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Sample records for fefe hydrogenase model

  1. Discovery of External Modulators of the Fe-Fe Hydrogenase Enzyme in Clostridium acetobutylicum

    DTIC Science & Technology

    2015-02-01

    ARL-TR-7189 ● FEB 2015 US Army Research Laboratory Discovery of External Modulators of the Fe-Fe Hydrogenase...ARL-TR-7189 ● FEB 2015 US Army Research Laboratory Discovery of External Modulators of the Fe-Fe Hydrogenase Enzyme in Clostridium...October 2014 4. TITLE AND SUBTITLE Discovery of External Modulators of the Fe-Fe Hydrogenase Enzyme in Clostridium acetobutylicum 5a. CONTRACT

  2. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases.

    PubMed

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-10-27

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

  3. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

    PubMed Central

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-01-01

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases. PMID:27786270

  4. Electronic structure of an [FeFe] hydrogenase model complex in solution revealed by X-ray absorption spectroscopy using narrow-band emission detection.

    PubMed

    Leidel, Nils; Chernev, Petko; Havelius, Kajsa G V; Schwartz, Lennart; Ott, Sascha; Haumann, Michael

    2012-08-29

    High-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection, supported by density functional theory calculations (XAES-DFT), was used to study a model complex, ([Fe(2)(μ-adt)(CO)(4)(PMe(3))(2)] (1, adt = S-CH(2)-(NCH(2)Ph)-CH(2)-S), of the [FeFe] hydrogenase active site. For 1 in powder material (1(powder)), in MeCN solution (1'), and in its three protonated states (1H, 1Hy, 1HHy; H denotes protonation at the adt-N and Hy protonation of the Fe-Fe bond to form a bridging metal hydride), relations between the molecular structures and the electronic configurations were determined. EXAFS analysis and DFT geometry optimization suggested prevailing rotational isomers in MeCN, which were similar to the crystal structure or exhibited rotation of the (CO) ligands at Fe1 (1(CO), 1Hy(CO)) and in addition of the phenyl ring (1H(CO,Ph), 1HHy(CO,Ph)), leading to an elongated solvent-exposed Fe-Fe bond. Isomer formation, adt-N protonation, and hydride binding caused spectral changes of core-to-valence (pre-edge of the Fe K-shell absorption) and of valence-to-core (Kß(2,5) emission) electronic transitions, and of Kα RIXS data, which were quantitatively reproduced by DFT. The study reveals (1) the composition of molecular orbitals, for example, with dominant Fe-d character, showing variations in symmetry and apparent oxidation state at the two Fe ions and a drop in MO energies by ~1 eV upon each protonation step, (2) the HOMO-LUMO energy gaps, of ~2.3 eV for 1(powder) and ~2.0 eV for 1', and (3) the splitting between iron d(z(2)) and d(x(2)-y(2)) levels of ~0.5 eV for the nonhydride and ~0.9 eV for the hydride states. Good correlations of reduction potentials to LUMO energies and oxidation potentials to HOMO energies were obtained. Two routes of facilitated bridging hydride binding thereby are suggested, involving ligand rotation at Fe1 for 1Hy(CO) or adt-N protonation for 1HHy(CO,Ph). XAES-DFT thus enables verification of the effects of ligand

  5. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    DOEpatents

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  6. A Cell-Free Microtiter Plate Screen for Improved [FeFe] Hydrogenases

    PubMed Central

    Stapleton, James A.; Swartz, James R.

    2010-01-01

    Background [FeFe] hydrogenase enzymes catalyze the production and dissociation of H2, a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms. Methodology/Principal Findings Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity ∼4 times that of the wild-type enzyme. Conclusions/Significance Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment. PMID:20479937

  7. The quest for a functional substrate access tunnel in FeFe hydrogenase.

    PubMed

    Lautier, Thomas; Ezanno, Pierre; Baffert, Carole; Fourmond, Vincent; Cournac, Laurent; Fontecilla-Camps, Juan C; Soucaille, Philippe; Bertrand, Patrick; Meynial-Salles, Isabelle; Léger, Christophe

    2011-01-01

    We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative "static" H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme's kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.

  8. [FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation.

    PubMed

    Peters, John W; Schut, Gerrit J; Boyd, Eric S; Mulder, David W; Shepard, Eric M; Broderick, Joan B; King, Paul W; Adams, Michael W W

    2015-06-01

    The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  9. Mechanism of O2 diffusion and reduction in FeFe hydrogenases

    NASA Astrophysics Data System (ADS)

    Kubas, Adam; Orain, Christophe; de Sancho, David; Saujet, Laure; Sensi, Matteo; Gauquelin, Charles; Meynial-Salles, Isabelle; Soucaille, Philippe; Bottin, Hervé; Baffert, Carole; Fourmond, Vincent; Best, Robert B.; Blumberger, Jochen; Léger, Christophe

    2017-01-01

    FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

  10. Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

    PubMed Central

    Stapleton, James A.; Swartz, James R.

    2010-01-01

    Background [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. Methodology/Principal Findings Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. Conclusions/Significance [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy. PMID:21151915

  11. Di/mono-nuclear iron(I)/(II) complexes as functional models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases: protonations, molecular structures and electrochemical properties.

    PubMed

    Gao, Shang; Fan, Jiangli; Sun, Shiguo; Song, Fengling; Peng, Xiaojun; Duan, Qian; Jiang, Dayong; Liang, Qingcheng

    2012-10-21

    Di/mono-nuclear iron(I)/(II) complexes containing conjugated and electron-withdrawing S-to-S linkers, [{(μ-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(6)] (1), [{(μ-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(5)(PMe(3))] (1P), and [{(μ-S)(2)(C(4)N(2)H(2))}Fe(CO)(2)(PMe(3))(2)] (2) were prepared as biomimetic models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases. The N atoms in the heterocyclic pyrazines of 1 and 2 were protonated in the presence of proton acid to generate one and two hydrides, [1(NH)](+) CF(3)SO(3)(-), [2(NH)](+) CF(3)SO(3)(-), and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2), respectively. The protonation processes were evidenced by in situ IR and NMR spectroscopy. The molecular structures of the protonated species [1(NH)](+) CF(3)SO(3)(-) and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2) together with their originating complexes and , and the mono-PMe(3) substituted diiron complex were identified by X-ray crystallography. The IR and single-crystal analysis data all suggested that the electron-withdrawing bridge, pyrazine, led to decreased electron density at the Fe centers of the model complexes, which was consistent with the electrochemical studies. The cyclic voltammograms indicated that complex exhibited a low primary reduction potential at -1.17 V vs. Fc-Fc(+) with a 270 mV positive shift compared with that of the benzene-1,2-dithiolate (bdt) bridged analogue [(μ-bdt)Fe(2)(CO)(6)]. Under the weak acid conditions, complexes 1 and 2 could electrochemically catalyze the proton reduction. More interestingly, the mononuclear ferrous complex 2 showed two catalytic peaks during the formation of hydrogen, confirming its potential as a catalyst for hydrogen production.

  12. A diferrous dithiolate as a model of the elusive H(ox)(inact) state of the [FeFe] hydrogenases: an electrochemical and theoretical dissection of its redox chemistry.

    PubMed

    Chouffai, Dounia; Capon, Jean-François; De Gioia, Luca; Pétillon, François Y; Schollhammer, Philippe; Talarmin, Jean; Zampella, Giuseppe

    2015-01-05

    The reduction of the Fe(II)Fe(II) complex [Fe2(CO)2{P(OMe)3}2(κ(2)-IMe-CH2-IMe)(μ-CO)(μ-pdt)](2+) (2P(2+); pdt = S(CH2)3S), which is a synthetic model of the H cluster of the [FeFe] hydrogenases in its inactive state, has been investigated electrochemically and theoretically (by density functional theory, DFT) in order to determine the mechanisms, intermediates, and products of the related processes. The electrochemical reduction of 2P(2+) occurs according to an ECE-type reaction where the intervening chemical step is the loss of one P(OMe)3 ligand. This outcome, which is based on cyclic voltammetric experiments, is strongly supported by DFT calculations that provide additional information on the intermediates and the energetics of the reactions involved. The electrochemical reoxidation of the neutral product of the reduction follows an EEC process where the chemical step is the binding of P(OMe)3 to a dicationic intermediate. DFT calculations reveal that this intermediate has an unusual geometry wherein one of the two C-H bonds of a side methylene from the pdt group forms an agostic interaction with one Fe center. This interaction is crucial to stabilize the 32e(-) diferrous center and concomitantly to preserve Fe(II) from binding of weakly coordinating species. Nonetheless, it could be displaced by a relatively stronger electron donor such as H2, which could be relevant for the design of new oxidation catalysts.

  13. Genomic analysis reveals multiple [FeFe] hydrogenases and hydrogen sensors encoded by treponemes from the H(2)-rich termite gut.

    PubMed

    Ballor, Nicholas R; Paulsen, Ian; Leadbetter, Jared R

    2012-02-01

    We have completed a bioinformatic analysis of the hydrogenases encoded in the genomes of three termite gut treponeme isolates: hydrogenotrophic, homoacetogenic Treponema primitia strains ZAS-1 and ZAS-2, and the hydrogen-producing, sugar-fermenting Treponema azotonutricium ZAS-9. H(2) is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The spirochetes encoded 4, 8, and 5 [FeFe] hydrogenase-like proteins, identified by their H domains, respectively, but no other recognizable hydrogenases. The [FeFe] hydrogenases represented many sequence families previously proposed in an analysis of termite gut metagenomic data. Each strain encoded both putative [FeFe] hydrogenase enzymes and evolutionarily related hydrogen sensor/transducer proteins likely involved in phosphorelay or methylation pathways, and possibly even chemotaxis. A new family of [FeFe] hydrogenases (FDH-Linked) is proposed that may form a multimeric complex with formate dehydrogenase to provide reducing equivalents for reductive acetogenesis in T. primitia. The many and diverse [FeFe] hydrogenase-like proteins encoded within the sequenced genomes of the termite gut treponemes has enabled the discovery of a putative new class of [FeFe] hydrogenase proteins potentially involved in acetogenesis and furthered present understanding of many families, including sensory, of H domain proteins beyond what was possible through the use of fragmentary termite gut metagenome sequence data alone, from which they were initially defined.

  14. Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster.

    PubMed

    Suess, Daniel L M; Bürstel, Ingmar; De La Paz, Liliana; Kuchenreuther, Jon M; Pham, Cindy C; Cramer, Stephen P; Swartz, James R; Britt, R David

    2015-09-15

    Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.

  15. Structural Characterization of the Novel and Thermal Stable Hydrogenases from the Purple Sulfur Bacteria Thiocapsa Roseopersicina and Lamprobacter Modestohalophilus

    DTIC Science & Technology

    2011-08-01

    fall into three main classes: [NiFe]-, [ FeFe ] and [Fe]- hydrogenases. [NiFe] – hydrogenases are represented in details as the most numerous class...of the effects of coupling model light harvesting complexes to both [NiFe]- and [ FeFe ]-hydrogenases that are the targets of our studies and in...Technical Report Page 20 / 20 characterization are the 1) [ FeFe ]-hydrogenase from Chlamydomonous reinhardtii and 2) the structural characterization of

  16. H₂-dependent azoreduction by Shewanella oneidensis MR-1: involvement of secreted flavins and both [Ni-Fe] and [Fe-Fe] hydrogenases.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Lorquin, Jean; Brugna, Myriam; Rousset, Marc

    2014-03-01

    In this paper, the hydrogen (H2)-dependent discoloration of azo dye amaranth by Shewanella oneidensis MR-1 was investigated. Experiments with hydrogenase-deficient strains demonstrated that periplasmic [Ni-Fe] hydrogenase (HyaB) and periplasmic [Fe-Fe] hydrogenase (HydA) are both respiratory hydrogenases of dissimilatory azoreduction in S. oneidensis MR-1. These findings suggest that HyaB and HydA can function as uptake hydrogenases that couple the oxidation of H2 to the reduction of amaranth to sustain cellular growth. This constitutes to our knowledge the first report of the involvement of [Fe-Fe] hydrogenase in a bacterial azoreduction process. Assays with respiratory inhibitors indicated that a menaquinone pool and different cytochromes were involved in the azoreduction process. High-performance liquid chromatography analysis revealed that flavin mononucleotide and riboflavin were secreted in culture supernatant by S. oneidensis MR-1 under H2-dependent conditions with concentration of 1.4 and 2.4 μmol g protein(-1), respectively. These endogenous flavins were shown to significantly accelerate the reduction of amaranth at micromolar concentrations acting as electron shuttles between the cell surface and the extracellular azo dye. This work may facilitate a better understanding of the mechanisms of azoreduction by S. oneidensis MR-1 and may have practical applications for microbiological treatments of dye-polluted industrial effluents.

  17. Biosynthesis of the [FeFe] Hydrogenase H Cluster: A Central Role for the Radical SAM Enzyme HydG.

    PubMed

    Suess, Daniel L M; Kuchenreuther, Jon M; De La Paz, Liliana; Swartz, James R; Britt, R David

    2016-01-19

    Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes-HydE, HydF, and HydG-in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe(2+), l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)](-) intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)](-) species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe(2+), Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN(-), and the identification of the final organometallic products of the reaction.

  18. Photo-induced hydrogen production in a helical peptide incorporating a [FeFe] hydrogenase active site mimic.

    PubMed

    Roy, Anindya; Madden, Christopher; Ghirlanda, Giovanna

    2012-10-11

    There is growing interest in the development of hydrogenase mimics for solar fuel production. Here, we present a bioinspired mimic designed by anchoring a diiron hexacarbonyl cluster to a model helical peptide via an artificial dithiol amino acid. The [FeFe]-peptide complex catalyses photo-induced production of hydrogen in water.

  19. The Cyanide Ligands of [FeFe] Hydrogenase: Pulse EPR Studies of 13C and 15N-Labeled H-Cluster

    PubMed Central

    2015-01-01

    The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN– ligands—a reporter of spin density on their respective coordinating iron ions—collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine. PMID:25133957

  20. Hydrogenase Enzymes and Their Synthetic Models: The Role of Metal Hydrides.

    PubMed

    Schilter, David; Camara, James M; Huynh, Mioy T; Hammes-Schiffer, Sharon; Rauchfuss, Thomas B

    2016-08-10

    Hydrogenase enzymes efficiently process H2 and protons at organometallic FeFe, NiFe, or Fe active sites. Synthetic modeling of the many H2ase states has provided insight into H2ase structure and mechanism, as well as afforded catalysts for the H2 energy vector. Particularly important are hydride-bearing states, with synthetic hydride analogues now known for each hydrogenase class. These hydrides are typically prepared by protonation of low-valent cores. Examples of FeFe and NiFe hydrides derived from H2 have also been prepared. Such chemistry is more developed than mimicry of the redox-inactive monoFe enzyme, although functional models of the latter are now emerging. Advances in physical and theoretical characterization of H2ase enzymes and synthetic models have proven key to the study of hydrides in particular, and will guide modeling efforts toward more robust and active species optimized for practical applications.

  1. Non-innocent bma ligand in a dissymetrically disubstituted diiron dithiolate related to the active site of the [FeFe] hydrogenases.

    PubMed

    Si, Youtao; Charreteur, Kévin; Capon, Jean-François; Gloaguen, Frederic; Pétillon, François Y; Schollhammer, Philippe; Talarmin, Jean

    2010-10-01

    The purpose of the present study was to evaluate the use of a non-innocent ligand as a surrogate of the anchored [4Fe4S] cubane in a synthetic mimic of the [FeFe] hydrogenase active site. Reaction of 2,3-bis(diphenylphosphino) maleic anhydride (bma) with [Fe(2)(CO)(6)(mu-pdt)] (propanedithiolate, pdt=S(CH(2))(3)S) in the presence of Me(3)NO-2H(2)O afforded the monosubstituted derivative [Fe(2)(CO)(5)(Me(2)NCH(2)PPh(2))(mu-pdt)] (1). This results from the decomposition of the bma ligand and the apparent C-H bond cleavage in the released trimethylamine. Reaction under photolytic conditions afforded [Fe(2)(CO)(4)(bma)(mu-pdt)] (2). Compounds 1 and 2 were characterized by IR, NMR and X-ray diffraction. Voltammetric study indicated that the primary reduction of 2 is centered on the bma ligand.

  2. Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases.

    PubMed

    Sommer, Constanze; Adamska-Venkatesh, Agnieszka; Pawlak, Krzysztof; Birrell, James A; Rüdiger, Olaf; Reijerse, Edward J; Lubitz, Wolfgang

    2017-02-01

    The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN(-) ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S]H(2+) ⇔ [4Fe-4S]H(+) and [Fe(I)Fe(II)]H ⇔ [Fe(I)Fe(I)]H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H(+) + 2e(-) ⇄ H2. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state Hred of the H-cluster actually consists of two species: Hred = [4Fe-4S]H(+) - [Fe(I)Fe(II)]H and HredH(+) = [4Fe-4S]H(2+) - [Fe(I)Fe(I)]H (H(+)) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S]H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (Hred/HredH(+)) has a pKa ≈ 7.2.

  3. Solution-phase photochemistry of a [FeFe]hydrogenase model compound: evidence of photoinduced isomerisation.

    PubMed

    Kania, Rafal; Frederix, Pim W J M; Wright, Joseph A; Ulijn, Rein V; Pickett, Christopher J; Hunt, Neil T

    2012-01-28

    The solution-phase photochemistry of the [FeFe] hydrogenase subsite model (μ-S(CH(2))(3)S)Fe(2)(CO)(4)(PMe(3))(2) has been studied using ultrafast time-resolved infrared spectroscopy supported by density functional theory calculations. In three different solvents, n-heptane, methanol, and acetonitrile, relaxation of the tricarbonyl intermediate formed by UV photolysis of a carbonyl ligand leads to geminate recombination with a bias towards a thermodynamically less stable isomeric form, suggesting that facile interconversion of the ligand groups at the Fe center is possible in the unsaturated species. In a polar or hydrogen bonding solvent, this process competes with solvent substitution leading to the formation of stable solvent adduct species. The data provide further insight into the effect of incorporating non-carbonyl ligands on the dynamics and photochemistry of hydrogenase-derived biomimetic compounds.

  4. Solution-phase photochemistry of a [FeFe]hydrogenase model compound: Evidence of photoinduced isomerisation

    SciTech Connect

    Kania, Rafal; Hunt, Neil T.; Frederix, Pim W. J. M.; Wright, Joseph A.; Pickett, Christopher J.; Ulijn, Rein V.

    2012-01-28

    The solution-phase photochemistry of the [FeFe] hydrogenase subsite model ({mu}-S(CH{sub 2}){sub 3}S)Fe{sub 2}(CO){sub 4}(PMe{sub 3}){sub 2} has been studied using ultrafast time-resolved infrared spectroscopy supported by density functional theory calculations. In three different solvents, n-heptane, methanol, and acetonitrile, relaxation of the tricarbonyl intermediate formed by UV photolysis of a carbonyl ligand leads to geminate recombination with a bias towards a thermodynamically less stable isomeric form, suggesting that facile interconversion of the ligand groups at the Fe center is possible in the unsaturated species. In a polar or hydrogen bonding solvent, this process competes with solvent substitution leading to the formation of stable solvent adduct species. The data provide further insight into the effect of incorporating non-carbonyl ligands on the dynamics and photochemistry of hydrogenase-derived biomimetic compounds.

  5. Solution-phase photochemistry of a [FeFe]hydrogenase model compound: Evidence of photoinduced isomerisation

    NASA Astrophysics Data System (ADS)

    Kania, Rafal; Frederix, Pim W. J. M.; Wright, Joseph A.; Ulijn, Rein V.; Pickett, Christopher J.; Hunt, Neil T.

    2012-01-01

    The solution-phase photochemistry of the [FeFe] hydrogenase subsite model (μ-S(CH2)3S)Fe2(CO)4(PMe3)2 has been studied using ultrafast time-resolved infrared spectroscopy supported by density functional theory calculations. In three different solvents, n-heptane, methanol, and acetonitrile, relaxation of the tricarbonyl intermediate formed by UV photolysis of a carbonyl ligand leads to geminate recombination with a bias towards a thermodynamically less stable isomeric form, suggesting that facile interconversion of the ligand groups at the Fe center is possible in the unsaturated species. In a polar or hydrogen bonding solvent, this process competes with solvent substitution leading to the formation of stable solvent adduct species. The data provide further insight into the effect of incorporating non-carbonyl ligands on the dynamics and photochemistry of hydrogenase-derived biomimetic compounds.

  6. 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.

  7. Engineering Oxidoreductases: Utilization of an Unnatural Amino Acid to Create Artificial Hydrogenases

    DTIC Science & Technology

    2011-11-30

    centers related to [ FeFe ]-hydrogenases, the biological catalysts for the reversible oxidation/production of hydrogen. Although a considerable number of...organometallic small molecule mimics of [ FeFe ]- hydrogenases have been reported, the exquisite functionality of the enzymes has yet to be replicated...metallocenters related to [ FeFe ]- hydrogenases to designed peptides/ proteins. Second, new approaches for interfacing peptides/proteins with electrodes

  8. Electron transfer and hydrogen generation from a molecular dyad: platinum(II) alkynyl complex anchored to [FeFe] hydrogenase subsite mimic.

