model, the Acetobacterium was the primary carbon fixer, and a keystone member of the community. Transcripts of soluble hydrogenases and ferredoxins from Acetobacterium and hydrogenases, formate dehydrogenase, and cytochromes of Desulfovibrio were found in high abundance near the electrode surface. Cytochrome c oxidases of facultative members of the community were highly expressed in the supernatant despite completely sealed reactors and constant flushing with anaerobic gases. These molecular discoveries and metabolic modeling now serve as a foundation for future examination and development of electrosynthetic microbial communities.

Streptomyces coelicolor SCO4226 Is a Nickel Binding Protein

PubMed Central

Jin, Hua; Zhang, Rong-Guang; Virolle, Marie-Joelle; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five β-strands flanked by two α-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification. PMID:25285530

The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.

PubMed

Heath, Caroline; Posner, Mareike G; Aass, Hans C; Upadhyay, Abhishek; Scott, David J; Hough, David W; Danson, Michael J

2007-10-01

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.

Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation.

PubMed

Ho, Chai-Ling; Teoh, Seddon; Teo, Swee-Sen; Rahim, Raha Abdul; Phang, Siew-Moi

2009-01-01

Light regulates photosynthesis, growth and reproduction, yield and properties of phycocolloids, and starch contents in seaweeds. Despite its importance as an environmental cue that regulates many developmental, physiological, and biochemical processes, the network of genes involved during light deprivation are obscure. In this study, we profiled the transcriptome of Gracilaria changii at two different irradiance levels using a cDNA microarray containing more than 3,000 cDNA probes. Microarray analysis revealed that 93 and 105 genes were up- and down-regulated more than 3-fold under light deprivation, respectively. However, only 50% of the transcripts have significant matches to the nonredundant peptide sequences in the database. The transcripts that accumulated under light deprivation include vanadium chloroperoxidase, thioredoxin, ferredoxin component, and reduced nicotinamide adenine dinucleotide dehydrogenase. Among the genes that were down-regulated under light deprivation were genes encoding light harvesting protein, light harvesting complex I, phycobilisome 7.8 kDa linker polypeptide, low molecular weight early light-inducible protein, and vanadium bromoperoxidase. Our findings also provided important clues to the functions of many unknown sequences that could not be annotated using sequence comparison.

Single gene insertion drives bioalcohol production by a thermophilic archaeon

PubMed Central

Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

2014-01-01

Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

Single gene insertion drives bioalcohol production by a thermophilic archaeon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basen, M; Schut, GJ; Nguyen, DM

2014-12-09

Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. Bymore » heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.« less

The CrIIL reduction of [2Fe-2S] ferredoxins and site of attachment of CrIII using 1H NMR and site-directed mutagenesis.

PubMed

Im, S C; Worrall, J A; Liu, G; Aliverti, A; Zanetti, G; Luchinat, C; Bertini, I; Sykes, A G

2000-04-17

The recently reported NMR solution structure of FeIIIFeIII parsley FdI has made possible 2D NOESY NMR studies to determine the point of attachment of CrIIIL in FeIIIFeIII...CrIIIL. The latter Cr-modified product was obtained by reduction of FeIIIFeIII parsley and spinach FdI forms with [Cr(15-aneN4) (H2O)2]2+ (15-aneN4 = 1,4,8,12-tetraazacyclopentadecane), referred to here as CrIIL, followed by air oxidation and chromatographic purification. From a comparison of NMR cross-peak intensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic CrIIIL broadening of a number of amino acid peaks, have been identified. The effects at site A (residues 19-22, 27, and 30) are greater than those at site B (residues 92-94 and 96), which is on the opposite side of the protein. From metal (ICP-AES) and electrospray ionization mass spectrometry (EIMS) analyses on the Cr-modified protein, attachment of a single CrIIIL only is confirmed for both parsley and spinach FdI and FdII proteins. Electrostatic interaction of the 3+ CrIIIL center covalently attached to one protein molecule (charge approximately -18) with a second (like) molecule provides an explanation for the involvement of two regions. Thus for 3-4 mM FeIIIFeIII...CrIIIL solutions used in NMR studies (CrIIIL attached at A), broadening effects due to electrostatic interactions at B on a second molecule are observed. Experiments with the Cys18Ala spinach FdI variant have confirmed that the previously suggested Cys-18 at site A is not the site of CrIIIL attachment. Line broadening at Val-22 of A gives the largest effect, and CrIIIL attachment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of CrIIL to bind in some (parsley and spinach) but not all cases (Anabaena variabilis) suggests that intramolecular H-bonding of acidic residues at A is relevant. The parsley and spinach FeIIFeIII...CrIIIL products undergo a second stage of reduction with the formation of FeIIFeII...CrIIIL. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appears that Glu-92 is required for the second stage of reduction to occur. A sample of CrIIIL-modified parsley FeIIIFeIII Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase.

Purification and Biochemical Properties of Phytochromobilin Synthase from Etiolated Oat Seedlings1

PubMed Central

McDowell, Michael T.; Lagarias, J. Clark

2001-01-01

Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (PΦB) for photoactivity. In planta, biliverdin IXα (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme PΦB synthase to yield 3Z-PΦB. Here, we describe the >50,000-fold purification of PΦB synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, PΦB synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s−1, which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat PΦB synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of PΦB synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A Km for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 μm. PΦB synthase has a high affinity for its bilin substrate, with a sub-micromolar Km for BV. PMID:11500553

Binding Thermodynamics of Ferredoxin:NADP+ Reductase: Two Different Protein Substrates and One Energetics

PubMed Central

Martínez-Júlvez, Marta; Medina, Milagros; Velázquez-Campoy, Adrián

2009-01-01

Abstract The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP+ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His299 FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics. PMID:19527656

Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

PubMed

Ghoshroy, Sohini; Robertson, Deborah L

2015-01-01

Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

Metabolic Reconstruction and Modeling Microbial Electrosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marshall, Christopher W.; Ross, Daniel E.; Handley, Kim M.

Microbial electrosynthesis is a renewable energy and chemical production platform that relies on microbial cells to capture electrons from a cathode and fix carbon. Yet despite the promise of this technology, the metabolic capacity of the microbes that inhabit the electrode surface and catalyze electron transfer in these systems remains largely unknown. Here, we assembled thirteen draft genomes from a microbial electrosynthesis system producing primarily acetate from carbon dioxide, and their transcriptional activity was mapped to genomes from cells on the electrode surface and in the supernatant. This allowed us to create a metabolic model of the predominant community membersmore » belonging to Acetobacterium, Sulfurospirillum, and Desulfovibrio. According to the model, the Acetobacterium was the primary carbon fixer, and a keystone member of the community. Transcripts of soluble hydrogenases and ferredoxins from Acetobacterium and hydrogenases, formate dehydrogenase, and cytochromes of Desulfovibrio were found in high abundance near the electrode surface. Cytochrome c oxidases of facultative members of the community were highly expressed in the supernatant despite completely sealed reactors and constant flushing with anaerobic gases. The resulting molecular discoveries and metabolic modeling now serve as a foundation for future examination and development of electrosynthetic microbial communities.« less

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

PubMed

Wagner, Tristan; Koch, Jürgen; Ermler, Ulrich; Shima, Seigo

2017-08-18

In methanogenic archaea, the carbon dioxide (CO 2 ) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Structural principles for computational and de novo design of 4Fe-4S metalloproteins

PubMed Central

Nanda, Vikas; Senn, Stefan; Pike, Douglas H.; Rodriguez-Granillo, Agustina; Hansen, Will; Khare, Sagar D.; Noy, Dror

2017-01-01

Iron-sulfur centers in metalloproteins can access multiple oxidation states over a broad range of potentials, allowing them to participate in a variety of electron transfer reactions and serving as catalysts for high-energy redox processes. The nitrogenase FeMoCO cluster converts di-nitrogen to ammonia in an eight-electron transfer step. The 2(Fe4S4) containing bacterial ferredoxin is an evolutionarily ancient metalloprotein fold and is thought to be a primordial progenitor of extant oxidoreductases. Controlling chemical transformations mediated by iron-sulfur centers such as nitrogen fixation, hydrogen production as well as electron transfer reactions involved in photosynthesis are of tremendous importance for sustainable chemistry and energy production initiatives. As such, there is significant interest in the design of iron-sulfur proteins as minimal models to gain fundamental understanding of complex natural systems and as lead-molecules for industrial and energy applications. Herein, we discuss salient structural characteristics of natural iron-sulfur proteins and how they guide principles for design. Model structures of past designs are analyzed in the context of these principles and potential directions for enhanced designs are presented, and new areas of iron-sulfur protein design are proposed. PMID:26449207

Metabolism and metronidazole uptake in Trichomonas vaginalis isolates with different metronidazole susceptibilities.

PubMed Central

Müller, M; Gorrell, T E

1983-01-01

Three Trichomonas vaginalis isolates with low in vivo susceptibilities to metronidazole (95% curative dose, greater than 3 X 100 mg kg-1 in subcutaneous infections in mice) were compared with strain ATCC 30001 and with four isolates exhibiting high in vivo susceptibilities (95% curative dose, less than 3 X 15 mg kg-1). Activity of pyruvate:ferredoxin oxidoreductase, anaerobic fermentation, and anaerobic intracellular accumulation of [14C]metronidazole label showed no significant isolate-dependent differences which could be correlated with drug susceptibility. The results suggest that processes providing electrons for metronidazole activation are not defective in the resistant strains. Aerobiosis, known to inhibit the antimicrobial action of metronidazole, inhibited accumulation of label more strongly in resistant isolates than in susceptible ones. No differences were detected, however, between resistant and susceptible isolates in respiration, aerobic fermentation, and the specific activity of NADH and NADPH oxidases, the main terminal oxidases of T. vaginalis. These findings suggest that the production of electrons is not diminished under aerobic conditions. The inhibitory effect of aerobic conditions on metronidazole activation, possibly due to competition for the electrons, is markedly enhanced in the resistant isolates compared to the susceptible ones. The mechanism of this effect, however, remains unknown. PMID:6607028

Proteomics of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates

PubMed Central

Goris, Tobias; Schiffmann, Christian L.; Gadkari, Jennifer; Schubert, Torsten; Seifert, Jana; Jehmlich, Nico; von Bergen, Martin; Diekert, Gabriele

2015-01-01

Organohalide respiration is an environmentally important but poorly characterized type of anaerobic respiration. We compared the global proteome of the versatile organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans grown with different electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). The most significant differences in protein abundance were found for gene products of the organohalide respiration region. This genomic region encodes the corrinoid and FeS cluster containing PCE reductive dehalogenase PceA and other proteins putatively involved in PCE metabolism such as those involved in corrinoid biosynthesis. The latter gene products as well as PceA and a putative quinol dehydrogenase were almost exclusively detected in cells grown with PCE. This finding suggests an electron flow from the electron donor such as formate or pyruvate via the quinone pool and a quinol dehydrogenase to PceA and the terminal electron acceptor PCE. Two putative accessory proteins, an IscU-like protein and a peroxidase-like protein, were detected with PCE only and might be involved in PceA maturation. The proteome of cells grown with pyruvate instead of formate as electron donor indicates a route of electrons from reduced ferredoxin via an Epsilonproteobacterial complex I and the quinone pool to PCE. PMID:26387727

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing,more » Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.« less

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.

PubMed

Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D; Proudfoot, Michael; Makarova, Kira S; Kudritska, Marina; Kochinyan, Samvel; Wang, Shuren; Chruszcz, Maksymilian; Minor, Wladek; Koonin, Eugene V; Edwards, Aled M; Savchenko, Alexei; Yakunin, Alexander F

2008-07-18

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

Chloroplast evolution, structure and functions

PubMed Central

Jensen, Poul Erik

2014-01-01

In this review, we consider a selection of recent advances in chloroplast biology. These include new findings concerning chloroplast evolution, such as the identification of Chlamydiae as a third partner in primary endosymbiosis, a second instance of primary endosymbiosis represented by the chromatophores found in amoebae of the genus Paulinella, and a new explanation for the longevity of captured chloroplasts (kleptoplasts) in sacoglossan sea slugs. The controversy surrounding the three-dimensional structure of grana, its recent resolution by tomographic analyses, and the role of the CURVATURE THYLAKOID1 (CURT1) proteins in supporting grana formation are also discussed. We also present an updated inventory of photosynthetic proteins and the factors involved in the assembly of thylakoid multiprotein complexes, and evaluate findings that reveal that cyclic electron flow involves NADPH dehydrogenase (NDH)- and PGRL1/PGR5-dependent pathways, both of which receive electrons from ferredoxin. Other topics covered in this review include new protein components of nucleoids, an updated inventory of the chloroplast proteome, new enzymes in chlorophyll biosynthesis and new candidate messengers in retrograde signaling. Finally, we discuss the first successful synthetic biology approaches that resulted in chloroplasts in which electrons from the photosynthetic light reactions are fed to enzymes derived from secondary metabolism. PMID:24991417

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

PubMed

Price, Morgan N; Zane, Grant M; Kuehl, Jennifer V; Melnyk, Ryan A; Wall, Judy D; Deutschbauer, Adam M; Arkin, Adam P

2018-01-01

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

Amixicile, a novel strategy for targeting oral anaerobic pathogens.

PubMed

Hutcherson, Justin A; Sinclair, Kathryn M; Belvin, Benjamin R; Gui, Qin; Hoffman, Paul S; Lewis, Janina P

2017-09-05

The oral microflora is composed of both health-promoting as well as disease-initiating bacteria. Many of the disease-initiating bacteria are anaerobic and include organisms such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. Here we investigated a novel therapeutic, amixicile, that targets pyruvate:ferredoxin oxidoreductase (PFOR), a major metabolic enzyme involved in energy generation through oxidative decarboxylation of pyruvate. PFOR is present in these anaerobic pathogenic bacteria and thus we hypothesized that amixicile would effectively inhibit their growth. In general, PFOR is present in all obligate anaerobic bacteria, while oral commensal aerobes, including aerotolerant ones, such as Streptococcus gordonii, use pyruvate dehydrogenase to decarboxylate pyruvate. Accordingly, we observed that growth of the PFOR-containing anaerobic periodontal pathogens, grown in both monospecies as well as multispecies broth cultures was inhibited in a dose-dependent manner while that of S. gordonii was unaffected. Furthermore, we also show that amixicile is effective against these pathogens grown as monospecies and multispecies biofilms. Finally, amixicile is the first selective therapeutic agent active against bacteria internalized by host cells. Together, the results show that amixicile is an effective inhibitor of oral anaerobic bacteria and as such, is a good candidate for treatment of periodontal diseases.

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Zane, Grant M.; Kuehl, Jennifer V.

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. Here, we studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fillmore » 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.« less

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

DOE PAGES

Price, Morgan N.; Zane, Grant M.; Kuehl, Jennifer V.; ...

2018-01-11

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. Here, we studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fillmore » 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.« less

Potential for microbial H2 and metal transformations associated with novel bacteria and archaea in deep terrestrial subsurface sediments.

PubMed

Hernsdorf, Alex W; Amano, Yuki; Miyakawa, Kazuya; Ise, Kotaro; Suzuki, Yohey; Anantharaman, Karthik; Probst, Alexander; Burstein, David; Thomas, Brian C; Banfield, Jillian F

2017-08-01

Geological sequestration in deep underground repositories is the prevailing proposed route for radioactive waste disposal. After the disposal of radioactive waste in the subsurface, H 2 may be produced by corrosion of steel and, ultimately, radionuclides will be exposed to the surrounding environment. To evaluate the potential for microbial activities to impact disposal systems, we explored the microbial community structure and metabolic functions of a sediment-hosted ecosystem at the Horonobe Underground Research Laboratory, Hokkaido, Japan. Overall, we found that the ecosystem hosted organisms from diverse lineages, including many from the phyla that lack isolated representatives. The majority of organisms can metabolize H 2 , often via oxidative [NiFe] hydrogenases or electron-bifurcating [FeFe] hydrogenases that enable ferredoxin-based pathways, including the ion motive Rnf complex. Many organisms implicated in H 2 metabolism are also predicted to catalyze carbon, nitrogen, iron and sulfur transformations. Notably, iron-based metabolism is predicted in a novel lineage of Actinobacteria and in a putative methane-oxidizing ANME-2d archaeon. We infer an ecological model that links microorganisms to sediment-derived resources and predict potential impacts of microbial activity on H 2 consumption and retardation of radionuclide migration.

A new metal binding domain involved in cadmium, cobalt and zinc transport

DOE PAGES

Smith, Aaron T.; Barupala, Dulmini; Stemmler, Timothy L.; ...

2015-07-20

The P 1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P 1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. In this paper, we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P 1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd 2+, Co 2+ or Zn 2+ ions in distinctmore » and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Finally, taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P 1B-ATPases.« less

Development of the photosynthetic apparatus of Cunninghamia lanceolata in light and darkness.

PubMed

Xue, Xian; Wang, Qi; Qu, Yanli; Wu, Hongyang; Dong, Fengqin; Cao, Haoyan; Wang, Hou-Ling; Xiao, Jianwei; Shen, Yingbai; Wan, Yinglang

2017-01-01

Here, we compared the development of dark- and light-grown Chinese fir (Cunninghamia lanceolata) cotyledons, which synthesize chlorophyll in the dark, representing a different phenomenon from angiosperm model plants. We determined that the grana lamellar membranes were well developed in both chloroplasts and etiochloroplasts. The accumulation of thylakoid membrane protein complexes was similar between chloroplasts and etiochloroplasts. Measurement of chlorophyll fluorescence parameters indicated that photosystem II (PSII) had low photosynthetic activities, whereas the photosystem I (PSI)-driven cyclic electron flow (CEF) rate exceeded the rate of PSII-mediated photon harvesting in etiochloroplasts. Analysis of the protein contents in etiochloroplasts indicated that the light-harvesting complex II remained mostly in its monomeric conformation. The ferredoxin NADP + oxidoreductase and NADH dehydrogenase-like complexes were relatively abundantly expressed in etiochloroplasts for Chinese fir. Our transcriptome analysis contributes a global expression database for Chinese fir cotyledons, providing background information on the regulatory mechanisms of different genes involved in the development of dark- and light-grown cotyledons. In conclusion, we provide a novel description of the early developmental status of the light-dependent and light-independent photosynthetic apparatuses in gymnosperms. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Metabolic Reconstruction and Modeling Microbial Electrosynthesis

DOE PAGES

Marshall, Christopher W.; Ross, Daniel E.; Handley, Kim M.; ...

2017-08-21

Microbial electrosynthesis is a renewable energy and chemical production platform that relies on microbial cells to capture electrons from a cathode and fix carbon. Yet despite the promise of this technology, the metabolic capacity of the microbes that inhabit the electrode surface and catalyze electron transfer in these systems remains largely unknown. Here, we assembled thirteen draft genomes from a microbial electrosynthesis system producing primarily acetate from carbon dioxide, and their transcriptional activity was mapped to genomes from cells on the electrode surface and in the supernatant. This allowed us to create a metabolic model of the predominant community membersmore » belonging to Acetobacterium, Sulfurospirillum, and Desulfovibrio. According to the model, the Acetobacterium was the primary carbon fixer, and a keystone member of the community. Transcripts of soluble hydrogenases and ferredoxins from Acetobacterium and hydrogenases, formate dehydrogenase, and cytochromes of Desulfovibrio were found in high abundance near the electrode surface. Cytochrome c oxidases of facultative members of the community were highly expressed in the supernatant despite completely sealed reactors and constant flushing with anaerobic gases. The resulting molecular discoveries and metabolic modeling now serve as a foundation for future examination and development of electrosynthetic microbial communities.« less

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

PubMed Central

Kuehl, Jennifer V.; Melnyk, Ryan A.; Deutschbauer, Adam M.; Arkin, Adam P.

2018-01-01

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes. PMID:29324779

Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

PubMed Central

Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

2011-01-01

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

Metabolism in anoxic permeable sediments is dominated by eukaryotic dark fermentation

NASA Astrophysics Data System (ADS)

Bourke, Michael F.; Marriott, Philip J.; Glud, Ronnie N.; Hasler-Sheetal, Harald; Kamalanathan, Manoj; Beardall, John; Greening, Chris; Cook, Perran L. M.

2017-01-01

Permeable sediments are common across continental shelves and are critical contributors to marine biogeochemical cycling. Organic matter in permeable sediments is dominated by microalgae, which as eukaryotes have different anaerobic metabolic pathways to bacteria and archaea. Here we present analyses of flow-through reactor experiments showing that dissolved inorganic carbon is produced predominantly as a result of anaerobic eukaryotic metabolic activity. In our experiments, anaerobic production of dissolved inorganic carbon was consistently accompanied by large dissolved H2 production rates, suggesting the presence of fermentation. The production of both dissolved inorganic carbon and H2 persisted following administration of broad spectrum bactericidal antibiotics, but ceased following treatment with metronidazole. Metronidazole inhibits the ferredoxin/hydrogenase pathway of fermentative eukaryotic H2 production, suggesting that pathway as the source of H2 and dissolved inorganic carbon production. Metabolomic analysis showed large increases in lipid production at the onset of anoxia, consistent with documented pathways of anoxic dark fermentation in microalgae. Cell counts revealed a predominance of microalgae in the sediments. H2 production was observed in dark anoxic cultures of diatoms (Fragilariopsis sp.) and a chlorophyte (Pyramimonas) isolated from the study site, substantiating the hypothesis that microalgae undertake fermentation. We conclude that microalgal dark fermentation could be an important energy-conserving pathway in permeable sediments.

Metabolism in anoxic permeable sediments is dominated by eukaryotic dark fermentation

PubMed Central

Bourke, Michael F; Marriott, Philip J.; Glud, Ronnie N.; Hasler-Sheetal, Harald; Kamalanathan, Manoj; Beardall, John; Greening, Chris; Cook, Perran L.M.

2016-01-01

Permeable sediments are common across continental shelves and are critical contributors to marine biogeochemical cycling. Organic matter in permeable sediments is dominated by microalgae, which as eukaryotes have different anaerobic metabolic pathways to prokaryotes such as bacteria and archaea. Here we present analyses of flow-through reactor experiments showing that dissolved inorganic carbon is produced predominantly as a result of anaerobic eukaryotic metabolic activity. In our experiments, anaerobic production of dissolved inorganic carbon was consistently accompanied by large dissolved H2 production rates, suggesting the presence of fermentation. The production of both dissolved inorganic carbon and H2 persisted following administration of broad spectrum bactericidal antibiotics, but ceased following treatment with metronidazole. Metronidazole inhibits the ferredoxin/hydrogenase pathway of fermentative eukaryotic H2 production, suggesting that pathway as the source of H2 and dissolved inorganic carbon production. Metabolomic analysis showed large increases in lipid production at the onset of anoxia, consistent with documented pathways of anoxic dark fermentation in microalgae. Cell counts revealed a predominance of microalgae in the sediments. H2 production was observed in dark anoxic cultures of diatoms (Fragilariopsis sp.) and a chlorophyte (Pyramimonas) isolated from the study site, substantiating the hypothesis that microalgae undertake fermentation. We conclude that microalgal dark fermentation could be an important energy-conserving pathway in permeable sediments. PMID:28070216

Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.

2013-03-05

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux throughmore » Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.« less

X-ray Structure of the Mature Ectodomain of Phogrin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noguera, M. E.; Primo, M.; Jakoncic, J.

2014-11-26

Phogrin/IA-2β and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet β-cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and β-cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure–function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7.4 and 4.6 has been solved at 1.95- and 2.01-Å resolution, respectively. Likewise to the ME of ICA512, ME phogrin adopts a ferredoxin-like fold:more » a sheet of four antiparallel β-strands packed against two α-helices. Furthermore, sequence conservation among vertebrates, plants and insects suggests that the structural similarity extends to all the receptor family. Crystallized ME phogrin is monomeric, in agreement with solution studies but in striking contrast with the behavior of homodimeric ME ICA512. The structural details that may cause the quaternary structure differences are analyzed. The results provide a basis for building models of the overall orientation and oligomerization state of the receptor in biological membranes.« less

Organization of genes responsible for the stereospecific conversion of hydantoins to alpha-amino acids in Arthrobacter aurescens DSM 3747.

PubMed

Wiese, A; Syldatk, C; Mattes, R; Altenbuchner, J

2001-09-01

Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5'-monosubstituted hydantoins to alpha-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.

All-atom molecular dynamics simulation of a photosystem i/detergent complex.