    PubMed

    Wang, Wen-Guang; Wang, Feng; Wang, Hong-Yan; Tung, Chen-Ho; Wu, Li-Zhu

    2012-02-28

    A PS-Fe(2)S(2) molecular dyad 1a directly anchoring a platinum(II) alkynyl complex to a Fe(2)S(2) active site of a [FeFe] H(2)ase mimic, and an intermolecular system of its reference complexes 1b and 2, have been successfully constructed. Time-dependence of H(2) evolution shows that PS-Fe(2)S(2)1a as well as complex 2 with 1b can produce H(2) in the presence of a proton source and sacrificial donor under visible light irradiation. Spectroscopic and electrochemical studies on the electron transfer event reveal that the reduced Fe(I)Fe(0) species generated by the first electron transfer from the excited platinum(II) complex to the Fe(2)S(2) active site in PS-Fe(2)S(2)1a and complex 2 with 1b is essential for photochemical H(2) evolution, while the second electron transfer from the excited platinum(II) complex to the protonated Fe(I)Fe(0) species is thermodynamically unfeasible, which might be an obstacle for the relatively small amount of H(2) obtained by PS-Fe(2)S(2) molecular dyads reported so far.

  9. Catalysis of H(2)/D(2) scrambling and other H/D exchange processes by [Fe]-hydrogenase model complexes.

    PubMed

    Zhao, Xuan; Georgakaki, Irene P; Miller, Matthew L; Mejia-Rodriguez, Rosario; Chiang, Chao-Yi; Darensbourg, Marcetta Y

    2002-07-29

    Protonation of the [Fe]-hydrogenase model complex (mu-pdt)[Fe(CO)(2)(PMe(3))](2) (pdt = SCH(2)CH(2)CH(2)S) produces a species with a high field (1)H NMR resonance, isolated as the stable [(mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)](+)[PF(6)](-) salt. Structural characterization found little difference in the 2Fe2S butterfly cores, with Fe.Fe distances of 2.555(2) and 2.578(1) A for the Fe-Fe bonded neutral species and the bridging hydride species, respectively (Zhao, X.; Georgakaki, I. P.; Miller, M. L.; Yarbrough, J. C.; Darensbourg, M. Y. J. Am. Chem. Soc. 2001, 123, 9710). Both are similar to the average Fe.Fe distance found in structures of three Fe-only hydrogenase active site 2Fe2S clusters: 2.6 A. A series of similar complexes (mu-edt)-, (mu-o-xyldt)-, and (mu-SEt)(2)[Fe(CO)(2)(PMe(3))](2) (edt = SCH(2)CH(2)S; o-xyldt = SCH(2)C(6)H(4)CH(2)S), (mu-pdt)[Fe(CO)(2)(PMe(2)Ph)](2), and their protonated derivatives likewise show uniformity in the Fe-Fe bond lengths of the neutral complexes and Fe.Fe distances in the cationic bridging hydrides. The positions of the PMe(3) and PMe(2)Ph ligands are dictated by the orientation of the S-C bonds in the (mu-SRS) or (mu-SR)(2) bridges and the subsequent steric hindrance of R. The Fe(II)(mu-H)Fe(II) complexes were compared for their ability to facilitate H/D exchange reactions, as have been used as assays of H(2)ase activity. In a reaction that is promoted by light but inhibited by CO, the [(mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)](+) complex shows H/D exchange activity with D(2), producing [(mu-D)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)](+) in CH(2)Cl(2) and in acetone, but not in CH(3)CN. In the presence of light, H/D scrambling between D(2)O and H(2) is also promoted by the Fe(II)(mu-H)Fe(II) catalyst. The requirement of an open site suggests that the key step in the reactions involves D(2) or H(2) binding to Fe(II) followed by deprotonation by the internal hydride base, or by external water. As indicated by similar catalytic efficiencies

  10. Heterolytic cleavage of hydrogen by an iron hydrogenase model: an Fe-H⋅⋅⋅H-N dihydrogen bond characterized by neutron diffraction.

    PubMed

    Liu, Tianbiao; Wang, Xiaoping; Hoffmann, Christina; DuBois, Daniel L; Bullock, R Morris

    2014-05-19

    Hydrogenase enzymes in nature use hydrogen as a fuel, but the heterolytic cleavage of H-H bonds cannot be readily observed in enzymes. Here we show that an iron complex with pendant amines in the diphosphine ligand cleaves hydrogen heterolytically. The product has a strong Fe-H⋅⋅⋅H-N dihydrogen bond. The structure was determined by single-crystal neutron diffraction, and has a remarkably short H⋅⋅⋅H distance of 1.489(10) Å between the protic N-H(δ+) and hydridic Fe-H(δ-) part. The structural data for [Cp(C5F4N)FeH(P(tBu)2N(tBu)2H)](+) provide a glimpse of how the H-H bond is oxidized or generated in hydrogenase enzymes. These results now provide a full picture for the first time, illustrating structures and reactivity of the dihydrogen complex and the product of the heterolytic cleavage of H2 in a functional model of the active site of the [FeFe] hydrogenase enzyme.

  11. The surprising diversity of clostridial hydrogenases: a comparative genomic perspective.

    PubMed

    Calusinska, Magdalena; Happe, Thomas; Joris, Bernard; Wilmotte, Annick

    2010-06-01

    Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermocellum and Clostridum phytofermentans.

  12. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production

    DOE PAGES

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; ...

    2016-08-17

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. Furthermore, this model system represents a unique method of developing hydrogenase-basedmore » H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.« less

  13. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation.

    PubMed

    Mulder, David W; Shepard, Eric M; Meuser, Jonathan E; Joshi, Neelambari; King, Paul W; Posewitz, Matthew C; Broderick, Joan B; Peters, John W

    2011-08-10

    Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

  14. Requirements for functional models of the iron hydrogenase active site: D2/H2O exchange activity in ((mu-SMe)(mu-pdt)[Fe(CO)2(PMe3)]2+)[BF4-].

    PubMed

    Georgakaki, Irene P; Miller, Matthew L; Darensbourg, Marcetta Y

    2003-04-21

    Hydrogen uptake in hydrogenase enzymes can be assayed by H/D exchange reactivity in H(2)/D(2)O or H(2)/D(2)/H(2)O mixtures. Diiron(I) complexes that serve as structural models for the active site of iron hydrogenase are not active in such isotope scrambling but serve as precursors to Fe(II)Fe(II) complexes that are functional models of [Fe]H(2)ase. Using the same experimental protocol as used previously for ((mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-H(+) (Zhao et al. J. Am. Chem. Soc. 2001, 123, 9710), we now report the results of studies of ((mu-SMe)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-SMe(+), toward H/D exchange. The 1-SMe(+) complex can take up H(2) and catalyze the H/D exchange reaction in D(2)/H(2)O mixtures under photolytic, CO-loss conditions. Unlike 1-H(+), it does not catalyze H(2)/D(2) scrambling under anhydrous conditions. The molecular structure of 1-SMe(+) involves an elongated Fe.Fe separation, 3.11 A, relative to 2.58 A in 1-H(+). It is proposed that the strong SMe(-) bridging ligand results in catalytic activity localized on a single Fe(II) center, a scenario that is also a prominent possibility for the enzyme active site. The single requirement is an open site on Fe(II) available for binding of D(2) (or H(2)), followed by deprotonation by the external base H(2)O (or D(2)O).

  15. Nickel-centred proton reduction catalysis in a model of [NiFe] hydrogenase

    NASA Astrophysics Data System (ADS)

    Brazzolotto, Deborah; Gennari, Marcello; Queyriaux, Nicolas; Simmons, Trevor R.; Pécaut, Jacques; Demeshko, Serhiy; Meyer, Franc; Orio, Maylis; Artero, Vincent; Duboc, Carole

    2016-11-01

    Hydrogen production through water splitting is one of the most promising solutions for the storage of renewable energy. [NiFe] hydrogenases are organometallic enzymes containing nickel and iron centres that catalyse hydrogen evolution with performances that rival those of platinum. These enzymes provide inspiration for the design of new molecular catalysts that do not require precious metals. However, all heterodinuclear NiFe models reported so far do not reproduce the Ni-centred reactivity found at the active site of [NiFe] hydrogenases. Here, we report a structural and functional NiFe mimic that displays reactivity at the Ni site. This is shown by the detection of two catalytic intermediates that reproduce structural and electronic features of the Ni-L and Ni-R states of the enzyme during catalytic turnover. Under electrocatalytic conditions, this mimic displays high rates for H2 evolution (second-order rate constant of 2.5 × 104 M-1 s-1 turnover frequency of 250 s-1 at 10 mM H+ concentration) from mildly acidic solutions.

  16. Femtosecond to microsecond photochemistry of a [FeFe]hydrogenase enzyme model compound.

    PubMed

    Kaziannis, Spyridon; Santabarbara, Stefano; Wright, Joseph A; Greetham, Gregory M; Towrie, Michael; Parker, Anthony W; Pickett, Christopher J; Hunt, Neil T

    2010-11-25

    The photochemistry and dynamics of a model compound of the active site of the [FeFe]hydrogenase enzyme system have been studied on a wide range of time scales using a unique combination of femtosecond time-resolved infrared spectroscopy, nanosecond time-resolved infrared spectroscopy, and steady-state UV-FTIR methods. Using three different solvents, heptane, acetonitrile, and cyanoheptane, we have observed the rapid formation of solvent adduct species from the first solvation shell of the solute following photolysis of a carbonyl ligand and global fitting techniques have been employed to provide new insights into the ultrafast dynamics of this process. In addition, the use of solvent mixtures has enabled the observation of competitive ligand substitution processes at the newly created coordination site on time scales of a few nanoseconds, shedding new light on the chemical behavior of these enzyme models.

  17. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production.

    PubMed

    Wecker, Matt S A; Beaton, Stephen E; Chado, Robert A; Ghirardi, Maria L

    2017-02-01

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al., 2009 Photobiological hydrogen-producing systems. Chem Soc Rev 38(1):52-61). Here, we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2 . The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. This model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2 . Biotechnol. Bioeng. 2017;114: 291-297. © 2016 Wiley Periodicals, Inc.

  18. O₂migration rates in [NiFe] hydrogenases. A joint approach combining free-energy calculations and kinetic modeling.

    PubMed

    Topin, Jérémie; Diharce, Julien; Fiorucci, Sébastien; Antonczak, Serge; Golebiowski, Jérôme

    2014-01-23

    Hydrogenases are promising candidates for the catalytic production of green energy by means of biological ways. The major impediment to such a production is rooted in their inhibition under aerobic conditions. In this work, we model dioxygen migration rates in mutants of a hydrogenase of Desulfovibrio fructusovorans. The approach relies on the calculation of the whole potential of mean force for O2 migration within the wild-type as well as in V74M, V74F, and V74Q mutant channels. The three free-energy barriers along the entire migration pathway are converted into chemical rates through modeling based on Transition State Theory. The use of such a model recovers the trend of O2 migration rates among the series.

  19. Influence of the Dithiolate Bridge on the Oxidative Processes of Diiron Models Related to the Active Site of [FeFe] Hydrogenases.

    PubMed

    Arrigoni, Federica; Mohamed Bouh, Salma; De Gioia, Luca; Elleouet, Catherine; Pétillon, François Y; Schollhammer, Philippe; Zampella, Giuseppe

    2017-03-28

    Electrochemical studies of [Fe2 (CO)4 (κ(2) -dmpe)(μ-dithiolate)] (dithiolate=adt(Bn) , pdt) and density functional theory (DFT) calculations reveal the striking influence of an amine functionality in the dithiolate bridge on their oxidative properties. [Fe2 (CO)4 (κ(2) -dmpe)(μ-adt(Bn) )] (1) undergoes two one-electron oxidation steps, with the first being partially reversible and the second irreversible. When the adt(Bn) bridge is replaced with pdt, a shift of 60 mV towards more positive potentials is observed for the first oxidation whereas 290 mV separate the oxidation potentials of the two cations. Under CO, oxidation of azadithiolate compound 1 occurs according to an ECE process whereas an EC mechanism takes place for the propanedithiolate species 2. The dication species [1-CO](2+) resulting from the two-electron oxidation of 1 has been spectroscopically and structurally characterized. The molecular details underlying the reactivity of oxidized species have been explored by DFT calculations. The differences in the behaviors of 1 and 2 are mainly due to the presence, or not, of favored interactions between the dithiolate bridge and the diiron site depending on the redox states, Fe(I) Fe(II) or Fe(II) Fe(II) , of the complexes.

  20. A functional [NiFe]-hydrogenase model compound that undergoes biologically relevant reversible thiolate protonation.

    PubMed

    Weber, Katharina; Krämer, Tobias; Shafaat, Hannah S; Weyhermüller, Thomas; Bill, Eckhard; van Gastel, Maurice; Neese, Frank; Lubitz, Wolfgang

    2012-12-26

    Two model compounds of the active site of [NiFe]-hydrogenases with an unusual {S(2)Ni(μ-S)(μ-CO)Fe(CO)(2)S}-coordination environment around the metals are reported. The neutral compound [Ni(xbsms)(μ-CO)(μ-S)Fe(CO)(2)('S')], (1) (H(2)xbsms = 1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) is converted to [1H][BF(4)] by reversible protonation using HBF(4)·Et(2)O. The protonation takes place at the terminal thiolate sulfur atom that is coordinated to nickel. Catalytic intermediates with a protonated terminal cysteinate were suggested for the native protein but have not yet been confirmed experimentally. [1H][BF(4)] is the first dinuclear [NiFe] model compound for such a species. Both complexes have been synthesized and characterized by X-ray crystallography, NMR-, FTIR-, and (57)Fe-Mössbauer spectroscopy as well as by electronic absorption and resonance Raman spectroscopy. The experimental results clearly show that the protonation has a significant impact on the electronic structure of the iron center, although it takes place at the nickel site. DFT calculations support the interpretation of the spectroscopic data and indicate the presence of a bonding interaction between the metal ions, which is relevant for the enzyme as well. Electrochemical experiments show that both 1 and [1H][BF(4)] are active for electrocatalytic proton reduction in aprotic solvents.

  1. Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

    PubMed Central

    Adams, M W; Eccleston, E; Howard, J B

    1989-01-01

    The iron and acid-labile sulfide contents and the electron paramagnetic resonance (EPR) properties of hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum (strain W5) have been determined on the basis of quantitative amino acid analyses. The iron and acid-labile sulfide values are approximately 20 and 18 atoms per molecule of hydrogenase I and 14 and 11 atoms per molecule of hydrogenase II, respectively. These amounts are substantially greater than previously reported values, which relied on protein concentration determined by colorimetric assay. The oxidized hydrogenases exhibit unusual EPR signals that originate from a novel type of iron-sulfur center, termed the hydrogenase or H cluster, which covalently binds the inhibitor CO. This EPR signal represents approximately one unpaired electron per molecule in each enzyme with and without bound CO, which is consistent with the presence of one oxidized H cluster (S = 1/2) per enzyme molecule. The two enzymes also contain ferredoxin-type four-iron centers or F clusters. The EPR signals from the F clusters observed in the reduced forms of hydrogenase I and hydrogenase II account for approximately four and one unpaired electron per molecule, respectively. We conclude from the iron determinations and the EPR results, together with a reevaluation of previous spectroscopic data, that in both hydrogenases the H cluster probably comprises six iron atoms. Mechanistic models of the two hydrogenases are presented that account for their cluster compositions and the dramatic differences in their catalytic activities. PMID:2544883

  2. Modeling the sublattice magnetizations for the layered bcc nanojunction … Fe[Fe1-cCoc ] ℓ Fe … systems

    NASA Astrophysics Data System (ADS)

    Ashokan, V.; Abou Ghantous, M.; Khater, A.

    2015-12-01

    Ferromagnetic nanojunctions … Fe[Fe1-cCoc ] ℓ Fe …, with ℓ is the number of layers which constitute the nanojunction, based on Fe/Co alloy are considered for the first time in this work. We model the salient magnetic properties of the layered ferromagnetic nanostructures between magnetically ordered iron leads. The effective field theory (EFT) Ising spin method is used to compute reliable Jav exchange values for the VCA Fe/Co alloy materials in comparison with experimental data and compared to existing DFT calculated exchange interactions. The new set of exchange interaction values between pairs of nearest neighbors atom in the alloy are deduced and agree with previous known measurement of lattice constant for this alloy. Using the combined EFT and mean field theory (MFT) spin methods, the sublattice magnetizations of the Fe and Co sites on the individual bcc basal planes of the layered nanostructures, are calculated and analyzed. The sublattice magnetizations, effective magnetic moments per site, and the possible ferromagnetic order of the layers [Fe1-cCoc ] ℓ on the individual bcc atomic planes of the embedded nanostructures for all temperatures and in particular for TcFe ≤ T ≤Tα→γ are presented as a function of temperature and thicknesses of the layered ferromagnetic nanostructures, for different stable concentrations c=0.25, 0.5 and 0.75. In the absence of first principles calculations for these basic physical variables for the layered nanostructures between iron leads, the combined EFT and MFT approach yields the only available information for them at present in the absence of a possible Curie temperature for these alloys. These variables are necessary for certain spin dynamic computations, as for the ballistic magnon transport across embedded nanojunctions in magnonics. The model is general, and may applied directly to other composite magnetic elements and embedded nanostructures.

  3. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  4. Immunological relationship among hydrogenases.

    PubMed Central

    Kovacs, K L; Seefeldt, L C; Tigyi, G; Doyle, C M; Mortenson, L E; Arp, D J

    1989-01-01

    We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed. Images PMID:2464579

  5. Key hydride vibrational modes in [NiFe] hydrogenase model compounds studied by resonance Raman spectroscopy and density functional calculations.

    PubMed

    Shafaat, Hannah S; Weber, Katharina; Petrenko, Taras; Neese, Frank; Lubitz, Wolfgang

    2012-11-05

    Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. While many enzymatic states of the [NiFe] hydrogenase have been studied extensively, there are multiple catalytically relevant EPR-silent states that remain poorly characterized. Analysis of model compounds using new spectroscopic techniques can provide a framework for the study of these elusive states within the protein. We obtained optical absorption and resonance Raman (RR) spectra of (dppe)Ni(μ-pdt)Fe(CO)(3) and [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)(3)][BF(4)], which are structural and functional model compounds for the EPR-silent Ni-SI and Ni-R states of the [NiFe] hydrogenase active site. The studies presented here use RR spectroscopy to probe vibrational modes of the active site, including metal-hydride stretching vibrations along with bridging ligand-metal and Fe-CO bending vibrations, with isotopic substitution used to identify key metal-hydride modes. The metal-hydride vibrations are essentially uncoupled and represent isolated, localized stretching modes; the iron-hydride vibration occurs at 1530 cm(-1), while the nickel-hydride vibration is observed at 945 cm(-1). The significant discrepancy between the metal-hydride vibrational frequencies reflects the slight asymmetry in the metal-hydride bond lengths. Additionally, time-dependent density functional theory (TD-DFT) calculations were carried out to obtain theoretical RR spectra of these compounds. On the basis of the detailed comparison of theory and experiment, the dominant electronic transitions and significant normal modes probed in the RR experiments were assigned; the primary transitions in the visible wavelengths represent metal-to-metal and metal-to-ligand charge transfer bands. Inherent properties of metal-hydride vibrational modes in resonance Raman spectra and DFT calculations are discussed together with the prospects of observing such vibrational modes in metal-hydride-containing proteins. Such a

  6. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production: A Model System for Optimizing H 2 Production

    DOE PAGES

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; ...

    2016-08-23

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al. 2009). Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. This model system represents a unique methodmore » of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.« less

  7. Hydride-Containing Models for the Active Site of the Nickel-Iron Hydrogenases

    PubMed Central

    Barton, Bryan E.; Rauchfuss, Thomas B.

    2010-01-01

    The [NiFe]-hydrogenase model complex NiFe(pdt)(dppe)(CO)3 (1) (pdt = 1,3-propanedithiolate) has been efficiently synthesized and found to be robust. This neutral complex sustains protonation to give the first nickel-iron hydride [1H]BF4. One CO ligand in [1H]BF4 is readily substituted by organophosphorus ligand to afford the substituted derivatives [HNiFe(pdt)(dppe)(CO)2(PR3)]BF4, where PR3 = P(OPh)3 ([2H]BF4); PPh3 ([3H]BF4); PPh2Py ([4H]BF4, where Py = 2-pyridyl). Variable temperature NMR measurements show that the neutral and protonated derivatives are dynamic on the NMR timescale, which partially symmetrizes the phosphine complex. The proposed stereodynamics involve twisting of the Ni(dppe) center, not rotation at the Fe(CO)2(PR3) center. In MeCN solution, 3, which can be prepared by deprotonation of [3H]BF4 with NaOMe, is about 104 stronger base than is 1. X-ray crystallographic analysis of [3H]BF4 revealed a highly unsymmetrical bridging hydride, the Fe-H bond being 0.40 Å shorter than the Ni-H distance. Complexes [2H]BF4, [3H]BF4, [4H]BF4 undergo reductions near −1.46 V vs Fc0/+. For [2H]BF4, this reduction process is reversible, and we assign it as a one-electron process. In the presence of trifluoroacetic acid, proton reduction catalysis coincides with this reductive event. The dependence of ic/ip on the concentration of the acid indicates that H2 evolution entails protonation of a reduced hydride. For [2H]+, [3H]+, and [4H]+, the acid-independent rate constants are 50-75 s−1. For [2H]+ and [3H]+, the overpotentials for H2 evolution are ~430 mV, whereas the overpotential for the N-protonated pyridinium complex [4H2]2+ is estimated to be 260 mV. The mechanism of H2 evolution is proposed to follow an ECEC sequence, where E and C correspond to one-electron reductions and protonations, respectively. On the basis of their values for its pKa and redox potentials, ΔGH• and ΔGH− are 57 and 79 kcal/mol for [1H]+ and [1]2+, respectively. PMID:20925337

  8. HydDB: A web tool for hydrogenase classification and analysis

    PubMed Central

    Søndergaard, Dan; Pedersen, Christian N. S.; Greening, Chris

    2016-01-01

    H2 metabolism is proposed to be the most ancient and diverse mechanism of energy-conservation. The metalloenzymes mediating this metabolism, hydrogenases, are encoded by over 60 microbial phyla and are present in all major ecosystems. We developed a classification system and web tool, HydDB, for the structural and functional analysis of these enzymes. We show that hydrogenase function can be predicted by primary sequence alone using an expanded classification scheme (comprising 29 [NiFe], 8 [FeFe], and 1 [Fe] hydrogenase classes) that defines 11 new classes with distinct biological functions. Using this scheme, we built a web tool that rapidly and reliably classifies hydrogenase primary sequences using a combination of k-nearest neighbors’ algorithms and CDD referencing. Demonstrating its capacity, the tool reliably predicted hydrogenase content and function in 12 newly-sequenced bacteria, archaea, and eukaryotes. HydDB provides the capacity to browse the amino acid sequences of 3248 annotated hydrogenase catalytic subunits and also contains a detailed repository of physiological, biochemical, and structural information about the 38 hydrogenase classes defined here. The database and classifier are freely and publicly available at http://services.birc.au.dk/hyddb/ PMID:27670643

  9. In search of metal hydrides: an X-ray absorption and emission study of [NiFe] hydrogenase model complexes.

    PubMed

    Hugenbruch, Stefan; Shafaat, Hannah S; Krämer, Tobias; Delgado-Jaime, Mario Ulises; Weber, Katharina; Neese, Frank; Lubitz, Wolfgang; DeBeer, Serena

    2016-04-28

    Metal hydrides are invoked as important intermediates in both chemical and biological H2 production. In the [NiFe] hydrogenase enzymes, pulsed EPR and high-resolution crystallography have argued that the hydride interacts primarily at the Ni site. In contrast, in [NiFe] hydrogenase model complexes, it is observed that the bridging hydride interacts primarily with the Fe. Herein, we utilize a combination of Ni and Fe X-ray absorption (XAS) and emission (XES) spectroscopies to examine the contribution of the bridging hydride to the observed spectral features in [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3](+). The corresponding data on (dppe)Ni(μ-pdt)Fe(CO)3 are used as a reference for the changes that occur in the absence of a hydride bridge. For further interpretation of the observed spectral features, all experimental spectra were calculated using a density functional theory (DFT) approach, with excellent agreement between theory and experiment. It is found that the iron valence-to-core (VtC) XES spectra reveal clear signatures for the presence of a Fe-H interaction in the hydride bridged model complex. In contrast, the Ni VtC XES spectrum largely reflects changes in the local Ni geometry and shows little contribution from a Ni-H interaction. A stepwise theoretical analysis of the hydride contribution and the Ni site symmetry provides insights into the factors, which govern the different metal-hydride interactions in both the model complexes and the enzyme. Furthermore, these results establish the utility of two-color XES to reveal important insights into the electronic structure of various metal-hydride species.

  10. Synthesis, Characterization, and Reactivity of Functionalized Trinuclear Iron-Sulfur Clusters - A New Class of Bioinspired Hydrogenase Models.

    PubMed

    Kaiser, Manuel; Knör, Günther

    2015-09-01

    The air- and moisture-stable iron-sulfur carbonyl clusters Fe3S2(CO)7(dppm) (1) and Fe3S2(CO)7(dppf) (2) carrying the bisphosphine ligands bis(diphenylphosphanyl)methane (dppm) and 1,1'-bis(diphenylphosphanyl)ferrocene (dppf) were prepared and fully characterized. Two alternative synthetic routes based on different thionation reactions of triiron dodecacarbonyl were tested. The molecular structures of the methylene-bridged compound 1 and the ferrocene-functionalized derivative 2 were determined by single-crystal X-ray diffraction. The catalytic reactivity of the trinuclear iron-sulfur cluster core for proton reduction in solution at low overpotential was demonstrated. These deeply colored bisphosphine-bridged sulfur-capped iron carbonyl systems are discussed as promising candidates for the development of new bioinspired model compounds of iron-based hydrogenases.

  11. Synthesis, Characterization, and Reactivity of Functionalized Trinuclear Iron–Sulfur Clusters – A New Class of Bioinspired Hydrogenase Models

    PubMed Central

    Kaiser, Manuel; Knör, Günther

    2015-01-01

    The air- and moisture-stable iron–sulfur carbonyl clusters Fe3S2(CO)7(dppm) (1) and Fe3S2(CO)7(dppf) (2) carrying the bisphosphine ligands bis(diphenylphosphanyl)methane (dppm) and 1,1′-bis(diphenylphosphanyl)ferrocene (dppf) were prepared and fully characterized. Two alternative synthetic routes based on different thionation reactions of triiron dodecacarbonyl were tested. The molecular structures of the methylene-bridged compound 1 and the ferrocene-functionalized derivative 2 were determined by single-crystal X-ray diffraction. The catalytic reactivity of the trinuclear iron–sulfur cluster core for proton reduction in solution at low overpotential was demonstrated. These deeply colored bisphosphine-bridged sulfur-capped iron carbonyl systems are discussed as promising candidates for the development of new bioinspired model compounds of iron-based hydrogenases. PMID:26512211

  12. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production

    SciTech Connect

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; Ghirardi, Maria L.

    2016-08-17

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. Furthermore, this model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.

  13. Density functional study of the thermodynamics of hydrogen production by tetrairon hexathiolate, Fe4[MeC(CH2S)3]2(CO)8, a hydrogenase model.

    PubMed

    Surawatanawong, Panida; Hall, Michael B

    2010-06-21

    The tetranuclear iron complex Fe(4)[MeC(CH(2)S)(3)](2)(CO)(8) (1) functions like a hydrogenase to catalyze proton reduction to H(2) in the presence of 2,6-dimethylpyridinium acid (LutH(+)). Experimentally, at the first reduction potential (-1.22 V vs Fc/Fc(+)), the concentration of LutH(+) decreases slowly, while at the second reduction potential, which is sufficient to reduce 1(-) (-1.58 V vs Fc/Fc(+)), the concentration of LutH(+) decreases more rapidly. Here, density functional theory predicts both reduction potentials (E(0)) and proton-transfer free energies relative to LutH(+) for numerous intermediates and several important transition states as a basis for developing thermodynamics cycles for routes to hydrogen production by 1. At the less negative potential, one-electron reduction of 1 is followed by protonation to form a bridging hydride complex; then, a second one-electron reduction is followed by a second protonation, an ECEC process. This doubly reduced and doubly protonated species has a structure with bridging hydrides between both outer Fe-Fe pairs and can produce H(2) and regenerate 1 only by bringing the two hydrogens into proximity through a high-energy intermediate or transition state, a result consistent with the experimentally slow uptake of LutH(+) at this potential. In contrast, at the more negative (lower) reduction potential the two-electron-reduced species, 1(2-), which has bridging carbonyls between both Fe-Fe pairs, is protonated at a terminal Fe position to form a species that produces H(2) by rapidly picking up a second proton and regenerating 1 in an EECC process. Thus, the latter route avoids the high-energy intermediates and transition states necessarily accessed by the former route, a result that explains the more rapid uptake of LutH(+) at the second more negative potential. Although both routes arrive at a doubly reduced, singly protonated species in the third step of these processes, the calculations predict that a high barrier

  14. Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase.

    PubMed

    Liebgott, Pierre-Pol; Leroux, Fanny; Burlat, Bénédicte; Dementin, Sébastien; Baffert, Carole; Lautier, Thomas; Fourmond, Vincent; Ceccaldi, Pierre; Cavazza, Christine; Meynial-Salles, Isabelle; Soucaille, Philippe; Fontecilla-Camps, Juan Carlos; Guigliarelli, Bruno; Bertrand, Patrick; Rousset, Marc; Léger, Christophe

    2010-01-01

    In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

  15. Studies of Hybrid Nano-Bio-System: Single-Walled Carbon Nanotubes and Hydrogenase

    SciTech Connect

    Svedruzic-Chang, D.; Blackburn, J. L.; McDonald, T. J.; Heben, M. J.; King, P. W.

    2008-01-01

    We have examined changes in single-walled carbon nanotubes (SWNT) optical signals upon addition of recombinant [FeFe] hydrogenases from Clostridium acetobutylicum or Chlamydomonas reinhardtii. We found evidence that novel and stable charge-transfer complexes are formed only under conditions of hydrogenase catalytic turnover. Formation of the complex sensitizes the nanotubes to the proton-to-hydrogen redox half-reaction. Thus, the experimental potential can be altered by changing the pH or molecular hydrogen concentration. In the presence of molecular hydrogen, hydrogenase mediates electron injection into the conduction band of semiconducting SWNT, which was observed as a quenching of the photoluminescence signals. Here, we will present recent Raman studies, which revealed that SWNTs in a complex with hydrogenase may undergo either oxidation or reduction, depending on the electronic structure of the SWNT and the oxidation state of the enzyme. In addition, we will describe our efforts to prepare stable, solubilized SWNT/hydrogenase complexes in the absence of detergent. This work shows that SWNT/hydrogenase complexes have potential applications as a component of an energy conversion device.

  16. Structure and function of photosystem I–[FeFe] hydrogenase protein fusions: An all-atom molecular dynamics study

    DOE PAGES

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

    2015-12-15

    All-atom molecular dynamics (MD) simulation was used to study the solution dynamics and protein protein interactions of protein fusions of photosystem I (PSI) from Thermosynechococcus elongatus and an [FeFe]-hydrogenase (FeFe H2ase) from Clostridium pasteurianum, a unique complex capable of photocatalytic hydrogen production. This study involved fusions of these two proteins via dithiol linkers of different length including decanedithiol, octanedithiol, and hexanedithiol, for which experimental data had previously been obtained. Evaluation of root-mean-squared deviations (RMSDs) relative to the respective crystal structures of PSI and the FeFe H2ase shows that these fusion complexes approach stable equilibrium conformations during the MD simulations. Investigatingmore » protein mobility via root-mean-squared fluctuations (RMSFs) reveals that tethering via the shortest hexanedithiol linker results in increased atomic fluctuations of both PSI and the hydrogenase in these fusion complexes. Furthermore, evaluation of the inter- and intraprotein electron transfer distances in these fusion complexes indicates that the structural changes in the FeFe H2ase arising from ligation to PSI via the shortest hexanedithiol linker may hinder electron transport in the hydrogenase, thus providing a molecular level explanation for the observation that the medium-length octanedithiol linker gives the highest hydrogen production rate.« less

  17. Structure and function of photosystem I–[FeFe] hydrogenase protein fusions: An all-atom molecular dynamics study

    SciTech Connect

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

    2015-12-15

    All-atom molecular dynamics (MD) simulation was used to study the solution dynamics and protein protein interactions of protein fusions of photosystem I (PSI) from Thermosynechococcus elongatus and an [FeFe]-hydrogenase (FeFe H2ase) from Clostridium pasteurianum, a unique complex capable of photocatalytic hydrogen production. This study involved fusions of these two proteins via dithiol linkers of different length including decanedithiol, octanedithiol, and hexanedithiol, for which experimental data had previously been obtained. Evaluation of root-mean-squared deviations (RMSDs) relative to the respective crystal structures of PSI and the FeFe H2ase shows that these fusion complexes approach stable equilibrium conformations during the MD simulations. Investigating protein mobility via root-mean-squared fluctuations (RMSFs) reveals that tethering via the shortest hexanedithiol linker results in increased atomic fluctuations of both PSI and the hydrogenase in these fusion complexes. Furthermore, evaluation of the inter- and intraprotein electron transfer distances in these fusion complexes indicates that the structural changes in the FeFe H2ase arising from ligation to PSI via the shortest hexanedithiol linker may hinder electron transport in the hydrogenase, thus providing a molecular level explanation for the observation that the medium-length octanedithiol linker gives the highest hydrogen production rate.

  18. Elucidating hydrogenase surfaces and tracing the intramolecular tunnels for hydrogenase inhibition in microalgal species

    PubMed Central

    Dixit, Kritika; Rahman, Md.Akhlaqur; Nath, Adi; Sundaram, Shanthy

    2016-01-01

    Intramolecular tunnels are majorly attracting attention as possible pathways for entry of inhibitors like oxygen and carbon monoxide to the active sites of the enzymes, hydrogenases. The results of homology modeling of the HydSL protein, a NiFe-hydrogenase from Chlamydomonas reinhardtii and Chlorella vulgaris are presented in this work. Here we identify and describe molecular tunnels observed in HydSL hydrogenase enzyme systems. The possible determinant of the oxygen stability of already studied hydrogenases could be the lack of several intramolecular tunnels. The possible tunnels were traced out using MOLE 2 software, which showed several intramolecular pathways that may be connecting the active sites of the enzyme. The RMSD value showed a great deal of significance in the enzyme homology. This is the first report of its kind in which mapping of the intramolecular tunnels in the four-hydrogenase enzymes disclosed potential variations between designed models and acknowledged structures. We are seeking out the explanations for oxygen sensitivity of studied hydrogenases within the structure of intramolecular tunnels. Local and Global RMSD (Root mean square deviation) was calculated for models and templates, which showed value of 1.284 indicating a successful homology model. The tunnel tracing study by Mole 2 indicated two tunnels joined into one in C. reinhardtii model whereas C. vulgaris model showed one tunnel almost like two tunnels. Templates of both the A. vinosum and D. vulgaris hydrogenase consisted of six tunnels. For HydSL from Chlamydomonas and Chlorella Species the maximal potential was set to 250 kcal/mol (1,046 kJ/mol) and the positive potential areas were marked. Electrostatic studies define electrostatic potential (ESP) that help shuttle protons to the active site. PMID:28149051

  19. Dependence of Localized Electronic Structure on Ligand Configuration in the [2Fe] Hydrogenase Catalytic Core^*

    NASA Astrophysics Data System (ADS)

    Chang, Christopher H.; Kim, Kwiseon

    2007-03-01

    The [FeFe] hydrogenase enzyme is found in a variety of organisms, including Archaea, Eubacteria, and green algae^1,2, and crystallographically determined atomic position data is available for two examples. The biologically unusual catalytic H-cluster, responsible for proton reduction to H2 in vivo, is conserved in the known structures and includes two bis-thiolato bridged iron ions with extensive cyano- and carbonyl ligation. To address the configurational specificity of the diatomic ligand ligation, density functional theoretical calculations were done on [2Fe] core models of the active center, with varying CO and CN^- ligation patterns. Bonding in each complex has been characterized within the Natural Bond Orbital formalism. The effect of ligand configuration on bonding and charge distribution as well as Kohn-Sham orbital structure will be presented. [1] M. Forestier, P. King, L. Zhang, M. Posewitz, S. Schwarzer, T. Happe, M.L. Ghirardi, and M. Seibert, Eur. J. Biochem. 270, 2750 (2003). [2] Posewitz, M.C., P.W. King, S.L. Smolinski, R.D. Smith, II, A.R. Ginley, M.L. Ghirardi, and M. Seibert, Biochem. Soc. Trans. 33, 102 (2005). ^*This work was supported by the US DOE-SC-BES Hydrogen Fuels Initiative, and done in collaboration with the NREL Chemical and Biosciences Center.

  20. [NiFe]-hydrogenases: spectroscopic and electrochemical definition of reactions and intermediates.

    PubMed

    Armstrong, Fraser A; Albracht, Simon P J

    2005-04-15

    Production and usage of di-hydrogen, H2, in micro-organisms is catalysed by highly active, 'ancient' metalloenzymes known as hydrogenases. Based on the number and identity of metal atoms in their active sites, hydrogenases fall into three main classes, [NiFe]-, [FeFe]- and [Fe]-. All contain the unusual ligand CO (and in most cases CN- as well) making them intriguing examples of 'organometallic' cofactors. These ligands render the active sites superbly 'visible' using infrared spectroscopy, which complements the use of electron paramagnetic resonance spectroscopy in studying mechanisms and identifying intermediates. Hydrogenases are becoming a focus of attention for research into future energy technologies, not only H2 production but also H2 oxidation in fuel cells. Hydrogenases immobilized on electrodes exhibit high electrocatalytic activity, providing not only an important new technique for their investigation, but also a basis for novel fuel cells either using the enzyme itself, or inspired synthetic catalysts. Favourable comparisons have been made with platinum electrocatalysts, an advantage of enzymes being their specificity for H2 and tolerance of CO. A challenge for exploiting hydrogenases is their sensitivity to O2, but some organisms are known to produce enzymes that overcome this problem by subtle alterations of the active site and gas access channels.

  1. Heterolytic Cleavage of Hydrogen by an Iron Hydrogenase Model: An Fe-H - - - H-N Dihydorgen Bond Characterized by Neutron Diffraction

    SciTech Connect

    Liu, Tianbiao L.; Wang, Xiaoping; Hoffmann, Christina; DuBois, Daniel L.; Bullock, R. Morris

    2014-05-19

    Use of hydrogen as a fuel by [FeFe]-hydrogenase enzymes in nature requires heterolytic cleavage of the H-H bond into a proton (H+) and hydride (H-), a reaction that is also a critical step in homogeneous catalysts for hydrogenation of C=O and C=N bonds. An understanding of the catalytic oxidation of H2 by hydrogenases provides insights into the design of synthetic catalysts that are sought as cost-effective alternatives to the use of the precious metal platinum in fuel cells. Crystallographic studies on the [FeFe]-hydrogenase enzyme were critical to understanding of its reactivity, but the key H-H cleavage step is not readily observed experimentally in natural hydrogenases. Synthetic biomimics have provided evidence for H2 cleavage leading to hydride transfer to the metal and proton transfer to an amine. Limitations on the precise location of hydrogen atoms by x-ray diffraction can be overcome by use of neutron diffraction, though its use is severely limited by the difficulty of obtaining suitable crystals and by the scarcity of neutron sources. Here we show that an iron complex with a pendant amine in the diphosphine ligand cleaves hydrogen heterolytically under mild conditions, leading to [CpC5F4NFeH(PtBu2NtBu2H)]+BArF4-, [PtBu2NtBu2 = 1,5-di(tert-butyl)-3,7-di(tert-butyl)-1,5-diaza-3,7-diphosphacyclooctane; ArF = 3,5-bis(trifluoromethyl)phenyl]. The Fe-H- - - H-N moiety has a strong dihydrogen bond, with a remarkably short H • • • H distance of 1.489(10) Å between the protic N-Hδ+ and hydridic Fe-Hδ-. The structural data for [CpC5F4NFeH(PtBu2NtBu2H)]+ provide a glimpse of how the H-H bond is oxidized or generated in hydrogenase enzymes, with the pendant amine playing a key role as a proton relay. The iron complex [CpC5F4NFeH(PtBu2NtBu2H)]+BArF4- is an electrocatalyst for oxidation of H2 (1 atm) at 22 °C, so the structural data are obtained on a complex that is a functional model for catalysis by [FeFe]-hydrogenase enzymes. This research was supported

  2. Computational Investigation of [FeFe]-Hydrogenase Models: Characterization of Singly and Doubly Protonated Intermediates and Mechanistic Insights

    PubMed Central

    2015-01-01

    The [FeFe]-hydrogenase enzymes catalyze hydrogen oxidation and production efficiently with binuclear Fe metal centers. Recently the bioinspired H2-producing model system Fe2(adt)(CO)2(dppv)2 (adt=azadithiolate and dppv=diphosphine) was synthesized and studied experimentally. In this system, the azadithiolate bridge facilitates the formation of a doubly protonated ammonium-hydride species through a proton relay. Herein computational methods are utilized to examine this system in the various oxidation states and protonation states along proposed mechanistic pathways for H2 production. The calculated results agree well with the experimental data for the geometries, CO vibrational stretching frequencies, and reduction potentials. The calculations illustrate that the NH···HFe dihydrogen bonding distance in the doubly protonated species is highly sensitive to the effects of ion-pairing between the ammonium and BF4– counterions, which are present in the crystal structure, in that the inclusion of BF4– counterions leads to a significantly longer dihydrogen bond. The non-hydride Fe center was found to be the site of reduction for terminal hydride species and unsymmetric bridging hydride species, whereas the reduced symmetric bridging hydride species exhibited spin delocalization between the Fe centers. According to both experimental measurements and theoretical calculations of the relative pKa values, the Fed center of the neutral species is more basic than the amine, and the bridging hydride species is more thermodynamically stable than the terminal hydride species. The calculations implicate a possible pathway for H2 evolution that involves an intermediate with H2 weakly bonded to one Fe, a short H2 distance similar to the molecular bond length, the spin density delocalized over the two Fe centers, and a nearly symmetrically bridged CO ligand. Overall, this study illustrates the mechanistic roles of the ammonium-hydride interaction, flexibility of the bridging CO

  3. Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism.

    PubMed

    Cha, Minseok; Chung, Daehwan; Westpheling, Janet

    2016-02-01

    The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.