PubMed

Harris, Bradley J; Cheng, Xiaolin; Frymier, Paul

2014-10-09

All-atom molecular dynamics (MD) simulation was used to investigate the solution structure and dynamics of the photosynthetic pigment-protein complex photosystem I (PSI) from Thermosynechococcus elongatus embedded in a toroidal belt of n-dodecyl-β-d-maltoside (DDM) detergent. Evaluation of root-mean-square deviations (RMSDs) relative to the known crystal structure show that the protein complex surrounded by DDM molecules is stable during the 200 ns simulation time, and root-mean-square fluctuation (RMSF) analysis indicates that regions of high local mobility correspond to solvent-exposed regions such as turns in the transmembrane α-helices and flexible loops on the stromal and lumenal faces. Comparing the protein-detergent complex to a pure detergent micelle, the detergent surrounding the PSI trimer is found to be less densely packed but with more ordered detergent tails, contrary to what is seen in most lipid bilayer models. We also investigated any functional implications for the observed conformational dynamics and protein-detergent interactions, discovering interesting structural changes in the psaL subunits associated with maintaining the trimeric structure of the protein. Importantly, we find that the docking of soluble electron mediators such as cytochrome c6 and ferredoxin to PSI is not significantly impacted by the solubilization of PSI in detergent.

A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D.

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure ofmore » SSO1404 was solved at 1.6{angstrom} resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.« less

Bioinformatics analysis to assess potential risks of allergenicity and toxicity of HRAP and PFLP proteins in genetically modified bananas resistant to Xanthomonas wilt disease.

PubMed

Jin, Yuan; Goodman, Richard E; Tetteh, Afua O; Lu, Mei; Tripathi, Leena

2017-11-01

Banana Xanthomonas wilt (BXW) disease threatens banana production and food security throughout East Africa. Natural resistance is lacking among common cultivars. Genetically modified (GM) bananas resistant to BXW disease were developed by inserting the hypersensitive response-assisting protein (Hrap) or/and the plant ferredoxin-like protein (Pflp) gene(s) from sweet pepper (Capsicum annuum). Several of these GM banana events showed 100% resistance to BXW disease under field conditions in Uganda. The current study evaluated the potential allergenicity and toxicity of the expressed proteins HRAP and PFLP based on evaluation of published information on the history of safe use of the natural source of the proteins as well as established bioinformatics sequence comparison methods to known allergens (www.AllergenOnline.org and NCBI Protein) and toxins (NCBI Protein). The results did not identify potential risks of allergy and toxicity to either HRAP or PFLP proteins expressed in the GM bananas that might suggest potential health risks to humans. We recognize that additional tests including stability of these proteins in pepsin assay, nutrient analysis and possibly an acute rodent toxicity assay may be required by national regulatory authorities. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The water-water cycle as alternative photon and electron sinks.

PubMed

Asada, K

2000-10-29

The water-water cycle in chloroplasts is the photoreduction of dioxygen to water in photosystem I (PS I) by the electrons generated in photosystem II (PS II) from water. In the water-water cycle, the rate of photoreduction of dioxygen in PS I is several orders of magnitude lower than those of the disproportionation of superoxide catalysed by superoxide dismutase, the reduction of hydrogen peroxide to water catalysed by ascorbate peroxidase, and the reduction of the resulting oxidized forms of ascorbate by reduced ferredoxin or catalysed by either dehydroascorbate reductase or monodehydroascorbate reductase. The water-water cycle therefore effectively shortens the lifetimes of photoproduced superoxide and hydrogen peroxide to suppress the production of hydroxyl radicals, their interactions with the target molecules in chloroplasts, and resulting photoinhibition. When leaves are exposed to photon intensities of sunlight in excess of that required to support the fixation of CO2, the intersystem electron carriers are over-reduced, resulting in photoinhibition. Under such conditions, the water-water cycle not only scavenges active oxygens, but also safely dissipates excess photon energy and electrons, in addition to downregulation of PS II and photorespiration. The dual functions of the water-water cycle for protection from photoinhibition under photon excess stress are discussed, along with its functional evolution.

All-atom molecular dynamics simulation of a photosystem I/detergent complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harris, Bradley J.; Cheng, Xiaolin; Frymier, Paul

2014-09-18

All-atom molecular dynamics (MD) simulation was used to investigate the solution structure and dynamics of the photosynthetic pigment protein complex photosystem I (PSI) from Thermosynechococcus elongatus embedded in a toroidal belt of n-dodecyl-β-d-maltoside (DDM) detergent. Evaluation of root-mean-square deviations (RMSDs) relative to the known crystal structure show that the protein complex surrounded by DDM molecules is stable during the 200 ns simulation time, and root-mean-square fluctuation (RMSF) analysis indicates that regions of high local mobility correspond to solvent-exposed regions such as turns in the transmembrane α-helices and flexible loops on the stromal and lumenal faces. Comparing the protein detergent complexmore » to a pure detergent micelle, the detergent surrounding the PSI trimer is found to be less densely packed but with more ordered detergent tails, contrary to what is seen in most lipid bilayer models. We also investigated any functional implications for the observed conformational dynamics and protein detergent interactions, discovering interesting structural changes in the psaL subunits associated with maintaining the trimeric structure of the protein. Moreover, we find that the docking of soluble electron mediators such as cytochrome c 6 and ferredoxin to PSI is not significantly impacted by the solubilization of PSI in detergent.« less

Sustained Photobiological Hydrogen Gas Production upon Reversible Inactivation of Oxygen Evolution in the Green Alga Chlamydomonas reinhardtii1

PubMed Central

Melis, Anastasios; Zhang, Liping; Forestier, Marc; Ghirardi, Maria L.; Seibert, Michael

2000-01-01

The work describes a novel approach for sustained photobiological production of H2 gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H2 production method circumvents the severe O2 sensitivity of the reversible hydrogenase by temporally separating photosynthetic O2 evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H2 production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O2 evolution. Under these conditions, oxidative respiration by the cells in the light depleted O2 and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H2 gas production was observed in the light but not in the dark. The mechanism of H2 production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b6-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H2 evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H2 in the chloroplast stroma. PMID:10631256

Potential for microbial H2 and metal transformations associated with novel bacteria and archaea in deep terrestrial subsurface sediments

PubMed Central

Hernsdorf, Alex W; Amano, Yuki; Miyakawa, Kazuya; Ise, Kotaro; Suzuki, Yohey; Anantharaman, Karthik; Probst, Alexander; Burstein, David; Thomas, Brian C; Banfield, Jillian F

2017-01-01

Geological sequestration in deep underground repositories is the prevailing proposed route for radioactive waste disposal. After the disposal of radioactive waste in the subsurface, H2 may be produced by corrosion of steel and, ultimately, radionuclides will be exposed to the surrounding environment. To evaluate the potential for microbial activities to impact disposal systems, we explored the microbial community structure and metabolic functions of a sediment-hosted ecosystem at the Horonobe Underground Research Laboratory, Hokkaido, Japan. Overall, we found that the ecosystem hosted organisms from diverse lineages, including many from the phyla that lack isolated representatives. The majority of organisms can metabolize H2, often via oxidative [NiFe] hydrogenases or electron-bifurcating [FeFe] hydrogenases that enable ferredoxin-based pathways, including the ion motive Rnf complex. Many organisms implicated in H2 metabolism are also predicted to catalyze carbon, nitrogen, iron and sulfur transformations. Notably, iron-based metabolism is predicted in a novel lineage of Actinobacteria and in a putative methane-oxidizing ANME-2d archaeon. We infer an ecological model that links microorganisms to sediment-derived resources and predict potential impacts of microbial activity on H2 consumption and retardation of radionuclide migration. PMID:28350393

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence[OPEN

PubMed Central

Christ, Bastien; Das, Aditi; Hörtensteiner, Stefan

2016-01-01

Chlorophyll degradation is the most obvious hallmark of leaf senescence. Phyllobilins, linear tetrapyrroles that are derived from opening of the chlorin macrocycle by the Rieske-type oxygenase PHEOPHORBIDE a OXYGENASE (PAO), are the end products of chlorophyll degradation. Phyllobilins carry defined modifications at several peripheral positions within the tetrapyrrole backbone. While most of these modifications are species-specific, hydroxylation at the C32 position is commonly found in all species analyzed to date. We demonstrate that this hydroxylation occurs in senescent chloroplasts of Arabidopsis thaliana. Using bell pepper (Capsicum annuum) chromoplasts, we establish that phyllobilin hydroxylation is catalyzed by a membrane-bound, molecular oxygen-dependent, and ferredoxin-dependent activity. As these features resemble the requirements of PAO, we considered membrane-bound Rieske-type oxygenases as potential candidates. Analysis of mutants of the two Arabidopsis Rieske-type oxygenases (besides PAO) uncovered that phyllobilin hydroxylation depends on TRANSLOCON AT THE INNER CHLOROPLAST ENVELOPE55 (TIC55). Our work demonstrates a catalytic activity for TIC55, which in the past has been considered as a redox sensor of protein import into plastids. Given the wide evolutionary distribution of both PAO and TIC55, we consider that chlorophyll degradation likely coevolved with land plants. PMID:27655840

Expression profiles of sugarcane under drought conditions: Variation in gene regulation.

PubMed

Andrade, Júlio César Farias de; Terto, Jackeline; Silva, José Vieira; Almeida, Cícero

2015-12-01

Drought is a major factor in decreased sugarcane productivity because of the resulting morphophysiological effects that it causes. Gene expression studies that have examined the influence of water stress in sugarcane have yielded divergent results, indicating the absence of a fixed pattern of changes in gene expression. In this work, we investigated the expression profiles of 12 genes in the leaves of a drought-tolerant genotype (RB72910) of sugarcane and compared the results with those of other studies. The genotype was subjected to 80-100% water availability (control condition) and 0-20% water availability (simulated drought). To analyze the physiological status, the SPAD index, Fv/Fm ratio, net photosynthesis (A), stomatal conductance (gs) and stomatal transpiration (E) were measured. Total RNA was extracted from leaves and the expression of SAMDC, ZmPIP2-1 protein, ZmTIP4-2 protein, WIP protein, LTP protein, histone H3, DNAj, ferredoxin I, β-tubulin, photosystem I, gene 1 and gene 2 was analyzed by quantitative real-time PCR (RT-PCR). Important differences in the expression profiles of these genes were observed when compared with other genotypes, suggesting that complex defense mechanisms are activated in response to water stress. However, there was no recognizable pattern for the changes in expression of the different proteins associated with tolerance to drought stress.

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum.

PubMed Central

Kenealy, W R; Thompson, T E; Schubert, K R; Zeikus, J G

1982-01-01

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting. PMID:6122678

Electron Bifurcation: Thermodynamics and Kinetics of Two-Electron Brokering in Biological Redox Chemistry.

PubMed

Zhang, Peng; Yuly, Jonathon L; Lubner, Carolyn E; Mulder, David W; King, Paul W; Peters, John W; Beratan, David N

2017-09-19

How can proteins drive two electrons from a redox active donor onto two acceptors at very different potentials and distances? And how can this transaction be conducted without dissipating very much energy or violating the laws of thermodynamics? Nature appears to have addressed these challenges by coupling thermodynamically uphill and downhill electron transfer reactions, using two-electron donor cofactors that have very different potentials for the removal of the first and second electron. Although electron bifurcation is carried out with near perfection from the standpoint of energy conservation and electron delivery yields, it is a biological energy transduction paradigm that has only come into focus recently. This Account provides an exegesis of the biophysical principles that underpin electron bifurcation. Remarkably, bifurcating electron transfer (ET) proteins typically send one electron uphill and one electron downhill by similar energies, such that the overall reaction is spontaneous, but not profligate. Electron bifurcation in the NADH-dependent reduced ferredoxin: NADP + oxidoreductase I (Nfn) is explored in detail here. Recent experimental progress in understanding the structure and function of Nfn allows us to dissect its workings in the framework of modern ET theory. The first electron that leaves the two-electron donor flavin (L-FAD) executes a positive free energy "uphill" reaction, and the departure of this electron switches on a second thermodynamically spontaneous ET reaction from the flavin along a second pathway that moves electrons in the opposite direction and at a very different potential. The singly reduced ET products formed from the bifurcating flavin are more than two nanometers distant from each other. In Nfn, the second electron to leave the flavin is much more reducing than the first: the potentials are said to be "crossed." The eventually reduced cofactors, NADH and ferredoxin in the case of Nfn, perform crucial downstream redox processes of their own. We dissect the thermodynamics and kinetics of electron bifurcation in Nfn and find that the key features of electron bifurcation are (1) spatially separated transfer pathways that diverge from a two-electron donor, (2) one thermodynamically uphill and one downhill redox pathway, with a large negative shift in the donor's reduction potential after departure of the first electron, and (3) electron tunneling and activation factors that enable bifurcation, producing a 1:1 partitioning of electrons onto the two pathways. Electron bifurcation is found in the CO 2 reducing pathways of methanogenic archaea, in the hydrogen pathways of hydrogenases, in the nitrogen fixing pathway of Fix, and in the mitochondrial charge transfer chain of complex III, cytochrome bc 1 . While crossed potentials may offer the biological advantage of producing tightly regulated high energy reactive species, neither kinetic nor thermodynamic considerations mandate crossed potentials to generate successful electron bifurcation. Taken together, the theoretical framework established here, focusing on the underpinning electron tunneling barriers and activation free energies, explains the logic of electron bifurcation that enables energy conversion and conservation in Nfn, points toward bioinspired schemes to execute multielectron redox chemistry, and establishes a roadmap for examining novel electron bifurcation networks in nature.

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aklujkar, Muktak; Krushkal, Julia; DiBartolo, Genevieve

Background. The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results. The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recentlymore » in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion. The genomic evidence suggests that metabolism, physiology Background. The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results. The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion. The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.« less

Sulfur K-Edge XAS Studies of the Effect of DNA Binding on the [Fe 4 S 4 ] Site in EndoIII and MutY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ha, Yang; Arnold, Anna R.; Nuñez, Nicole N.

S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe 4S 4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe–S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron–thiolate and iron–sulfide bonds would stabilize the oxidized state of the [Fe 4S 4] clusters. Our results are compared to those on previously studied [Fe 4S 4] model complexes, ferredoxin (Fd), and to new data on high-potential iron–sulfur protein (HiPIP). A limited decrease in covalency ismore » observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe 4S 4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron–sulfur bonds. Furthermore, in EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.« less

Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA.

PubMed

Zubieta, Chloe; Joseph, Rosanne; Krishna, S Sri; McMullan, Daniel; Kapoor, Mili; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elias, Ylva; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Murphy, Kevin D; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; Trout, Christina V; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2007-11-01

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases. (c) 2007 Wiley-Liss, Inc.

The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria.

PubMed

Horiuchi, Mayumi; Nakamura, Kinu; Kojima, Kouji; Nishiyama, Yoshitaka; Hatakeyama, Wakako; Hisabori, Toru; Hihara, Yukako

2010-10-01

The redox state of the photosynthetic electron transport chain acts as a critical sensing mechanism by regulating the transcription of key genes involved in the acclimation response to a change in the environment. In the present study we show that the small LuxR-type regulator PedR interacts with Trx (thioredoxin) to achieve photosynthetic electron-transport-dependent transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. TrxM, an isoform of Trx, was isolated as an interacting factor of PedR by pull-down assays. In vitro analysis revealed that the intermolecular disulfide bond formed between Cys80 residues of the PedR homodimer was reduced by both TrxM and TrxX. It has been shown previously that, although PedR is active under low-light conditions, it becomes transiently inactivated following a shift to high-light conditions, with a concomitant conformational change [Nakamura and Hihara (2006) J. Biol. Chem. 281, 36758-36766]. In the present study, we found that the conformational change of PedR and the change in the transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin-Trx reductase or NADPH-Trx reductase were exposed to high levels of light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions.

Metaproteomics analysis of the functional insights into microbial communities of combined hydrogen and methane production by anaerobic fermentation from reed straw.

PubMed

Jia, Xuan; Xi, Bei-Dou; Li, Ming-Xiao; Yang, Yang; Wang, Yong

2017-01-01

A metaproteomic approach was used to analyse the proteins expressed and provide functional evidence of key metabolic pathways in the combined production of hydrogen and methane by anaerobic fermentation (CHMP-AF) for reed straw utilisation. The functions and structures of bacteria and archaea populations show significant succession in the CHMP-AF process. There are many kinds of bacterial functional proteins, mainly belonging to phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, that are involved in carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Ferredoxin-NADP reductase, present in bacteria in genus Azotobacter, is an important enzyme for NADH/NAD+ equilibrium regulation in hydrogen production. The archaeal functional proteins are mainly involved in methane metabolism in energy metabolism, such as acetyl-CoA decarboxylase, and methyl-coenzyme M reductase, and the acetic acid pathway exhibited the highest proportion of the total. The archaea of genus Methanosarcina in phylum Euryarchaeota can produce methane under the effect of multi-functional proteins through acetic acid, CO2 reduction, and methyl nutrient pathways. The study demonstrates metaproteomics as a new way of uncovering community functional and metabolic activity. The combined information was used to identify the metabolic pathways and organisms crucial for lignocellulosic biomass degradation and biogas production. This also regulates the process from its protein levels and improves the efficiency of biogas production using reed straw biomass.

Metaproteomics analysis of the functional insights into microbial communities of combined hydrogen and methane production by anaerobic fermentation from reed straw

PubMed Central

Yang, Yang; Wang, Yong

2017-01-01

A metaproteomic approach was used to analyse the proteins expressed and provide functional evidence of key metabolic pathways in the combined production of hydrogen and methane by anaerobic fermentation (CHMP-AF) for reed straw utilisation. The functions and structures of bacteria and archaea populations show significant succession in the CHMP-AF process. There are many kinds of bacterial functional proteins, mainly belonging to phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, that are involved in carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Ferredoxin-NADP reductase, present in bacteria in genus Azotobacter, is an important enzyme for NADH/NAD+ equilibrium regulation in hydrogen production. The archaeal functional proteins are mainly involved in methane metabolism in energy metabolism, such as acetyl-CoA decarboxylase, and methyl-coenzyme M reductase, and the acetic acid pathway exhibited the highest proportion of the total. The archaea of genus Methanosarcina in phylum Euryarchaeota can produce methane under the effect of multi-functional proteins through acetic acid, CO2 reduction, and methyl nutrient pathways. The study demonstrates metaproteomics as a new way of uncovering community functional and metabolic activity. The combined information was used to identify the metabolic pathways and organisms crucial for lignocellulosic biomass degradation and biogas production. This also regulates the process from its protein levels and improves the efficiency of biogas production using reed straw biomass. PMID:28817657

Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis.

PubMed

Pi, Hualiang; Helmann, John D

2017-11-28

Bacterial cells modulate transcription in response to changes in iron availability. The ferric uptake regulator (Fur) senses intracellular iron availability and plays a central role in maintaining iron homeostasis in Bacillus subtilis Here we utilized FrvA, a high-affinity Fe 2+ efflux transporter from Listeria monocytogenes , as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, uptake systems for elemental iron ( efeUOB ), ferric citrate ( fecCDEF ), and petrobactin ( fpbNOPQ ) are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin ( dhbACEBF ) and turns on bacillibactin ( feuABC ) and hydroxamate siderophore ( fhuBCGD ) uptake systems to scavenge iron from the environment and flavodoxins ( ykuNOP ) to replace ferredoxins. Third, as iron levels decline further, an "iron-sparing" response ( fsrA , fbpAB , and fbpC ) is induced to block the translation of abundant iron-utilizing proteins and thereby permit the most essential iron-dependent enzymes access to the limited iron pools. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher-affinity binding of Fur to the "late"-induced genes.

Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts.

PubMed

Row, P E; Gray, J C

2001-01-01

In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP(+) reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the gamma subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 microM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein.

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability.

PubMed

Yoshida, Keisuke; Hisabori, Toru

2016-07-05

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions.

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron-Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis.

PubMed

Artz, Jacob H; Mulder, David W; Ratzloff, Michael W; Lubner, Carolyn E; Zadvornyy, Oleg A; LeVan, Axl X; Williams, S Garrett; Adams, Michael W W; Jones, Anne K; King, Paul W; Peters, John W

2017-07-19

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H 2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (∼ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd ox /Fd red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd red that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.

Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase.

PubMed

Boll, Matthias; Einsle, Oliver; Ermler, Ulrich; Kroneck, Peter M H; Ullmann, G Matthias

2016-01-01

In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry. © 2016 S. Karger AG, Basel.

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

PubMed Central

Yoshida, Keisuke; Hisabori, Toru

2016-01-01

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions. PMID:27335455

A Redox Basis for Metronidazole Resistance in Helicobacter pylori▿

PubMed Central

Kaakoush, N. O.; Asencio, C.; Mégraud, F.; Mendz, G. L.

2009-01-01

Metronidazole resistance in Helicobacter pylori has been attributed to mutations in rdxA or frxA. Insufficient data correlating RdxA and/or FrxA with the resistant phenotype, and the emergence of resistant strains with no mutations in either rdxA or frxA, indicated that the molecular basis of H. pylori resistance to metronidazole required further characterization. The rdxA and frxA genes of four matched pairs of metronidazole-susceptible and -resistant strains were sequenced. The resistant strains had mutations in either rdxA, frxA, neither gene, or both genes. The reduction rates of five substrates suggested that metabolic differences between susceptible and resistant strains cannot be explained only by mutations in rdxA and/or frxA. A more global approach to understanding the resistance phenotype was taken by employing two-dimensional gel electrophoresis combined with tandem mass spectrometry analyses to identify proteins differentially expressed by the matched pair of strains with no mutations in rdxA or frxA. Proteins involved in the oxireduction of ferredoxin were downregulated in the resistant strain. Other redox enzymes, such as thioredoxin reductase, alkyl hydroperoxide reductase, and superoxide dismutase, showed a pI change in the resistant strain. The data suggested that metronidazole resistance involved more complex metabolic changes than specific gene mutations, and they provided evidence of a role for the intracellular redox potential in the development of resistance. PMID:19223619

Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium

PubMed Central

Hu, Jing; Aitken, Michael D.

2012-01-01

A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene. PMID:22427500

Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Catalanotti, C.; Dubini, A.; Subramanian, V.

2012-02-01

Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a doublemore » mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.« less

Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carte, Jason; Wang, Ruiying; Li, Hong

An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targetingmore » RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.« less

Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7

PubMed Central

Li, Ping; Wang, Lei

2013-01-01

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. PMID:23417490

Design and engineering of a man-made diffusive electron-transport protein

PubMed Central

Fry, Bryan A.; Solomon, Lee A.; Dutton, P. Leslie

2016-01-01

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7 × 106 M−1s−1 to 1.2 × 109 M−1s−1 follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and −19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. PMID:26423266

Physiological and Biochemical Response of the Photosynthetic Apparatus of Two Marine Diatoms to Fe Stress.

PubMed Central

McKay, R. M. L.; Geider, R. J.; LaRoche, J.

1997-01-01

Flavodoxin is a small electron-transfer protein capable of replacing ferredoxin during periods of Fe deficiency. When evaluating the suitability of flavodoxin as a diagnostic indicator for Fe limitation of phytoplankton growth, we examined its expression in two marine diatoms we cultured using trace-metal-buffered medium. Thalassio-sira weissflogii and Phaeodactylum tricornutum were cultured in ethylenediaminetetraacetic acid-buffered Sargasso Sea water containing from 10 to 1000 nM added Fe. Trace-metal-buffered cultures of each diatom maintained high growth rates across the entire range of Fe additions. Similarly, declines in chlorophyll/cell and in the ratio of photosystem II variable-to-maximum fluorescence were negligible (P. tricornutum) to moderate (T. weissflogii; 54% decline in chlorophyll/cell and 22% decrease in variable-to-maximum fluorescence). Moreover, only minor variations in photosynthetic parameters were observed across the range of additions. In contrast, flavodoxin was expressed to high levels in low-Fe cultures. Despite the inverse relationship between flavodoxin expression and Fe content of the medium, its expression was seemingly independent of any of the indicators of cell physiology that were assayed. It appears that flavodoxin is expressed as an early-stage response to Fe stress and that its accumulation need not be intimately connected to limitations imposed by Fe on the growth rate of these diatoms. PMID:12223732

Short-term effects of salt exposure on the maize chloroplast protein pattern.

PubMed

Zörb, Christian; Herbst, Ramona; Forreiter, Christoph; Schubert, Sven

2009-09-01

It is of fundamental importance to understand the physiological differences leading to salt resistance and to get access to the molecular mechanisms underlying this physiological response. The aim of this work was to investigate the effects of short-term salt exposure on the proteome of maize chloroplasts in the initial phase of salt stress (up to 4 h). It could be shown that sodium ions accumulate quickly and excessively in chloroplasts in the initial phase of moderate salt stress. A change in the chloroplast protein pattern was observed without a change in water potential of the leaves. 2-DE revealed that 12 salt-responsive chloroplast proteins increased while eight chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1e and a Ca(2+)-sensing receptor show a rather transient response for the first 4 h of salt exposure. The enhanced abundance of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the photosystem II, and the FtsH-like protein might reflect mechanism to attenuate the detrimental effects of Na(+) on the photosynthetic machinery. The observed transient increase and subsequent decrease of selected proteins may exhibit a counterbalancing effect of target proteins in this context. Intriguingly, several subunits of the CF1-CF0 complex are unequally affected, whereas others do not respond at all.