  4. International Hydrogenase Conference (7th) Held at the University of Reading on August 24th to 29th 2004.

    DTIC Science & Technology

    2007-11-02

    FeFe ]Hlase Si and electrocatalysis: as good as platinum or as . . . . .. . . d e . . . . . . . ..goo as it g ts.] .... 11.40 12.00 T6. Chris Pickett PB...Asymmetric Models of the Active Site of [ FeFe ]H ase and p2 5 Electrocatalysis: As Good as Platinum or as Good as It Gets? Marcetta Y. Darensbourg...4th to 29’", 2004 T5. Asymmetric Models of the Active Site of [ FeFe ]H2ase and Electrocatalysis: As Good as Platinum or as Good as It Gets? Marcetta Y

  5. Favorable Protonation of the (μ-edt)[Fe(2)(PMe(3))(4)(CO)(2)(H-terminal)](+) Hydrogenase Model Complex Over Its Bridging μ-H Counterpart: A Spectroscopic and DFT Study.

    PubMed

    Galinato, Mary Grace I; Whaley, C Matthew; Roberts, Dean; Wang, Peng; Lehnert, Nicolai

    2011-03-01

    The mechanism of hydrogen production in [FeFe] hydrogenase remains elusive. However, a species featuring a terminal hydride bound to the distal Fe is thought to be the key intermediate leading to hydrogen production. In this study, density functional theory (DFT) calculations on the terminal (H-term) and bridging (μ-H) hydride isomers of (μ-edt)-[Fe(2)(PMe(3))(4)(CO)(2)H](+) are presented in order to understand the factors affecting their propensity for protonation. Relative to H-term, μ-H is 12.7 kcal/mol more stable, which contributes to its decreased reactivity towards an acid. Potential energy surface (PES) calculations for the reaction of the H-term isomer with 4-nitropyridinium, a proton source, further reveal a lower activation energy barrier (14.5 kcal/mol) for H-term than for μ-H (29 kcal/mol). Besides these energetic considerations, the H-term isomer displays a key molecular orbital (MO <139>) that has a relatively strong hydride (1s) contribution (23%), which is not present in the μ-H isomer. This indicates a potential orbital control of the reaction of the hydride complexes with acid. The lower activation energy barrier and this key MO together control the overall catalytic activity of (μ-edt)[Fe(2)(PMe(3))(4)(CO)(2)(H-term)](+). Lastly, Raman and IR spectroscopy were performed in order to probe the ν(Fe-H) stretching mode of the two isomers and their deuterated counterparts. A ν(Fe-H) stretching mode was observed for the μ-H complex at 1220 cm(-1). However, the corresponding mode is not observed for the less stable H-term isomer.

  6. Intramolecular iron-mediated C-H bond heterolysis with an assist of pendant base in a [FeFe]-hydrogenase model.

    PubMed

    Zheng, Dehua; Wang, Ning; Wang, Mei; Ding, Shengda; Ma, Chengbing; Darensbourg, Marcetta Y; Hall, Michael B; Sun, Licheng

    2014-12-03

    Although many metalloenzymes containing iron play a prominent role in biological C-H activation processes, to date iron-mediated C(sp(3))-H heterolysis has not been reported for synthetic models of Fe/S-metalloenzymes. In contrast, ample precedent has established that nature's design for reversible hydrogen activation by the diiron hydrogenase ([FeFe]-H2ase) active site involves multiple irons, sulfur bridges, a redox switch, and a pendant amine base, in an intricate arrangement to perform H-H heterolytic cleavage. In response to whether this strategy might be extended to C-H activation, we report that a [FeFe]-H2ase model demonstrates iron-mediated intramolecular C-H heterolytic cleavage via an agostic C-H interaction, with proton removal by a nearby pendant amine, affording Fe(II)-[Fe'(II)-CH-S] three-membered-ring products, which can be reduced back to 1 by Cp2Co in the presence of HBF4. The function of the pendant base as a proton shuttle was confirmed by the crystal structures of the N-protonated intermediate and the final deprotonated product in comparison with that of a similar but pendant-amine-free complex that does not show evidence of C-H activation. The mechanism of the process was backed up by DFT calculations.

  7. Hydrogenase electrodes for fuel cells.

    PubMed

    Karyakin, A A; Morozov, S V; Karyakina, E E; Zorin, N A; Perelygin, V V; Cosnier, S

    2005-02-01

    Considering crucial problems that limit use of platinum-based fuel cells, i.e. cost and availability, poisoning by fuel impurities and low selectivity, we propose electrocatalysis by enzymes as a valuable alternative to noble metals. Hydrogenase electrodes in neutral media achieve hydrogen equilibrium potential (providing 100% energy conversion), and display high activity in H(2) electrooxidation, which is similar to that of Pt-based electrodes in sulphuric acid. In contrast with platinum, enzyme electrodes are highly selective for their substrates, and are not poisoned by fuel impurities. Hydrogenase electrodes are capable of consuming hydrogen directly from microbial media, which ensures their use as fuel electrodes in treatment of organic wastes.

  8. Roles of Fe-Nx and Fe-Fe3C@C Species in Fe-N/C Electrocatalysts for Oxygen Reduction Reaction.

    PubMed

    Kim, Jae Hyung; Sa, Young Jin; Jeong, Hu Young; Joo, Sang Hoon

    2017-03-22

    Iron and nitrogen codoped carbons (Fe-N/C) have emerged as promising nonprecious metal catalysts for the oxygen reduction reaction (ORR). While Fe-Nx sites have been widely considered as active species for Fe-N/C catalysts, very recently, iron and/or iron carbide encased with carbon shells (Fe-Fe3C@C) has been suggested as a new active site for the ORR. However, most of synthetic routes to Fe-N/C catalysts involve high-temperature pyrolysis, which unavoidably yield both Fe-Nx and Fe-Fe3C@C species, hampering the identification of exclusive role of each species. Herein, in order to establish the respective roles of Fe-Nx and Fe-Fe3C@C sites we rationally designed model catalysts via the phase conversion reactions of Fe3O4 nanoparticles supported on carbon nanotubes. The resulting catalysts selectively contained Fe-Nx, Fe-Fe3C@C, and N-doped carbon (C-Nx) sites. It was revealed that Fe-Nx sites dominantly catalyze ORR via 4-electron (4 e(-)) pathway, exerting a major role for high ORR activity, whereas Fe-Fe3C@C sites mainly promote 2 e(-) reduction of oxygen followed by 2 e(-) peroxide reduction, playing an auxiliary role.

  9. Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum

    SciTech Connect

    Biswas, Ranjita; Zheng, Tianyong; Olson, Daniel G.; Lynd, Lee R.; Guss, Adam M.

    2015-02-01

    The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl-CoA reduction to ethanol. C. thermocellum encodes four hydrogenases and rather than delete each individually, we targeted a hydrogenase maturase gene (hydG), involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in hydG ech was undetectable and ethanol yield increased nearly 2-fold compared to wild type. Interestingly, mutant growth improved upon the addition of acetate, which led to increased expression of genes related to sulfate metabolism, suggesting these mutants may use sulfate as a terminal electron acceptor to balance redox reactions. Genomic analysis of hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation is found in ethanol tolerant C. thermocellum strain E50C, hydG and hydG ech are not more ethanol tolerant than wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. The dramatic increase in ethanol production here suggests that targeting protein post-translational modification is a promising new approach for inactivation of multiple enzymes simultaneously for metabolic engineering.

  10. Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum

    DOE PAGES

    Biswas, Ranjita; Zheng, Tianyong; Olson, Daniel G.; ...

    2015-02-01

    The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl-CoA reduction to ethanol. C. thermocellum encodes four hydrogenases and rather than delete each individually, we targeted a hydrogenase maturase gene (hydG), involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe]more » hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in hydG ech was undetectable and ethanol yield increased nearly 2-fold compared to wild type. Interestingly, mutant growth improved upon the addition of acetate, which led to increased expression of genes related to sulfate metabolism, suggesting these mutants may use sulfate as a terminal electron acceptor to balance redox reactions. Genomic analysis of hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation is found in ethanol tolerant C. thermocellum strain E50C, hydG and hydG ech are not more ethanol tolerant than wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. The dramatic increase in ethanol production here suggests that targeting protein post-translational modification is a promising new approach for inactivation of multiple enzymes simultaneously for metabolic engineering.« less

  11. Cyanide inactivation of hydrogenase from Azotobacter vinelandii

    SciTech Connect

    Seefeldt, L.C.; Arp, D.J. )

    1989-06-01

    The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M{sup {minus}1} min{sup {minus}1} for CN{sup {minus}}). The rate of inactivation decreased with decreasing pH. ({sup 14}C)cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.

  12. Cyanide inactivation of hydrogenase from Azotobacter vinelandii.

    PubMed Central

    Seefeldt, L C; Arp, D J

    1989-01-01

    The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state. PMID:2656648

  13. Synthesis, characterization, and electrochemical properties of diiron propaneditellurolate (PDTe) complexes as active site models of [FeFe]-hydrogenases.

    PubMed

    Song, Li-Cheng; Li, Qian-Li; Feng, Zhan-Heng; Sun, Xiao-Jing; Xie, Zhao-Jun; Song, Hai-Bin

    2013-02-07

    Parent complex (μ-PDTe)Fe(2)(CO)(6) (1, PDTe = μ-TeCH(2)CH(2)CH(2)Te-μ) is prepared via a new synthetic route involving the reaction of (μ-Te(2))Fe(2)(CO)(6) with Et(3)BHLi, followed by treatment of (μ-LiTe)(2)Fe(2)(CO)(6) with Br(CH(2))(3)Br in a 43% yield. Further reactions of 1 with 1 equiv of monophosphines in the presence of the decarbonylating agent Me(3)NO afford the corresponding monophosphine-substituted complexes (μ-PDTe)Fe(2)(CO)(5)(L) (2, L = PPh(3); 3, PPh(2)H; 4, PMe(3)) in 37%-47% yields, whereas the N-heterocyclic carbene I(Mes)-monosubstituted complex (μ-PDTe)Fe(2)(CO)(5)(I(Mes)) (5) can be prepared in a 26% yield by treatment of 1 with the in situ generated I(Mes) from the 1,3-bis(mesityl)imidazolium salt I(Mes)·HCl and n-BuLi. While the diphosphine-bridged single-butterfly complexes (μ-PDTe)Fe(2)(CO)(4)(dppm) (6) and (μ-PDTe)Fe(2)(CO)(4)(dppn) (7) can be prepared in 28% and 21% yields by treatment of 1 with 1 equiv of the corresponding diphosphines in refluxing xylene, treatment of 1 with 0.5 equiv of diphosphines in the presence of Me(3)NO results in the formation of the corresponding diphosphine-bridged double-butterfly complexes [(μ-PDTe)Fe(2)(CO)(5)](2)(dppp) (8), [(μ-PDTe)Fe(2)(CO)(5)](2)(dppb) (9), and [(μ-PDTe)Fe(2)(CO)(5)](2)(dppf) (10) in 25-37% yields. All the new substituted model complexes 2-10 are characterized by combustion analysis and spectroscopy, and particularly for 2, 3, 5, and 7-10, by X-ray crystallography. In addition, a comparative study on the electrochemical and electrocatalytic properties of the PDTe-type model complexes 1 and 7 with their corresponding selenium and sulfur analogs are reported.

  14. Artificial hydrogenase: biomimetic approaches controlling active molecular catalysts.

    PubMed

    Onoda, Akira; Hayashi, Takashi

    2015-04-01

    Hydrogenase catalyses reversible transformation of H2 to H(+) using an active site which includes an iron or nickel atom. Synthetic model complexes and molecular catalysts inspired by nature have unveiled the structural and functional basis of the active site with remarkable accuracy and this has led to the discovery of active synthetic catalysts. To further improve the activity of such molecular catalysts, both the first and outer coordination spheres should be well-organized and harmonized for an efficient shuttling of H(+), electrons, and H2. This article reviews recent advances in the design and catalytic properties of artificial enzymes that mimic the hydrogenase active site and the outer coordination sphere in combination with a peptide or protein scaffold.

  15. Hydrogenase polypeptide and methods of use

    DOEpatents

    Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

    2016-02-02

    Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

  16. Photocatalytic hydrogen evolution by a diiron hydrogenase model based on a peptide fragment of cytochrome c556 with an attached diiron carbonyl cluster and an attached ruthenium photosensitizer.

    PubMed

    Sano, Yohei; Onoda, Akira; Hayashi, Takashi

    2012-03-01

    It is of particular interest to mimic the process of intramolecular electron relay at the active site of [FeFe]-hydrogenase in order to understand the mechanism of the catalytic activity of H(2) evolution. We have recently focused on using the native CXXCH peptide sequence of the C-terminal segment of cytochrome c(556) as a platform which holds a diiron carbonyl cluster via two cysteines and have attached a ruthenium photosensitizer via a histidine. The modified peptide with the two metal moieties is found to act as the photocatalyst for H(2) evolution with a turnover number of ~9 over 2h at pH 8.5 in the presence of ascorbate as a sacrificial reagent.

  17. Effects of metal ions on the reactivity and corrosion electrochemistry of Fe/FeS nanoparticles.

    PubMed

    Kim, Eun-Ju; Kim, Jae-Hwan; Chang, Yoon-Seok; Turcio-Ortega, David; Tratnyek, Paul G

    2014-04-01

    Nano-zerovalent iron (nZVI) formed under sulfidic conditions results in a biphasic material (Fe/FeS) that reduces trichloroethene (TCE) more rapidly than nZVI associated only with iron oxides (Fe/FeO). Exposing Fe/FeS to dissolved metals (Pd(2+), Cu(2+), Ni(2+), Co(2+), and Mn(2+)) results in their sequestration by coprecipitation as dopants into FeS and FeO and/or by electroless precipitation as zerovalent metals that are hydrogenation catalysts. Using TCE reduction rates to probe the effect of metal amendments on the reactivity of Fe/FeS, it was found that Mn(2+) and Cu(2+) decreased TCE reduction rates, while Pd(2+), Co(2+), and Ni(2+) increased them. Electrochemical characterization of metal-amended Fe/FeS showed that aging caused passivation by growth of FeO and FeS phases and poisoning of catalytic metal deposits by sulfide. Correlation of rate constants for TCE reduction (kobs) with electrochemical parameters (corrosion potentials and currents, Tafel slopes, and polarization resistance) and descriptors of hydrogen activation by metals (exchange current density for hydrogen reduction and enthalpy of solution into metals) showed the controlling process changed with aging. For fresh Fe/FeS, kobs was best described by the exchange current density for activation of hydrogen, whereas kobs for aged Fe/FeS correlated with electrochemical descriptors of electron transfer.

  18. CO and CN- syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events.

    PubMed

    Pagnier, Adrien; Martin, Lydie; Zeppieri, Laura; Nicolet, Yvain; Fontecilla-Camps, Juan C

    2016-01-05

    The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN(-) ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN(-) and the CO precursor (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN(-), and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold.

  19. CO and CN− syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

    PubMed Central

    Pagnier, Adrien; Martin, Lydie; Zeppieri, Laura; Nicolet, Yvain; Fontecilla-Camps, Juan C.

    2016-01-01

    The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN− ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN− and the CO precursor −:CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN−, and −:CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN−, and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold. PMID:26699472

  20. Photocatalytic H2 production using a hybrid assembly of an [FeFe]-hydrogenase model and CdSe quantum dot linked through a thiolato-functionalized cyclodextrin.

    PubMed

    Cheng, Minglun; Wang, Mei; Zhang, Shuai; Liu, Fengyuan; Yang, Yong; Wan, Boshun; Sun, Licheng

    2017-03-07

    It is a great challenge to develop iron-based highly-efficient and durable catalytic systems for the hydrogen evolution reaction (HER) by understanding and learning from [FeFe]-hydrogenases. Here we report photocatalytic H2 production by a hybrid assembly of a sulfonate-functionalized [FeFe]-hydrogenase mimic (1) and CdSe quantum dot (QD), which is denoted as 1/β-CD-6-S-CdSe (β-CD-6-SH = 6-mercapto-β-cyclodextrin). In this assembly, thiolato-functionalized β-CD acts not only as a stabilizing reagent of CdSe QDs but also as a host compound for the diiron catalyst, so as to confine CdSe QDs to the space near the site of diiron catalyst. In addition, another two reference systems comprising MAA-CdSe QDs (HMAA = mercaptoacetic acid) and 1 in the presence and absence of β-CD, denoted as 1/β-CD/MAA-CdSe and 1/MAA-CdSe, were studied for photocatalytic H2 evolution. The influences of β-CD and the stabilizing reagent β-CD-6-S(-) on the stability of diiron catalyst, the fluorescence lifetime of CdSe QDs, the apparent electron transfer rate, and the photocatalytic H2-evolving efficiency were explored by comparative studies of the three hybrid systems. The 1/β-CD-6-S-CdSe system displayed a faster apparent rate for electron transfer from CdSe QDs to the diiron catalyst compared to that observed for MAA-CdSe-based systems. The total TON for visible-light driven H2 evolution by the 1/β-CD-6-S-CdSe QDs in water at pH 4.5 is about 2370, corresponding to a TOF of 150 h(-1) in the initial 10 h of illumination, which is 2.7- and 6.6-fold more than the amount of H2 produced from the reference systems 1/β-CD/MAA-CdSe and 1/MAA-CdSe. Additionally, 1/β-CD-6-S-CdSe gave 2.4-5.1 fold enhancement in the apparent quantum yield and significantly improved the stability of the system for photocatalytic H2 evolution.

  1. De novo modeling of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy

    PubMed Central

    Mills, Deryck J; Vitt, Stella; Strauss, Mike; Shima, Seigo; Vonck, Janet

    2013-01-01

    Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI: http://dx.doi.org/10.7554/eLife.00218.001 PMID:23483797

  2. Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector.

    PubMed

    Allegretti, Matteo; Mills, Deryck J; McMullan, Greg; Kühlbrandt, Werner; Vonck, Janet

    2014-02-25

    The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct electron detector in video mode, we have reconstructed a map at 3.36 Å resolution of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from only 320,000 asymmetric units. Videos frames were aligned by a combination of image and particle alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map shows all secondary structure as well as clear side chain densities for most residues. The full coordination of all cofactors in the electron transfer chain (a [NiFe] center, four [4Fe4S] clusters and an FAD) is clearly visible along with a well-defined substrate access channel. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes. DOI: http://dx.doi.org/10.7554/eLife.01963.001.

  3. Microstructure, mechanical property, biodegradation behavior, and biocompatibility of biodegradable Fe-Fe2O3 composites.

    PubMed

    Cheng, J; Huang, T; Zheng, Y F

    2014-07-01

    In this study, the effects of Fe2O3 (addition, 2, 5, 10, and 50 wt %) on the microstructure, mechanical properties, corrosion behaviors, and in vitro biocompatibility of Fe-Fe2O3 composites fabricated by spark plasma sintering were systematically investigated as a novel-structure biodegradable metallic material. The results of X-ray diffraction analysis and optical microscopy indicated that Fe-Fe2O3 composite is composed of α-Fe and FeO instead of Fe2O3. Both eletrochemical measurements and immersion test showed a faster degradation rate of Fe-2Fe2O3 and Fe-5Fe2O3 composites than pure iron and Fe-5Fe2O3 exhibited the fastest corrosion rate among these composites. Besides, the effect of Fe2O3 on the corrosion behavior of Fe-Fe2O3 composites was discussed. The extracts of Fe-Fe2O3 composite exhibited no cytotoxicity to both ECV304 and L929 cells, whereas greatly reduced cell viabilities of vascular smooth muscle cells. In addition, good hemocompatibility of all Fe-Fe2O3 composites and pure iron was obtained. To sum up, Fe-5Fe2O3 composite is a promising alternative for biodegradable stent material with elevated corrosion rate, enhanced mechanical properties, as well as excellent biocompatibility.

  4. Improving cyanobacterail O2-tolerance using CBS hydrogenase for hydrogen production

    SciTech Connect

    Maness, Pin-Ching; Eckert, Carrie; Wawrousek, Karen; Noble, Scott; Pennington, Grant; Yu, Jianping

    2016-11-11

    Cyanobacterial H2 production is a viable path to renewable H2 with water serving as the electron donor and sunlight the energy source. A grand challenge is the sensitivity of the underlying hydrogenase to O2, the latter an inherent byproduct of oxygenic photosynthesis. This challenge has been identified as a technical barrier in the Fuel Cell Technologies Office (FCTO) Multi-year Research, Development and Deployment Plan. One solution is to express in cyanobacterium an O2-tolerant hydrogenase to circumvent this barrier. We have uncovered an O2-tolerant hydrogenase from a photosynthetic bacterium Rubrivivax gelatinosus CBS (Casa Bonita Strain; hereafter “CBS”) with a half-life near 21 h when exposed to ambient O2. We sequenced the CBS genome and identified two sets of maturation machineries hyp1 and hyp2. Transcripts expression analysis and mutagenesis revealed that hyp1 is responsible for the assembly of the O2-tolerant CO-oxidation (Coo) hydrogenase and hyp2 is involved in the maturation of a H2-uptake hydrogenase. The structural genes encoding the O2-tolerant hydrogenase (cooLXUH) and maturation genes hyp1FABCDE were therefore cloned and expressed in the model cyanobacterium Synechocystis sp. PCC 6803. We obtained several recombinants displaying hydrogenase activity in a Synechocystis host lacking background activity, suggesting that the CBS hydrogenase is active in Synechocystis. Yet the activity is extremely low. To ensure balanced protein expression, we systematically optimized heterologous expression of 10 CBS genes by using stronger promoters and better ribosome binding site. Moreover we attempted the expression of cooM and cooK genes, verified to be important in CBS to afford activity. CooM is a very large protein and both CooM and CooK are membrane-associated. These properties limited our success in expressing both genes in Synechocystis, although they

  5. Evolutionary Significance of an Algal Gene Encoding an [FeFe]-Hydrogenase with F-Domain Homology and Hydrogenase Activity in Chlorella Variabilis NC64A

    SciTech Connect

    Meuser, J. E.; Boyd, E. S.; Ananyev, G.; Karns, D.; Radakovits, R.; Murthy, U. M. N.; Ghirardi, M. L.; Dismukes, G. C.; Peters, J. W.; Posewitz, M. C.

    2011-10-01

    [FeFe]-hydrogenases (HYDA) link the production of molecular H{sub 2} to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H{sub 2}-photoproduction activity observed is as O2 sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

  6. Structure and function of [NiFe] hydrogenases.

    PubMed

    Ogata, Hideaki; Lubitz, Wolfgang; Higuchi, Yoshiki

    2016-11-01

    Hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons via a heterolytic splitting mechanism. The active sites of [NiFe] hydrogenases comprise a dinuclear Ni-Fe center carrying CO and CN(-) ligands. The catalytic activity of the standard (O2-sensitive) [NiFe] hydrogenases vanishes under aerobic conditions. The O2-tolerant [NiFe] hydrogenases can sustain H2 oxidation activity under atmospheric conditions. These hydrogenases have very similar active site structures that change the ligand sphere during the activation/catalytic process. An important structural difference between these hydrogenases has been found for the proximal iron-sulphur cluster located in the vicinity of the active site. This unprecedented [4Fe-3S]-6Cys cluster can supply two electrons, which lead to rapid recovery of the O2 inactivation, to the [NiFe] active site.

  7. Hydrogenases and Hydrogen Metabolism of Cyanobacteria

    PubMed Central

    Tamagnini, Paula; Axelsson, Rikard; Lindberg, Pia; Oxelfelt, Fredrik; Wünschiers, Röbbe; Lindblad, Peter

    2002-01-01

    Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included. PMID:11875125

  8. A Bacterial Electron-bifurcating Hydrogenase*

    PubMed Central

    Schuchmann, Kai; Müller, Volker

    2012-01-01

    The Wood-Ljungdahl pathway of anaerobic CO2 fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD+ for this reaction. NAD+ was also reduced but only in the presence of ferredoxin. NAD+ and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD+ and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD+ reduction. PMID:22810230

  9. Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants

    PubMed Central

    Abou Hamdan, Abbas; Liebgott, Pierre-Pol; Fourmond, Vincent; Gutiérrez-Sanz, Oscar; De Lacey, Antonio L.; Infossi, Pascale; Rousset, Marc; Dementin, Sébastien; Léger, Christophe

    2012-01-01

    Nickel-containing hydrogenases, the biological catalysts of oxidation and production, reversibly inactivate under anaerobic, oxidizing conditions. We aim at understanding the mechanism of (in)activation and what determines its kinetics, because there is a correlation between fast reductive reactivation and oxygen tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnology. Direct electrochemistry is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here we describe simple analytical models that we used to characterize and compare 16 mutants, obtained by substituting the position-74 valine of the -sensitive NiFe hydrogenase from Desulfovibrio fructosovorans. We observed that this substitution can accelerate reactivation up to 1,000-fold, depending on the polarity of the position 74 amino acid side chain. In terms of kinetics of anaerobic (in)activation and oxygen tolerance, the valine-to-histidine mutation has the most spectacular effect: The V74H mutant compares favorably with the -tolerant hydrogenase from Aquifex aeolicus, which we use here as a benchmark. PMID:23169623

  10. Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS.

    PubMed

    Tengölics, Roland; Mészáros, Lívia; Győri, E; Doffkay, Zsolt; Kovács, Kornél L; Rákhely, Gábor

    2014-10-01

    Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.

  11. Effect of Bridgehead Steric Bulk on the Intramolecular C-H Heterolysis of [FeFe]-Hydrogenase Active Site Models Containing a P2N2 Pendant Amine Ligand.

    PubMed

    Zheng, Dehua; Wang, Mei; Wang, Ning; Cheng, Minglun; Sun, Licheng

    2016-01-19

    A series of pendant amine-containing [FeFe]-hydrogenase models, [X(CH2S-μ)2{Fe(CO)3}{Fe(CO)(P2(Ph)N2(Bn))}] (1H, X = CH2; 2Me, C(CH3)2; 3Et, C(CH2CH3)2; and P2(Ph)N2(Bn) = 1,5-dibenzyl-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane) with different groups at the bridgehead carbon of the S-to-S linker were synthesized. The oxidations of these complexes as well as the reverse reduction reaction were studied by cyclic voltammetry and in situ IR spectroscopy. Regardless of the bridgehead steric bulk, all three complexes demonstrate intramolecular iron-mediated C(sp(3))-H bond heterolytic cleavage with the assistance of the pendant amine base within the chelating diphosphine ligand in the two-electron oxidation process. X-ray crystallographic analysis shows that the doubly oxidized products, [1'H](+), [2'Me](+), and [3'Et](+), all have a rigid FeSC three-membered ring at the open apical site of the rotated iron center. The most noticeable difference in structures of the oxidized complexes is that the single CO ligand of the rotated Fe(P2(Ph)N2(Bn))(CO) unit in [1'H](+) and [2'Me](+) is found below the Fe···Fe vector, while in [3'Et](+) an unusually rotated Fe(P2(Ph)N2(Bn))(CO) moiety positions one of the P donors within the bidentate ligand under the Fe···Fe vector. The starting Fe(I)Fe(I) complexes can be recovered from their corresponding doubly oxidized complexes by reduction in the presence of Brönsted acid.

  12. Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron–copper crosstalk

    PubMed Central

    Waters, Brian M.; McInturf, Samuel A.; Amundsen, Keenan

    2014-01-01

    Summary Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu regulated genes and stimulated Cu accumulation. The C940-fe (fefe) Fe uptake mutant of melon (Cucumis melo) was characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu deficient plants was independent of the normal Fe uptake system. One of the four FRO genes in the melon and cucumber (Cucumis sativus) genomes was Fe regulated, and one was Cu regulated. Simultaneous Fe and Cu deficiency synergistically upregulated Fe uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe– Cu crosstalk in metal homeostasis. The fefe gene is not orthologous to FIT, thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants. PMID:24975482

  13. Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii1

    PubMed Central

    Bailleul, Benjamin; Berne, Nicolas

    2015-01-01

    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. PMID:25931521

  14. Hydrogenase in actinorhizal root nodules and root nodule homogenates.

    PubMed Central

    Benson, D R; Arp, D J; Burris, R H

    1980-01-01

    Hydrogenases were measured in intact actinorhizal root nodules and from disrupted nodules of Alnus glutinosa, Alnus rhombifolia, Alnus rubra, and Myrica pensylvanica. Whole nodules took up H2 in an O2-dependent reaction. Endophyte preparations oxidized H2 through the oxyhydrogen reaction, but rates were enhanced when hydrogen uptake was coupled to artificial electron acceptors. Oxygen inhibited artifical acceptor-dependent H2 uptake. The hydrogenase system from M. pensylvanica had a different pattern of coupling to various electron acceptors than the hydrogenase systems from the alders; only the bayberry system evolved H2 from reduced viologen dyes. PMID:6989799

  15. Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.

    PubMed

    Adam, Nicole; Perner, Mirjam

    2017-01-01

    Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.

  16. Comparison of carbon materials as electrodes for enzyme electrocatalysis: hydrogenase as a case study.

    PubMed

    Quinson, Jonathan; Hidalgo, Ricardo; Ash, Philip A; Dillon, Frank; Grobert, Nicole; Vincent, Kylie A

    2014-01-01

    We present a study of electrocatalysis by an enzyme adsorbed on a range of carbon materials, with different size, surface area, morphology and graphitic structure, which are either commercially available or prepared via simple, established protocols. We choose as our model enzyme the hydrogenase I from E. coli (Hyd-1), which is an active catalyst for H2 oxidation, is relatively robust and has been demonstrated in H2 fuel cells and H2-driven chemical synthesis. The carbon materials were characterised according to their surface area, surface morphology and graphitic character, and we use the electrocatalytic H2 oxidation current for Hyd-1 adsorbed on these materials to evaluate their effectiveness as enzyme electrodes. Here, we show that a variety of carbon materials are suitable for adsorbing hydrogenases in an electroactive configuration. This unified study provides insight into selection and design of carbon materials for study of redox enzymes and different applications of enzyme electrocatalysis.

  17. Photosensitized Production of Hydrogen by Hydrogenase in Reversed Micelles

    NASA Astrophysics Data System (ADS)

    Hilhorst, Riet; Laane, Colja; Veeger, Cees

    1982-06-01

    Hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from Desulfovibrio vulgaris was encapsulated in reversed micelles with cetyltrimethylammonium bromide as surfactant and a chloroform/octane mixture as solvent. Reducing equivalents for hydrogenase-catalyzed hydrogen production were provided by vectorial photosensitized electron transfer from a donor (thiophenol) in the organic phase through a surfactant-Ru2+ sensitizer located in the interphase to methyl viologen concentrated in the aqueous core of the reversed micelle. The results show that reversed micelles provide a microenvironment that (i) stabilizes hydrogenase against inactivation and (ii) allows an efficient vectorial photosensitized electron and proton flow from the organic phase to hydrogenase in the aqueous phase.

  18. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    SciTech Connect

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    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, 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.

  19. Hydrogenases in sulfate-reducing bacteria function as chromium reductase.

    PubMed

    Chardin, B; Giudici-Orticoni, M-T; De Luca, G; Guigliarelli, B; Bruschi, M

    2003-12-01

    The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H(2)S produced by the bacteria, and enzymatically, by polyhemic cytochromes c(3). We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K(m) towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.

  20. Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk.

    PubMed

    Waters, Brian M; McInturf, Samuel A; Amundsen, Keenan

    2014-09-01

    Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu-regulated genes and stimulated Cu accumulation. The C940-fe (fefe) Fe-uptake mutant of melon (Cucumis melo) was characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild-type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe-regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu-deficient plants was independent of the normal Fe-uptake system. One of the four FRO genes in the melon and cucumber (Cucumis sativus) genomes was Fe-regulated, and one was Cu-regulated. Simultaneous Fe and Cu deficiency synergistically up-regulated Fe-uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe-Cu crosstalk in metal homeostasis. The fefe gene is not orthologous to FIT, and thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants.

  1. Electron microscopy of nickel-containing methanogenic enzymes: methyl reductase and F420-reducing hydrogenase.

    PubMed Central

    Wackett, L P; Hartwieg, E A; King, J A; Orme-Johnson, W H; Walsh, C T

    1987-01-01

    Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum delta H have been determined by electron microscopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, indicating a holoenzyme molecular weight of 8.0 X 10(5). Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis. Images PMID:3804976

  2. Modeling the active site of [NiFe] hydrogenases and the [NiFeu] subsite of the C-cluster of carbon monoxide dehydrogenases: low-spin iron(II) versus high-spin iron(II).

    PubMed

    Weber, Katharina; Erdem, Özlen F; Bill, Eckhard; Weyhermüller, Thomas; Lubitz, Wolfgang

    2014-06-16

    A series of four [S2Ni(μ-S)2FeCp*Cl] compounds with different tetradentate thiolate/thioether ligands bound to the Ni(II) ion is reported (Cp* = C5Me5). The {S2Ni(μ-S)2Fe} core of these compounds resembles structural features of the active site of [NiFe] hydrogenases. Detailed analyses of the electronic structures of these compounds by Mössbauer and electron paramagnetic resonance spectroscopy, magnetic measurements, and density functional theory calculations reveal the oxidation states Ni(II) low spin and Fe(II) high spin for the metal ions. The same electronic configurations have been suggested for the Cred1 state of the C-cluster [NiFeu] subsite in carbon monoxide dehydrogenases (CODH). The Ni-Fe distance of ∼3 Å excludes a metal-metal bond between nickel and iron, which is in agreement with the computational results. Electrochemical experiments show that iron is the redox active site in these complexes, performing a reversible one-electron oxidation. The four complexes are discussed with regard to their similarities and differences both to the [NiFe] hydrogenases and the C-cluster of Ni-containing CODH.

  3. Fe@Fe2O3 promoted electrochemical mineralization of atrazine via a triazinon ring opening mechanism.

    PubMed

    Ding, Xing; Wang, Shengyao; Shen, Wanqiu; Mu, Yi; Wang, Li; Chen, Hao; Zhang, Lizhi

    2017-04-01

    In this study, an electrochemical/electro-Fenton oxidation (EC/EF) system was designed to degrade atrazine, by utilizing boron-doped diamond (BDD) and Fe@Fe2O3 core-shell nanowires loaded active carbon fiber (Fe@Fe2O3/ACF) as the anode and the cathode, respectively. This EC/EF system exhibited much higher degradation rate, decholorination and mineralization efficiency of atrazine than the electrochemical (EC) and electrochemical/traditional electro-Fenton (EC/TEF) oxidation counterpart systems without Fe@Fe2O3 core-shell nanowires. Active species trapping experiment revealed that Fe@Fe2O3 could activate molecular oxygen to produce more OH through Fenton reaction, which favored the atrazine degradation. High performance liquid chromatography, high performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry were applied to probe the decomposition and mineralization of atrazine during this novel EC/EF process, which revealed that two intermediates of triazinons (the isomerization of hydroxylated atrazine) were generated during the electrochemical/electro-Fenton oxidation of atrazine in the presence of Fe@Fe2O3 core-shell nanowires. The experimental and theoretical calculation results suggested that atrazine might be degraded via a triazinon ring opening mechanism, while the presence of Fe@Fe2O3 notably accelerated the decholorination process, and produced more hydroxylated products to promote the generation of trazinons and the subsequent ring cleavage as well as the final complete mineralization. This work provides a deep insight into the triazine ring opening mechanism and the design of efficient electrochemical advanced oxidation technologies (EAOTs) for persistent organic pollutant removal.

  4. Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii.

    PubMed

    Sawyer, Anne; Bai, Yu; Lu, Yinghua; Hemschemeier, Anja; Happe, Thomas

    2017-03-13

    Molecular hydrogen (H2 ) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H2 production. The transformants expressing HYDA1 from the chloroplast genome displayed H2 production levels comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H2 -producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression. This article is protected by copyright. All rights reserved.

  5. Solar powered biohydrogen production requires specific localization of the hydrogenase

    SciTech Connect

    Burroughs, Nigel J.; Boehm, Marko; Eckert, Carrie; Mastroianni, Giulia; Spence, Edward M.; Yu, Jianfeng; Nixon, Peter J.; Appel, Jens; Mullineaux, Conrad W.; Bryan, Samantha J.

    2014-09-04

    Cyanobacteria contain a bidirectional [NiFe] hydrogenase which transiently produces hydrogen upon exposure of anoxic cells to light, potentially acting as a “valve” releasing excess electrons from the electron transport chain. However, its interaction with the photosynthetic electron transport chain remains unclear. By GFP-tagging the HoxF diaphorase subunit we show that the hydrogenase is thylakoid associated, comprising a population dispersed uniformly through the thylakoids and a subpopulation localized to discrete puncta in the distal thylakoid. Thylakoid localisation of both the HoxH and HoxY hydrogenase subunits is confirmed by immunogold electron microscopy. The diaphorase HoxE subunit is essential for recruitment to the dispersed thylakoid population, potentially anchoring the hydrogenase to the membrane, but aggregation to puncta occurs through a distinct HoxE-independent mechanism. Membrane association does not require NDH-1. Localization is dynamic on a scale of minutes, with anoxia and high light inducing a significant redistribution between these populations in favour of puncta. Lastly, since HoxE is essential for access to its electron donor, electron supply to the hydrogenase depends on a physiologically controlled localization, potentially offering a new avenue to enhance photosynthetic hydrogen production by exploiting localization/aggregation signals.

  6. Solar powered biohydrogen production requires specific localization of the hydrogenase

    DOE PAGES

    Burroughs, Nigel J.; Boehm, Marko; Eckert, Carrie; ...

    2014-09-04

    Cyanobacteria contain a bidirectional [NiFe] hydrogenase which transiently produces hydrogen upon exposure of anoxic cells to light, potentially acting as a “valve” releasing excess electrons from the electron transport chain. However, its interaction with the photosynthetic electron transport chain remains unclear. By GFP-tagging the HoxF diaphorase subunit we show that the hydrogenase is thylakoid associated, comprising a population dispersed uniformly through the thylakoids and a subpopulation localized to discrete puncta in the distal thylakoid. Thylakoid localisation of both the HoxH and HoxY hydrogenase subunits is confirmed by immunogold electron microscopy. The diaphorase HoxE subunit is essential for recruitment to themore » dispersed thylakoid population, potentially anchoring the hydrogenase to the membrane, but aggregation to puncta occurs through a distinct HoxE-independent mechanism. Membrane association does not require NDH-1. Localization is dynamic on a scale of minutes, with anoxia and high light inducing a significant redistribution between these populations in favour of puncta. Lastly, since HoxE is essential for access to its electron donor, electron supply to the hydrogenase depends on a physiologically controlled localization, potentially offering a new avenue to enhance photosynthetic hydrogen production by exploiting localization/aggregation signals.« less

  7. Oxygen-tolerant hydrogenases in hydrogen-based technologies.

    PubMed

    Friedrich, Bärbel; Fritsch, Johannes; Lenz, Oliver

    2011-06-01

    To develop a viable H2 technology, production of H2 has to be significantly enlarged by using renewable resources. One option of generating H2 is the photosynthetic conversion of sunlight and water directly to H2 and O2. Photosystems and hydrogenases are currently being exploited for the design of efficient H2-producing systems that require highly active and O2-tolerant biocatalysts. This communication focuses on two challenging features: hydrogenases that produce H2 in the presence of O2, and direct electron transfer between photosystem I (PS I) and hydrogenase. The latter is accomplished by connecting both modules through a protein fusion or a synthetic molecular wire. These are first steps toward a photosynthetic microbial cell or a semi-synthetic system that may be employed in future H2-based technologies.

  8. Distribution and activity of hydrogenase enzymes in subsurface sediments

    NASA Astrophysics Data System (ADS)

    Adhikari, R.; Nickel, J.; Glombitza, C.; Spivack, A. J.; D'Hondt, S. L.; Kallmeyer, J.

    2013-12-01

    Metabolically active microbial communities are present in a wide range of subsurface environments. Techniques like enumeration of microbial cells, activity measurements with radiotracer assays and the analysis of porewater constituents are currently being used to explore the subsurface biosphere, alongside with molecular biological analyses. However, many of these techniques reach their detection limits due to low microbial activity and abundance. Direct measurements of microbial turnover not just face issues of insufficient sensitivity, they only provide information about a single specific process rather than an overall microbial activity. Since hydrogenase enzymes are intracellular and ubiquitous in subsurface microbial communities, the enzyme activity represents a measure of total activity of the entire microbial community. A hydrogenase activity assay could quantify total metabolic activity without having to identify specific processes. This would be a major advantage in subsurface biosphere studies, where several metabolic processes can occur simultaneously. We quantified hydrogenase enzyme activity and distribution in sediment samples from different aquatic subsurface environments (Lake Van, Barents Sea, Equatorial Pacific and Gulf of Mexico) using a tritium-based assay. We found enzyme activity at all sites and depths. Volumetric hydrogenase activity did not show much variability between sites and sampling depths, whereas cell-specific activity ranged from 10-5 to 1 nmol H2 cell-1 d-1. Activity was lowest in sediment layers where nitrate was detected. Higher activity was associated with samples in which sulfate was the predominant electron acceptor. We found highest activity in samples from environments with >10 ppm methane in the pore water. The results show that cell-specific hydrogenase enzyme activity increases with decreasing energy yield of the electron acceptor used. It is not possible to convert volumetric or cell-specific hydrogenase activity into a

  9. Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum.