Sulfur K-Edge XAS Studies of the Effect of DNA Binding on the [Fe 4 S 4 ] Site in EndoIII and MutY

DOE PAGES

Ha, Yang; Arnold, Anna R.; Nuñez, Nicole N.; ...

2017-07-18

S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe 4S 4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe–S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron–thiolate and iron–sulfide bonds would stabilize the oxidized state of the [Fe 4S 4] clusters. Our results are compared to those on previously studied [Fe 4S 4] model complexes, ferredoxin (Fd), and to new data on high-potential iron–sulfur protein (HiPIP). A limited decrease in covalency ismore » observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe 4S 4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron–sulfur bonds. Furthermore, in EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.« less

Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

PubMed

Lallemand, Mathilde; Login, Frédéric H; Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E

2013-01-01

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

PubMed Central

Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E.

2013-01-01

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine. PMID:24223969

Insights into the ecology, evolution, and metabolism of the widespread Woesearchaeotal lineages.

PubMed

Liu, Xiaobo; Li, Meng; Castelle, Cindy J; Probst, Alexander J; Zhou, Zhichao; Pan, Jie; Liu, Yang; Banfield, Jillian F; Gu, Ji-Dong

2018-06-08

As a recently discovered member of the DPANN superphylum, Woesearchaeota account for a wide diversity of 16S rRNA gene sequences, but their ecology, evolution, and metabolism remain largely unknown. Here, we assembled 133 global clone libraries/studies and 19 publicly available genomes to profile these patterns for Woesearchaeota. Phylogenetic analysis shows a high diversity with 26 proposed subgroups for this recently discovered archaeal phylum, which are widely distributed in different biotopes but primarily in inland anoxic environments. Ecological patterns analysis and ancestor state reconstruction for specific subgroups reveal that oxic status of the environments is the key factor driving the distribution and evolutionary diversity of Woesearchaeota. A selective distribution to different biotopes and an adaptive colonization from anoxic to oxic environments can be proposed and supported by evidence of the presence of ferredoxin-dependent pathways in the genomes only from anoxic biotopes but not from oxic biotopes. Metabolic reconstructions support an anaerobic heterotrophic lifestyle with conspicuous metabolic deficiencies, suggesting the requirement for metabolic complementarity with other microbes. Both lineage abundance distribution and co-occurrence network analyses across diverse biotopes confirmed metabolic complementation and revealed a potential syntrophic relationship between Woesearchaeota and methanogens, which is supported by metabolic modeling. If correct, Woesearchaeota may impact methanogenesis in inland ecosystems. The findings provide an ecological and evolutionary framework for Woesearchaeota at a global scale and indicate their potential ecological roles, especially in methanogenesis.

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

PubMed

Hannemann, Frank; Virus, Cornelia; Bernhardt, Rita

2006-06-25

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Interactions of iron-bound frataxin with ISCU and ferredoxin on the cysteine desulfurase complex leading to Fe-S cluster assembly.

PubMed

Cai, Kai; Frederick, Ronnie O; Tonelli, Marco; Markley, John L

2018-06-01

Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe 2+ but not Fe 3+ . While FXN (with or without bound Fe 2+ ) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the cysteine desulfurase complex ([NFS1] 2 :[ISD11] 2 :[Acp] 2 ), abbreviated as (NIA) 2 , where "N" represents the cysteine desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA) 2 weakly in the absence of ISCU but more strongly in its presence. Fe 2+ -FXN binds to the (NIA) 2 -ISCU 2 complex without release of iron. However, upon the addition of both l-cysteine and a reductant (either reduced FDX2 or DTT), Fe 2+ is released from FXN as consistent with Fe 2+ -FXN being the proximal source of iron for Fe-S cluster assembly. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Glutathione-complexed [2Fe-2S] clusters function in Fe-S cluster storage and trafficking.

PubMed

Fidai, Insiya; Wachnowsky, Christine; Cowan, J A

2016-10-01

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS4) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe-S cluster transfer reactions. UV-vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS4) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS4) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe-S cluster proteins, thereby supporting a possible physiological role for such centers.

Evolution and Cellular Function of Monothiol Glutaredoxins: Involvement in Iron-Sulphur Cluster Assembly

PubMed Central

Vilella, Felipe; Alves, Rui; Rodríguez-Manzaneque, María Teresa; Bellí, Gemma; Swaminathan, Swarna; Sunnerhagen, Per

2004-01-01

A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron–sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron–sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell. PMID:18629168

Genome-scale stoichiometry analysis to elucidate the innate capability of the cyanobacterium Synechocystis for electricity generation.

PubMed

Mao, Longfei; Verwoerd, Wynand S

2013-10-01

Synechocystis sp. PCC 6803 has been considered as a promising biocatalyst for electricity generation in recent microbial fuel cell research. However, the innate maximum current production potential and underlying metabolic pathways supporting the high current output are still unknown. This is mainly due to the fact that the high-current production cell phenotype results from the interaction among hundreds of reactions in the metabolism and it is impossible for reductionist methods to characterize the pathway selection in such a metabolic state. In this study, we employed computational metabolic techniques, flux balance analysis, and flux variability analysis, to exploit the maximum current outputs of Synechocystis sp. PCC 6803, in five electron transfer cases, namely, ferredoxin- and plastoquinol-dependent electron transfers under photoautotrophic cultivation, and NADH-dependent mediated electron transfer under photoautotrophic, heterotrophic, and mixotrophic conditions. In these five modes, the maximum current outputs were computed as 0.198, 0.7918, 0.198, 0.4652, and 0.4424 A gDW⁻¹, respectively. Comparison of the five operational modes suggests that plastoquinol-/c-type cytochrome-targeted electricity generation had an advantage of liberating the highest current output achievable for Synechocystis sp. PCC 6803. On the other hand, the analysis indicates that the currency metabolite, NADH-, dependent electricity generation can rely on a number of reactions from different pathways, and is thus more robust against environmental perturbations.

Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II.

PubMed

Takagi, Daisuke; Ifuku, Kentaro; Nishimura, Taishi; Miyake, Chikahiro

2018-05-22

The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b 559 (Cyt b 559 ) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b 559 , which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b 559 was oxidized by P680 + upon light illumination and re-reduced in the dark, mainly by the electron from the Q B site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b 559 and induced its reduction under the illumination. This inhibition of Cyt b 559 oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b 559 , rather than that of CEF-PSI in photosynthetic organisms.

Redox Regulation of Plant Development

PubMed Central

Considine, Michael J.

2014-01-01

Abstract Significance: We provide a conceptual framework for the interactions between the cellular redox signaling hub and the phytohormone signaling network that controls plant growth and development to maximize plant productivity under stress-free situations, while limiting growth and altering development on exposure to stress. Recent Advances: Enhanced cellular oxidation plays a key role in the regulation of plant growth and stress responses. Oxidative signals or cycles of oxidation and reduction are crucial for the alleviation of dormancy and quiescence, activating the cell cycle and triggering genetic and epigenetic control that underpin growth and differentiation responses to changing environmental conditions. Critical Issues: The redox signaling hub interfaces directly with the phytohormone network in the synergistic control of growth and its modulation in response to environmental stress, but a few components have been identified. Accumulating evidence points to a complex interplay of phytohormone and redox controls that operate at multiple levels. For simplicity, we focus here on redox-dependent processes that control root growth and development and bud burst. Future Directions: The multiple roles of reactive oxygen species in the control of plant growth and development have been identified, but increasing emphasis should now be placed on the functions of redox-regulated proteins, along with the central roles of reductants such as NAD(P)H, thioredoxins, glutathione, glutaredoxins, peroxiredoxins, ascorbate, and reduced ferredoxin in the regulation of the genetic and epigenetic factors that modulate the growth and vigor of crop plants, particularly within an agricultural context. Antioxid. Redox Signal. 21, 1305–1326. PMID:24180689

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Artz, Jacob H.; Mulder, David W.; Ratzloff, Michael W.

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H 2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentialsmore » for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (~ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd ox/Fd red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd redthat accumulates during fermentation. In conclusion, subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winterbourn, C.C.; Sutton, H.C.

O2- was produced by gamma irradiation of formate solutions, by the action of xanthine oxidase on hypoxanthine and O2, and by the action of ferredoxin reductase on NADPH and paraquat in the presence of O2. Its reaction with H2O2 and various iron chelates was studied. Oxidation of deoxyribose to thiobarbituric acid-reactive products that was appropriately inhibited by OH. scavengers, or formate oxidation to CO2, was used to detect OH(.). With each source of O2-, and by these criteria, Fe(EDTA) efficiently catalyzed this (Haber-Weiss) reaction, but little catalysis was detectable with iron bound to DTPA, citrate, ADP, ATP, or pyrophosphate, ormore » without chelator in phosphate buffer. O2- produced from xanthine oxidase, but not from the other sources, underwent another iron-dependent reaction with H2O2, to produce an oxidant that did not behave as free OH(.). It was formed in phosphate or bicarbonate buffer, and caused deoxyribose oxidation that was readily inhibited by mannitol or Tris, but not by benzoate, formate, or dimethyl sulfoxide. It did not oxidize formate to CO2. Addition of EDTA changed the pattern of inhibition to that expected for a reaction of OH(.). The other chelators all inhibited deoxyribose oxidation, provided their concentrations were high enough. The results are compatible with iron bound to xanthine oxidase catalyzing production of a strong oxidant (which is not free OH.) from H2O2 and O2- produced by the enzyme.« less

Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

PubMed Central

O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

1994-01-01

The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

The genome analysis of Oleiphilus messinensis ME102 (DSM 13489T) reveals backgrounds of its obligate alkane-devouring marine lifestyle.

PubMed

Toshchakov, Stepan V; Korzhenkov, Alexei A; Chernikova, Tatyana N; Ferrer, Manuel; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N

2017-12-01

Marine bacterium Oleiphilus messinensis ME102 (DSM 13489 T ) isolated from the sediments of the harbor of Messina (Italy) is a member of the order Oceanospirillales, class Gammaproteobacteria, representing the physiological group of marine obligate hydrocarbonoclastic bacteria (OHCB) alongside the members of the genera Alcanivorax, Oleispira, Thalassolituus, Cycloclasticus and Neptunomonas. These organisms play a crucial role in the natural environmental cleanup in marine systems. Despite having the largest genome (6.379.281bp) among OHCB, O. messinensis exhibits a very narrow substrate profile. The alkane metabolism is pre-determined by three loci encoding for two P450 family monooxygenases, one of which formed a cassette with ferredoxin and alcohol dehydrogenase encoding genes and alkane monoxygenase (AlkB) gene clustered with two genes for rubredoxins and NAD + -dependent rubredoxin reductase. Its genome contains the largest numbers of genomic islands (15) and mobile genetic elements (140), as compared with more streamlined genomes of its OHCB counterparts. Among hydrocarbon-degrading Oceanospirillales, O. messinensis encodes the largest array of proteins involved in the signal transduction for sensing and responding to the environmental stimuli (345 vs 170 in Oleispira antarctica, the bacterium with the second highest number). This must be an important trait to adapt to the conditions in marine sediments with a high physico-chemical patchiness and heterogeneity as compared to those in the water column. Copyright © 2017. Published by Elsevier B.V.

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

PubMed

Lancaster, J R; Vega, J M; Kamin, H; Orme-Johnson, N R; Orme-Johnson, W H; Krueger, R J; Siegel, L M

1979-02-25

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.

Ru–protein–Co biohybrids designed for solar hydrogen production: understanding electron transfer pathways related to photocatalytic function† †Electronic supplementary information (ESI) available: Time traces of photocatalysis, additional EPR spectra and parameters, UV-visible spectroscopy data, and kinetic fits of TA traces. See DOI: 10.1039/c6sc03121h Click here for additional data file.

PubMed Central

Soltau, Sarah R.; Dahlberg, Peter D.; Niklas, Jens; Poluektov, Oleg G.; Mulfort, Karen L.

2016-01-01

A series of Ru–protein–Co biohybrids have been prepared using the electron transfer proteins ferredoxin (Fd) and flavodoxin (Fld) as scaffolds for photocatalytic hydrogen production. The light-generated charge separation within these hybrids has been monitored by transient optical and electron paramagnetic resonance spectroscopies. Two distinct electron transfer pathways are observed. The Ru–Fd–Co biohybrid produces up to 650 turnovers of H2 utilizing an oxidative quenching mechanism for Ru(ii)* and a sequential electron transfer pathway via the native [2Fe–2S] cluster to generate a Ru(iii)–Fd–Co(i) charge separated state that lasts for ∼6 ms. In contrast, a direct electron transfer pathway occurs for the Ru–ApoFld–Co biohybrid, which lacks an internal electron relay, generating Ru(i)–ApoFld–Co(i) charge separated state that persists for ∼800 μs and produces 85 turnovers of H2 by a reductive quenching mechanism for Ru(ii)*. This work demonstrates the utility of protein architectures for linking donor and catalytic function via direct or sequential electron transfer pathways to enable stabilized charge separation which facilitates photocatalysis for solar fuel production. PMID:28451142

Ferrous Ion and Medium Composition Effects on Acidogenic Phase in Biobutanol Production from Molasses

NASA Astrophysics Data System (ADS)

Restiawaty, E.; Grinanda, D.

2017-07-01

Clostridium acetobutylicum B530 has ability to convert sugar into biobutanol through two phases, i.e. acidogenic and solventogenic. This fermentation process is often hampered by high raw material cost and low product yield. In order to suppress the production cost, the molasses, a byproduct of sugar cane process production, was used as carbon source in this research. Molasses has nitrogen content in a small amount, thus could be negating the beef extract component, which is expected not to affect the growth of C. acetobutylicum B530 and also can reduce the production cost. In addition, a certain amount of Fe2+ (ferrous ion), a precursor in the formation of the enzyme ferredoxin, was added to the fermentation medium to contribute in the synthesis of acetyl-CoA, so that the formation of acidogenic products such as butyric acid and acetic acid is affected. This study aimed to investigate the effect of ferrous ion and the medium composition in acidogenic phase. The addition of 20 ppm FeSO4.7H2O in the fermentation medium without beef extract can increase the concentration of butyric acid by 20% at a temperature of 35°C, while acetic acid concentration decreased by 6%. According to those results, it is expected that the product selectivity of butanol will increase in solventogenic phase. In addition, the removal of beef extract in the fermentation medium does not affect the kinetics of growth of C. acetobutylicum B530.

Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe-2S] cluster optical spectra and transfer chemistry.

PubMed

Sen, Sambuddha; Bonfio, Claudia; Mansy, Sheref S; Cowan, J A

2018-03-01

Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.

Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp{sup 143} and Arg{sup 232} play divergent roles toward different substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xiuhua; Yuan, Zenglin; Wang, Jiaxu

YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His{sup 215}, Asp{sup 143} and Arg{sup 232}. His{sup 215} functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp{sup 143} and Arg{sup 232} depends on the substrate types and that YfeX may adopt various catalytic mechanismsmore » toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H{sub 2}O{sub 2}. - Highlights: • We determined the crystal structure of YfeX bound to heme at 2.09 Å resolution. • The conserved Asp{sup 143} and Arg{sup 232} play different roles toward divergent substrates. • The H{sub 2}O{sub 2} access tunnel spans from the protein molecular surface to the heme distal region.« less

The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough.

PubMed

Ramos, Ana Raquel; Grein, Fabian; Oliveira, Gonçalo P; Venceslau, Sofia S; Keller, Kimberly L; Wall, Judy D; Pereira, Inês A C

2015-07-01

Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H 2 and CO 2 compared to organotrophic growth with fructose

DOE PAGES

Aklujkar, Muktak; Leang, Ching; Shrestha, Pravin M.; ...

2017-10-13

Clostridium ljungdahlii derives energy by lithotrophic and organotrophic acetogenesis. C. ljungdahlii was grown organotrophically with fructose and also lithotrophically, either with syngas - a gas mixture containing hydrogen (H 2), carbon dioxide (CO 2), and carbon monoxide (CO), or with H 2 and CO 2. Gene expression was compared quantitatively by microarrays using RNA extracted from all three conditions. Gene expression with fructose and with H 2/CO 2 was compared by RNA-Seq. Upregulated genes with both syngas and H 2/CO 2 (compared to fructose) point to the urea cycle, uptake and degradation of peptides and amino acids, response to sulfurmore » starvation, potentially NADPH-producing pathways involving (S)-malate and ornithine, quorum sensing, sporulation, and cell wall remodeling, suggesting a global and multicellular response to lithotrophic conditions. With syngas, the upregulated (R)-lactate dehydrogenase gene represents a route of electron transfer from ferredoxin to NAD. With H 2/CO 2, flavodoxin and histidine biosynthesis genes were upregulated. Downregulated genes corresponded to an intracytoplasmic microcompartment for disposal of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several cytoplasmic and membrane-associated redox-active protein genes were differentially regulated. In conclusion, the transcriptomic profiles of C. ljungdahlii in lithotrophic and organotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide biofuel and commodity chemical production with this species.« less

Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

PubMed Central

Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

2013-01-01

Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05), and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates. PMID:23340676

Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H 2 and CO 2 compared to organotrophic growth with fructose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aklujkar, Muktak; Leang, Ching; Shrestha, Pravin M.

Clostridium ljungdahlii derives energy by lithotrophic and organotrophic acetogenesis. C. ljungdahlii was grown organotrophically with fructose and also lithotrophically, either with syngas - a gas mixture containing hydrogen (H 2), carbon dioxide (CO 2), and carbon monoxide (CO), or with H 2 and CO 2. Gene expression was compared quantitatively by microarrays using RNA extracted from all three conditions. Gene expression with fructose and with H 2/CO 2 was compared by RNA-Seq. Upregulated genes with both syngas and H 2/CO 2 (compared to fructose) point to the urea cycle, uptake and degradation of peptides and amino acids, response to sulfurmore » starvation, potentially NADPH-producing pathways involving (S)-malate and ornithine, quorum sensing, sporulation, and cell wall remodeling, suggesting a global and multicellular response to lithotrophic conditions. With syngas, the upregulated (R)-lactate dehydrogenase gene represents a route of electron transfer from ferredoxin to NAD. With H 2/CO 2, flavodoxin and histidine biosynthesis genes were upregulated. Downregulated genes corresponded to an intracytoplasmic microcompartment for disposal of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several cytoplasmic and membrane-associated redox-active protein genes were differentially regulated. In conclusion, the transcriptomic profiles of C. ljungdahlii in lithotrophic and organotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide biofuel and commodity chemical production with this species.« less

H2 metabolism is widespread and diverse among human colonic microbes

PubMed Central

Wolf, Patricia G.; Biswas, Ambarish; Morales, Sergio E.; Greening, Chris; Gaskins, H. Rex

2016-01-01

ABSTRACT Microbial molecular hydrogen (H2) cycling is central to metabolic homeostasis and microbial composition in the human gastrointestinal tract. Molecular H2 is produced as an endproduct of carbohydrate fermentation and is reoxidised primarily by sulfate-reduction, acetogenesis, and methanogenesis. However, the enzymatic basis for these processes is incompletely understood and the hydrogenases responsible have not been investigated. In this work, we surveyed the genomic and metagenomic distribution of hydrogenase-encoding genes in the human colon to infer dominant mechanisms of H2 cycling. The data demonstrate that 70% of gastrointestinal microbial species listed in the Human Microbiome Project encode the genetic capacity to metabolise H2. A wide variety of anaerobically-adapted hydrogenases were present, with [FeFe]-hydrogenases predominant. We subsequently analyzed the hydrogenase gene content of stools from 20 healthy human subjects. The hydrogenase gene content of all samples was overwhelmingly dominated by fermentative and electron-bifurcating [FeFe]-hydrogenases emerging from the Bacteroidetes and Firmicutes. This study supports that H2 metabolism in the human gut is driven by fermentative H2 production and interspecies H2 transfer. However, it suggests that electron-bifurcation rather than respiration is the dominant mechanism of H2 reoxidation in the human colon, generating reduced ferredoxin to sustain carbon-fixation (e.g. acetogenesis) and respiration (via the Rnf complex). This work provides the first comprehensive bioinformatic insight into the mechanisms of H2 metabolism in the human colon. PMID:27123663

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

NASA Technical Reports Server (NTRS)

Henry, R. L.; Takemoto, L. J.; Murphy, J.; Gallegos, G. L.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

DOE PAGES

Artz, Jacob H.; Mulder, David W.; Ratzloff, Michael W.; ...

2017-06-21

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H 2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentialsmore » for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (~ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd ox/Fd red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd redthat accumulates during fermentation. In conclusion, subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.« less

The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin

PubMed Central

Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M.; Battaile, Kevin P.; Vakser, Ilya A.; Rivera, Mario

2012-01-01

Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage, or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis. PMID:22812654

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation.

PubMed Central

Wright, D. P.; Huppe, H. C.; Turpin, D. H.

1997-01-01

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids. PMID:12223780

Enzymatic redox properties of novel nitrotriazole explosives implications for their toxicity.

PubMed

Sarlauskas, Jonas; Nemeikaite-Ceniene, Ausra; Anusevicius, Zilvinas; Miseviciene, Lina; Maroziene, Audrone; Markevicius, Arvydas; Cenas, Narimantas

2004-01-01

The toxicity of conventional nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) is caused by their enzymatic free radical formation with the subsequent oxidative stress, the formation of alkylating nitroso and/or hydroxylamino metabolites, and oxyhemoglobin oxidation into methemoglobin. In order to get an insight into the mechanisms of toxicity of the novel explosives NTO (5-nitro-1,2,4-triazol-3-one) and ANTA (5-nitro-1,2,4-triazol-3-amine), we examined their reactions with the single-electron transferring flavoenzymes NADPH: cytochrome P-450 reductase and ferredoxin:NADP+ reductase, two-electron transferring flavoenzymes mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase), and Enterobacter cloacae NAD(P)H:nitroreductase, and their reactions with oxyhemoglobin. The reactivity of NTO and ANTA in the above reactions was markedly lower than that of TNT. The toxicity of NTO and ANTA in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. This points to the involvement of oxidative stress in their cytotoxicity, presumably to the redox cycling of free radicals. The FLK cell line cytotoxicity and the methemoglobin formation in isolated human erythrocytes of NTO and ANTA were also markedly lower than those of TNT, and similar to those of nitrobenzene. Taken together, our data demonstrate that the low toxicity of nitrotriazole explosives may be attributed to their low electron-accepting properties.

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.

1988-08-25

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by (14C)iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two (14C)carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, themore » analysis of the tryptic digest of light-activated (14C)carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.« less

Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

DOE PAGES

Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.; ...

2015-02-11

We report that microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detectedmore » were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. In conclusion, the proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.« less

Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.

We report that microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detectedmore » were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. In conclusion, the proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.« less

Electrical signals as mechanism of photosynthesis regulation in plants.

PubMed

Sukhov, Vladimir

2016-12-01

This review summarizes current works concerning the effects of electrical signals (ESs) on photosynthesis, the mechanisms of the effects, and its physiological role in plants. Local irritations of plants induce various photosynthetic responses in intact leaves, including fast and long-term inactivation of photosynthesis, and its activation. Irritation-induced ESs, including action potential, variation potential, and system potential, probably causes the photosynthetic responses in intact leaves. Probable mechanisms of induction of fast inactivation of photosynthesis are associated with Ca 2+ - and (or) H + -influxes during ESs generation; long-term inactivation of photosynthesis might be caused by Ca 2+ - and (or) H + -influxes, production of abscisic and jasmonic acids, and inactivation of phloem H + -sucrose symporters. It is probable that subsequent development of inactivation of photosynthesis is mainly associated with decreased CO 2 influx and inactivation of the photosynthetic dark reactions, which induces decreased photochemical quantum yields of photosystems I and II and increased non-photochemical quenching of photosystem II fluorescence and cyclic electron flow around photosystem I. However, other pathways of the ESs influence on the photosynthetic light reactions are also possible. One of them might be associated with ES-connected acidification of chloroplast stroma inducing ferredoxin-NADP + reductase accumulation at the thylakoids in Tic62 and TROL complexes. Mechanisms of ES-induced activation of photosynthesis require further investigation. The probable ultimate effect of ES-induced photosynthetic responses in plant life is the increased photosynthetic machinery resistance to stressors, including high and low temperatures, and enhanced whole-plant resistance to environmental factors at least during 1 h after irritation.

NRVS and EPR Spectroscopy of 57Fe-enriched [FeFe] Hydrogenase Indicate Stepwise Assembly of the H-cluster†

PubMed Central

Kuchenreuther, Jon M.; Guo, Yisong; Wang, Hongxin; Myers, William K.; George, Simon J.; Boyke, Christine A.; Yoda, Yoshitaka; Alp, E. Ercan; Zhao, Jiyong; Britt, R. David; Swartz, James R.; Cramer, Stephen P.