    PubMed Central

    Minamisawa, K; Fukai, K; Asami, T

    1990-01-01

    Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein. Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression. Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression. The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine. These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B. japonicum. Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell. Images PMID:2198262

  10. The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

    PubMed Central

    Sayavedra-Soto, L A; Arp, D J

    1992-01-01

    The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain. Images PMID:1644756

  11. The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein.

    PubMed Central

    Maier, T; Jacobi, A; Sauter, M; Böck, A

    1993-01-01

    The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released. Images PMID:8423137

  12. From enzyme maturation to synthetic chemistry: the case of hydrogenases.

    PubMed

    Artero, Vincent; Berggren, Gustav; Atta, Mohamed; Caserta, Giorgio; Roy, Souvik; Pecqueur, Ludovic; Fontecave, Marc

    2015-08-18

    Water splitting into oxygen and hydrogen is one of the most attractive strategies for storing solar energy and electricity. Because the processes at work are multielectronic, there is a crucial need for efficient and stable catalysts, which in addition have to be cheap for future industrial developments (electrolyzers, photoelectrochemicals, and fuel cells). Specifically for the water/hydrogen interconversion, Nature is an exquisite source of inspiration since this chemistry contributes to the bioenergetic metabolism of a number of living organisms via the activity of fascinating metalloenzymes, the hydrogenases. In this Account, we first briefly describe the structure of the unique dinuclear organometallic active sites of the two classes of hydrogenases as well as the complex protein machineries involved in their biosynthesis, their so-called maturation processes. This knowledge allows for the development of a fruitful bioinspired chemistry approach, which has already led to a number of interesting and original catalysts mimicking the natural active sites. More specifically, we describe our own attempts to prepare artificial hydrogenases. This can be achieved via the standard bioinspired approach using the combination of a synthetic bioinspired catalyst and a polypeptide scaffold. Such hybrid complexes provide the opportunity to optimize the system by manipulating both the catalyst through chemical synthesis and the protein component through mutagenesis. We also raise the possibility to reach such artificial systems via an original strategy based on mimicking the enzyme maturation pathways. This is illustrated in this Account by two examples developed in our laboratory. First, we show how the preparation of a lysozyme-{Mn(I)(CO)3} hybrid and its clean reaction with a nickel complex led us to generate a new class of binuclear Ni-Mn H2-evolving catalysts mimicking the active site of [NiFe]-hydrogenases. Then we describe how we were able to rationally design and

  13. Rates and Routes of Electron Transfer of [NiFe]-Hydrogenase in an Enzymatic Fuel Cell.

    PubMed

    Petrenko, Alexander; Stein, Matthias

    2015-10-29

    Hydrogenase enzymes are being used in enzymatic fuel cells immobilized on a graphite or carbon electrode surface, for example. The enzyme is used for the anodic oxidation of molecular hydrogen (H2) to produce protons and electrons. The association and orientation of the enzyme at the anode electrode for a direct electron transfer is not completely resolved. The distal FeS-cluster in [NiFe]-hydrogenases contains a histidine residue which is known to play a critical role in the intermolecular electron transfer between the enzyme and the electrode surface. The [NiFe]-hydrogenase graphite electrode association was investigated using Brownian Dynamics simulations. Residues that were shown to be in proximity to the electrode surface were identified (His184, Ser196, Glu461, Glu464), and electron transfer routes connecting the distal FeS-cluster with the surface residues were investigated. Several possible pathways for electron transfer between the distal FeS-cluster and the terminal amino acid residues were probed in terms of their rates of electron transfer using DFT methods. The reorganization energies λ of the distal iron-sulfur cluster and coronene as a molecular model for graphite were calculated. The reorganization energy of the distal (His)(Cys)3 cluster was found to be not very different from that of a standard cubane clusters with a (Cys)4 coordination. Electronic coupling matrix elements and rates of electron transfer for the different pathways were calculated according to the Marcus equation. The rates for glutamate-mediated electrode binding were found to be incompatible with experimental data. A direct electron transfer from the histidine ligand of the distal FeS-cluster to the electrode yielded rates of electron transfer in excellent agreement with experiment. A second pathway, however, from the distal FeS-cluster to the Ser196 residue was found to be equally efficient and feasible.

  14. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre.

    PubMed

    Fritsch, Johannes; Scheerer, Patrick; Frielingsdorf, Stefan; Kroschinsky, Sebastian; Friedrich, Bärbel; Lenz, Oliver; Spahn, Christian M T

    2011-10-16

    Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(2)-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H(2) oxidation and as an electron-delivering device upon O(2) attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H(2)-converting catalysts that are capable of cycling H(2) in air.

  15. Guiding Principles of Hydrogenase Catalysis Instigated and Clarified by Protein Film Electrochemistry.

    PubMed

    Armstrong, Fraser A; Evans, Rhiannon M; Hexter, Suzannah V; Murphy, Bonnie J; Roessler, Maxie M; Wulff, Philip

    2016-05-17

    Protein film electrochemistry (PFE) is providing cutting-edge insight into the chemical principles underpinning biological hydrogen. Attached to an electrode, many enzymes exhibit "reversible" electrocatalytic behavior, meaning that a catalyzed redox reaction appears reversible or quasi-reversible when viewed by cyclic voltammetry. This efficiency is most relevant for enzymes that are inspiring advances in renewable energy, such as hydrogen-activating and CO2-reducing enzymes. Exploiting the rich repertoire of available instrumental methods, PFE experiments yield both a general snapshot and fine detail, all from tiny samples of enzyme. The dynamic electrochemical investigations blaze new trails and add exquisite detail to the information gained from structural and spectroscopic studies. This Account describes recent investigations of hydrogenases carried out in Oxford, including ideas initiated with PFE and followed through with complementary techniques, all contributing to an eventual complete picture of fast and efficient H2 activation without Pt. By immobilization of an enzyme on an electrode, catalytic electron flow and the chemistry controlling it can be addressed at the touch of a button. The buried nature of the active site means that structures that have been determined by crystallography or spectroscopy are likely to be protected, retained, and fully relevant in a PFE experiment. An electrocatalysis model formulated for the PFE of immobilized enzymes predicts interesting behavior and gives insight into why some hydrogenases are H2 producers and others are H2 oxidizers. Immobilization also allows for easy addition and removal of inhibitors along with precise potential control, one interesting outcome being that formaldehyde forms a reversible complex with reduced [FeFe]-hydrogenases, thereby providing insight into the order of electron and proton transfers. Experiments on O2-tolerant [NiFe]-hydrogenases show that O2 behaves like a reversible inhibitor: it

  16. Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme.

    PubMed Central

    Sawers, R G; Ballantine, S P; Boxer, D H

    1985-01-01

    The cellular contents of the nickel-containing, membrane-bound hydrogenase isoenzymes 1 and 2 (hydrogenases 1 and 2) were analyzed by crossed immunoelectrophoresis. Their expression was differentially influenced by nutritional and genetic factors. Hydrogenase 2 content was enhanced after growth with either hydrogen and fumarate or glycerol and fumarate and correlated reasonably with cellular hydrogen uptake capacity. Hydrogenase 1 content was negligible under the above conditions but was enhanced by exogenous formate. Its expression was greatly reduced in a pfl mutant, which is unable to synthesise formate, but was restored to normal levels when the growth medium included formate. A mutation in the anaerobic regulatory gene, fnr, led to low overall hydrogenase activity and greatly reduced levels of both isoenzymes and abolished the formate enhancement of hydrogenase 1 content. Formate hydrogenlyase activity was similarly reduced in the fnr strain but, in contrast, was restored, as was overall hydrogenase activity, to normal levels by growth in the presence of formate. Low H2 uptake activity was found for the fnr strain under all growth conditions examined. Hydrogenase 1 content, therefore, does not correlate with formate hydrogenlyase activity and its role is unclear. A third hydrogenase isoenzyme, immunologically distinct from hydrogenases 1 and 2, whose expression is enhanced by formate, is present and forms part of the formate hydrogenlyase. We suggest that the effect of the fnr gene product on formate hydrogenlyase expression is mediated via internal formate. Images PMID:3905769

  17. Nanocrystalline Fe-Fe2O3 particle-deposited N-doped graphene as an activity-modulated Pt-free electrocatalyst for oxygen reduction reaction

    NASA Astrophysics Data System (ADS)

    Dhavale, Vishal M.; Singh, Santosh K.; Nadeema, Ayasha; Gaikwad, Sachin S.; Kurungot, Sreekumar

    2015-11-01

    The size-controlled growth of nanocrystalline Fe-Fe2O3 particles (2-3 nm) and their concomitant dispersion on N-doped graphene (Fe-Fe2O3/NGr) could be attained when the mutually assisted redox reaction between NGr and Fe3+ ions could be controlled within the aqueous droplets of a water-in-oil emulsion. The synergistic interaction existing between Fe-Fe2O3 and NGr helped the system to narrow down the overpotential for the oxygen reduction reaction (ORR) by bringing a significant positive shift to the reduction onset potential, which is just 15 mV higher than its Pt-counterpart. In addition, the half-wave potential (E1/2) of Fe-Fe2O3/NGr is found to be improved by a considerable amount of 135 mV in comparison to the system formed by dispersing Fe-Fe2O3 nanoparticles on reduced graphene oxide (Fe-Fe2O3/RGO), which indicates the presence of a higher number of active sites in Fe-Fe2O3/NGr. Despite this, the ORR kinetics of Fe-Fe2O3/NGr are found to be shifted significantly to the preferred 4-electron-transfer pathway compared to NGr and Fe-Fe2O3/RGO. Consequently, the H2O2% was found to be reduced by 78.3% for Fe-Fe2O3/NGr (13.0%) in comparison to Fe-Fe2O3/RGO (51.2%) and NGr (41.0%) at -0.30 V (vs. Hg/HgO). This difference in the yield of H2O2 formed between the systems along with the improvements observed in terms of the oxygen reduction onset and E1/2 in the case of Fe-Fe2O3/NGr reveals the activity modulation achieved for the latter is due to the coexistence of factors such as the presence of the mixed valancies of iron nanoparticles, small size and homogeneous distribution of Fe-Fe2O3 nanoparticles and the electronic modifications induced by the doped nitrogen in NGr. A controlled interplay of these factors looks like worked favorably in the case of Fe-Fe2O3/NGr. As a realistic system level validation, Fe-Fe2O3/NGr was employed as the cathode electrode of a single cell in a solid alkaline electrolyte membrane fuel cell (AEMFC). The system could display an open

  18. Anoxic and oxic removal of humic acids with Fe@Fe2O3 core-shell nanowires: a comparative study.

    PubMed

    Wu, Hao; Ai, Zhihui; Zhang, Lizhi

    2014-04-01

    In this study we comparatively investigate the removal of humic acids with Fe@Fe2O3 core-shell nanowires under anoxic and oxic conditions. The products of humic acids after reacting with Fe@Fe2O3 core-shell nanowires under anoxic and oxic conditions were carefully examined with three-dimensional excitation emission matrix fluorescence spectroscopy and gas chromatography mass spectrometry. It was found that humic acids were removed by Fe@Fe2O3 core-shell nanowires via adsorption under anoxic condition. Langmuir adsorption isotherm was applicable to describe the adsorption processes. Kinetics of humic acids adsorption onto Fe@Fe2O3 core-shell nanowires was found to follow pseudo-second-order rate equation. By contrast, the oxic removal of humic acids with Fe@Fe2O3 core-shell nanowires involved adsorption and subsequent oxidation of humic acids because Fe@Fe2O3 core-shell nanowires could activate molecular oxygen to produce reactive oxygen species to oxidize humic acids. This subsequent oxidation of humic acids could improve the oxic removal rate to 2.5 times that of anoxic removal, accompanying with about 8.4% of mineralization. This study provides a new method for humic acids removal and also sheds light on the effects of humic acids on the pollutant removal by nano zero-valent iron.

  19. Force-field development and molecular dynamics simulations of ferrocene-peptide conjugates as a scaffold for hydrogenase mimics

    SciTech Connect

    De Hatten, Xavier; Cournia, Zoe; Smith, Jeremy C; Huc, I; Metzler-Nolte, Nils

    2007-08-01

    The increasing importance of hydrogenase enzymes in the new energy research field has led us to examine the structure and dynamics of potential hydrogenase mimics, based on a ferrocene-peptide scaffold, using molecular dynamics (MD) simulations. To enable this MD study, a molecular mechanics force field for ferrocene-bearing peptides was developed and implemented in the CHARMM simulation package, thus extending the usefulness of the package into peptide-bioorganometallic chemistry. Using the automated frequency-matching method (AFMM), optimized intramolecular force-field parameters were generated through quantum chemical reference normal modes. The partial charges for ferrocene were derived by fitting point charges to quantum-chemically computed electrostatic potentials. The force field was tested against experimental X-ray crystal structures of dipeptide derivatives of ferrocene-1,1'-dicarboxylic acid. The calculations reproduce accurately the molecular geometries, including the characteristic C{sub 2}-symmetrical intramolecular hydrogen-bonding pattern, that were stable over 0.1 {micro}s MD simulations. The crystal packing properties of ferrocene-1-(D)alanine-(D)proline-1'-(D)alanine-(D)proline were also accurately reproduced. The lattice parameters of this crystal were conserved during a 0.1 {micro}s MD simulation and match the experimental values almost exactly. Simulations of the peptides in dichloromethane are also in good agreement with experimental NMR and circular dichroism (CD) data in solution. The developed force field was used to perform MD simulations on novel, as yet unsynthesized peptide fragments that surround the active site of [Ni-Fe] hydrogenase. The results of this simulation lead us to propose an improved design for synthetic peptide-based hydrogenase models. The presented MD simulation results of metallocenes thereby provide a convincing validation of our proposal to use ferrocene-peptides as minimal enzyme mimics.

  20. Force-field development and molecular dynamics simulations of ferrocene-peptide conjugates as a scaffold for hydrogenase mimics.

    SciTech Connect

    De Hatten, Xavier; Cournia, Zoe; Smith, Jeremy C; Metzler-Nolte, Nils

    2007-08-01

    The increasing importance of hydrogenase enzymes in the new energy research field has led us to examine the structure and dynamics of potential hydrogenase mimics, based on a ferrocene-peptide scaffold, using molecular dynamics (MD) simulations. To enable this MD study, a molecular mechanics force field for ferrocene-bearing peptides was developed and implemented in the CHARMM simulation package, thus extending the usefulness of the package into peptide-bioorganometallic chemistry. Using the automated frequency-matching method (AFMM), optimized intramolecular force-field parameters were generated through quantum chemical reference normal modes. The partial charges for ferrocene were derived by fitting point charges to quantum-chemically computed electrostatic potentials. The force field was tested against experimental X-ray crystal structures of dipeptide derivatives of ferrocene-1,1{prime}-dicarboxylic acid. The calculations reproduce accurately the molecular geometries, including the characteristic C2-symmetrical intramolecular hydrogen-bonding pattern, that were stable over 0.1{micro}s MD simulations. The crystal packing properties of ferrocene-1-(D)alanine-(D)proline{prime}-1-(D)alanine-(D)proline were also accurately reproduced. The lattice parameters of this crystal were conserved during a 0.1 s MD simulation and match the experimental values almost exactly. Simulations of the peptides in dichloromethane are also in good agreement with experimental NMR and circular dichroism (CD) data in solution. The developed force field was used to perform MD simulations on novel, as yet unsynthesized peptide fragments that surround the active site of [Ni-Fe] hydrogenase. The results of this simulation lead us to propose an improved design for synthetic peptide-based hydrogenase models. The presented MD simulation results of metallocenes thereby provide a convincing validation of our proposal to use ferrocene-peptides as minimal enzyme mimics.

  1. Identification of a catalytic iron-hydride at the H-cluster of [FeFe]-hydrogenase

    SciTech Connect

    Mulder, David W.; Guo, Yisong; Ratzloff, Michael W.; King, Paul W.

    2016-12-14

    Hydrogenases couple electrochemical potential to the reversible chemical transformation of H2 and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (Hhyd) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]H) with CO and CN- ligands. Mossbauer analysis and density functional theory (DFT) calculations show that Hhyd consists of a reduced [4Fe-4S]H+ coupled to a diferrous [2Fe]H with a terminally bound Fe-hydride. The existence of the Fe-hydride in Hhyd was demonstrated by an unusually low Mossbauer isomer shift of the distal Fe of the [2Fe]H subcluster. As a result, a DFT model of Hhyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.

  2. Identification of a catalytic iron-hydride at the H-cluster of [FeFe]-hydrogenase

    DOE PAGES

    Mulder, David W.; Guo, Yisong; Ratzloff, Michael W.; ...

    2016-12-14

    Hydrogenases couple electrochemical potential to the reversible chemical transformation of H2 and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (Hhyd) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]H) with CO and CN- ligands. Mossbauer analysis and density functional theory (DFT) calculations show that Hhyd consists of a reduced [4Fe-4S]H+ coupled to a diferrous [2Fe]H with a terminally bound Fe-hydride. The existence ofmore » the Fe-hydride in Hhyd was demonstrated by an unusually low Mossbauer isomer shift of the distal Fe of the [2Fe]H subcluster. As a result, a DFT model of Hhyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.« less

  3. Chemical synthesis of Fe/Fe3O4 core-shell composites with enhanced soft magnetic performances

    NASA Astrophysics Data System (ADS)

    Yang, Bai; Li, Xiaopan; Yang, Xueying; Yu, Ronghai

    2017-04-01

    The large-grain Fe/Fe3O4 composite particles with average size of about 1.2 μm have been fabricated by a facile one-step solvothermal method. The formation of high-purity Fe3O4 as the shells (90.14 wt%) and α-Fe as the cores (9.86 wt%) in the Fe/Fe3O4 composites leads to their high saturation magnetization of 119.6 A m2 Kg-1. Very low coercivity of 30 Oe is obtained in the composites due to their uniform cubic-shaped morphologies. Compared with Fe-based nanosized particles, these micron-sized magnetic Fe/Fe3O4 composites exhibit high air stability and good compactibility with high compressed density of 5.9 g cm-3. The fully compacted sample shows good soft magnetic properties including high magnetic induction B1.2k (H=1200 A/m) of 540 mT and good frequency-dependent magnetic properties with operating frequency up to 50 MHz superior to those of the most traditional soft magnetic ferrites, which promotes their potential applications in high-frequency and high-power magnetic devices.

  4. Isotopic fractionation associated with [NiFe]- and [FeFe]-hydrogenases

    SciTech Connect

    Yang, Hui; Gandhi, Hasand; Cornish, Adam J.; Moran, James J.; Kreuzer, Helen W.; Ostrom, Nathaniel; Hegg, Eric L.

    2016-01-30

    Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. In this manuscript, we used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27-0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41-0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing us with a more complete picture of the three reactions catalyzed by hydrogenases. The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.

  5. Oxygen-resistant hydrogenases and methods for designing and making same

    DOEpatents

    King, Paul; Ghirardi, Maria L; Seibert, Michael

    2009-03-10

    The invention provides oxygen- resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H.sub.2 in a light catalyzed reaction having water as the reactant.

  6. Oxygen-resistant hydrogenases and methods for designing and making same

    DOEpatents

    King, Paul; Ghirardi, Maria Lucia; Seibert, Michael

    2014-03-04

    The invention provides oxygen-resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H.sub.2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H.sub.2 in a light catalyzed reaction having water as the reactant.

  7. Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic

    PubMed Central

    Esselborn, Julian; Berggren, Gustav; Noth, Jens; Siebel, Judith; Hemschemeier, Anja; Artero, Vincent; Reijerse, Edward; Fontecave, Marc; Lubitz, Wolfgang; Happe, Thomas

    2013-01-01

    Hydrogenases catalyze the formation of hydrogen. The cofactor (H-cluster) of [FeFe]-hydrogenases consists of a [4Fe-4S]-cluster bridged to a unique [2Fe]-subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe]-subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing novel artificial H2-producing catalysts. PMID:23934246

  8. Selenium makes the difference: protonation of [FeFe]-hydrogenase mimics with diselenolato ligands.

    PubMed

    Abul-Futouh, Hassan; El-Khateeb, Mohammad; Görls, Helmar; Asali, Khalil Jamil; Weigand, Wolfgang

    2017-02-28

    The synthetic models of the active site of an [FeFe]-hydrogenase containing a Sn atom in the bridgehead of the diselenato ligand, namely [Fe2(CO)6{μ-(SeCH2Se)SnMe2}], 3 and [Fe2(CO)6{μ-(SeCH2)2SnMe2}], 4 have been synthesized and characterized by different spectroscopic methods. The protonation properties of complex 4 have been investigated by monitoring the IR spectra in the carbonyl stretching region, (1)H NMR in the hydride region as well as the (77)Se{H} NMR upon addition of strong and moderate acids wherein the protonation of the active site of the [FeFe]-hydrogenase at one of its internal basic sites is considered an essential step in the catalytic cycle. Furthermore, we investigated the redox properties and the catalytic behaviour of complexes 3 and 4 in the presence of AcOH as a source of protons suggesting an ECE (E = electrochemical process, C = chemical process) mechanism.

  9. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

    PubMed Central

    Wu, Chang-Hao; McTernan, Patrick M.; Walter, Mary E.; Adams, Michael W. W.

    2015-01-01

    Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed. PMID:26543406

  10. Melting experiments on Fe-Fe3S system to 254 GPa

    NASA Astrophysics Data System (ADS)

    Mori, Yuko; Ozawa, Haruka; Hirose, Kei; Sinmyo, Ryosuke; Tateno, Shigehiko; Morard, Guillaume; Ohishi, Yasuo

    2017-04-01

    Melting experiments were performed on the Fe-Fe3S system at high pressures between 34 and 254 GPa in a laser-heated diamond-anvil cell (DAC), using starting materials of fine-grained homogeneous mixtures of Fe and FeS (<500 nm) prepared by induction melting and rapid quenching techniques. Melting phase relations including the liquid/solid partitioning of sulfur were examined on the basis of textural and chemical characterizations of recovered samples using a focused ion beam (FIB) and electron microprobes. The results demonstrate that the sulfur content in eutectic liquid decreases substantially with increasing pressure. The eutectic liquid Fe with 5.7 (± 0.3) wt.% S coexisted with both solid Fe3S and Fe containing 3.9 (± 0.4) wt.% S at 254 GPa and 3550 K. The eutectic liquid at inner core boundary (ICB) pressure includes less sulfur than is required to account for the density deficit of the outer core (≥10 wt.% S). Furthermore, the difference in sulfur concentration between coexisting liquid and solid is not sufficient to account for the observed density jump across the ICB. These indicate that sulfur cannot be a predominant light element in the core.

  11. De novo design of functional proteins: Toward artificial hydrogenases.

    PubMed

    Faiella, Marina; Roy, Anindya; Sommer, Dayn; Ghirlanda, Giovanna

    2013-11-01

    Over the last 25 years, de novo design has proven to be a valid approach to generate novel, well-folded proteins, and most recently, functional proteins. In response to societal needs, this approach is been used increasingly to design functional proteins developed with an eye toward sustainable fuel production. This review surveys recent examples of bioinspired de novo designed peptide based catalysts, focusing in particular on artificial hydrogenases.

  12. Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism.

    PubMed Central

    van den Berg, W A; van Dongen, W M; Veeger, C

    1991-01-01

    To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed. Images PMID:1711025

  13. Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting.

    PubMed

    Mersch, Dirk; Lee, Chong-Yong; Zhang, Jenny Zhenqi; Brinkert, Katharina; Fontecilla-Camps, Juan C; Rutherford, A William; Reisner, Erwin

    2015-07-08

    In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions.

  14. EPR Spectroscopic Studies of [FeFe]-Hydrogenase Maturation.

    PubMed

    Suess, Daniel L M; Britt, R David

    2015-09-01

    Proton reduction and H2 oxidation are key elementary reactions for solar fuel production. Hydrogenases interconvert H(+) and H2 with remarkable efficiency and have therefore received much attention in this context. For [FeFe]-hydrogenases, catalysis occurs at a unique cofactor called the H-cluster. In this article, we discuss ways in which EPR spectroscopy has elucidated aspects of the bioassembly of the H-cluster, with a focus on four case studies: EPR spectroscopic identification of a radical en route to the CO and CN(-) ligands of the H-cluster, tracing (57)Fe from the maturase HydG into the H-cluster, characterization of the auxiliary Fe-S cluster in HydG, and isotopic labeling of the CN(-) ligands of HydA for electronic structure studies of its Hox state. Advances in cell-free maturation protocols have enabled several of these mechanistic studies, and understanding H-cluster maturation may in turn provide insights leading to improvements in hydrogenase production for biotechnological applications.

  15. Investigation of Fe-FeS phase diagram and liquid structure at high pressure and high temperature

    NASA Astrophysics Data System (ADS)

    Morard, G.; Sanloup, C.; Fiquet, G.; Mezouar, M.; Andrault, D.; Guignot, N.

    2007-12-01

    Sulfur is believed to be an alloying light element in iron-rich planetary cores such as those of the Earth and Mars 1, 2. Recent studies have suggested that Mars, like the Earth, could have a liquid metallic outer core together with a solid inner core 3. Hence, it is important to investigate the evolution of the Fe-FeS phase diagram and of the structural properties of the liquid Fe-FeS alloys in respect to pressure, temperature and sulphur content. A new cell assembly has been developed to heat samples to more than 1300 K at 17 GPa using the Paris Edinburgh Press4. This allows us to conduct detailed structural investigations of the Fe-FeS eutectic liquid by in situ X-ray diffraction5 . Analysis of these data highlights an increase of the liquid compacity with increasing pressure. We also show that the eutectic liquid structure is closer to that of FeSi, explaining the closure of the miscibility gap in the Fe-S-Si system 6. The evolution of the Fe-FeS eutectic liquid structure at high pressure could have significant effect on extrapolated wave speed of metallic Fe-FeS alloy at core pressures. We have used a double-sided laser-heated diamond-anvil cell 7 to study the Fe-FeS phase diagram up to 65 GPa and 2500 K8. We used laser heated diamond anvil cell coupled with synchrotron radiation and confirm a S- solubility below 4 at% (2.3 %wt) up to 65 GPa. The eutectic temperatures present a uniform increase, with a rate of ~15K/GPa, up to 65 GPa and 2200 K. Finally, we present new constraints on the phase diagram evolution to very high pressures which provide unambiguous evidence for an upper limit of 4-8 %wt for the inner core S- content. Therefore, sulphur is not favoured to be the major light element in the Earth's core. 1. Allegre, C. J., Poirier, J. P., Humler, E. & Hofmann, A. W. The chemical composition of the Earth. Earth Planet. Sc. Lett. 134, 515-526 (1995). 2. Sohl, F. & Spohn, T. The interior structure of Mars : Implications from SNC meteorites. J. Geophys. Res

  16. 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.

  17. Synthesis, characterization, and H/D exchange of μ-hydride-containing [FeFe]-hydrogenase subsite models formed by protonation reactions of (μ-TDT)Fe2(CO)4(PMe3)2 (TDT = SCH2SCH2S) with protic acids.

    PubMed

    Song, Li-Cheng; Zhu, An-Guo; Guo, Yuan-Qiang

    2016-03-28

    As [FeFe]-hydrogenase models, the first thiodithiolate (TDT) ligand-containing μ-hydride complexes [(μ-TDT)Fe2(CO)4(PMe3)2(μ-H)](+)Y(-) (2-7, Y = Cl, ClO4, PF6, BF4, CF3CO2, CF3SO3) have been prepared by protonation reactions of (μ-TDT)Fe2(CO)4(PMe3)2 (1) with the corresponding HY acids. While the protonation reactions are monitored by in situ(1)H and (31)P{(1)H} NMR spectroscopy to show the isomer type and stability of 2-7, the structures of the isolated 2-7 are characterized by elemental analysis, spectroscopy and for some of them by X-ray crystallography. Although the H/D exchange of μ-hydride complex 7 (Y = CF3SO3) with D2 or D2O has been proved not to occur under the studied conditions, the H/D exchange of 7 with DCl gives the μ-deuterium complex [(μ-TDT)Fe2(CO)4(PMe3)2(μ-D)](+)[CF3SO3](-) (8) in a nearly quantitative yield. To our knowledge, 8 is the first crystallographically characterized μ-deuterium-containing butterfly [2Fe2S] complex produced by H/D exchange reaction.

  18. Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits.

    PubMed Central

    Brito, B; Palacios, J M; Hidalgo, E; Imperial, J; Ruiz-Argüeso, T

    1994-01-01

    Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels. Images PMID:8071205

  19. Distribution Analysis of Hydrogenases in Surface Waters of Marine and Freshwater Environments

    PubMed Central

    Barz, Martin; Beimgraben, Christian; Staller, Torsten; Germer, Frauke; Opitz, Friederike; Marquardt, Claudia; Schwarz, Christoph; Gutekunst, Kirstin; Vanselow, Klaus Heinrich; Schmitz, Ruth; LaRoche, Julie; Schulz, Rüdiger; Appel, Jens

    2010-01-01

    Background Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism in marine environments is nitrogen fixation. Principal Findings We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H2 uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean. Significance This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H2-evolving

  20. Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase.

    PubMed

    Lo, Shou-Chen; Shih, Shau-Hua; Chang, Jui-Jen; Wang, Chun-Ying; Huang, Chieh-Chen

    2012-08-01

    The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 °C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 °C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.

  1. An [Fe] hydrogenase from the anaerobic hydrogenosome-containing fungus Neocallimastix frontalis L2.

    PubMed

    Davidson, Elizabeth A; van der Giezen, Mark; Horner, David S; Embley, T Martin; Howe, Christopher J

    2002-08-21

    Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic eukaryotes scattered throughout the eukaryotic tree. All of the eukaryotic enzymes characterized so far are iron-only [Fe] hydrogenases. In contrast, it has previously been suggested that hydrogenosomes of the best-studied anaerobic fungus Neocallimastix frontalis L2 contain an unrelated iron-nickel-selenium [NiFeSe] hydrogenase. We have isolated a gene from strain L2 that encodes a putative protein containing all of the characteristic features of an iron-only [Fe] hydrogenase, including the cysteine residues required for the co-ordination of the unique 'hydrogen cluster'. As is the case for experimentally verified hydrogenosomal matrix enzymes from N. frontalis, the [Fe] hydrogenase encodes a plausible amino terminal extension that resembles mitochondrial targeting signals. Phylogenetic analyses of an expanded [Fe] hydrogenase dataset reveal a complicated picture that is difficult to interpret in the light of current ideas of species relationships. Nevertheless, our analyses cannot reject the hypothesis that the novel [Fe] hydrogenase gene of Neocallimastix is specifically related to other eukaryote [Fe] hydrogenases, and thus ultimately might be traced to the same ancestral source.

  2. Enhanced Hydrogen Production by Co-cultures of Hydrogenase and Nitrogenase in Escherichia coli.

    PubMed

    Lee, Hyun Jeong; Sekhon, Simranjeet Singh; Kim, Young Su; Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2016-03-01

    Rhodobacter sphaeroides is a bacterium that can produce hydrogen by interaction with hydrogenase and nitrogenase. We report a hydrogen production system using co-cultivation of hydrogenase in liquid medium and immobilized nitrogenase in Escherichia coli. The recombinant plasmid has been constructed to analyze the effect of hydrogen production on the expression of hupSL hydrogenase and nifHDK nitrogenase isolated from R. sphaeroides. All recombinant E. coli strains were cultured anaerobically, and cells for nitrogenase were immobilized in agar gel, whereas cells for hydrogenase were supplemented on the nitrogenase agar gel. The hupSL hydrogenase has been observed to enhance hydrogen production and hydrogenase activity under co-culture with nifHDK nitrogenase. The maximum hydrogen production has been obtained at an agar gel concentration and a cell concentration for co-culture of 2 % and 6.4 × 10(8) CFU. Thus, co-culture of hupSL hydrogenase and nifHDK nitrogenase provides a promising route for enhancing the hydrogen production and hydrogenase activity.

  3. Evidence for a Third Uptake Hydrogenase Phenotype among the Soybean Bradyrhizobia

    PubMed Central

    van Berkum, Peter

    1990-01-01

    The existence of a hydrogen uptake host-regulated (Hup-hr) phenotype was established among the soybean bradyrhizobia. The Hup-hr phenotype is characterized by the expression of uptake hydrogenase activity in symbiosis with cowpea but not soybean. Uptake hydrogenase induction is not possible under free-living cultural conditions by using techniques developed for uptake hydrogenase-positive (Hup+) Bradyrhizobium japonicum. Hydrogen oxidation by Hup-hr phenotype USDA 61 in cowpea symbioses was significant because hydrogen evolution from nitrogen-fixing nodules was not detected. An examination for uptake hydrogenase activity in soybean and cowpea with 123 strains diverse in origin and serology identified 16 Hup+ and 28 Hup-hr phenotype strains; the remainder appeared to be Hup−. The Hup-hr phenotype was associated with serogroups 31, 76, and 94, while strains belonging to serogroups 6, 31, 110, 122, 123, and 38/115 were Hup+. Hup+ strains of the 123 serogroup typed positive with USDA 129-specific antiserum. The presence of the uptake hydrogenase protein in cowpea bacteroids of Hup+ strains was demonstrated with immunoblot analyses by using antibodies against the 65-kDa subunit of uptake hydrogenase purified from strain SR470. However, the hydrogenase protein of Hup-hr strains was not detected. Results of Southern hybridization analyses with pHU1 showed the region of DNA with hydrogenase genes among Hup+ strains to be similar. Hybridization was also obtained with Hup-hr strains by using a variety of cloned DNA as probes including hydrogenase structural genes. Both hydrogenase structural genes also hybridized with the DNA of four Hup− strains. Images PMID:16348383

  4. H, not O or pressure, causes eutectic T depression in the Fe-FeS System to 8 GPa

    NASA Astrophysics Data System (ADS)

    Buono, Antonio S.; Walker, David

    2015-04-01

    The Fe-FeS system maintains a eutectic temperature of 990 ± 10 °C to at least 8 GPa if starting materials and pressure media are rigorously dehydrated. Literature reports of pressure-induced freezing point depression of the eutectic for the Fe-FeS system are not confirmed. Modest addition of oxygen alone is confirmed to cause negligible freezing point depression at 6 GPa. Addition of H alone causes a progressive decrease in the eutectic temperature with P in the Fe-FeS-H system to below 965 °C at 6 GPa to below 950 °C at 8 GPa. It is our hypothesis that moisture contamination in unrigorously dried experiments may be an H source for freezing point depression. O released from H2O disproportionation reacts with Fe and is sequestered as ferropericlase along the sample capsules walls, leaving the H to escape the system and/or enter the Fe-FeS mixture. The observed occurrence of ferropericlase on undried MgO capsule margins is otherwise difficult to explain, because an alternate source for the oxygen in the ferropericlase layer is difficult to identify. This study questions the use of pressure-depressed Fe-S eutectic temperatures and suggests that the lower eutectic temperatures sometimes reported are achieved by moving into the ternary Fe-S-H system. These results adjust slightly the constraints on eutectic temperatures allowed for partly solidified cores on small planets. H substantially diminishes the temperature extent of the melting interval in Fe-S by reducing the melting points of the crystalline phases more than it depresses the eutectic.

  5. Melting relations in the Fe-rich portion of the system FeFeS at 30 kb pressure

    USGS Publications Warehouse

    Brett, R.; Bell, P.M.

    1969-01-01

    The melting relations of FeFeS mixtures covering the composition range from Fe to Fe67S33 have been determined at 30 kb pressure. The phase relations are similar to those at low pressure. The eutectic has a composition of Fe72.9S27.1 and a temperature of 990??C. Solubility of S in Fe at elevated temperatures at 30 kb is of the same order of magnitude as at low pressure. Sulfur may have significantly lowered the melting point of iron in the upper mantle during the period of coalescence of metal prior to core formation in the primitive earth. ?? 1969.

  6. Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

    PubMed Central

    Tamura, Takashi; Tsunekawa, Naoki; Nemoto, Michiko; Inagaki, Kenji; Hirano, Toshiyuki; Sato, Fumitoshi

    2016-01-01

    Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni–Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions. PMID:26818780

  7. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  8. Structural differences of oxidized iron-sulfur and nickel-iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy.

    PubMed

    Sigfridsson, Kajsa G V; Leidel, Nils; Sanganas, Oliver; Chernev, Petko; Lenz, Oliver; Yoon, Ki-Seok; Nishihara, Hirofumi; Parkin, Alison; Armstrong, Fraser A; Dementin, Sébastien; Rousset, Marc; De Lacey, Antonio L; Haumann, Michael

    2015-02-01

    The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.

  9. Intracellular Location and O2 Sensitivity of Uptake Hydrogenase in Azospirillum spp

    PubMed Central

    Fu, Changlin; Knowles, Roger

    1989-01-01

    Uptake hydrogenase activity of Azospirillum brasilense in vitro (cell-free extract) was very much more sensitive to O2 than was that of A. amazonense, and the O2 pressure optima for uptake hydrogenase activities were 0.01 and 0.4 to 3 kPa for A. brasilense and A. amazonense, respectively. The addition of superoxide dismutase did not increase uptake hydrogenase activity of A. brasilense either in vivo or in vitro. The O2 uptake rates of A. brasilense and A. amazonense were nearly the same. Inhibition of A. brasilense O2-dependent uptake hydrogenase activity by O2 was highly reversible under the conditions tested. O2 also markedly inhibited in vitro methylene blue-dependent uptake hydrogenase activity of A. brasilense, and this inhibition was highly reversible. It is concluded that the difference in O2 tolerance of the uptake hydrogenases is not due to a difference in respiratory protection in the two species and may be due to inherent differences in the two enzymes. For the three species, A. brasilense, A. amazonense, and A. lipoferum, almost all the recovered methylene blue-dependent uptake hydrogenase activity was associated with the membrane fraction. PMID:16348011

  10. The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis.

    PubMed

    Marques, Marta C; Tapia, Cristina; Gutiérrez-Sanz, Oscar; Ramos, Ana Raquel; Keller, Kimberly L; Wall, Judy D; De Lacey, Antonio L; Matias, Pedro M; Pereira, Inês A C

    2017-03-20

    Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 production. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

  11. The Role of Frozen Spins in the Exchange Anisotropy of Core–Shell Fe@Fe3O4 Nanoparticles

    PubMed Central

    Ong, Quy Khac; Lin, Xiao-Min; Wei, Alexander

    2011-01-01

    Core–shell Fe@Fe3O4 nanoparticles exhibit substantial exchange bias at low temperatures, mediated by unidirectionally aligned moments at the core–shell interface. These spins are frozen into magnetic alignment with field cooling, and are depinned in a temperature-dependent manner. The population of such frozen spins has a direct impact on both coercivity (HC) and the exchange-bias field (HE), which are modulated by external physical parameters such as the strength of the applied cooling field and the cycling history of magnetic field sweeps (training effect). Aging of the core–shell nanoparticles under ambient conditions results in a gradual decrease in magnetization but overall retention of HC and HE, as well as a large increase in the population of frozen spins. These changes are accompanied by a structural evolution from well-defined core–shell structures to particles containing multiple voids, attributable to the Kirkendall effect. Energy-filtered and high-resolution transmission electron microscopy both indicate further oxidation of the shell layer, but the Fe core is remarkably well preserved. The increase in frozen spin population with age is responsible for the overall retention of exchange bias, despite void formation and other oxidation-dependent changes. The exchange-bias field becomes negligible upon deliberate oxidation of Fe@Fe3O4 nanoparticles into yolk–shell particles, with a nearly complete physical separation of core and shell. PMID:21321674

  12. Uptake Hydrogenase (Hup) in Common Bean (Phaseolus vulgaris) Symbioses

    PubMed Central

    Navarro, Rosangela B.; Vargas, Alvaro A. T.; Schröder, Eduardo C.; van Berkum, Peter

    1993-01-01

    Strains of Rhizobium forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (hup) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the hup structural genes of Bradyrhizobium japonicum was present in 100 of 248 strains examined. EcoRI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal SacI fragment, which contains part of both bradyrhizobial hup structural genes. The DNA with homology to the hup genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the hup structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup+ strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains. Images PMID:16349115

  13. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

    DOE PAGES

    Wu, Chang-Hao; McTernan, Patrick M.; Walter, Mary E.; ...

    2015-01-01

    Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus ,more » a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.« less

  14. Physiological Factors Determining Hydrogenase Activity in Nitrogen-Fixing Heterocystous Cyanobacteria 1

    PubMed Central

    Chen, Pei-Chung; Almon, Helmar; Böger, Peter

    1989-01-01

    Four species of nitrogen-fixing heterocystous cyanobacteria were compared with respect to induction of hydrogenase activity. Two of the strains contained phycoerythrin and built up high levels of carbohydrate storage material when grown in batch culture under nitrogen-fixing conditions and continuous illumination. These strains did not exhibit hydrogenase activity. Lack of activity in the phycoerythrin-containing species was determined by cell-free assays measuring both hydrogen-evolving and hydrogen-uptake activities. Apparently, expression of hydrogenase is negatively correlated with the carbohydrate pool present and concurrent respiration. Furthermore, there is an apparent relationship between the presence of phycoerythrin, carbohydrate accumulation, and the absence of hydrogenase activity. PMID:16666659

  15. Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

    PubMed

    Caffrey, Sean M; Park, Hyung-Soo; Voordouw, Johanna K; He, Zhili; Zhou, Jizhong; Voordouw, Gerrit

    2007-09-01

    The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.

  16. Function of Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough▿ †

    PubMed Central

    Caffrey, Sean M.; Park, Hyung-Soo; Voordouw, Johanna K.; He, Zhili; Zhou, Jizhong; Voordouw, Gerrit

    2007-01-01

    The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen > 50% hydrogen > lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase. PMID:17601789

  17. (Catalytic mechanism of hydrogenase from aerobic N sub 2 -fixing microorganisms)

    SciTech Connect

    Arp, D.J.

    1991-01-01

    The results of this DOE-sponsored project have contributed to our understanding of the catalytic mechanism of A. vinelandii hydrogenase. A group of inhibitors have been characterized. These provide information about the different types of redox clusters involved in catalysis and the roles of each. One group has already used acetylene in a study of three desulfovibrian hydrogenases and shown that only the NiFe hydrogenases are inhibited. We have characterized a number of spectral properties of A. vinelandii hydrogenase. The EPR signals associated with this hydrogenase in the reduced state are reminiscent of other NiFe dimeric hydrogenases such as A. eutrophus, but distinctly difference from others such as D. gigas and Chromatium vinosum. Thus, while the NiFe dimeric hydrogenases are now recognized as a large group of similar enzymes, there are differences in the spectral and catalytic properties which are not explained by their similar redox inventories, identical subunit structures, immunological cross reactivity and conserved sequences. The inhibitors we have characterized are also proving of value in the spectral characterizations. Surprisingly, we only see a significant EP signal attributable to Ni after the enzyme has been inactivated with O{sub 2} and then reduced (though not reactivated). No spectral perterbations (EPR or UV-V is) of active enzyme can be attributed to binding of H{sub 2}, even though H{sub 2} clearly binds to this form of the enzyme. Acetylene, which does not substantially perterb the EPR signal of active hydrogenase, does result in a new absorption envelope in the UV-V is spectrum. Overall, the results of this project have revealed the complex interactions of the redox clusters in catalysis through studies of inhibitor mechanisms and spectral properties. 14 refs., 9 figs.

  18. Catalytic mechanism of hydrogenase from Azotobacter vinelandii. Final technical report, August 1, 1994--July 31, 1997

    SciTech Connect

    Arp, D.J.

    1997-10-01

    This project is focused on investigations of the catalytic mechanism of the hydrogenase found in the aerobic, N{sub 2}-fixing microorganism Azotobacter vinelandii. This report summarizes the progress during the first two years of the current project and include the anticipated course of the research for the remaining year of the current project. Because the current proposal represents a change in direction, the authors also include a brief progress report of prior DOE-sponsored research dealing with hydrogenases.

  19. Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.