2013-01-01

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe–S clusters that facilitate electron transfer to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H2. Here, we have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe–4S] ferredoxin as well as a model complex resembling the [2Fe]H catalytic domain of the H-cluster. In order to enrich the hydrogenase with 57Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with 56Fe-labeled Fe–S clusters was activated in vitro using 57Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe]H subcluster with 57Fe, which NRVS confirms by detecting 57Fe–CO and 57Fe–CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second 57Fe–S cluster. EPR spectroscopy indicates that this 57Fe-enriched metal center is not the [4Fe– 4S]H subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an 56Fe-enriched [4Fe–4S]H cluster during in vitro maturation, providing unambiguous evidence for stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases. PMID:23249091

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging.

PubMed

Haque, Emdadul; Abe, Fumitaka; Mori, Masahiko; Nanjo, Yohei; Komatsu, Setsuko; Oyanagi, Atsushi; Kawaguchi, Kentaro

2014-11-04

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 ( TaBWPR-1.2 ), TaBWPR-1.2#2 and TaBWPR-1.2# 13. Using microprojectile bombardment, both cDNAs were introduced into "Bobwhite". Despite low transformation efficiency, four independent T₂ homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi: TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.

Copper stress-induced changes in leaf soluble proteome of Cu-sensitive and tolerant Agrostis capillaris L. populations.

PubMed

Hego, Elena; Vilain, Sébastien; Barré, Aurélien; Claverol, Stéphane; Dupuy, Jean-William; Lalanne, Céline; Bonneu, Marc; Plomion, Christophe; Mench, Michel

2016-05-01

Changes in leaf soluble proteome were explored in 3-month-old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC-MS/MS. In both populations, Cu excess impacted both light-dependent (OEE, cytochrome b6-f complex, and chlorophyll a-b binding protein), and -independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin-NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S-containing amino-acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 (http//proteomecentral.proteomexchange.org/dataset/PXD001930). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

PubMed Central

2009-01-01

Background The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second putative succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae. PMID:19473543

Self-Assembling Peptide Detergents Stabilize Isolated Photosystem Ion a Dry Surface for an Extended Time

PubMed Central

Kiley, Patrick; Zhao, Xiaojun; Vaughn, Michael; Baldo, Marc A; Bruce, Barry D

2005-01-01

We used a class of designed peptide detergents to stabilize photosystem I (PS-I) upon extended drying under N2 on a gold-coated-Ni-NTA glass surface. PS-I is a chlorophyll-containing membrane protein complex that is the primary reducer of ferredoxin and the electron acceptor of plastocyanin. We isolated the complex from the thylakoids of spinach chloroplasts using a chemical detergent. The chlorophyll molecules associated with the PS-I complex provide an intrinsic steady-state emission spectrum between 650 and 800 nm at −196.15 °C that reflects the organization of the pigment-protein interactions. In the absence of detergents, a large blue shift of the fluorescence maxima from approximately 735 nm to approximately 685 nm indicates a disruption in light-harvesting subunit organization, thus revealing chlorophyll−protein interactions. The commonly used membrane protein-stabilizing detergents, N-dodecyl-β-D-maltoside and N-octyl-β-D-glucoside, only partially stabilized the approximately 735-nm complex with approximately 685-nm spectroscopic shift. However, prior to drying, addition of the peptide detergent acetyl- AAAAAAK at increasing concentration significantly stabilized the PS-I complex. Moreover, in the presence of acetyl- AAAAAAK, the PS-I complex is stable in a dried form at room temperature for at least 3 wk. Another peptide detergent, acetyl-VVVVVVD, also stabilized the complex but to a lesser extent. These observations suggest that the peptide detergents may effectively stabilize membrane proteins in the solid-state. These designed peptide detergents may facilitate the study of diverse types of membrane proteins. PMID:15954800

Effects of halophilic peptide fusion on solubility, stability, and catalytic performance of D-phenylglycine aminotransferase.

PubMed

Javid, Hossein; Jomrit, Juntratip; Chantarasiri, Aiya; Isarangkul, Duangnate; Meevootisom, Vithaya; Wiyakrutta, Suthep

2014-05-01

D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of D-PhgAT was genetically fused with short peptides (A1 α- helix, A2 α-helix, and ALAL, which is a hybrid of A1 and A2) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes A1-D-PhgAT, A2-D-PhgAT, and ALAL-D-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, 23.82 ± 1.47 mM/h, was that obtained from having ALAL-D-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.

Energetics of Respiration and Oxidative Phosphorylation in Mycobacteria.

PubMed

Cook, Gregory M; Hards, Kiel; Vilchèze, Catherine; Hartman, Travis; Berney, Michael

2014-06-01

Mycobacteria inhabit a wide range of intracellular and extracellular environments. Many of these environments are highly dynamic and therefore mycobacteria are faced with the constant challenge of redirecting their metabolic activity to be commensurate with either replicative growth or a non-replicative quiescence. A fundamental feature in this adaptation is the ability of mycobacteria to respire, regenerate reducing equivalents and generate ATP via oxidative phosphorylation. Mycobacteria harbor multiple primary dehydrogenases to fuel the electron transport chain and two terminal respiratory oxidases, an aa3 -type cytochrome c oxidase and cytochrome bd-type menaquinol oxidase, are present for dioxygen reduction coupled to the generation of a protonmotive force. Hypoxia leads to the downregulation of key respiratory complexes, but the molecular mechanisms regulating this expression are unknown. Despite being obligate aerobes, mycobacteria have the ability to metabolize in the absence of oxygen and a number of reductases are present to facilitate the turnover of reducing equivalents under these conditions (e.g. nitrate reductase, succinate dehydrogenase/fumarate reductase). Hydrogenases and ferredoxins are also present in the genomes of mycobacteria suggesting the ability of these bacteria to adapt to an anaerobic-type of metabolism in the absence of oxygen. ATP synthesis by the membrane-bound F1FO-ATP synthase is essential for growing and non-growing mycobacteria and the enzyme is able to function over a wide range of protonmotive force values (aerobic to hypoxic). The discovery of lead compounds that target respiration and oxidative phosphorylation in Mycobacterium tuberculosis highlights the importance of this area for the generation of new front line drugs to combat tuberculosis.

Design and engineering of a man-made diffusive electron-transport protein.

PubMed

Fry, Bryan A; Solomon, Lee A; Leslie Dutton, P; Moser, Christopher C

2016-05-01

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7×10(6) M(-1) s(-1) to 1.2×10(9) M(-1) s(-1) follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and -19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2015 Elsevier B.V. All rights reserved.

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

PubMed Central

Zeikus, J. G.; Fuchs, G.; Kenealy, W.; Thauer, R. K.

1977-01-01

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent Vmax and KM values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO2. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed. PMID:914779

In Silico Analysis of the Regulation of the Photosynthetic Electron Transport Chain in C3 Plants1[OPEN

PubMed Central

Kramer, David M.

2018-01-01

We present a new simulation model of the reactions in the photosynthetic electron transport chain of C3 species. We show that including recent insights about the regulation of the thylakoid proton motive force, ATP/NADPH balancing mechanisms (cyclic and noncyclic alternative electron transport), and regulation of Rubisco activity leads to emergent behaviors that may affect the operation and regulation of photosynthesis under different dynamic environmental conditions. The model was parameterized with experimental results in the literature, with a focus on Arabidopsis (Arabidopsis thaliana). A dataset was constructed from multiple sources, including measurements of steady-state and dynamic gas exchange, chlorophyll fluorescence, and absorbance spectroscopy under different light intensities and CO2, to test predictions of the model under different experimental conditions. Simulations suggested that there are strong interactions between cyclic and noncyclic alternative electron transport and that an excess capacity for alternative electron transport is required to ensure adequate redox state and lumen pH. Furthermore, the model predicted that, under specific conditions, reduction of ferredoxin by plastoquinol is possible after a rapid increase in light intensity. Further analysis also revealed that the relationship between ATP synthesis and proton motive force was highly regulated by the concentrations of ATP, ADP, and inorganic phosphate, and this facilitated an increase in nonphotochemical quenching and proton motive force under conditions where metabolism was limiting, such as low CO2, high light intensity, or combined high CO2 and high light intensity. The model may be used as an in silico platform for future research on the regulation of photosynthetic electron transport. PMID:28924017

Expression profile of desiccation tolerance factors in intertidal seaweed species during the tidal cycle.

PubMed

Fierro, Camila; López-Cristoffanini, Camilo; Meynard, Andrés; Lovazzano, Carlos; Castañeda, Francisco; Guajardo, Eduardo; Contreras-Porcia, Loretto

2017-06-01

The transcriptional modulation of desiccation tolerance factors in P. orbicularis explains its successful recuperation after water deficit. Differential responses to air exposure clarify seaweed distribution along intertidal rocky zones. Desiccation-tolerant seaweed species, such as Pyropia orbicularis, can tolerate near 96% water loss during air exposure. To understand the phenotypic plasticity of P. orbicularis to desiccation, several tolerance factors were assessed by RT-qPCR, Western-blot analysis, and enzymatic assays during the natural desiccation-rehydration cycle. Comparative enzymatic analyses were used to evidence differential responses between P. orbicularis and desiccation-sensitive species. The results showed that during desiccation, the relative mRNA levels of genes associated with basal metabolism [trehalose phosphate synthase (tps) and pyruvate dehydrogenase (pdh)] were overexpressed in P. orbicularis. Transcript levels related to antioxidant metabolism [peroxiredoxin (prx); thioredoxin (trx); catalase (cat); lipoxygenase (lox); ferredoxin (fnr); glutathione S-transferase (gst)], cellular detoxification [ABC transporter (abc) and ubiquitin (ubq)], and signal transduction [calmodulin (cam)] increased approximately 15- to 20-fold, with the majority returning to basal levels during the final hours of rehydration. In contrast, actin (act) and transcription factor 1 (tf1) transcripts were down-regulated. ABC transporter protein levels increased in P. orbicularis during desiccation, whereas PRX transcripts decreased. The antioxidant enzymes showed higher specific activity in P. orbicularis under desiccation, and sensitive species exhibited enzymatic inactivation and scarce ABC and PRX protein detection following prolonged desiccation. In conclusion, the reported findings contribute towards understanding the ecological distribution of intertidal seaweeds at the molecular and functional levels.

Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses.

PubMed

Vuorijoki, Linda; Tiwari, Arjun; Kallio, Pauli; Aro, Eva-Mari

2017-05-01

Iron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygen-evolving photosynthetic cyanobacteria. The aim here was to elucidate protein-level effects of the transcriptional repressor SufR involved in the regulation of Fe-S cluster biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. The approach was to quantitate 94 pre-selected target proteins associated with various metabolic functions using SRM in Synechocystis. The evaluation was conducted in response to sufR deletion under different iron conditions, and complemented with EPR analysis on the functionality of the photosystems I and II as well as with RT-qPCR to verify the effects of SufR also on transcript level. The results on both protein and transcript levels show that SufR acts not only as a repressor of the suf operon when iron is available but also has other direct and indirect functions in the cell, including maintenance of the expression of pyruvate:ferredoxin oxidoreductase NifJ and other Fe-S cluster proteins under iron sufficient conditions. Furthermore, the results imply that in the absence of iron the suf operon is repressed by some additional regulatory mechanism independent of SufR. The study demonstrates that Fe-S cluster metabolism in Synechocystis is stringently regulated, and has complex interactions with multiple primary functions in the cell, including photosynthesis and central carbon metabolism. The study provides new insight into the regulation of Fe-S cluster biogenesis via suf operon, and the associated wide-ranging protein-level changes in photosynthetic cyanobacteria. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Photosynthetic Fractionation of the Stable Isotopes of Oxygen and Carbon.

PubMed Central

Guy, R. D.; Fogel, M. L.; Berry, J. A.

1993-01-01

Isotope discrimination during photosynthetic exchange of O2 and CO2 was measured using enzyme, thylakoid, and whole cell preparations. Evolved oxygen from isolated spinach thylakoids was isotopically identical (within analytical error) to its source water. Similar results were obtained with Anacystis nidulans Richter and Phaeodactylum tricornutum Bohlin cultures purged with helium. For consumptive reactions, discrimination ([delta], where 1 + [delta]/1000 equals the isotope effect, k16/k18 or k12/k13) was determined by analysis of residual substrate (O2 or CO2). The [delta] for the Mehler reaction, mediated by ferredoxin or methylviologen, was 15.3[per mille (thousand) sign]. Oxygen isotope discrimination during oxygenation of ribulose-1,5-bisphosphate (RuBP) catalyzed by RuBP carboxylase/oxygenase (Rubisco) was 21.3[per mille (thousand) sign] and independent of enzyme source, unlike carbon isotope discrimination: 30.3[per mille (thousand) sign] for spinach enzyme and 19.6 to 23[per mille (thousand) sign] for Rhodospirillum rubrum and A. nidulans enzymes, depending on reaction conditions. The [delta] for O2 consumption catalyzed by glycolate oxidase was 22.7[per mille (thousand) sign]. The expected overall [delta] for photorespiration is about 21.7[per mille (thousand) sign]. Consistent with this, when Asparagus sprengeri Regel mesophyll cells approached the compensation point within a sealed vessel, the [delta]18O of dissolved O2 came to a steady-state value of about 21.5[per mille (thousand) sign] relative to the source water. The results provide improved estimates of discrimination factors in several reactions prominent in the global O cycle and indicate that photorespiration plays a significant part in determining the isotopic composition of atmospheric oxygen. PMID:12231663

The dimer formed by the periplasmic domain of EpsL from the Type 2 Secretion System of Vibrio parahaemolyticus

PubMed Central

Abendroth, Jan; Kreger, Allison C.; Hol, Wim G. J.

2010-01-01

The Type 2 Secretion System (T2SS), occurring in many Gram-negative bacteria, is responsible for the transport of a diversity of proteins from the periplasm across the outer membrane into the extracellular space. In Vibrio cholerae, the T2SS secretes several unrelated proteins including the major virulence factor cholera toxin. The T2SS consists of three subassemblies, one of which is the Inner Membrane Complex which contains multiple copies of five proteins, including the bitopic membrane protein EpsL. Here we report the 2.3 Å resolution crystal structure of the periplasmic domain of EpsL (peri-EpsL) from V. parahaemolyticus, which is 56 % identical in sequence to its homolog in V. cholerae. The domain adopts a circular permutation of the “common” ferredoxin fold with two contiguous sub-domains. Remarkably, this permutation has so far only been observed once before: in the periplasmic domain of EpsM (peri-EpsM), another T2SS protein which interacts with EpsL. These two domains are 18 % identical in sequence which may indicate a common evolutionary origin. Both peri-EpsL and peri-EpsM form dimers, but the organization of the subunits in these dimers appears to be entirely different. We have previously shown that the cytoplasmic domain of EpsL is also dimeric and forms a heterotetramer with the first domain of the “secretion ATPase” EpsE. The latter enzyme is most likely hexameric. The possible consequences of the combination of the different symmetries of EpsE and EpsL for the architecture of the T2SS are discussed. PMID:19646531

Identification of surface-exposed domains on the reducing side of photosystem I

NASA Technical Reports Server (NTRS)

Xu, Q.; Guikema, J. A.; Chitnis, P. R.; Spooner, B. S. (Principal Investigator)

1994-01-01

Photosystem I (PSI) is a multisubunit enzyme that catalyzes the light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobacterium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low concentration of endoproteinase glutamic acid (Glu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. Glu63 and Glu67, located near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cleaved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Glu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, several secondary cleavage sites on PsaD, PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD and PsaE were intact. Glu88 and Glu124 of PsaF became susceptible to endoproteinase Glu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase Glu-C or thermolysin showed that the intact PsaE and PsaD, but not their major degradation products lacking C-terminal domains, were heavily biotinylated. Therefore, lysine-74 at the C terminus of PsaE was accessible for biotinylation. Similarly, lysine-107, or lysine-118, or both in PsaD could be modified by NHS-biotin.

Characterization of Ferredoxin-Dependent Glutamine-Oxoglutarate Amidotransferase (Fd-GOGAT) Genes and Their Relationship with Grain Protein Content QTL in Wheat

PubMed Central

2014-01-01

Background In higher plants, inorganic nitrogen is assimilated via the glutamate synthase cycle or GS-GOGAT pathway. GOGAT enzyme occurs in two distinct forms that use NADH (NADH-GOGAT) or Fd (Fd-GOGAT) as electron carriers. The goal of the present study was to characterize wheat Fd-GOGAT genes and to assess the linkage with grain protein content (GPC), an important quantitative trait controlled by multiple genes. Results We report the complete genomic sequences of the three homoeologous A, B and D Fd-GOGAT genes from hexaploid wheat (Triticum aestivum) and their localization and characterization. The gene is comprised of 33 exons and 32 introns for all the three homoeologues genes. The three genes show the same exon/intron number and size, with the only exception of a series of indels in intronic regions. The partial sequence of the Fd-GOGAT gene located on A genome was determined in two durum wheat (Triticum turgidum ssp. durum) cvs Ciccio and Svevo, characterized by different grain protein content. Genomic differences allowed the gene mapping in the centromeric region of chromosome 2A. QTL analysis was conducted in the Svevo×Ciccio RIL mapping population, previously evaluated in 5 different environments. The study co-localized the Fd-GOGAT-A gene with the marker GWM-339, identifying a significant major QTL for GPC. Conclusions The wheat Fd-GOGAT genes are highly conserved; both among the three homoeologous hexaploid wheat genes and in comparison with other plants. In durum wheat, an association was shown between the Fd-GOGAT allele of cv Svevo with increasing GPC - potentially useful in breeding programs. PMID:25099972

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apoferredoxin in an ATP-dependent reaction.

PubMed

Chandramouli, Kala; Johnson, Michael K

2006-09-19

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.

Strain-resolved Metatranscriptomic Analysis Reveals Unexpectedly Diverse Heterotrophic and Lithoautotrophic Microbial Metabolism in Naturally Reduced Aquifer Sediments

NASA Astrophysics Data System (ADS)

Beller, H. R.; Jewell, T. N. M.; Karaoz, U.; Bill, M.; Chakraborty, R.; Brodie, E.; Williams, K. H.

2016-12-01

In this study, we sought to better understand how natural organic matter fuels microbial communities in the anoxic subsurface at the Rifle (CO) site. We conducted a 20-day microcosm experiment with naturally reduced zone (NRZ) sediments and collected samples every 5 days for omics (metagenome and metatranscriptome) and geochemical measurements. No electron donors were added other than the NRZ sediment, which is enriched in buried woody plant material. The microcosms were constructed and incubated under anaerobic conditions in serum bottles with a N2 headspace. Biogeochemical measurements indicated that the decomposition of native organic matter occurred in different phases, including mineralization of dissolved organic carbon (DOC) to CO2 during the first week of incubation, followed by a pulse of acetogenesis that dominated carbon flux after 2 weeks. The depletion of DOC over time was strongly correlated with increases in expression of many genes associated with heterotrophy (e.g., amino acid, fatty acid, and carbohydrate metabolism) belonging to a Hydrogenophaga strain that accounted for a relatively large percentage ( 8%) of the metatranscriptome. This Hydrogenophaga strain also expressed genes indicative of chemolithoautotrophy, including CO2 fixation (RubisCO), H2 oxidation, S-compound oxidation, and denitrification. The pulse of acetogenesis appears to have been collectively catalyzed by a number of different organisms and metabolisms, most prominently pyruvate:ferredoxin oxidoreductase. Unexpected genes were identified among the most highly expressed (>98th percentile) transcripts, including acetone carboxylase and cell wall-associated hydrolases, some of which are known to act on peptidoglycan. Many of the most highly expressed hydrolases belonged to a Ca. Bathyarchaeota strain and may have been associated with scavenging of bacterial biomass. Overall, observed metabolism ranged far beyond the expected fermentation of plant-derived organic matter.

Thioredoxin Redox Regulates ATPase Activity of Magnesium Chelatase CHLI Subunit and Modulates Redox-Mediated Signaling in Tetrapyrrole Biosynthesis and Homeostasis of Reactive Oxygen Species in Pea Plants1[C][W][OA

PubMed Central

Luo, Tao; Fan, Tingting; Liu, Yinan; Rothbart, Maxi; Yu, Jing; Zhou, Shuaixiang; Grimm, Bernhard; Luo, Meizhong

2012-01-01

The chloroplast thioredoxins (TRXs) function as messengers of redox signals from ferredoxin to target enzymes. In this work, we studied the regulatory impact of pea (Pisum sativum) TRX-F on the magnesium (Mg) chelatase CHLI subunit and the enzymatic activation of Mg chelatase in vitro and in vivo. In vitro, reduced TRX-F activated the ATPase activity of pea CHLI and enhanced the activity of Mg chelatase reconstituted from the three recombinant subunits CHLI, CHLD, and CHLH in combination with the regulator protein GENOMES UNCOUPLED4 (GUN4). Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that TRX-F physically interacts with CHLI but not with either of the other two subunits or GUN4. In vivo, virus-induced TRX-F gene silencing (VIGS-TRX-F) in pea plants did not result in an altered redox state of CHLI. However, simultaneous silencing of the pea TRX-F and TRX-M genes (VIGS-TRX-F/TRX-M) resulted in partially and fully oxidized CHLI in vivo. VIGS-TRX-F/TRX-M plants demonstrated a significant reduction in Mg chelatase activity and 5-aminolevulinic acid synthesizing capacity as well as reduced pigment content and lower photosynthetic capacity. These results suggest that, in vivo, TRX-M can compensate for a lack of TRX-F and that both TRXs act as important redox regulators of Mg chelatase. Furthermore, the silencing of TRX-F and TRX-M expression also affects gene expression in the tetrapyrrole biosynthesis pathway and leads to the accumulation of reactive oxygen species, which may also serve as an additional signal for the transcriptional regulation of photosynthesis-associated nuclear genes. PMID:22452855

CO 2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

DOE PAGES

Xiong, Wei; Lin, Paul P.; Magnusson, Lauren; ...

2016-10-28

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO 2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO 2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO 2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO 2 to formate serves as a CO 2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO 2 via two biochemical reactions:more » the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO 2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO 2 uptake, and provided physical evidence of a distinct in vivo 'rPFOR-PFL shunt' to reduce CO 2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO 2 fixation via the reductive C1 metabolic pathway in C. thermocellum. Lastly, these findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO 2 as well.« less

NTRC-dependent redox balance of 2-Cys peroxiredoxins is needed for optimal function of the photosynthetic apparatus.

PubMed

Pérez-Ruiz, Juan Manuel; Naranjo, Belén; Ojeda, Valle; Guinea, Manuel; Cejudo, Francisco Javier

2017-11-07

Thiol-dependent redox regulation allows the rapid adaptation of chloroplast function to unpredictable changes in light intensity. Traditionally, it has been considered that chloroplast redox regulation relies on photosynthetically reduced ferredoxin (Fd), thioredoxins (Trxs), and an Fd-dependent Trx reductase (FTR), the Fd-FTR-Trxs system, which links redox regulation to light. More recently, a plastid-localized NADPH-dependent Trx reductase (NTR) with a joint Trx domain, termed NTRC, was identified. NTRC efficiently reduces 2-Cys peroxiredoxins (Prxs), thus having antioxidant function, but also participates in redox regulation of metabolic pathways previously established to be regulated by Trxs. Thus, the NTRC, 2-Cys Prxs, and Fd-FTR-Trxs redox systems may act concertedly, but the nature of the relationship between them is unknown. Here we show that decreased levels of 2-Cys Prxs suppress the phenotype of the Arabidopsis thaliana ntrc KO mutant. The excess of oxidized 2-Cys Prxs in NTRC-deficient plants drains reducing power from chloroplast Trxs, which results in low efficiency of light energy utilization and impaired redox regulation of Calvin-Benson cycle enzymes. Moreover, the dramatic phenotype of the ntrc-trxf1f2 triple mutant, lacking NTRC and f -type Trxs, was also suppressed by decreased 2-Cys Prxs contents, as the ntrc-trxf1f2-Δ2cp mutant partially recovered the efficiency of light energy utilization and exhibited WT rate of CO 2 fixation and growth phenotype. The suppressor phenotype was not caused by compensatory effects of additional chloroplast antioxidant systems. It is proposed that the Fd-FTR-Trx and NTRC redox systems are linked by the redox balance of 2-Cys Prxs, which is crucial for chloroplast function. Copyright © 2017 the Author(s). Published by PNAS.

Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

PubMed

Kimata-Ariga, Yoko; Hase, Toshiharu

2014-01-01

Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

Seahawk: moving beyond HTML in Web-based bioinformatics analysis.

PubMed

Gordon, Paul M K; Sensen, Christoph W

2007-06-18

Traditional HTML interfaces for input to and output from Bioinformatics analysis on the Web are highly variable in style, content and data formats. Combining multiple analyses can therefore be an onerous task for biologists. Semantic Web Services allow automated discovery of conceptual links between remote data analysis servers. A shared data ontology and service discovery/execution framework is particularly attractive in Bioinformatics, where data and services are often both disparate and distributed. Instead of biologists copying, pasting and reformatting data between various Web sites, Semantic Web Service protocols such as MOBY-S hold out the promise of seamlessly integrating multi-step analysis. We have developed a program (Seahawk) that allows biologists to intuitively and seamlessly chain together Web Services using a data-centric, rather than the customary service-centric approach. The approach is illustrated with a ferredoxin mutation analysis. Seahawk concentrates on lowering entry barriers for biologists: no prior knowledge of the data ontology, or relevant services is required. In stark contrast to other MOBY-S clients, in Seahawk users simply load Web pages and text files they already work with. Underlying the familiar Web-browser interaction is an XML data engine based on extensible XSLT style sheets, regular expressions, and XPath statements which import existing user data into the MOBY-S format. As an easily accessible applet, Seahawk moves beyond standard Web browser interaction, providing mechanisms for the biologist to concentrate on the analytical task rather than on the technical details of data formats and Web forms. As the MOBY-S protocol nears a 1.0 specification, we expect more biologists to adopt these new semantic-oriented ways of doing Web-based analysis, which empower them to do more complicated, ad hoc analysis workflow creation without the assistance of a programmer.

Seahawk: moving beyond HTML in Web-based bioinformatics analysis

PubMed Central

Gordon, Paul MK; Sensen, Christoph W

2007-01-01

Background Traditional HTML interfaces for input to and output from Bioinformatics analysis on the Web are highly variable in style, content and data formats. Combining multiple analyses can therfore be an onerous task for biologists. Semantic Web Services allow automated discovery of conceptual links between remote data analysis servers. A shared data ontology and service discovery/execution framework is particularly attractive in Bioinformatics, where data and services are often both disparate and distributed. Instead of biologists copying, pasting and reformatting data between various Web sites, Semantic Web Service protocols such as MOBY-S hold out the promise of seamlessly integrating multi-step analysis. Results We have developed a program (Seahawk) that allows biologists to intuitively and seamlessly chain together Web Services using a data-centric, rather than the customary service-centric approach. The approach is illustrated with a ferredoxin mutation analysis. Seahawk concentrates on lowering entry barriers for biologists: no prior knowledge of the data ontology, or relevant services is required. In stark contrast to other MOBY-S clients, in Seahawk users simply load Web pages and text files they already work with. Underlying the familiar Web-browser interaction is an XML data engine based on extensible XSLT style sheets, regular expressions, and XPath statements which import existing user data into the MOBY-S format. Conclusion As an easily accessible applet, Seahawk moves beyond standard Web browser interaction, providing mechanisms for the biologist to concentrate on the analytical task rather than on the technical details of data formats and Web forms. As the MOBY-S protocol nears a 1.0 specification, we expect more biologists to adopt these new semantic-oriented ways of doing Web-based analysis, which empower them to do more complicated, ad hoc analysis workflow creation without the assistance of a programmer. PMID:17577405

Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine1[W

PubMed Central

Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

2012-01-01

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways. PMID:22523226

Early bioenergetic evolution

PubMed Central

Sousa, Filipa L.; Thiergart, Thorsten; Landan, Giddy; Nelson-Sathi, Shijulal; Pereira, Inês A. C.; Allen, John F.; Lane, Nick; Martin, William F.

2013-01-01

Life is the harnessing of chemical energy in such a way that the energy-harnessing device makes a copy of itself. This paper outlines an energetically feasible path from a particular inorganic setting for the origin of life to the first free-living cells. The sources of energy available to early organic synthesis, early evolving systems and early cells stand in the foreground, as do the possible mechanisms of their conversion into harnessable chemical energy for synthetic reactions. With regard to the possible temporal sequence of events, we focus on: (i) alkaline hydrothermal vents as the far-from-equilibrium setting, (ii) the Wood–Ljungdahl (acetyl-CoA) pathway as the route that could have underpinned carbon assimilation for these processes, (iii) biochemical divergence, within the naturally formed inorganic compartments at a hydrothermal mound, of geochemically confined replicating entities with a complexity below that of free-living prokaryotes, and (iv) acetogenesis and methanogenesis as the ancestral forms of carbon and energy metabolism in the first free-living ancestors of the eubacteria and archaebacteria, respectively. In terms of the main evolutionary transitions in early bioenergetic evolution, we focus on: (i) thioester-dependent substrate-level phosphorylations, (ii) harnessing of naturally existing proton gradients at the vent–ocean interface via the ATP synthase, (iii) harnessing of Na+ gradients generated by H+/Na+ antiporters, (iv) flavin-based bifurcation-dependent gradient generation, and finally (v) quinone-based (and Q-cycle-dependent) proton gradient generation. Of those five transitions, the first four are posited to have taken place at the vent. Ultimately, all of these bioenergetic processes depend, even today, upon CO2 reduction with low-potential ferredoxin (Fd), generated either chemosynthetically or photosynthetically, suggesting a reaction of the type ‘reduced iron → reduced carbon’ at the beginning of bioenergetic evolution. PMID:23754820

Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

PubMed Central

Diekert, Gabriele B.; Thauer, Rudolf K.

1978-01-01

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO2. Rates up to 0.4 μmol min−1 mg of wet cells−1 were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO2 reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min−1 mg of protein−1 (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO2 to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO2 do not possess a CO-oxidizing system. PMID:711675

The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota

PubMed Central

Siebers, Bettina; Zaparty, Melanie; Raddatz, Guenter; Tjaden, Britta; Albers, Sonja-Verena; Bell, Steve D.; Blombach, Fabian; Kletzin, Arnulf; Kyrpides, Nikos; Lanz, Christa; Plagens, André; Rampp, Markus; Rosinus, Andrea; von Jan, Mathias; Makarova, Kira S.; Klenk, Hans-Peter; Schuster, Stephan C.; Hensel, Reinhard

2011-01-01

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078T) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea. PMID:22003381

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production.

PubMed

Kannuchamy, Saranyah; Mukund, Nisha; Saleena, Lilly M

2016-05-11

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis. The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain. Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

Fluorescent probes for tracking the transfer of iron–sulfur cluster and other metal cofactors in biosynthetic reaction pathways

DOE PAGES

Vranish, James N.; Russell, William K.; Yu, Lusa E.; ...

2014-12-05

Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S clustermore » transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. Lastly, we anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways.« less

Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis.

PubMed

Bradic, Martina; Warring, Sally D; Tooley, Grace E; Scheid, Paul; Secor, William E; Land, Kirkwood M; Huang, Po-Jung; Chen, Ting-Wen; Lee, Chi-Ching; Tang, Petrus; Sullivan, Steven A; Carlton, Jane M

2017-06-01

Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis

PubMed Central

Bradic, Martina; Warring, Sally D.; Tooley, Grace E.; Scheid, Paul; Secor, William E.; Land, Kirkwood M.; Huang, Po-Jung; Chen, Ting-Wen; Lee, Chi-Ching; Tang, Petrus; Sullivan, Steven A.

2017-01-01

Abstract Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool. PMID:28633446

High rates of double-stranded RNA viruses and Mycoplasma hominis in Trichomonas vaginalis clinical isolates in South Brazil.

PubMed

da Luz Becker, Débora; dos Santos, Odelta; Frasson, Amanda Piccoli; de Vargas Rigo, Graziela; Macedo, Alexandre José; Tasca, Tiana

2015-08-01

Trichomonas vaginalis is the etiological agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in world, with 276.4 million new cases each year. T. vaginalis can be naturally infected with Mycoplasma hominis and Trichomonasvirus species. This study aimed to evaluate the prevalence of T. vaginalis infected with four distinct T. vaginalis viruses (TVVs) and M. hominis among isolates from patients in Porto Alegre city, South Brazil. An additional goal of this study was to investigate whether there is association between metronidazole resistance and the presence of M. hominis during TVV infection. The RNA expression level of the pyruvate ferredoxin oxidoreductase (PFOR) gene was also evaluated among metronidazole-resistant and metronidazole-sensitive T. vaginalis isolates. A total of 530 urine samples were evaluated, and 5.7% samples were positive for T. vaginalis infection. Among them, 4.51% were isolated from female patients and 1.12% were from male patients. Remarkably, the prevalence rates of M. hominis and TVV-positive T. vaginalis isolates were 56.7% and 90%, respectively. Most of the T. vaginalis isolates were metronidazole-sensitive (86.7%), and only four isolates (13.3%) were resistant. There is no statistically significant association between infection by M. hominis and infection by TVVs. Our results refute the hypothesis that the presence of the M. hominis and TVVs is enough to confer metronidazole resistance to T. vaginalis isolates. Additionally, the role of PFOR RNA expression levels in metronidazole resistance as the main mechanism of resistance to metronidazole could not be established. This study is the first report of the T. vaginalis infection by M. hominis and TVVs in a large collection of isolates from South Brazil. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of the HSPA9/HSC20 chaperone pair in promoting directional human iron-sulfur cluster exchange involving monothiol glutaredoxin 5.

PubMed

Olive, Joshua A; Cowan, J A

2018-07-01

Iron‑sulfur clusters are essential cofactors found across all domains of life. Their assembly and transfer are accomplished by highly conserved protein complexes and partners. In eukaryotes a [2Fe-2S] cluster is first assembled in the mitochondria on the iron‑sulfur cluster scaffold protein ISCU in tandem with iron, sulfide, and electron donors. Current models suggest that a chaperone pair interacts with a cluster-bound ISCU to facilitate cluster transfer to a monothiol glutaredoxin. In humans this protein is glutaredoxin 5 (GLRX5) and the cluster can then be exchanged with a variety of target apo proteins. By use of circular dichroism spectroscopy, the kinetics of cluster exchange reactivity has been evaluated for human GLRX5 with a variety of cluster donor and acceptor partners, and the role of chaperones determined for several of these. In contrast to the prokaryotic model, where heat-shock type chaperone proteins HscA and HscB are required for successful and efficient transfer of a [2Fe-2S] cluster from the ISCU scaffold to a monothiol glutaredoxin. However, in the human system the chaperone homologs, HSPA9 and HSC20, are not necessary for human ISCU to promote cluster transfer to GLRX5, and appear to promote the reverse transfer. Cluster exchange with the human iron‑sulfur cluster carrier protein NFU1 and ferredoxins (FDX's), and the role of chaperones, has also been evaluated, demonstrating in certain cases control over the directionality of cluster transfer. In contrast to other prokaryotic and eukaryotic organisms, NFU1 is identified as a more likely physiological donor of [2Fe-2S] cluster to human GLRX5 than ISCU. Copyright © 2018 Elsevier Inc. All rights reserved.

Role of protein-glutathione contacts in defining glutaredoxin-3 [2Fe-2S] cluster chirality, ligand exchange and transfer chemistry.

PubMed

Sen, Sambuddha; Cowan, J A

2017-10-01

Monothiol glutaredoxins (Grx) serve as intermediate cluster carriers in iron-sulfur cluster trafficking. The [2Fe-2S]-bound holo forms of Grx proteins display cysteinyl coordination from exogenous glutathione (GSH), in addition to contact from protein-derived Cys. Herein, we report mechanistic studies that investigate the role of exogenous glutathione in defining cluster chirality, ligand exchange, and the cluster transfer chemistry of Saccharomyces cerevisiae Grx3. Systematic perturbations were introduced to the glutathione-binding site by substitution of conserved charged amino acids that form crucial electrostatic contacts with the glutathione molecule. Native Grx3 could also be reconstituted in the absence of glutathione, with either DTT, BME or free L-cysteine as the source of the exogenous Fe-S ligand contact, while retaining full functional reactivity. The delivery of the [2Fe-2S] cluster to Grx3 from cluster donor proteins such as Isa, Nfu, and a [2Fe-2S](GS) 4 complex, revealed that electrostatic contacts are of key importance for positioning the exogenous glutathione that in turn influences the chiral environment of the cluster. All Grx3 derivatives were reconstituted by standard chemical reconstitution protocols and found to transfer cluster to apo ferredoxin 1 (Fdx1) at rates comparable to native protein, even when using DTT, BME or free L-cysteine as a thiol source in place of GSH during reconstitution. Kinetic analysis of cluster transfer from holo derivatives to apo Fdx1 has led to a mechanistic model for cluster transfer chemistry of native holo Grx3, and identification of the likely rate-limiting step for the reaction.

Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus.

PubMed

Tovar-Méndez, Alejandro; Matamoros, Manuel A; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

2011-07-01

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

Metabolic Interfaces of Mercury Methylation Proteins in Desulfovibrio sp. ND132

NASA Astrophysics Data System (ADS)

Wall, J. D.; Bridou, R.; Smith, S. D.; Mok, K.; Widner, F.; Johs, A.; Parks, J.; Pierce, E. M.; Elias, D. A.; Gilmour, C. C.; Taga, M.

2015-12-01

Two genes necessary for microbial production of the neurotoxin methylmercury have been identified; hgcA encoding a corrinoid methyltransferase and hgcB, a ferredoxin-like protein. To date, all microbes possessing orthologs of these genes that have been tested are capable of methylating mercury; whereas, organisms lacking hgcA and hgcB are not. Also of interest is the observation that confirmed mercury-methylating microbes are all considered anaerobes although not members of a specific phylogenetic group. They are found scattered in the genomes of methanogens, Firmicutes, and Deltaproteobacteria. Methylation has not been demonstrated to provide protection of the microbes to mercury exposure. To determine the source of evolutionary pressure for acquisition and maintenance of these genes, we are seeking to understand whether there is a second function of the proteins. We are seeking evidence for the metabolic source(s) of the methyl group and for competing reactions. We have found that deletion of the metH gene encoding a tetrahydrofolate methyltransferase in Desulfovibrio sp. ND132 decreases the mercury methylation capacity by ca. 95%, consistent with an interpretation that this enzyme is involved in the pathway for the methyl group for HgcA. In addition, the corrinoid present in HgcA and the MetH of ND132 is strictly dependent on nicotinate nucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase encoded by the cobT gene, linking methionine biosynthesis with mercury methylation at a second level. Additional methyl transferases have not been found to be necessary for this function. While earlier evidence was provided for an involvement of the CO dehydrogenase/acetylCoA synthase, this enzyme is not universally present in methylating strains unlike the pathway for methionine synthesis.

The Methanosarcina barkeri genome: comparative analysis withMethanosarcina acetivorans and Methanosarcina mazei reveals extensiverearrangement within methanosarcinal genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maeder, Dennis L.; Anderson, Iain; Brettin, Thomas S.

2006-05-19

We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. All three genomes share a conserved double origin of replication and many gene clusters. M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcinae in the region proximal to the origin of replication with interspecies gene similarities as high as 95%. However it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the proximal semi-genome. Of the 3680 open reading frames in M. barkeri,more » 678 had paralogs with better than 80% similarity to both M. acetivorans and M. mazei while 128 nonhypothetical orfs were unique (non-paralogous) amongst these species including a complete formate dehydrogenase operon, two genes required for N-acetylmuramic acid synthesis, a 14 gene gas vesicle cluster and a bacterial P450-specific ferredoxin reductase cluster not previously observed or characterized in this genus. A cryptic 36 kbp plasmid sequence was detected in M. barkeri that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143 nt motif. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the large M. acetivorans is the result of multiple gene-scale insertions and duplications uniformly distributed in that genome, while M. barkeri is characterized by localized inversions associated with the loss of gene content. In contrast, the relatively short M. mazei most closely approximates the ancestral organizational state.« less

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Ordine, Robert L.; Rydel, Timothy J.; Storek, Michael J.

2009-09-08

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O{sub 2} into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer ({alpha}{sub 3}) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While themore » Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co{sup 2+}, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 {angstrom}, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.« less

The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PubMed

Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

2016-06-01

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of Photochemical Processes for H2 Production by CdS Nanorod-[FeFe] Hydrogenase Complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, K. A.; Wilker, M. B.; Boehm, M.

2012-03-28

We have developed complexes of CdS nanorods capped with 3-mercaptopropionic acid (MPA) and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) that photocatalyze reduction of H{sup +} to H{sub 2} at a CaI turnover frequency of 380-900 s{sup -1} and photon conversion efficiencies of up to 20% under illumination at 405 nm. In this paper, we focus on the compositional and mechanistic aspects of CdS:CaI complexes that control the photochemical conversion of solar energy into H{sub 2}. Self-assembly of CdS with CaI was driven by electrostatics, demonstrated as the inhibition of ferredoxin-mediated H{sub 2} evolution by CaI. Production of H{sub 2} by CdS:CaImore » was observed only under illumination and only in the presence of a sacrificial donor. We explored the effects of the CdS:CaI molar ratio, sacrificial donor concentration, and light intensity on photocatalytic H{sub 2} production, which were interpreted on the basis of contributions to electron transfer, hole transfer, or rate of photon absorption, respectively. Each parameter was found to have pronounced effects on the CdS:CaI photocatalytic activity. Specifically, we found that under 405 nm light at an intensity equivalent to total AM 1.5 solar flux, H{sub 2} production was limited by the rate of photon absorption ({approx}1 ms{sup -1}) and not by the turnover of CaI. Complexes were capable of H{sub 2} production for up to 4 h with a total turnover number of 106 before photocatalytic activity was lost. This loss correlated with inactivation of CaI, resulting from the photo-oxidation of the CdS capping ligand MPA.« less

The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aklujkar, Muktak; Haveman, Shelley; DiDonatoJr, Raymond

2012-01-01

Background: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species. Results: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to theirmore » structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate: ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires. Conclusions: Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.« less

Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

PubMed Central

Kim, Dockyu; Chae, Jong-Chan; Zylstra, Gerben J.; Kim, Young-Soo; Kim, Seong-Ki; Nam, Myung Hee; Kim, Young Min; Kim, Eungbin

2004-01-01

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively. PMID:15574904

Synthesis, structural characterization and conversion of dinuclear iron-sulfur clusters containing the disulfide ligand: [Cp*Fe(μ-η2:η2-bdt)(cis-μ-η1:η1-S2)FeCp*], [Cp*Fe(μ-S(C6H4S2))(cis-μ-η1:η1-S2)FeCp*], and [{Cp*Fe(bdt)}2(trans-μ-η1:η1-S2)].

PubMed

Ji, Xiaoxiao; Tong, Peng; Yang, Dawei; Wang, Baomin; Zhao, Jinfeng; Li, Yang; Qu, Jingping

2017-03-21

The treatment of [Cp*Fe(μ-η 2 :η 4 -bdt)FeCp*] (1, Cp* = η 5 -C 5 Me 5 , bdt = benzene-1,2-dithiolate) with 1/4 equiv. of elemental sulfur (S 8 ) gave a dinuclear iron-sulfur cluster [Cp*Fe(μ-η 2 :η 2 -bdt)(cis-μ-η 1 :η 1 -S 2 )FeCp*] (2), which contains a cis-1,2-disulfide ligand. When complex 2 further interacted with 1/8 equiv. of S 8 , another sulfur atom inserted into an Fe-S bond to give a rare product [Cp*Fe(μ-S(C 6 H 4 S 2 ))(cis-μ-η 1 :η 1 -S 2 )FeCp*] (3). Unexpectedly, a trans-1,2 disulfide-bridged diiron complex [{Cp*Fe(bdt)} 2 (trans-μ-η 1 :η 1 -S 2 )] (4) was isolated from the reaction of complex 1 with 1/2 equiv. of S 8 , which represents a structural isomer of [2Fe-2S] ferredoxin-type clusters. In addition, cis-1,2-disulfide-bridged complex 3 can slowly convert into trans-1,2-disulfide-bridged complex 4 and the complex [Cp*Fe(μ-η 2 :η 2 -S 2 )(cis-μ-η 1 :η 1 -S 2 )FeCp*] (5) by self-assembly reaction at ambient temperature, which is evidenced by time-dependent 1 H NMR spectroscopy.

Analysis of Photosystem I Donor and Acceptor Sides with a New Type of Online-Deconvoluting Kinetic LED-Array Spectrophotometer.

PubMed

Schreiber, Ulrich; Klughammer, Christof

2016-07-01

The newly developed Dual/KLAS-NIR spectrophotometer, technical details of which were reported very recently, is used in measuring redox changes of P700, plastocyanin (PC) and ferredoxin (Fd) in intact leaves of Hedera helix, Taxus baccata and Brassica napus An overview of various light-/dark-induced changes of deconvoluted P700 + , PC + and Fd - signals is presented demonstrating the wealth of novel information and the consistency of the obtained results. Fd - changes are particularly large after dark adaptation. PC oxidation precedes P700 oxidation during dark-light induction and in steady-state light response curves. Fd reoxidation during induction correlates with the secondary decline of simultaneously measured fluorescence yield, both of which are eliminated by removal of O 2 By determination of 100% redox changes, relative contents of PC/P700 and Fd/P700 can be assessed, which show considerable variations between different leaves, with a trend to higher values in sun leaves. Based on deconvoluted P700 + signals, the complementary quantum yields of PSI, Y(I) (photochemical energy use), Y(ND) (non-photochemical loss due to oxidized primary donor) and Y(NA) (non-photochemical loss due to reduced acceptor) are determined as a function of light intensity and compared with the corresponding complementary quantum yields of PSII, Y(II) (photochemical energy use), Y(NPQ) (regulated non-photochemical loss) and Y(NO) (non-regulated non-photochemical loss). The ratio Y(I)/Y(II) increases with increasing intensities. In the low intensity range, a two-step increase of PC + is indicative of heterogeneous PC pools. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis.

PubMed

Ferrández, Julia; González, Maricruz; Cejudo, Francisco Javier

2012-09-01

Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

PubMed

Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

2010-07-01

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

The delivery of copper for thylakoid import observed by NMR

PubMed Central

Banci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Kandias, Nikolaos G.; Robinson, Nigel J.; Spyroulias, Georgios A.; Su, Xun-Cheng; Tottey, Stephen; Vanarotti, Murugendra

2006-01-01

The thylakoid compartments of plant chloroplasts are a vital destination for copper. Copper is needed to form holo-plastocyanin, which must shuttle electrons between photosystems to convert light into biologically useful chemical energy. Copper can bind tightly to proteins, so it has been hypothesized that copper partitions onto ligand-exchange pathways to reach intracellular locations without inflicting damage en route. The copper metallochaperone Atx1 of chloroplast-related cyanobacteria (ScAtx1) engages in bacterial two-hybrid interactions with N-terminal domains of copper-transporting ATPases CtaA (cell import) and PacS (thylakoid import). Here we visualize copper delivery. The N-terminal domain PacSN has a ferredoxin-like fold that forms copper-dependent heterodimers with ScAtx1. Removal of copper, by the addition of the cuprous-ion chelator bathocuproine disulfonate, disrupts this heterodimer, as shown from a reduction of the overall tumbling rate of the protein mixture. The NMR spectral changes of the heterodimer versus the separate proteins reveal that loops 1, 3, and 5 (the carboxyl tail) of the ScAtx1 Cu(I) site switch to an apo-like configuration in the heterodimer. NMR data (2JNH couplings in the imidazole ring of 15N ScAtx1 His-61) also show that His-61, bound to copper(I) in [Cu(I)ScAtx1]2, is not coordinated to copper in the heterodimer. A model for the PacSN/Cu(I)/ScAtx1 complex is presented. Contact with PacSN induces change to the ScAtx1 copper-coordination sphere that drives copper release for thylakoid import. These data also elaborate on the mechanism to keep copper(I) out of the ZiaAN ATPase zinc sites. PMID:16707580

RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea*S⃞

PubMed Central

Ferguson, Tsuneo; Soares, Jitesh A.; Lienard, Tanja; Gottschalk, Gerhard; Krzycki, Joseph A.

2009-01-01

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function. PMID:19043046

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

PubMed Central

Hennig, Sandra E.; Jeoung, Jae-Hun; Goetzl, Sebastian; Dobbek, Holger

2012-01-01

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe4S4] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins. PMID:22431597

Production of bioplastics and hydrogen gas by photosynthetic microorganisms

NASA Astrophysics Data System (ADS)

Yasuo, Asada; Masato, Miyake; Jun, Miyake

1998-03-01

Our efforts have been aimed at the technological basis of photosynthetic-microbial production of materials and an energy carrier. We report here accumulation of poly-(3-hydroxybutyrate) (PHB), a raw material of biodegradable plastics and for production of hydrogen gas, and a renewable energy carrier by photosynthetic microorganisms (tentatively defined as cyanobacteria plus photosynthetic bateria, in this report). A thermophilic cyanobacterium, Synechococcus sp. MA19 that accumulates PHB at more than 20% of cell dry wt under nitrogen-starved conditions was isolated and microbiologically identified. The mechanism of PHB accumulation was studied. A mesophilic Synechococcus PCC7942 was transformed with the genes encoding PHB-synthesizing enzymes from Alcaligenes eutrophus. The transformant accumulated PHB under nitrogen-starved conditions. The optimal conditions for PHB accumulation by a photosynthetic bacterium grown on acetate were studied. Hydrogen production by photosynthetic microorganisms was studied. Cyanobacteria can produce hydrogen gas by nitrogenase or hydrogenase. Hydrogen production mediated by native hydrogenase in cyanobacteria was revealed to be in the dark anaerobic degradation of intracellular glycogen. A new system for light-dependent hydrogen production was targeted. In vitro and in vivo coupling of cyanobacterial ferredoxin with a heterologous hydrogenase was shown to produce hydrogen under light conditions. A trial for genetic trasformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum is going on. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Co-culture of Rhodobacter and Clostriumdium was applied to produce hydrogen from glucose. Conversely in the case of cyanobacteria, genetic regulation of photosynthetic proteins was intended to improve conversion efficiency in hydrogen production by the photosynthetic bacterium, Rhodobacter sphaeroides RV. A mutant acquired by UV irradiation will be characterized for the mutation and for hydrogen productivity in comparison with the wild type strain. Some basic studies to develop photobioreactors are also introduced.

The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes.

PubMed

Santiago-Martínez, Michel Geovanni; Encalada, Rusely; Lira-Silva, Elizabeth; Pineda, Erika; Gallardo-Pérez, Juan Carlos; Reyes-García, Marco Antonio; Saavedra, Emma; Moreno-Sánchez, Rafael; Marín-Hernández, Alvaro; Jasso-Chávez, Ricardo

2016-05-01

Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production. © 2016 Federation of European Biochemical Societies.

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

PubMed Central

2011-01-01

Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

Proton gradient regulation 5 supports linear electron flow to oxidize photosystem I.

PubMed

Takagi, Daisuke; Miyake, Chikahiro

2018-03-31

In higher plants, light drives the linear photosynthetic electron transport reaction from H 2 O to electron sinks, which is called as linear electron flow (LEF). LEF activity should be regulated depending on electron sinks; otherwise excess electrons accumulate in the thylakoid membranes and stimulate reactive oxygen species (ROS) production in photosystem I (PSI), which causes oxidative damage to PSI. To prevent ROS production in PSI, PSI should be oxidized during photosynthesis, and PROTON GRADIENT REGULATION 5 (PGR5) and PGR like 1 (PGRL1) are important to oxidized PSI. PGR5 and PGRL1 are recognized as a component of ferredoxin-dependent cyclic electron flow around PSI (Fd-CEF-PSI), however there is no direct evidence for the significant operation of Fd-CEF-PSI during photosynthesis in wild-type (WT) plants. Thus, electron distribution by PGR5 and PGRL1 between Fd-CEF-PSI and LEF is still elusive. Here, we show direct evidence that Fd-CEF-PSI activity is minor during steady state photosynthesis by measuring the Fd redox state in vivo in Arabidopsis thaliana. We found that Fd oxidation rate is determined by LEF activity during steady state photosynthesis in WT. On the other hand, pgr5 and pgrl1 showed lower electron transport efficiency from PSI to electron sinks through Fd during steady state photosynthesis. These results demonstrate that electrons are exclusively consumed in electron sinks through Fd, and the phenotypes of pgr5 and pgrl1 are likely caused by the disturbance of the LEF between PSI and electron sinks. We suggest that PGR5 and PGRL1 modulate the LEF according to electron sink activities around PSI. This article is protected by copyright. All rights reserved.

On the influence of crystal size and wavelength on native SAD phasing.

PubMed

Liebschner, Dorothee; Yamada, Yusuke; Matsugaki, Naohiro; Senda, Miki; Senda, Toshiya

2016-06-01

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g. air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.

A new method to evaluate the unfolding activity of chaperone unit ClpA based on Fe-S cluster disruption.

PubMed

Ohgita, Takashi; Okuno, Takashi; Hama, Susumu; Tsuchiya, Hiroyuki; Kogure, Kentaro

2011-01-01

ATP-dependent proteases unfold their substrates and then refold (via chaperone activity) or degrade (via protease activity) them. The proteases choose between these two activities by selecting their substrates; however, little is known about their substrate selection mechanism. The present study attempts to clarify this mechanism by investigating the role of the Escherichia coli (E. coli) ATP-dependent protease ClpAP. To address this, a reaction system that can measure both chaperone and protease activities simultaneously must be constructed. However, the chaperone activities cannot be evaluated in the presence of protease units. Green fluorescent protein (GFP) is usually used as a model substrate of ClpAP; the fluorescence decrease reflects the degradation of substrates. However, it is difficult to evaluate the chaperone activity of ClpAP using this system, because it cannot distinguish between intact and refolded substrates. Therefore, it is necessary to evaluate the exact unfolding activity while avoiding restoration of substrate spectroscopic characteristics due to chaperone activity. In this study, E. coli Ferredoxin (Fd) was used as a new model substrate for ClpAP to evaluate its unfolding activity. Intact and refolded substrates may be distinguished by the existence of an Fd Fe-S cluster. To verify this hypothesis, the absorption spectrum of Fd complexed with ClpA, the chaperone unit of ClpAP, was measured. A decrease in two peaks derived from the Fe-S cluster was observed, indicating that the Fe-S cluster of Fd was disrupted by the ClpA chaperone. This reaction system should prove useful to evaluate the exact unfolding activity of ATP-dependent proteases.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

PubMed

Cory, Seth A; Van Vranken, Jonathan G; Brignole, Edward J; Patra, Shachin; Winge, Dennis R; Drennan, Catherine L; Rutter, Jared; Barondeau, David P

2017-07-03

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

The SUFBC2 D complex is required for the biogenesis of all major classes of plastid Fe-S proteins.

PubMed

Hu, Xueyun; Kato, Yukako; Sumida, Akihiro; Tanaka, Ayumi; Tanaka, Ryouichi

2017-04-01

Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC 2 D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC 2 D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC 2 D is the major, and possibly sole scaffold in plastids. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Malate valves: Old shuttles with new perspectives.

PubMed

Selinski, Jennifer; Scheibe, Renate

2018-06-22

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyze the reversible interconversion of malate and oxaloacetate and their transport. Depending on the coenzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes: Activities of NAD-dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids, respectively. In addition, chloroplasts possess a NADP-dependent MDH isoform. The NADP-MDH as part of the "light malate valve" plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post-translational redox-modification mediated via the ferredoxin-thioredoxin system and fine control via the NADP + /NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD-MDH ("dark malate valve") is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, the knowledge about regulation of the other two cytosolic MDHs as well as NAD-MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria, and peroxisomes have been characterized, but not much is known about cytosolic NAD-MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange focusing on the various metabolic functions of these valves. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

CO 2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Wei; Lin, Paul P.; Magnusson, Lauren

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO 2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO 2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO 2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO 2 to formate serves as a CO 2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO 2 via two biochemical reactions:more » the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO 2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO 2 uptake, and provided physical evidence of a distinct in vivo 'rPFOR-PFL shunt' to reduce CO 2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO 2 fixation via the reductive C1 metabolic pathway in C. thermocellum. Lastly, these findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO 2 as well.« less

In Silico Analysis of the Regulation of the Photosynthetic Electron Transport Chain in C3 Plants.

PubMed

Morales, Alejandro; Yin, Xinyou; Harbinson, Jeremy; Driever, Steven M; Molenaar, Jaap; Kramer, David M; Struik, Paul C

2018-02-01

We present a new simulation model of the reactions in the photosynthetic electron transport chain of C3 species. We show that including recent insights about the regulation of the thylakoid proton motive force, ATP/NADPH balancing mechanisms (cyclic and noncyclic alternative electron transport), and regulation of Rubisco activity leads to emergent behaviors that may affect the operation and regulation of photosynthesis under different dynamic environmental conditions. The model was parameterized with experimental results in the literature, with a focus on Arabidopsis ( Arabidopsis thaliana ). A dataset was constructed from multiple sources, including measurements of steady-state and dynamic gas exchange, chlorophyll fluorescence, and absorbance spectroscopy under different light intensities and CO 2 , to test predictions of the model under different experimental conditions. Simulations suggested that there are strong interactions between cyclic and noncyclic alternative electron transport and that an excess capacity for alternative electron transport is required to ensure adequate redox state and lumen pH. Furthermore, the model predicted that, under specific conditions, reduction of ferredoxin by plastoquinol is possible after a rapid increase in light intensity. Further analysis also revealed that the relationship between ATP synthesis and proton motive force was highly regulated by the concentrations of ATP, ADP, and inorganic phosphate, and this facilitated an increase in nonphotochemical quenching and proton motive force under conditions where metabolism was limiting, such as low CO 2 , high light intensity, or combined high CO 2 and high light intensity. The model may be used as an in silico platform for future research on the regulation of photosynthetic electron transport. © 2018 American Society of Plant Biologists. All Rights Reserved.

Peroxiredoxins and NADPH-Dependent Thioredoxin Systems in the Model Legume Lotus japonicus1[W][OA

PubMed Central

Tovar-Méndez, Alejandro; Matamoros, Manuel A.; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

2011-01-01

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules. PMID:21562331

Microbial electron transport and energy conservation – the foundation for optimizing bioelectrochemical systems

PubMed Central

Kracke, Frauke; Vassilev, Igor; Krömer, Jens O.

2015-01-01

Microbial electrochemical techniques describe a variety of emerging technologies that use electrode–bacteria interactions for biotechnology applications including the production of electricity, waste and wastewater treatment, bioremediation and the production of valuable products. Central in each application is the ability of the microbial catalyst to interact with external electron acceptors and/or donors and its metabolic properties that enable the combination of electron transport and carbon metabolism. And here also lies the key challenge. A wide range of microbes has been discovered to be able to exchange electrons with solid surfaces or mediators but only a few have been studied in depth. Especially electron transfer mechanisms from cathodes towards the microbial organism are poorly understood but are essential for many applications such as microbial electrosynthesis. We analyze the different electron transport chains that nature offers for organisms such as metal respiring bacteria and acetogens, but also standard biotechnological organisms currently used in bio-production. Special focus lies on the essential connection of redox and energy metabolism, which is often ignored when studying bioelectrochemical systems. The possibility of extracellular electron exchange at different points in each organism is discussed regarding required redox potentials and effect on cellular redox and energy levels. Key compounds such as electron carriers (e.g., cytochromes, ferredoxin, quinones, flavins) are identified and analyzed regarding their possible role in electrode–microbe interactions. This work summarizes our current knowledge on electron transport processes and uses a theoretical approach to predict the impact of different modes of transfer on the energy metabolism. As such it adds an important piece of fundamental understanding of microbial electron transport possibilities to the research community and will help to optimize and advance bioelectrochemical techniques. PMID:26124754

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum

DOE PAGES

Thompson, R. Adam; Layton, Donovan S.; Guss, Adam M.; ...

2015-10-21

Clostridium thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of C. thermocellum, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for C. thermocellum which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model smore » capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. In silico predictions of a redox-imbalanced genotype incapable of growth were confirmed in vivo, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of C. thermocellum and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain C. thermocellum to achieve high ethanol yields while requiring minimal genetic manipulations. Furthermore, the model can be applied to design C. thermocellum as a platform microbe for consolidated bioprocessing to produce ethanol and other reduced metabolites.« less

Proteomic and enzymatic response under Cr(VI) overload in yeast isolated from textile-dye industry effluent.

PubMed

Irazusta, Verónica; Bernal, Anahí Romina; Estévez, María Cristina; de Figueroa, Lucía I C

2018-02-01

Cyberlindnera jadinii M9 and Wickerhamomyces anomalus M10 isolated from textile-dye liquid effluents has shown capacity for chromium detoxification via Cr(VI) biological reduction. The aim of the study was to evaluate the effect of hexavalent chromium on synthesis of novel and/or specific proteins involved in chromium tolerance and reduction in response to chromium overload in two indigenous yeasts. A study was carried out following a proteomic approach with W. anomalus M10 and Cy. jadinii M9 strains. For this, proteins extracts belonging to total cell extracts, membranes and mitochondria were analyzed. When Cr(VI) was added to culture medium there was an over-synthesis of 39 proteins involved in different metabolic pathways. In both strains, chromium supplementation changed protein biosynthesis by upregulating proteins involved in stress response, methionine metabolism, energy production, protein degradation and novel oxide-reductase enzymes. Moreover, we observed that Cy. jadinii M9 and W. anomalus M10 displayed ability to activate superoxide dismutase, catalase and chromate reductase activity. Two enzymes from the total cell extracts, type II nitroreductase (Frm2) and flavoprotein wrbA (Ycp4), were identified as possibly responsible for inducing crude chromate-reductase activity in cytoplasm of W. anomalus M10 under chromium overload. In Cy.jadinii M9, mitochondrial Ferredoxine-NADP reductase (Yah1) and membrane FAD flavoprotein (Lpd1) were identified as probably involved in Cr(VI) reduction. To our knowledge, this is the first study proposing chromate reductase activity of these four enzymes in yeast and reporting a relationship between protein synthesis, enzymatic response and chromium biospeciation in Cy. jadinii and W. anomalus. Copyright © 2017 Elsevier Inc. All rights reserved.

Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase.

PubMed Central

Gangeswaran, R; Eady, R R

1996-01-01

Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not. PMID:8694750

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: the qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side.

PubMed

Schansker, Gert; Tóth, Szilvia Z; Strasser, Reto J

2006-07-01

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700(+) and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP(+)-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 mumol photons m(-2) s(-1) did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to 'close' all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q(A) a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q(B)-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.

Degradation of Acetaldehyde and Its Precursors by Pelobacter carbinolicus and P. acetylenicus

PubMed Central

Schmidt, Alexander; Frensch, Marco; Schleheck, David; Schink, Bernhard; Müller, Nicolai

2014-01-01

Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost “Methanobacillus omelianskii” coculture. During growth on ethanol, both Pelobacter species exhibited NAD+-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD+-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be EH2 = -358±12 mV and EHCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was −35 to −28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically. PMID:25536080

Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.).

PubMed

Andrés-Bordería, Amparo; Andrés, Fernando; Garcia-Molina, Antoni; Perea-García, Ana; Domingo, Concha; Puig, Sergi; Peñarrubia, Lola

2017-09-01

Copper deficiency and excess differentially affect iron homeostasis in rice and overexpression of the Arabidopsis high-affinity copper transporter COPT1 slightly increases endogenous iron concentration in rice grains. Higher plants have developed sophisticated mechanisms to efficiently acquire and use micronutrients such as copper and iron. However, the molecular mechanisms underlying the interaction between both metals remain poorly understood. In the present work, we study the effects produced on iron homeostasis by a wide range of copper concentrations in the growth media and by altered copper transport in Oryza sativa plants. Gene expression profiles in rice seedlings grown under copper excess show an altered expression of genes involved in iron homeostasis compared to standard control conditions. Thus, ferritin OsFER2 and ferredoxin OsFd1 mRNAs are down-regulated whereas the transcriptional iron regulator OsIRO2 and the nicotianamine synthase OsNAS2 mRNAs rise under copper excess. As expected, the expression of OsCOPT1, which encodes a high-affinity copper transport protein, as well as other copper-deficiency markers are down-regulated by copper. Furthermore, we show that Arabidopsis COPT1 overexpression (C1 OE ) in rice causes root shortening in high copper conditions and under iron deficiency. C1 OE rice plants modify the expression of the putative iron-sensing factors OsHRZ1 and OsHRZ2 and enhance the expression of OsIRO2 under copper excess, which suggests a role of copper transport in iron signaling. Importantly, the C1 OE rice plants grown on soil contain higher endogenous iron concentration than wild-type plants in both brown and white grains. Collectively, these results highlight the effects of rice copper status on iron homeostasis, which should be considered to obtain crops with optimized nutrient concentrations in edible parts.

Terminal oxidase diversity and function in "Metallosphaera yellowstonensis": gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales.

PubMed

Kozubal, M A; Dlakic, M; Macur, R E; Inskeep, W P

2011-03-01

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

PubMed

Argüello-García, Raúl; Cruz-Soto, Maricela; Romero-Montoya, Lydia; Ortega-Pierres, Guadalupe

2009-12-01

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

2012-04-01

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus,more » led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.« less

Suppression of progesterone synthesis in human trophoblast cells by fine particulate matter primarily derived from industry.

PubMed

Wang, Cui; Yang, Jinhuan; Hao, Zhengliang; Gong, Chenxue; Tang, Lihua; Xu, Yingling; Lu, Dezhao; Li, Zhuoyu; Zhao, Meirong

2017-12-01

Epidemiological studies have exhibited a positive association between fine particulate matter (PM 2.5 ) exposure and adverse pregnancy outcome (APO). However, source-related effect and the potential mechanism have not been thoroughly elucidated in toxicology. In this study, PM 2.5 was collected during a severe winter haze episode in an energy-base city of China. We coupled this approach with the source appointment by applying the Lagrangian Integrated Trajectory and Concentration Weighted Trajectory model. We observed that the primary trajectory with high polluted air mass came from the northwest of the sampling site. Approximately 90% or more of PM 2.5 was derived from the industry at this haze period. Next, the sampled PM 2.5 was used to study the classical hormone synthesis pathway on trophoblast JEG-3 cells. PM 2.5 induced the secretion of human chorionic gonadotrophin (HCG) and the proliferation of JEG-3 cells at a noncytotoxic concentration. However, the synthesis of progesterone was significantly suppressed, even if both hCG and cyclic adenosine monophosphate (cAMP) were increased, suggesting that PM 2.5 may interfere the downstream of cAMP. As expected, the phosphorylated activity of protein kinase A (PKA) was attenuated. Subsequently, the downstream molecules of steroidogenesis, such as ferredoxin reductase (FDXR), CYP11A1 (encoded P450scc), and 3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1), were inhibited. Therefore, PM 2.5, primarily derived from industry, may directly inhibit the phosphorylation status of PKA in JEG-3 which, in turn, inhibited the proteins expression in progesterone-synthesis to suppress progesterone levels. Considering the pivotal role of progesterone in pregnancy maintenance, the mechanism on hormone synthesis may provide a better understanding for PM 2.5 -caused APO. Industry-emanated PM 2.5 , though not specific, could threaten the placenta, which needs to be verified by further epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

PubMed Central

2011-01-01

Background Elucidation of molecular mechanism of silver nanoparticles (SNPs) biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution) of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro) and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro) SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using C. reinhardtii as a model system. PMID:22152042

Redox regulation of carbon storage and partitioning in response to light and sugars.

PubMed

Geigenberger, Peter; Kolbe, Anna; Tiessen, Axel

2005-06-01

Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.

Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side-chain-cleavage enzyme, P450scc [corrected], in human steroidogenic tissues.

PubMed Central

Voutilainen, R; Miller, W L

1987-01-01

Insulin-like growth factors (IGFs) are single-chain polypeptides important for cell proliferation and growth. IGFs are produced in several tissues, suggesting that they function in a paracrine or autocrine fashion as well as functioning as endocrine hormones. We studied the hormonal regulation of IGF-I and IGF-II mRNA in human steroidogenic tissues. In cultured human ovarian granulosa cells, follicle-stimulating hormone, human chorionic gonadotropin, and dibutyryl cAMP increased IGF-II mRNA, but corticotropin [adrenocorticotropic hormone (ACTH)], chorionic somatomammotropin, growth hormone, prolactin, dexamethasone, estradiol, and progesterone had no effect. In cultured human fetal adrenal cells, ACTH and dibutyryl cAMP increased IGF-II mRNA accumulation, but human chorionic gonadotropin and angiotensin II did not. The same five size species of IGF-II mRNA were detected in transfer blots of RNA from granulosa cells and fetal adrenal cells, and all of these increased after hormonal stimuli. Dibutyryl cAMP also increased IGF-II mRNA accumulation in cultured human placental cells. Accumulation of mRNA for the cholesterol side-chain-cleavage monooxygenase [P450scc [corrected]; cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.6] was regulated in parallel with IGF-II mRNA in all these steroidogenic tissues. IGF-I mRNA was not detected in transfer blots of these RNAs, and the minimal amounts detected in dot blots showed no detectable change after any of the hormonal stimuli studied. The data indicate that the IGF-II gene is expressed in human steroidogenic tissues and is regulated by cAMP. These data suggest that IGF-II may act in an autocrine or paracrine fashion to stimulate the adrenal and gonadal growth stimulated by ACTH and gonadotropins, respectively. Images PMID:3031644

Characterization and Evolution of Anthranilate 1,2-Dioxygenase from Acinetobacter sp. Strain ADP1

PubMed Central

Eby, D. Matthew; Beharry, Zanna M.; Coulter, Eric D.; Kurtz, Donald M.; Neidle, Ellen L.

2001-01-01

The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O2 and consumption of one NADH. The terminal oxygenase component formed an α3β3 hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O2 consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O2 consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1,2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23°C) or nonpermissive (39°C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the α and β subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component. PMID:11114907

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum.

PubMed

Thompson, R Adam; Layton, Donovan S; Guss, Adam M; Olson, Daniel G; Lynd, Lee R; Trinh, Cong T

2015-11-01

Clostridium thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of C. thermocellum, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for C. thermocellum which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model's capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. In silico predictions of a redox-imbalanced genotype incapable of growth were confirmed in vivo, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of C. thermocellum and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain C. thermocellum to achieve high ethanol yields while requiring minimal genetic manipulations. The model can be applied to design C. thermocellum as a platform microbe for consolidated bioprocessing to produce ethanol and other reduced metabolites. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

PubMed Central

Ebert, Matthias; Laaß, Sebastian; Burghartz, Melanie; Petersen, Jörn; Koßmehl, Sebastian; Wöhlbrand, Lars; Rabus, Ralf; Wittmann, Christoph; Jahn, Dieter

2013-01-01

Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na+/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies. PMID:23974024

Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni.

PubMed

Hoffman, Paul S; Sisson, Gary; Croxen, Matthew A; Welch, Kevin; Harman, W Dean; Cremades, Nunilo; Morash, Michael G

2007-03-01

Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K(i), 2 to 10 microM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO(2)) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and (1)H nuclear magnetic resonance analyses determined that the spectral shift (A(418) to A(351)) of NTZ was due to protonation of the anion (NTZ(-)) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK(a) = 6.18). We propose that NTZ(-) intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the "activated cofactor" of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.

Response of the seagrass Posidonia oceanica to different light environments: Insights from a combined molecular and photo-physiological study.

PubMed

Dattolo, E; Ruocco, M; Brunet, C; Lorenti, M; Lauritano, C; D'Esposito, D; De Luca, P; Sanges, R; Mazzuca, S; Procaccini, G

2014-10-01

Here we investigated mechanisms underlying the acclimation to light in the marine angiosperm Posidonia oceanica, along its bathymetric distribution (at -5 m and -25 m), combining molecular and photo-physiological approaches. Analyses were performed during two seasons, summer and autumn, in a meadow located in the Island of Ischia (Gulf of Naples, Italy), where a genetic distinction between plants growing above and below the summer thermocline was previously revealed. At molecular level, analyses carried out using cDNA-microarray and RT-qPCR, revealed the up-regulation of genes involved in photoacclimation (RuBisCO, ferredoxin, chlorophyll binding proteins), and photoprotection (antioxidant enzymes, xanthophyll-cycle related genes, tocopherol biosynthesis) in the upper stand of the meadow, indicating that shallow plants are under stressful light conditions. However, the lack of photo-damage, indicates the successful activation of defense mechanisms. This conclusion is also supported by several responses at physiological level as the lower antenna size, the higher number of reaction centers and the higher xanthophyll cycle pigment pool, which are common plant responses to high-light adaptation/acclimation. Deep plants, despite the lower available light, seem to be not light-limited, thanks to some shade-adaptation strategies (e.g. higher antenna size, lower Ek values). Furthermore, also at the molecular level there were no signs of stress response, indicating that, although the lower energy available, low-light environments are more favorable for P. oceanica growth. Globally, results of whole transcriptome analysis displayed two distinct gene expression signatures related to depth distribution, reflecting the different light-adaptation strategies adopted by P. oceanica along the depth gradient. This observation, also taking into account the genetic disjunction of clones along the bathymetry, might have important implications for micro-evolutionary processes happening at meadow scale. Further investigations in controlled conditions must be performed to respond to these questions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genome-Guided Analysis of Clostridium ultunense and Comparative Genomics Reveal Different Strategies for Acetate Oxidation and Energy Conservation in Syntrophic Acetate-Oxidising Bacteria

PubMed Central

Manzoor, Shahid; Schnürer, Anna; Müller, Bettina

2018-01-01

Syntrophic acetate oxidation operates close to the thermodynamic equilibrium and very little is known about the participating organisms and their metabolism. Clostridium ultunense is one of the most abundant syntrophic acetate-oxidising bacteria (SAOB) that are found in engineered biogas processes operating with high ammonia concentrations. It has been proven to oxidise acetate in cooperation with hydrogenotrophic methanogens. There is evidence that the Wood-Ljungdahl (WL) pathway plays an important role in acetate oxidation. In this study, we analysed the physiological and metabolic capacities of C. ultunense strain Esp and strain BST on genome scale and conducted a comparative study of all the known characterised SAOB, namely Syntrophaceticus schinkii, Thermacetogenium phaeum, Tepidanaerobacter acetatoxydans, and Pseudothermotoga lettingae. The results clearly indicated physiological robustness to be beneficial for anaerobic digestion environments and revealed unexpected metabolic diversity with respect to acetate oxidation and energy conservation systems. Unlike S. schinkii and Th. phaeum, C. ultunense clearly does not employ the oxidative WL pathway for acetate oxidation, as its genome (and that of P. lettingae) lack important key genes. In both of those species, a proton motive force is likely formed by chemical protons involving putative electron-bifurcating [Fe-Fe] hydrogenases rather than proton pumps. No genes encoding a respiratory Ech (energy-converting hydrogenase), as involved in energy conservation in Th. phaeum and S. schinkii, were identified in C. ultunense and P. lettingae. Moreover, two respiratory complexes sharing similarities to the proton-translocating ferredoxin:NAD+ oxidoreductase (Rnf) and the Na+ pumping NADH:quinone hydrogenase (NQR) were predicted. These might form a respiratory chain that is involved in the reduction of electron acceptors rather than protons. However, involvement of these complexes in acetate oxidation in C. ultunense and P. lettingae needs further study. This genome-based comparison provides a solid platform for future meta-proteomics and meta-transcriptomics studies and for metabolic engineering, control, and monitoring of SAOB. PMID:29690652

Determination of the rate of photoreduction of O2 in the water-water cycle in watermelon leaves and enhancement of the rate by limitation of photosynthesis.

PubMed

Miyake, C; Yokota, A

2000-03-01

A study was performed to determine how the electron fluxes for the photosynthetic carbon reduction (PCR) and the photorespiratory carbon oxidation (PCO) cycles affect the photoreduction of O2 at PSI, which is the limiting step in the water-water cycle. Simultaneous measurements were made of CO2-gas exchange, transpiration and quantum yield of PSII [phi(PSII)] using leaves of watermelon (Citrullus lanatus). The total electron flux in PSII[Je(PSII)], as estimated from phi(PSII), was always larger than the total electron flux required for the PCR and PCO cycles at various partial pressures of CO2 and O2 and 1,100 micromol photons m(-2)s(-1). This observation suggested the existence of an alternative electron flux (Ja). Ja was divided into O2-dependent [Ja(O2-depend)] and O2-independent [Ja(O2-independ)] components. The magnitude of half Ja(O2-depend), 7.5 to 9.5 micromol e- m(-2)s(-1), and its apparent Km for O2, about 8.0 kPa, could be accounted for by the photoreduction of O2 at PSI either mediated by ferredoxin or catalyzed by monodehydroascorbate reductase. The results indicated that Ja(O2-depend) was driven by the water-water cycle. A decrease in the intercellular partial pressure of CO2 from 23 to 5.0 Pa at 21 kPa O2 enhanced Ja(O2-depend) by a factor of 1.3. Saturation of the activities of both the PCR and PCO cycles by increasing the photon flux density induced Ja. These results indicate the electron flux in PSII that exceeds the flux required for the PCR and PCO cycles induces the photoreduction of O2 in the water-water cycle.

Bacterial Diversity and Nitrogen Utilization Strategies in the Upper Layer of the Northwestern Pacific Ocean.

PubMed

Li, Yuan-Yuan; Chen, Xiao-Huang; Xie, Zhang-Xian; Li, Dong-Xu; Wu, Peng-Fei; Kong, Ling-Fen; Lin, Lin; Kao, Shuh-Ji; Wang, Da-Zhi

2018-01-01

Nitrogen (N) is a primary limiting nutrient for bacterial growth and productivity in the ocean. To better understand bacterial community and their N utilization strategy in different N regimes of the ocean, we examined bacterial diversity, diazotrophic diversity, and N utilization gene expressions in the northwestern Pacific Ocean (NWPO) using a combination of high-throughput sequencing and real-time qPCR methods. 521 and 204 different operational taxonomic units (OTUs) were identified in the 16s rRNA and nifH libraries from nine surface samples. Of the 16s rRNA gene OTUs, 11.9% were observed in all samples while 3.5 and 15.9% were detected only in N-sufficient and N-deficient samples. Proteobacteria, Cyanobacteria and Bacteroidetes dominated the bacterial community. Prochlorococcus and Pseudoalteromonas were the most abundant at the genus level in N-deficient regimes, while SAR86, Synechococcus and SAR92 were predominant in the Kuroshio-Oyashio confluence region. The distribution of the nifH gene presented great divergence among sampling stations: Cyanobacterium_UCYN-A dominated the N-deficient stations, while clusters related to the Alpha-, Beta- , and Gamma-Proteobacteria were abundant in other stations. Temperature was the main factor that determined bacterial community structure and diversity while concentration of NO X -N was significantly correlated with structure and distribution of N 2 -fixing microorganisms. Expression of the ammonium transporter was much higher than that of urea transporter subunit A ( urtA ) and ferredoxin-nitrate reductase , while urtA had an increased expression in N-deficient surface water. The predicted ammonium transporter and ammonium assimilation enzymes were most abundant in surface samples while urease and nitrogenase were more abundant in the N-deficient regions. These findings underscore the fact that marine bacteria have evolved diverse N utilization strategies to adapt to different N habitats, and that urea metabolism is of vital ecological importance in N-deficient regimes.

delta 6 Hexadecenoic acid is synthesized by the activity of a soluble delta 6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.

PubMed

Cahoon, E B; Cranmer, A M; Shanklin, J; Ohlrogge, J B

1994-11-04

delta 6 Hexadecenoic acid (16:1 delta 6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble delta 6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, delta 6 16:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in delta 9 stearoyl (18:0)- and delta 4 16:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor delta 9 18:0-ACP desaturase and 57% identity with the mature coriander delta 4 16:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the delta 6 desaturation of 16:0-ACP. These results demonstrate that 16:1 delta 6 in T. alata endosperm is formed by the activity of a soluble delta 6 16:0-ACP desaturase that is structurally related to the delta 9 18:0- and delta 4 16:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer.

PubMed

Hannemann, Frank; Guyot, Arnaud; Zöllner, Andy; Müller, Jürgen J; Heinemann, Udo; Bernhardt, Rita

2009-07-01

Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

Thylakoid membrane model of the Chl a fluorescence transient and P700 induction kinetics in plant leaves.

PubMed

Belyaeva, N E; Bulychev, A A; Riznichenko, G Yu; Rubin, A B

2016-12-01

A new Thylakoid model is presented, which describes in detail the electron/proton transfer reactions between membrane protein complexes including photosystems II and I (PSII, PSI), cytochrome (Cyt) b 6 f, mobile plastoquinone PQ pool in the thylakoid membrane, plastocyanin in lumen and ferredoxin in stroma, reduction of NADP via FNR and cyclic electron transfer. The Thylakoid model parameters were fitted both to Chl fluorescence induction data (FI) and oxido-reductions of P700 (ΔA 810 ) measured from 20 μs up to 20 s in pea leaves. The two-wave kinetics of FI and ΔA 810 (O(JI)PSM and OABCDE) were described quantitatively, provided that the values of membrane electrochemical potential components ΔΨ(t), pH L (t)/pH S (t) are in physiologically relevant ranges. The time courses on the time scale from nanoseconds to tens of seconds of oxido-reduction changes of ET components as well as concentrations of proton/ions (K + , Cl - ) were calculated. We assume a low constant FNR activity over this period. Charge movements across the thylakoid membrane by passive leakage and active ATPase transport and proton buffer reactions are simulated. The dynamics of charge fluxes during photosynthetic induction under low light (PFD 200 μmol photons m -2  s -1 ) were analyzed. The initial wave of P700 oxidation within 20 ms during independent operation of PSI and PSII was followed after 50 ms by PSI donor-side reduction from reduced PQ pool via Cyt b 6 f site. The Cyt b 6 f reactions contribute to the stabilization of fluxes in the time range 1 s < t < 10 s. The detailed analysis of Chl a fluorescence at the PSM stage (t > 10 s) would need the investigation of FNR activation effect in order to explain the transitions between cyclic and linear electron transport.

Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

PubMed Central

Kitagawa, Wataru; Takami, Sachiko; Miyauchi, Keisuke; Masai, Eiji; Kamagata, Yoichi; Tiedje, James M.; Fukuda, Masao

2002-01-01

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC. PMID:11751829

Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.

PubMed

Rodrigues, Andria V; Kandegedara, Ashoka; Rotondo, John A; Dancis, Andrew; Stemmler, Timothy L

2015-06-01

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

The phn Genes of Burkholderia sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism

PubMed Central

Laurie, Andrew D.; Lloyd-Jones, Gareth

1999-01-01

Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. This phn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenanthrene-degrading Burkholderia sp. strain RP007. The phn genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in Escherichia coli the recombinant phn enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein α and β subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the phn catabolic genes are two putative regulatory genes, phnR and phnS. Sequence homology suggests that phnS is a LysR-type transcriptional activator and that phnR, which is divergently transcribed with respect to phnSFECDAcAdB, is a member of the ς54-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS. PMID:9882667

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin.

PubMed

Bruntner, C; Lauer, B; Schwarz, W; Möhrle, V; Bormann, C

1999-08-01

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins Cx and Cz. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins Lx and Lz, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine, the putative activities of each of their products are discussed.

The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.

PubMed

Schut, Gerrit J; Zadvornyy, Oleg; Wu, Chang-Hao; Peters, John W; Boyd, Eric S; Adams, Michael W W

2016-07-01

Complex I or NADH quinone oxidoreductase (NUO) is an integral component of modern day respiratory chains and has a close evolutionary relationship with energy-conserving [NiFe]-hydrogenases of anaerobic microorganisms. Specifically, in all of biology, the quinone-binding subunit of Complex I, NuoD, is most closely related to the proton-reducing, H2-evolving [NiFe]-containing catalytic subunit, MbhL, of membrane-bound hydrogenase (MBH), to the methanophenzine-reducing subunit of a methanogenic respiratory complex (FPO) and to the catalytic subunit of an archaeal respiratory complex (MBX) involved in reducing elemental sulfur (S°). These complexes also pump ions and have at least 10 homologous subunits in common. As electron donors, MBH and MBX use ferredoxin (Fd), FPO uses either Fd or cofactor F420, and NUO uses either Fd or NADH. In this review, we examine the evolutionary trajectory of these oxidoreductases from a proton-reducing ancestral respiratory complex (ARC). We hypothesize that the diversification of ARC to MBH, MBX, FPO and eventually NUO was driven by the larger energy yields associated with coupling Fd oxidation to the reduction of oxidants with increasing electrochemical potential, including protons, S° and membrane soluble organic compounds such as phenazines and quinone derivatives. Importantly, throughout Earth's history, the availability of these oxidants increased as the redox state of the atmosphere and oceans became progressively more oxidized as a result of the origin and ecological expansion of oxygenic photosynthesis. ARC-derived complexes are therefore remarkably stable respiratory systems with little diversity in core structure but whose general function appears to have co-evolved with the redox state of the biosphere. This article is part of a Special Issue entitled Respiratory Complex I, edited by Volker Zickermann and Ulrich Brandt. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-Guided Analysis of Clostridium ultunense and Comparative Genomics Reveal Different Strategies for Acetate Oxidation and Energy Conservation in Syntrophic Acetate-Oxidising Bacteria.

PubMed

Manzoor, Shahid; Schnürer, Anna; Bongcam-Rudloff, Erik; Müller, Bettina

2018-04-23

Syntrophic acetate oxidation operates close to the thermodynamic equilibrium and very little is known about the participating organisms and their metabolism. Clostridium ultunense is one of the most abundant syntrophic acetate-oxidising bacteria (SAOB) that are found in engineered biogas processes operating with high ammonia concentrations. It has been proven to oxidise acetate in cooperation with hydrogenotrophic methanogens. There is evidence that the Wood-Ljungdahl (WL) pathway plays an important role in acetate oxidation. In this study, we analysed the physiological and metabolic capacities of C. ultunense strain Esp and strain BS T on genome scale and conducted a comparative study of all the known characterised SAOB, namely Syntrophaceticus schinkii , Thermacetogenium phaeum , Tepidanaerobacter acetatoxydans , and Pseudothermotoga lettingae . The results clearly indicated physiological robustness to be beneficial for anaerobic digestion environments and revealed unexpected metabolic diversity with respect to acetate oxidation and energy conservation systems. Unlike S. schinkii and Th. phaeum , C. ultunense clearly does not employ the oxidative WL pathway for acetate oxidation, as its genome (and that of P. lettingae ) lack important key genes. In both of those species, a proton motive force is likely formed by chemical protons involving putative electron-bifurcating [Fe-Fe] hydrogenases rather than proton pumps. No genes encoding a respiratory Ech (energy-converting hydrogenase), as involved in energy conservation in Th. phaeum and S. schinkii, were identified in C. ultunense and P. lettingae . Moreover, two respiratory complexes sharing similarities to the proton-translocating ferredoxin:NAD⁺ oxidoreductase (Rnf) and the Na⁺ pumping NADH:quinone hydrogenase (NQR) were predicted. These might form a respiratory chain that is involved in the reduction of electron acceptors rather than protons. However, involvement of these complexes in acetate oxidation in C. ultunense and P. lettingae needs further study. This genome-based comparison provides a solid platform for future meta-proteomics and meta-transcriptomics studies and for metabolic engineering, control, and monitoring of SAOB.

Thioredoxins Play a Crucial Role in Dynamic Acclimation of Photosynthesis in Fluctuating Light.

PubMed

Thormählen, Ina; Zupok, Arkadiusz; Rescher, Josephin; Leger, Jochen; Weissenberger, Stefan; Groysman, Julia; Orwat, Anne; Chatel-Innocenti, Gilles; Issakidis-Bourguet, Emmanuelle; Armbruster, Ute; Geigenberger, Peter

2017-01-09

Sunlight represents the energy source for photosynthesis and plant growth. When growing in the field, plant photosynthesis has to manage strong fluctuations in light intensities. Regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of photosynthetic reactions in the chloroplast. However, direct evidence for a role of this system in regulating dynamic acclimation of photosynthesis in fluctuating conditions is largely lacking. In this report we show that the ferredoxin-dependent Trxs m1 and m2 as well as the NADPH-dependent NTRC are both indispensable for photosynthetic acclimation in fluctuating light intensities. Arabidopsis mutants with combined deficiency in Trxs m1 and m2 show wild-type growth and photosynthesis under constant light condition, while photosynthetic parameters are strongly modified in rapidly alternating high and low light. Two independent trxm1m2 mutants show lower photosynthetic efficiency in high light, but surprisingly significantly higher photosynthetic efficiency in low light. Our data suggest that a main target of Trx m1 and m2 is the NADP-malate dehydrogenase involved in export of excess reductive power from the chloroplast. The decreased photosynthetic efficiency in the high-light peaks may thus be explained by a reduced capacity of the trxm1m2 mutants in the rapid light activation of this enzyme. In the ntrc mutant, dynamic responses of non-photochemical quenching of excitation energy and plastoquinone reduction state both were strongly attenuated in fluctuating light intensities, leading to a massive decrease in PSII quantum efficiency and a specific decrease in plant growth under these conditions. This is likely due to the decreased ability of the ntrc mutant to control the stromal NADP(H) redox poise. Taken together, our results indicate that NTRC is indispensable in ensuring the full range of dynamic responses of photosynthesis to optimize photosynthesis and maintain growth in fluctuating light, while Trxs m1 and m2 are indispensable for full activation of photosynthesis in the high-light periods but negatively affect photosynthetic efficiency in the low-light periods of fluctuating light. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes

PubMed Central

Sewell, Holly L.; Kaster, Anne-Kristin

2017-01-01

ABSTRACT The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi. PMID:29259088

Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C

PubMed Central

Crnovčić, Ivana; Rückert, Christian; Semsary, Siamak; Lang, Manuel; Kalinowski, Jörn; Keller, Ullrich

2017-01-01

Sequencing the actinomycin (acm) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus. PMID:28435299

Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C.

PubMed

Crnovčić, Ivana; Rückert, Christian; Semsary, Siamak; Lang, Manuel; Kalinowski, Jörn; Keller, Ullrich

2017-01-01

Sequencing the actinomycin ( acm ) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN , encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus .

Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis

PubMed Central

Bengelsdorf, Frank R.; Poehlein, Anja; Linder, Sonja; Erz, Catarina; Hummel, Tim; Hoffmeister, Sabrina; Daniel, Rolf; Dürre, Peter

2016-01-01

Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood–Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (PthlA) from C. acetobutylicum or native pta-ack promoter (Ppta-ack) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2-propanol. Furthermore, the ClosTronTM system was used to construct an adhE1 integration mutant. These results provide extensive insights into genetic features of industrially relevant bacterial biocatalysts and expand the toolbox for metabolic engineering of acetogenic bacteria able to ferment syngas. PMID:27458439

Unraveling the molecular mechanisms of nitrogenase conformational protection against oxygen in diazotrophic bacteria.

PubMed

Lery, Letícia M S; Bitar, Mainá; Costa, Mauricio G S; Rössle, Shaila C S; Bisch, Paulo M

2010-12-22

G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms. Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved "fer2" domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface. The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.

Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co-factors in zebrafish.

PubMed

Weger, M; Diotel, N; Weger, B D; Beil, T; Zaucker, A; Eachus, H L; Oakes, J A; do Rego, J L; Storbeck, K-H; Gut, P; Strähle, U; Rastegar, S; Müller, F; Krone, N

2018-04-01

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co-factors during embryonic development and stress, as well as in brain homeostasis and function. © 2018 British Society for Neuroendocrinology.

Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere.

PubMed

Fullerton, Heather; Moyer, Craig L

2016-05-15

Chloroflexi small-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylum Chloroflexi is represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurface Chloroflexi Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurface Chloroflexi single amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nine Chloroflexi SAGs (seven of which are from the order Anaerolineales) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of the Chloroflexi SAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the seven Anaerolineales SAGs contains a distinct reductive dehalogenase homologous (rdhA) gene. Through the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, the Chloroflexi These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examined Chloroflexi SAGs, our genomes (several are from the order Anaerolineales) were recovered from a hydrothermally driven system and therefore provide a unique window into the metabolic potential of this type of habitat. In addition, a reductive dehalogenase gene (rdhA) has been directly linked to marine subsurface Chloroflexi, suggesting that reductive dehalogenation is not limited to the class Dehalococcoidia This discovery expands the nutrient-cycling and metabolic potential present within the deep subsurface and provides functional gene information relating to this enigmatic group. Copyright © 2016 Fullerton and Moyer.

Update on Nitazoxanide: A Multifunctional Chemotherapeutic Agent.

PubMed

Shakya, Anshul; Bhat, Hans Raj; Ghosh, Surjit Kumar

2017-07-27

The thiazolide nitazoxanide (NTZ) is a broad-spectrum anti-infective drug that adversely affects viability, growth, and proliferation of a range of extracellular and intracellular protozoan, helminths, anaerobic and microaerophilic bacteria, and viruses. Current review compiled the potential chemotherapeutic efficacy of NTZ against a variety of such disease-causing macro and/or micro-organisms as well as neoplastic cells, using various search engines viz. Web of Science, Scopus and PubMed up to February 2017. The most accepted anti-infective mechanism of NTZ involves impairment of the energy metabolism in anaerobic pathogens by inhibition of the pyruvate: ferredoxin/flavodoxin oxidoreductase (PFOR). In parasitic-protozoan NTZ also induces lesions/voids in the cell membrane and depolarises the mitochondrial membrane along with the inhibition of quinone oxidoreductase NQO1, nitroreductase-1 and protein disulphide isomerase. NTZ also inhibits the glutathione-S-transferase (a major detoxifying enzyme) and modulates a gene (avr-14 gene) encoding for the alpha-type subunit of glutamate-gated chloride ion channel present in the nematodes. Apart from well recognized non-competitive inhibition of the PFOR in anaerobic bacteria, NTZ also showed a variety of other antibacterial mechanisms viz. inhibits pyruvate dehydrogenase in the Escherichia coli, disrupts the membrane potential and pH homeostasis in the Mycobacterium tuberculosis, suppresses the chaperone/usher (CU) pathway of the gram-negative bacteria and stimulates host macrophage autophagy in the tubercular patients. NTZ also suppresses the viral replication by inhibiting maturation of the viral hemagglutinin and the viral transcription factor immediate early 2 (IE2) as well as by activating the eukaryotic translation initiation factor 2α (an antiviral intracellular protein). Additionally, NTZ expresses inhibitory effect on the tumour cell progression by modulating drug detoxification (glutathione-S-transferase P1), unfolded protein response, autophagy, anti-cytokines activities and c-Myc inhibition. These potentially versatile molecular interactions of NTZ within invading pathogen(s) and immunomodulatory efficacy over the hosts, justify the multifunctional chemotherapeutic significance of this chemical agent. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Iron catalysis at the origin of life.

PubMed

Camprubi, Eloi; Jordan, Sean F; Vasiliadou, Rafaela; Lane, Nick

2017-06-01

Iron-sulphur proteins are ancient and drive fundamental processes in cells, notably electron transfer and CO 2 fixation. Iron-sulphur minerals with equivalent structures could have played a key role in the origin of life. However, the 'iron-sulphur world' hypothesis has had a mixed reception, with questions raised especially about the feasibility of a pyrites-pulled reverse Krebs cycle. Phylogenetics suggests that the earliest cells drove carbon and energy metabolism via the acetyl CoA pathway, which is also replete in Fe(Ni)S proteins. Deep differences between bacteria and archaea in this pathway obscure the ancestral state. These differences make sense if early cells depended on natural proton gradients in alkaline hydrothermal vents. If so, the acetyl CoA pathway diverged with the origins of active ion pumping, and ancestral CO 2 fixation might have been equivalent to methanogens, which depend on a membrane-bound NiFe hydrogenase, energy converting hydrogenase. This uses the proton-motive force to reduce ferredoxin, thence CO 2 . The mechanism suggests that pH could modulate reduction potential at the active site of the enzyme, facilitating the difficult reduction of CO 2 by H 2 . This mechanism could be generalised under abiotic conditions so that steep pH differences across semi-conducting Fe(Ni)S barriers drives not just the first steps of CO 2 fixation to C1 and C2 organics such as CO, CH 3 SH and CH 3 COSH, but a series of similar carbonylation and hydrogenation reactions to form longer chain carboxylic acids such as pyruvate, oxaloacetate and α-ketoglutarate, as in the incomplete reverse Krebs cycle found in methanogens. We suggest that the closure of a complete reverse Krebs cycle, by regenerating acetyl CoA directly, displaced the acetyl CoA pathway from many modern groups. A later reliance on acetyl CoA and ATP eliminated the need for the proton-motive force to drive most steps of the reverse Krebs cycle. © 2017 IUBMB Life, 69(6):373-381, 2017. © 2017 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.

Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

PubMed Central

Yen, K M; Karl, M R; Blatt, L M; Simon, M J; Winter, R B; Fausset, P R; Lu, H S; Harcourt, A A; Chen, K K

1991-01-01

Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the offO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SacI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene. Images PMID:1885512

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum

PubMed Central

Carlson, Ellinor D.

2017-01-01

ABSTRACT With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO2 to CO and oxidize CO during growth on glucose. PMID:28625981

In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals.

PubMed

Holland, Jason P; Giansiracusa, Jeffrey H; Bell, Stephen G; Wong, Luet-Lok; Dilworth, Jonathan R

2009-04-07

The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [(60/62/64)Cu(II)ATSM] and [(60/62/64)Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO(2)-dependent in vitro cellular uptake and retention of [(64)Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k(1) = 9.8 +/- 0.59 x 10(-4) s(-1) and k(2) = 2.9 +/- 0.17 x 10(-3) s(-1)), intracellular reduction (k(3) = 5.2 +/- 0.31 x 10(-2) s(-1)), reoxidation (k(4) = 2.2 +/- 0.13 mol(-1) dm(3) s(-1)) and proton-mediated ligand dissociation (k(5) = 9.0 +/- 0.54 x 10(-5) s(-1)). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the structure of the ligand and the results confirm that the proposed reduction step in the mechanism of hypoxia selectivity is likely to be mediated by NADH-dependent enzymes. Further understanding of the mechanism of hypoxia selectivity may facilitate the development of new imaging and radiotherapeutic agents with increased specificity for tumour hypoxia.

In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals

NASA Astrophysics Data System (ADS)

Holland, Jason P.; Giansiracusa, Jeffrey H.; Bell, Stephen G.; Wong, Luet-Lok; Dilworth, Jonathan R.

2009-04-01

The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [60/62/64Cu(II)ATSM] and [60/62/64Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO2-dependent in vitro cellular uptake and retention of [64Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k1 = 9.8 ± 0.59 × 10-4 s-1 and k2 = 2.9 ± 0.17 × 10-3 s-1), intracellular reduction (k3 = 5.2 ± 0.31 × 10-2 s-1), reoxidation (k4 = 2.2 ± 0.13 mol-1 dm3 s-1) and proton-mediated ligand dissociation (k5 = 9.0 ± 0.54 × 10-5 s-1). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the structure of the ligand and the results confirm that the proposed reduction step in the mechanism of hypoxia selectivity is likely to be mediated by NADH-dependent enzymes. Further understanding of the mechanism of hypoxia selectivity may facilitate the development of new imaging and radiotherapeutic agents with increased specificity for tumour hypoxia.

Molecular and Structural Characterization of the Tegumental 20.6-kDa Protein in Clonorchis sinensis as a Potential Druggable Target.

PubMed

Kim, Yu-Jung; Yoo, Won Gi; Lee, Myoung-Ro; Kang, Jung-Mi; Na, Byoung-Kuk; Cho, Shin-Hyeong; Park, Mi-Yeoun; Ju, Jung-Won

2017-03-04

The tegument, representing the membrane-bound outer surface of platyhelminth parasites, plays an important role for the regulation of the host immune response and parasite survival. A comprehensive understanding of tegumental proteins can provide drug candidates for use against helminth-associated diseases, such as clonorchiasis caused by the liver fluke Clonorchis sinensis . However, little is known regarding the physicochemical properties of C. sinensis teguments. In this study, a novel 20.6-kDa tegumental protein of the C. sinensis adult worm (CsTegu20.6) was identified and characterized by molecular and in silico methods. The complete coding sequence of 525 bp was derived from cDNA clones and encodes a protein of 175 amino acids. Homology search using BLASTX showed CsTegu20.6 identity ranging from 29% to 39% with previously-known tegumental proteins in C. sinensis . Domain analysis indicated the presence of a calcium-binding EF-hand domain containing a basic helix-loop-helix structure and a dynein light chain domain exhibiting a ferredoxin fold. We used a modified method to obtain the accurate tertiary structure of the CsTegu20.6 protein because of the unavailability of appropriate templates. The CsTegu20.6 protein sequence was split into two domains based on the disordered region, and then, the structure of each domain was modeled using I-TASSER. A final full-length structure was obtained by combining two structures and refining the whole structure. A refined CsTegu20.6 structure was used to identify a potential CsTegu20.6 inhibitor based on protein structure-compound interaction analysis. The recombinant proteins were expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity chromatography. In C. sinensis , CsTegu20.6 mRNAs were abundant in adult and metacercariae, but not in the egg. Immunohistochemistry revealed that CsTegu20.6 localized to the surface of the tegument in the adult fluke. Collectively, our results contribute to a better understanding of the structural and functional characteristics of CsTegu20.6 and homologs of flukes. One compound is proposed as a putative inhibitor of CsTegu20.6 to facilitate further studies for anthelmintics.

Electron transfer to nitrogenase in different genomic and metabolic backgrounds.

PubMed

Poudel, Saroj; Colman, Daniel R; Fixen, Kathryn R; Ledbetter, Rhesa N; Zheng, Yanning; Pence, Natasha; Seefeldt, Lance C; Peters, John W; Harwood, Caroline S; Boyd, Eric S

2018-02-26

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) using low potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O 2 ) sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd/Fld-reducing enzymes in 359 genomes of putative N 2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified and their distributions largely corresponded to differences in the host cells' ability to integrate O 2 or light into energy metabolism. Predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the level of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N 2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation. IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O 2 or light into their energy metabolism. Acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex. Copyright © 2018 American Society for Microbiology.

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum.

PubMed

Carlson, Ellinor D; Papoutsakis, Eleftherios T

2017-08-15

With recent advances in synthetic biology, CO 2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO 2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO 2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO 2 , and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO 2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum , which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO 2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO 2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO 2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum , which is natively incapable of CO 2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO 2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13 C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO 2 to CO and oxidize CO during growth on glucose. Copyright © 2017 American Society for Microbiology.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism.

PubMed

Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

A proteomic approach to the analysis of the components of the phycobilisomes from two cyanobacteria with complementary chromatic adaptation: Fremyella diplosiphon UTEX B590 and Tolypothrix PCC 7601.

PubMed

Pérez-Gómez, Bertha; Mendoza-Hernández, Guillermo; Cabellos-Avelar, Tecilli; Leyva-Castillo, Lourdes Elizabeth; Gutiérrez-Cirlos, Emma Berta; Gómez-Lojero, Carlos

2012-10-01

Tolypothrix PCC 7601 and Fremyella diplosiphon UTEX B590 can produce two alternative phycobilisome (PBS) rods. PE-PBSs with one phycocyanin (PC) disk and multiple phycoerythrin (PE) disks are found in cells grown under green light (GL). PC-PBSs with only PC disks are obtained from cells grown under red light (RL). In this manuscript, we show the localization of the linker proteins and ferredoxin-NADP(+) oxidoreductase (FNR) in the PC-PBS and of PE-PBS rods using visible spectroscopy and mass spectrometry. PE-PBSs with different [PE]/[PC] ratios and PC-PBSs with different [PC]/[AP] (AP, allophycocyanin) ratios were isolated. CpeC was the primary rod linker protein found in the PBSs with a [PE]/[PC] ratio of 1.1, which indicates that this is the rod linker at the interphase PC-PE. CpeC and CpeD were identified in the PBSs with a [PE]/[PC] ratio of 1.6, which indicates that CpcD is the linker between the first and the second PE hexamers. Finally, CpeC, CpeD, and CpeE were found in the PBSs with a [PE]/[PC] ratio of 2.9, indicating the position of CpeE between the second and third PE moieties. CpcI2 was identified in the two PC-PBSs obtained from cells grown under RL, which indicates that CpcI2 is the linker between the first and second PC hexamers. CpcH2 was identified only in the PC-PBSs from Tolypothrix with a high [PC]/[AP] ratio of 1.92, which indicates that CpcH2 is the linker between the second and third PC hexamers. The PC-PBSs contained the rod cap protein L(R)(10) (CpcD), but this protein was absent in the PE-PBSs. PE-PBSs (lacking L(R)(10)) incorporated exogenous rFNR in a stoichiometry of up to five FNRs per PBS. A maximum of two FNRs per PBS were found in PC-PBSs (with L(R)(10)). These observations support the hypothesis that FNR binds at the distal ends of the PBS rods in the vacant site of CpcD L(R)(10). Finally, the molecular mass of the core membrane linker (L(CM)) was determined to be 102 kDa from a mass spectrometry analysis.

Metatranscriptomic Analysis Reveals Unexpectedly Diverse Microbial Metabolism in a Biogeochemical Hot Spot in an Alluvial Aquifer

DOE PAGES

Jewell, Talia N. M.; Karaoz, Ulas; Bill, Markus; ...

2017-01-25

Organic matter deposits in alluvial aquifers have been shown to result in the formation of naturally reduced zones (NRZs), which can modulate aquifer redox status and influence the speciation and mobility of metals, affecting groundwater geochemistry. In this study, we sought to better understand how natural organic matter fuels microbial communities within anoxic biogeochemical hot spots (NRZs) in a shallow alluvial aquifer at the Rifle (CO) site. We conducted a 20-day microcosm experiment in which NRZ sediments, which were enriched in buried woody plant material, served as the sole source of electron donors and microorganisms. The microcosms were constructed andmore » incubated under anaerobic conditions in serum bottles with an initial N 2 headspace and were sampled every 5 days for metagenome and metatranscriptome profiles in combination with biogeochemical measurements. Biogeochemical data indicated that the decomposition of native organic matter occurred in different phases, beginning with mineralization of dissolved organic matter (DOM) to CO 2 during the first week of incubation, followed by a pulse of acetogenesis that dominated carbon flux after 2 weeks. A pulse of methanogenesis co-occurred with acetogenesis, but only accounted for a small fraction of carbon flux. The depletion of DOM over time was strongly correlated with increases in expression of many genes associated with heterotrophy (e.g., amino acid, fatty acid, and carbohydrate metabolism) belonging to a Hydrogenophaga strain that accounted for a relatively large percentage (~8%) of the metatranscriptome. This Hydrogenophaga strain also expressed genes indicative of chemolithoautotrophy, including CO 2 fixation, H 2 oxidation, S-compound oxidation, and denitrification. The pulse of acetogenesis appears to have been collectively catalyzed by a number of different organisms and metabolisms, most prominently pyruvate:ferredoxin oxidoreductase. Unexpected genes were identified among the most highly expressed (>98th percentile) transcripts, including acetone carboxylase and cell-wall-associated hydrolases with unknown substrates (numerous lesser expressed cell-wall-associated hydrolases targeted peptidoglycan). Many of the most highly expressed hydrolases belonged to a Ca. Bathyarchaeota strain and may have been associated with recycling of bacterial biomass. Overall, these results highlight the complex nature of organic matter transformation in NRZs and the microbial metabolic pathways that interact to mediate redox status and elemental cycling.« less

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

PubMed

Clark, Melinda E; He, Zhili; Redding, Alyssa M; Joachimiak, Marcin P; Keasling, Jay D; Zhou, Jizhong Z; Arkin, Adam P; Mukhopadhyay, Aindrila; Fields, Matthew W

2012-04-16

Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

PubMed Central

Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle. PMID:29491838

Metatranscriptomic Analysis Reveals Unexpectedly Diverse Microbial Metabolism in a Biogeochemical Hot Spot in an Alluvial Aquifer

PubMed Central

Jewell, Talia N. M.; Karaoz, Ulas; Bill, Markus; Chakraborty, Romy; Brodie, Eoin L.; Williams, Kenneth H.; Beller, Harry R.

2017-01-01

Organic matter deposits in alluvial aquifers have been shown to result in the formation of naturally reduced zones (NRZs), which can modulate aquifer redox status and influence the speciation and mobility of metals, affecting groundwater geochemistry. In this study, we sought to better understand how natural organic matter fuels microbial communities within anoxic biogeochemical hot spots (NRZs) in a shallow alluvial aquifer at the Rifle (CO) site. We conducted a 20-day microcosm experiment in which NRZ sediments, which were enriched in buried woody plant material, served as the sole source of electron donors and microorganisms. The microcosms were constructed and incubated under anaerobic conditions in serum bottles with an initial N2 headspace and were sampled every 5 days for metagenome and metatranscriptome profiles in combination with biogeochemical measurements. Biogeochemical data indicated that the decomposition of native organic matter occurred in different phases, beginning with mineralization of dissolved organic matter (DOM) to CO2 during the first week of incubation, followed by a pulse of acetogenesis that dominated carbon flux after 2 weeks. A pulse of methanogenesis co-occurred with acetogenesis, but only accounted for a small fraction of carbon flux. The depletion of DOM over time was strongly correlated with increases in expression of many genes associated with heterotrophy (e.g., amino acid, fatty acid, and carbohydrate metabolism) belonging to a Hydrogenophaga strain that accounted for a relatively large percentage (~8%) of the metatranscriptome. This Hydrogenophaga strain also expressed genes indicative of chemolithoautotrophy, including CO2 fixation, H2 oxidation, S-compound oxidation, and denitrification. The pulse of acetogenesis appears to have been collectively catalyzed by a number of different organisms and metabolisms, most prominently pyruvate:ferredoxin oxidoreductase. Unexpected genes were identified among the most highly expressed (>98th percentile) transcripts, including acetone carboxylase and cell-wall-associated hydrolases with unknown substrates (numerous lesser expressed cell-wall-associated hydrolases targeted peptidoglycan). Many of the most highly expressed hydrolases belonged to a Ca. Bathyarchaeota strain and may have been associated with recycling of bacterial biomass. Overall, these results highlight the complex nature of organic matter transformation in NRZs and the microbial metabolic pathways that interact to mediate redox status and elemental cycling. PMID:28179898

Separate Fe-S Scaffold And Carrier Functions For SufB2C2 And SufA During In Vitro Maturation Of [2Fe-2S] Fdx

PubMed Central

Chahal, Harsimranjit K.; Outten, F. Wayne

2012-01-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of Fe-S clusters proceeds via complex pathways involving multiple protein complexes. In the Suf Fe-S cluster biogenesis system, SufB may be a scaffold for nascent Fe-S cluster assembly whereas SufA is proposed to act as either a scaffold or an Fe-S cluster carrier from the scaffold to target apo-proteins. However, SufB can form multiple stable complexes with other Suf proteins, such as SufB2C2 and SufBC2D and the specific functions of these complexes in Fe-S cluster assembly are not clear. Here we compare the ability of the SufB2C2 and SufBC2D complexes as well as SufA to promote in vitro maturation of the [2Fe-2S] ferredoxin (Fdx). We found that SufB2C2 was most proficient as a scaffold for de novo assembly of holo-Fdx using sulfide and iron as freely available building blocks while SufA was best at direct transfer of a pre-formed Fe-S cluster to Fdx. Furthermore, cluster transfer from [4Fe-4S] SufB2C2 or SufBC2D to Fdx will proceed through a SufA intermediate to Fdx is SufA is present. Finally, addition of ATP repressed cluster transfer from [4Fe-4S] SufB2C2 to Fdx and from SufBC2D to [2Fe-2S] SufA or Fdx. These studies indicate that SufB2C2 can serve as a terminal scaffold to load the SufA Fe-S cluster carrier for in vitro maturation of [2Fe-2S] enzymes like Fdx. This work is the first to systematically compare the cluster transfer rates of a scaffold (SufB) to the transfer rates of a carrier (SufA) under the same conditions to the same target enzyme and is also the first to reconstitute the full transfer pathway (from scaffold to carrier to target enzyme) in a single reaction. PMID:23018275

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: Carbon and energy flow contribute to the distinct biofilm growth state

PubMed Central

2012-01-01

Background Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. Results The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Conclusions Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion. PMID:22507456

Metatranscriptomic Analysis Reveals Unexpectedly Diverse Microbial Metabolism in a Biogeochemical Hot Spot in an Alluvial Aquifer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jewell, Talia N. M.; Karaoz, Ulas; Bill, Markus

Organic matter deposits in alluvial aquifers have been shown to result in the formation of naturally reduced zones (NRZs), which can modulate aquifer redox status and influence the speciation and mobility of metals, affecting groundwater geochemistry. In this study, we sought to better understand how natural organic matter fuels microbial communities within anoxic biogeochemical hot spots (NRZs) in a shallow alluvial aquifer at the Rifle (CO) site. We conducted a 20-day microcosm experiment in which NRZ sediments, which were enriched in buried woody plant material, served as the sole source of electron donors and microorganisms. The microcosms were constructed andmore » incubated under anaerobic conditions in serum bottles with an initial N 2 headspace and were sampled every 5 days for metagenome and metatranscriptome profiles in combination with biogeochemical measurements. Biogeochemical data indicated that the decomposition of native organic matter occurred in different phases, beginning with mineralization of dissolved organic matter (DOM) to CO 2 during the first week of incubation, followed by a pulse of acetogenesis that dominated carbon flux after 2 weeks. A pulse of methanogenesis co-occurred with acetogenesis, but only accounted for a small fraction of carbon flux. The depletion of DOM over time was strongly correlated with increases in expression of many genes associated with heterotrophy (e.g., amino acid, fatty acid, and carbohydrate metabolism) belonging to a Hydrogenophaga strain that accounted for a relatively large percentage (~8%) of the metatranscriptome. This Hydrogenophaga strain also expressed genes indicative of chemolithoautotrophy, including CO 2 fixation, H 2 oxidation, S-compound oxidation, and denitrification. The pulse of acetogenesis appears to have been collectively catalyzed by a number of different organisms and metabolisms, most prominently pyruvate:ferredoxin oxidoreductase. Unexpected genes were identified among the most highly expressed (>98th percentile) transcripts, including acetone carboxylase and cell-wall-associated hydrolases with unknown substrates (numerous lesser expressed cell-wall-associated hydrolases targeted peptidoglycan). Many of the most highly expressed hydrolases belonged to a Ca. Bathyarchaeota strain and may have been associated with recycling of bacterial biomass. Overall, these results highlight the complex nature of organic matter transformation in NRZs and the microbial metabolic pathways that interact to mediate redox status and elemental cycling.« less

Transformation of dinitrosyl iron complexes [(NO)2Fe(SR)2]- (R = Et, Ph) into [4Fe-4S] Clusters [Fe4S4(SPh)4]2-: relevance to the repair of the nitric oxide-modified ferredoxin [4Fe-4S] clusters.

PubMed

Tsou, Chih-Chin; Lin, Zong-Sian; Lu, Tsai-Te; Liaw, Wen-Feng

2008-12-17

Transformation of dinitrosyl iron complexes (DNICs) [(NO)(2)Fe(SR)(2)](-) (R = Et, Ph) into [4Fe-4S] clusters [Fe(4)S(4)(SPh)(4)](2-) in the presence of [Fe(SPh)(4)](2-/1-) and S-donor species S(8) via the reassembling process ([(NO)(2)Fe(SR)(2)](-) --> [Fe(4)S(3)(NO)(7)](-) (1)/[Fe(4)S(3)(NO)(7)](2-) (2) --> [Fe(4)S(4)(NO)(4)](2-) (3) --> [Fe(4)S(4)(SPh)(4)](2-) (5)) was demonstrated. Reaction of [(NO)(2)Fe(SR)(2)](-) (R = Et, Ph) with S(8) in THF, followed by the addition of HBF(4) into the mixture solution, yielded complex [Fe(4)S(3)(NO)(7)](-) (1). Complex [Fe(4)S(3)(NO)(7)](2-) (2), obtained from reduction of complex 1 by [Na][biphenyl], was converted into complex [Fe(4)S(4)(NO)(4)](2-) (3) along with byproduct [(NO)(2)Fe(SR)(2)](-) via the proposed [Fe(4)S(3)(SPh)(NO)(4)](2-) intermediate upon treating complex 2 with 1.5 equiv of [Fe(SPh)(4)](2-) and the subsequent addition of 1/8 equiv of S(8) in CH(3)CN at ambient temperature. Complex 3 was characterized by IR, UV-vis, and single-crystal X-ray diffraction. Upon addition of complex 3 to the CH(3)CN solution of [Fe(SPh)(4)](-) in a 1:2 molar ratio at ambient temperature, the rapid NO radical-thiyl radical exchange reaction between complex 3 and the biomimetic oxidized form of rubredoxin [Fe(SPh)(4)](-) occurred, leading to the simultaneous formation of [4Fe-4S] cluster [Fe(4)S(4)(SPh)(4)](2-) (5) and DNIC [(NO)(2)Fe(SPh)(2)](-). This result demonstrates a successful biomimetic reassembly of [4Fe-4S] cluster [Fe(4)S(4)(SPh)(4)](2-) from NO-modified [Fe-S] clusters, relevant to the repair of DNICs derived from nitrosylation of [4Fe-4S] clusters of endonuclease III back to [4Fe-4S] clusters upon addition of ferrous ion, cysteine, and IscS.

Dynamics of an [Fe4S4(SPh)4]2- cluster explored via IR, Raman, and nuclear resonance vibrational spectroscopy (NRVS)-analysis using 36S substitution, DFT calculations, and empirical force fields.

PubMed

Xiao, Yuming; Koutmos, Markos; Case, David A; Coucouvanis, Dimitri; Wang, Hongxin; Cramer, Stephen P

2006-05-14

We have used four vibrational spectroscopies--FT-IR, FT-Raman, resonance Raman, and 57Fe nuclear resonance vibrational spectroscopy (NRVS)--to study the normal modes of the Fe-S cluster in [(n-Bu)4N]2[Fe4S4(SPh)4]. This [Fe4S4(SR)4]2- complex serves as a model for the clusters in 4Fe ferredoxins and high-potential iron proteins (HiPIPs). The IR spectra exhibited differences above and below the 243 K phase transition. Significant shifts with 36S substitution into the bridging S positions were also observed. The NRVS results were in good agreement with the low temperature data from the conventional spectroscopies. The NRVS spectra were interpreted by normal mode analysis using optimized Urey-Bradley force fields (UBFF) as well as from DFT theory. For the UBFF calculations, the parameters were refined by comparing calculated and observed NRVS frequencies and intensities. The frequency shifts after 36S substitution were used as an additional constraint. A D 2d symmetry Fe4S4S'4 model could explain most of the observed frequencies, but a better match to the observed intensities was obtained when the ligand aromatic rings were included for a D 2d Fe4S4(SPh)4 model. The best results were obtained using the low temperature structure without symmetry constraints. In addition to stretching and bending vibrations, low frequency modes between approximately 50 and 100 cm(-1) were observed. These modes, which have not been seen before, are interpreted as twisting motions with opposing sides of the cube rotating in opposite directions. In contrast with a recent paper on a related Fe4S4 cluster, we find no need to assign a large fraction of the low frequency NRVS intensity to 'rotational lattice modes'. We also reassign the 430 cm(-1) band as primarily an elongation of the thiophenolate ring, with approximately 10% terminal Fe-S stretch character. This study illustrates the benefits of combining NRVS with conventional Raman and IR analysis for characterization of Fe-S centers. DFT theory is shown to provide remarkable agreement with the experimental NRVS data. These results provide a reference point for the analysis of more complex Fe-S clusters in proteins.

Modular Homogeneous Chromophore–Catalyst Assemblies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mulfort, Karen L.; Utschig, Lisa M.

2016-05-17

Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate thatmore » molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule–nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.« less

The alkaline solution to the emergence of life: energy, entropy and early evolution.

PubMed

Russell, Michael J

2007-01-01

The Earth agglomerates and heats. Convection cells within the planetary interior expedite the cooling process. Volcanoes evolve steam, carbon dioxide, sulfur dioxide and pyrophosphate. An acidulous Hadean ocean condenses from the carbon dioxide atmosphere. Dusts and stratospheric sulfurous smogs absorb a proportion of the Sun's rays. The cooled ocean leaks into the stressed crust and also convects. High temperature acid springs, coupled to magmatic plumes and spreading centers, emit iron, manganese, zinc, cobalt and nickel ions to the ocean. Away from the spreading centers cooler alkaline spring waters emanate from the ocean floor. These bear hydrogen, formate, ammonia, hydrosulfide and minor methane thiol. The thermal potential begins to be dissipated but the chemical potential is dammed. The exhaling alkaline solutions are frustrated in their further attempt to mix thoroughly with their oceanic source by the spontaneous precipitation of biomorphic barriers of colloidal iron compounds and other minerals. It is here we surmise that organic molecules are synthesized, filtered, concentrated and adsorbed, while acetate and methane--separate products of the precursor to the reductive acetyl-coenzyme-A pathway-are exhaled as waste. Reactions in mineral compartments produce acetate, amino acids, and the components of nucleosides. Short peptides, condensed from the simple amino acids, sequester 'ready-made' iron sulfide clusters to form protoferredoxins, and also bind phosphates. Nucleotides are assembled from amino acids, simple phosphates carbon dioxide and ribose phosphate upon nanocrystalline mineral surfaces. The side chains of particular amino acids register to fitting nucleotide triplet clefts. Keyed in, the amino acids are polymerized, through acid-base catalysis, to alpha chains. Peptides, the tenuous outer-most filaments of the nanocrysts, continually peel away from bound RNA. The polymers are concentrated at cooler regions of the mineral compartments through thermophoresis. RNA is reproduced through a convective polymerase chain reaction operating between 40 and 100 degrees C. The coded peptides produce true ferredoxins, the ubiquitous proteins with the longest evolutionary pedigree. They take over the role of catalyst and electron transfer agent from the iron sulfides. Other iron-nickel sulfide clusters, sequestered now by cysteine residues as CO-dehydrogenase and acetyl-coenzyme-A synthase, promote further chemosynthesis and support the hatchery--the electrochemical reactor--from which they sprang. Reactions and interactions fall into step as further pathways are negotiated. This hydrothermal circuitry offers a continuous supply of material and chemical energy, as well as electricity and proticity at a potential appropriate for the onset of life in the dark, a rapidly emerging kinetic structure born to persist, evolve and generate entropy while the sun shines.

Cloning, Expression, and Nucleotide Sequence of the Pseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates

PubMed Central

Tsoi, Tamara V.; Plotnikova, Elena G.; Cole, James R.; Guerin, William F.; Bagdasarian, Michael; Tiedje, James M.

1999-01-01

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997. PMID:10224014