    PubMed Central

    Eberz, G; Friedrich, B

    1991-01-01

    Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes. Images PMID:2001989

  20. Function of Periplasmic Hydrogenases in the Sulfate-ReducingBacterium Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Caffrey, Sean M.; Park, Hyung-Soo; Voordouw, Johanna K.; He,Zhili; Zhou, Jizhong; Voordouw, Gerrit

    2007-09-24

    The sulfate-reducing bacterium Desulfovibrio vulgarisHildenborough possesses four periplasmic hydrogenases to facilitate theoxidation of molecular hydrogen. These include an [Fe]hydrogenase, an[NiFeSe]hydrogenase, and two [NiFe]hydrogenases encoded by the hyd,hys, hyn1, and hyn2 genes, respectively. In order to understand theircellular functions, we have compared the growth rates of existing (hydand hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those ofthe wild type in defined media in which lactate or hydrogen at either 5or 50 percent (vol/vol) was used as the sole electron donor for sulfatereduction. Only strains missing the [Fe]hydrogenase were significantlyaffected during growth with lactate or with 50 percent (vol/vol) hydrogenas the sole electron donor. When the cells were grown at low (5 percent[vol/vol]) hydrogen concentrations, those missing the [NiFeSe]hydrogenase suffered the greatest impairment. The growth rate datacorrelated strongly with gene expression results obtained from microarrayhybridizations and real-time PCR using mRNA extracted from cells grownunder the three conditions. Expression of the hys genes followed theorder 5 percent hydrogen>50 percent hydrogen>lactate, whereasexpression of the hyd genes followed the reverse order. These resultssuggest that growth with lactate and 50 percent hydrogen is associatedwith high intracellular hydrogen concentrations, which are best capturedby the higher activity, lower affinity [Fe]hydrogenase. In contrast,growth with 5 percent hydrogen is associated with a low intracellularhydrogen concentration, requiring the lower activity, higher affinity[NiFeSe]hydrogenase.

  1. Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

    PubMed Central

    Kelly, Ciarán L.; Pinske, Constanze; Murphy, Bonnie J.; Parkin, Alison; Armstrong, Fraser; Palmer, Tracy; Sargent, Frank

    2015-01-01

    Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme. PMID:26839796

  2. (Catalytic mechanism of hydrogenase from aerobic N2-fixing microorganisms). [Azotobacter vinelandii:a1

    SciTech Connect

    Not Available

    1991-01-01

    The results of this DOE-sponsored project have contributed to our understanding of the catalytic mechanism of A. vinelandii hydrogenase. A group of inhibitors have been characterized. These provide information about the different types of redox clusters involved in catalysis and the roles of each. One group has already used acetylene in a study of three desulfovibrian hydrogenases and shown that onbly the NiFe hydrogenases are inhibited. The inhibitor studies are also being extended to other enzymes. We have characterized a number of special properties of A. vinelandii hydrogenase. While the NiFe dimeric hydrogenases are now recognized as a large group of similar enzymes, there are differences in the spectral and catalytic properties which are not explained by their similar redox inventories, identical subunit structures, immunological cross reactivity and conserved sequences. Surprisingly, we only see a significant EPR signal attributable to Ni after the enzyme has been inactivated with O{sub 2} and then re-reduced (though not reactivated). Acetylene, which does not substantially perterb the EPR signal of active hydrogenase, does result in a new absorption envelope in the UV-Vis spectrum. Overall, the results of this project have revealed the complex interactions of the redox clusters in catalysis through studies of inhibitor mechanisms and spectral properties. 14 refs., 9 figs.

  3. Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy.

    PubMed

    Reijerse, Edward J; Pham, Cindy C; Pelmenschikov, Vladimir; Gilbert-Wilson, Ryan; Adamska-Venkatesh, Agnieszka; Siebel, Judith F; Gee, Leland B; Yoda, Yoshitaka; Tamasaku, Kenji; Lubitz, Wolfgang; Rauchfuss, Thomas B; Cramer, Stephen P

    2017-03-29

    [FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN(-) and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.

  4. Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase.

    PubMed

    Greene, Brandon L; Wu, Chang-Hao; Vansuch, Gregory E; Adams, Michael W W; Dyer, R Brian

    2016-03-29

    Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.

  5. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

    PubMed Central

    Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady

    2016-01-01

    Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression. PMID:27547572

  6. Hydrogenase Activity of Mineral-Associated and Suspended Populations of Desulfovibrio desulfuricans Essex 6

    SciTech Connect

    C.L. Reardon; T.S. Magnuson; E.S. Boyd; W.D. Leavitt; D.W. Reed; G.G. Geesey

    2014-02-01

    The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell–mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl2 and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.

  7. The Investigation and Characterization of the Group 3 [Nickel-Iron]-Hydrogenases Using Protein Film Electrochemistry

    NASA Astrophysics Data System (ADS)

    McIntosh, Chelsea Lee

    Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and catalyze hydrogen oxidation in vivo. In contrast, the group 3 [NiFe]-hydrogenases are heteromultimeric, bifunctional enzymes that fulfill various cellular roles. In this dissertation, protein film electrochemistry (PFE) is used to characterize the catalytic properties of two group 3 [NiFe]-hydrogenases: HoxEFUYH from Synechocystsis sp. PCC 6803 and SHI from Pyrococcus furiosus. First, HoxEFUYH is shown to be biased towards hydrogen production. Upon exposure to oxygen, HoxEFUYH inactivates to two states, both of which can be reactivated on the timescale of seconds. Second, we show that PfSHI is the first example of an oxygen tolerant [NiFe]-hydrogenase that produces two inactive states upon exposure to oxygen. Both inactive states are analogous to those characterized for HoxEFUYH, but oxygen exposed PfSHI produces a greater fraction that reactivates at high potentials, enabling hydrogen oxidation in the presence of oxygen. Third, it is shown that removing the NAD(P)-reducing subunits from PfSHI leads to a decrease in bias towards hydrogen oxidation and renders the enzyme oxygen sensitive. Both traits are likely due to impaired intramolecular electron transfer. Mechanistic hypotheseses for these functional differences are considered.

  8. Identification of a locus within the hydrogenase gene cluster involved in intracellular nickel metabolism in Bradyrhizobium japonicum

    SciTech Connect

    Changlin Fu; Maier, R.J. )

    1991-12-01

    A 0.6-kb fragment of DNA involved in intracellular Ni metabolism was isolated and cloned from a cosmid containing 23.2 kb of hydrogenase-related genes of Bradyrhizobium japonicum. This locus is located 8.3 kb upstream of the hydrogenase structural genes. The hydrogenase activity of a mutant with a gene-directed mutation at this locus (strain JHK7) showed dependency on nickel provided during hydrogenase depression. The hydrogenase activity was only 20% of that in the wild-type strain, JH, at a concentration of 0.5 {mu}M NiCl{sub 2}. The hydrogenase activity in JH reached its maximum at 3 {mu}M NiCl{sub 2}, whereas the mutant (JHK7) reached wild-type levels of hydrogenase activity when derepressed in 50 {mu}M NiCl{sub 2}. Studies with the hup-lacZ transcriptional fusion plasmid pSY7 in JHK7 showed that the mutant JHK7 expressed less promoter activity under low-nickel conditions than did strain JH. The mutant accumulated less nickel during a 45-h hydrogenase under low-nickel conditions than did strain JH. The mutant accumulated less nickel during a 45-h hydrogenase derepression period than did the wild type. However, both JHK7 and the JH wild-type strain had the same short-term Ni transport rates, and the K{sub m}s for Ni of both strains were about 62 {mu}M. When incubated under non-hydrogenase-derepression conditions, the mutant accumulated Ni at the same rate as strain JH. However, this stored source of nickel was unable to restore hydrogenase expression ability of the mutant to wild-type levels during derepression without nickel. The results that the locus identified in B. japonicum is not involved in nickel-specific transport.

  9. Spectroscopic and electrochemical characterization of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Miyazaki F: reversible redox behavior and interactions between electron transfer centers.

    PubMed

    Riethausen, Jana; Rüdiger, Olaf; Gärtner, Wolfgang; Lubitz, Wolfgang; Shafaat, Hannah S

    2013-09-23

    Characterizing a new hydrogenase: The newly isolated [NiFeSe] hydrogenase from Desulfovibrio vulgaris Miyazaki F displays catalytic properties distinct from other hydrogenase proteins. Here we apply site-specific spectroscopic and electrochemical techniques to characterize these unique features at the molecular level.

  10. Hydrogenase Gene Distribution and H2 Consumption Ability within the Thiomicrospira Lineage

    PubMed Central

    Hansen, Moritz; Perner, Mirjam

    2016-01-01

    Thiomicrospira were originally characterized as sulfur-oxidizing chemolithoautotrophs. Attempts to grow them on hydrogen failed for many years. Only recently we demonstrated hydrogen consumption among two of three tested Thiomicrospira and posited that hydrogen consumption may be more widespread among Thiomicrospira than previously assumed. Here, we investigate and compare the hydrogen consumption ability and the presence of group 1 [NiFe]-hydrogenase genes (enzyme catalyzes H2↔2H+ + 2e-) for sixteen different Thiomicrospira species. Seven of these Thiomicrospira species encoded group 1 [NiFe]-hydrogenase genes and five of these species could also consume hydrogen. All Thiomicrospira species exhibiting hydrogen consumption were from hydrothermal vents along the Mid-Atlantic ridge or Eastern Pacific ridges. The tested Thiomicrospira from Mediterranean and Western Pacific vents could not consume hydrogen. The [NiFe]-hydrogenase genes were categorized into two clusters: those resembling the hydrogenase from Hydrogenovibrio are in cluster I and are related to those from Alpha- and other Gammaproteobacteria. In cluster II, hydrogenases found exclusively in Thiomicrospira crunogena strains are combined and form a monophyletic group with those from Epsilonproteobacteria suggesting they were acquired through horizontal gene transfer. Hydrogen consumption appears to be common among some Thiomicrospira, given that five of the tested sixteen strains carried this trait. The hydrogen consumption ability expands their competitiveness within an environment. PMID:26903978

  11. Characterization and cloning of oxygen-tolerant hydrogenase from Klebsiella oxytoca HP1.

    PubMed

    Wu, Xiaobing; Liang, Yi; Li, Qianyi; Zhou, Juan; Long, Minnan

    2011-04-01

    Hydrogenase from a hot spring bacterium Klebsiella oxytoca HP1 was purified and found to have a specific activity of 199.8 U/mg of protein and a yield of 7.3%. The purified enzyme was determined to consist of six subunits (65, 33, 28, 23, 21 and 18 kDa), similar to hydrogenase-3 from Escherichia coli, and therefore it was named Hyd3. The enzyme displayed remarkable oxygen tolerance. For the purified enzyme, 50% maximal activity was maintained following incubation for 24 h in air at room temperature. The hydrogenase gene cluster (hyc) was cloned and found to consist of hycD, hycE, hycF, hycdG, hycH and hycI genes. hycE and hycG genes encode for the large and small subunit of the hydrogenase, respectively. A hycE gene deletion mutant, ΔhycE, was constructed for elucidating the function of the hyc-operon in hydrogen metabolism. Compared with the wild type strain HP1, the mutant strain showed a dramatic decrease in hydrogen production in the presence of formate, sodium pyruvate and glucose under O(2)-stressed conditions, while substantial activity was detected under anaerobic conditions. This strongly suggests that K. oxytoca HP1 carries a number of hydrogenases or hydrogen metabolic pathways independently of Hyd3. However, Hyd3 is the main factor responsible for hydrogen production under O(2) stress conditions.

  12. A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

    PubMed Central

    Kortlüke, C; Horstmann, K; Schwartz, E; Rohde, M; Binsack, R; Friedrich, B

    1992-01-01

    One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units. Images PMID:1383192

  13. The eutectic liquid composition in the Fe-Fe3S binary system at the core pressure range

    NASA Astrophysics Data System (ADS)

    Mori, Y.; Hirose, K.; Tateno, S.; Morard, G.; Ohishi, Y.

    2015-12-01

    Sulfur is considered to be an important component in the Earth's core because it is depleted in the crust and mantle compared to other volatile elements and often included in iron meteorites. Here we examine the liquidus phase relations in the Fe-Fe3S binary system between 38 and 138 GPa based on characterization of a sample recovered from a melting experiment at high pressure and temperature in a laser-heated diamond-anvil cell. Both Fe-8wt.%S and Fe-14wt.%S samples were employed as starting materials, which were homogeneous mixtures of fine-grain Fe and FeS (<1 μm) prepared by induction melting and rapid quenching technique [Morard et al., 2011 PCM]. We used a focused ion beam (FIB) equipped with energy dispersive X-ray spectrometry for textural and chemical characterizations of recovered samples. The samples exhibited a melting texture with quenched liquid alloy at the hottest part and solid Fe or Fe3S at its outside. In a couple of samples, the quenched liquid was in direct contact with solid Fe3S, and solid Fe was also present right next to Fe3S, suggesting that the composition of such liquid is close to a eutectic composition (~10 wt.% S at 66 GPa and ~12 wt.% S at 138 GPa). Indeed, this interpretation is consistent with the results of other experiments obtained in this study. Our data demonstrate that the eutectic liquid composition in the Fe-Fe3S binary system decreases its sulfur concentration with increasing pressure, which is in agreement with previous studies [Morard et al., 2008 EPSL; Kamada et al., 2012 EPSL]. The sulfur content in the eutectic liquid composition may be less than 10 wt.% at the inner core boundary pressure. The recent shock-wave study by Huang et al.[2013 GRL] suggested 10 wt.% S in the outer core, but the present study indicates that such liquid alloy with 10 wt.% S crystalizes the B2 phase of Fe-S alloy and thus does not support the sulfur-rich outer core.

  14. Production of nearly monodisperse Fe3O4 and Fe@Fe3O4 nanoparticles in aqueous medium and their surface modification for biomedical applications

    NASA Astrophysics Data System (ADS)

    Tegafaw, Tirusew; Xu, Wenlong; Lee, Sang Hyup; Chae, Kwon Seok; Chang, Yongmin; Lee, Gang Ho

    2017-02-01

    Iron (Fe)-based nanoparticles are extremely valuable in biomedical applications owing to their low toxicity and high magnetization values at room temperature. In this study, we synthesized nearly monodisperse iron oxide (Fe3O4) and Fe@Fe3O4 (core: Fe, shell: Fe3O4) nanoparticles in aqueous medium under argon flow and then, coated them with various biocompatible ligands and silica. In this study, eight types of surface-modified nanoparticles were investigated, namely, Fe3O4@PAA (PAA = polyacrylic acid; Mw of PAA = 5100 amu and 15,000 amu), Fe3O4@PAA-FA (FA = folic acid; Mw of PAA = 5100 amu and 15,000 amu), Fe3O4@PEI-fluorescein (PEI = polyethylenimine; Mw of PEI = 1300 amu), Fe@Fe3O4@PEI (Mw of PEI = 10,000 amu), Fe3O4@SiO2 and Fe@Fe3O4@SiO2 nanoparticles. We characterized the prepared surface-modified nanoparticles using high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) absorption spectroscopy, a superconducting quantum interference device (SQUID), X-ray photoelectron spectroscopy (XPS), photoluminescence (PL) spectroscopy and confocal microscopy. Finally, we measured the cytotoxicity of the samples. The results indicate that the surface-modified nanoparticles are biocompatible and are potential candidates for various biomedical applications.

  15. Enhanced dewatering of excess activated sludge through decomposing its extracellular polymeric substances by a Fe@Fe2O3-based composite conditioner.

    PubMed

    He, Dong-Qin; Luo, Hong-Wei; Huang, Bao-Cheng; Qian, Chen; Yu, Han-Qing

    2016-10-01

    Efficient sludge dewatering methods are highly desired by municipal wastewater treatment plants. In this study, Fe@Fe2O3 nanomaterial, combined with polydiallyldimethylammonium chloride (PDMDAAC) and H2SO4, was used for sludge dewatering. This composite conditioner exhibited an excellent dewatering capability. By using uniform design, the optimized dosages of Fe@Fe2O3, H2SO4 and PDMDAAC were determined to be 40, 136 and 4.8mg/gDS (dry solids), respectively. The moisture content of sludge cake decreased from 78.1% to 64.8%, and the capillary suction time from 56 to 21s. The sludge extracellular polymeric substances (EPS) were decomposed, resulting in greater conversion of the bound water into free water and the release of free water. The electron spin resonance results show that the molecular oxygen activation process induced by Fe@Fe2O3 produced hydroxyl radicals, which were mainly responsible for the EPS decomposition. In this way, an efficient composite conditioner for enhancing sludge dewatering was developed.

  16. 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 (TONHydA1 ) yet reported for an enzyme-based in vitro system (2.9×10(6)  mol(H2 ) mol(cat)(-1) ) and a maximum turnover frequency (TOFHydA1 ) of 550 mol(H2 ) 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.

  17. Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity.

    PubMed Central

    Bonam, D; Lehman, L; Roberts, G P; Ludden, P W

    1989-01-01

    Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase. Images PMID:2498285

  18. 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

  19. Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum

    PubMed Central

    2012-01-01

    Background [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. Results HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ΔhupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. Conclusions The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen. PMID:23136881

  20. Crystallographic studies of nitrogenase and hydrogenase. Progress report, June 1, 1992--April 1, 1994

    SciTech Connect

    Bolin, J.T.

    1994-05-01

    The long term goal of this project is to obtain detailed knowledge of the structure and function of nitrogenase and hydrogenase through the analysis of physical, chemical, and biological data with reference to three-dimensional, atomic resolution crystal structures of components of the enzyme and/or complexes of the components. The current objectives to determine the crystal structure of wild-type Av1, the nitrogenase MoFe protein from Azotobacter vinelandii; to refine this structure at high resolution; and to initiate studies of mutant MoFe proteins that express altered chemical and physical properties. Further we seek to determine the crystal structure of the bi-directional all-Fe hydrogenase from C. pasteurianum, Cp-hydrI, and to initiate studies of the uptake hydrogenase from the same organism, Cp-hydrII.

  1. Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase

    PubMed Central

    Zheng, Yanning; Kahnt, Jörg; Kwon, In Hyuk; Mackie, Roderick I.

    2014-01-01

    Ruminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO2, and 2.6 H2 when growing in batch culture and to 2 acetate, 2 CO2, and 4 H2 when growing in continuous culture in syntrophic association with H2-consuming microorganisms that keep the H2 partial pressure low. The organism uses NAD+ and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO2, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H2. Of all the enzymes involved, only the enzyme catalyzing the formation of H2 from NADH remained unknown. Here, we report that R. albus 7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]-hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H2 from NADH to the exergonic formation of H2 from reduced ferredoxin. Interestingly, hydA2 is adjacent to the hydS gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that hydS and hydA2 are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when R. albus grows at high H2 partial pressures, HydS could be a H2-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis. PMID:25157086

  2. Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

    PubMed Central

    Schäfer, Caspar; Friedrich, Bärbel

    2013-01-01

    Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2. PMID:23793632

  3. Respiratory Membrane endo-Hydrogenase Activity in the Microaerophile Azorhizobium caulinodans Is Bidirectional

    PubMed Central

    Sprecher, Brittany N.; Gittings, Margo E.; Ludwig, Robert A.

    2012-01-01

    Background The microaerophilic bacterium Azorhizobium caulinodans, when fixing N2 both in pure cultures held at 20 µM dissolved O2 tension and as endosymbiont of Sesbania rostrata legume nodules, employs a novel, respiratory-membrane endo-hydrogenase to oxidize and recycle endogenous H2 produced by soluble Mo-dinitrogenase activity at the expense of O2. Methods and Findings From a bioinformatic analysis, this endo-hydrogenase is a core (6 subunit) version of (14 subunit) NADH:ubiquinone oxidoreductase (respiratory complex I). In pure A. caulinodans liquid cultures, when O2 levels are lowered to <1 µM dissolved O2 tension (true microaerobic physiology), in vivo endo-hydrogenase activity reverses and continuously evolves H2 at high rates. In essence, H+ ions then supplement scarce O2 as respiratory-membrane electron acceptor. Paradoxically, from thermodynamic considerations, such hydrogenic respiratory-membrane electron transfer need largely uncouple oxidative phosphorylation, required for growth of non-phototrophic aerobic bacteria, A. caulinodans included. Conclusions A. caulinodans in vivo endo-hydrogenase catalytic activity is bidirectional. To our knowledge, this study is the first demonstration of hydrogenic respiratory-membrane electron transfer among aerobic (non-fermentative) bacteria. When compared with O2 tolerant hydrogenases in other organisms, A. caulinodans in vivo endo-hydrogenase mediated H2 production rates (50,000 pmol 109·cells−1 min−1) are at least one-thousandfold higher. Conceivably, A. caulinodans respiratory-membrane hydrogenesis might initiate H2 crossfeeding among spatially organized bacterial populations whose individual cells adopt distinct metabolic states in response to variant O2 availability. Such organized, physiologically heterogeneous cell populations might benefit from augmented energy transduction and growth rates of the populations, considered as a whole. PMID:22662125

  4. Evolutionary and Biotechnological Implications of Robust Hydrogenase Activity in Halophilic Strains of Tetraselmis

    PubMed Central

    D'Adamo, Sarah; Jinkerson, Robert E.; Boyd, Eric S.; Brown, Susan L.; Baxter, Bonnie K.; Peters, John W.; Posewitz, Matthew C.

    2014-01-01

    Although significant advances in H2 photoproduction have recently been realized in fresh water algae (e.g. Chlamydomonas reinhardtii), relatively few studies have focused on H2 production and hydrogenase adaptations in marine or halophilic algae. Salt water organisms likely offer several advantages for biotechnological H2 production due to the global abundance of salt water, decreased H2 and O2 solubility in saline and hypersaline systems, and the ability of extracellular NaCl levels to influence metabolism. We screened unialgal isolates obtained from hypersaline ecosystems in the southwest United States and identified two distinct halophilic strains of the genus Tetraselmis (GSL1 and QNM1) that exhibit both robust fermentative and photo H2-production activities. The influence of salinity (3.5%, 5.5% and 7.0% w/v NaCl) on H2 production was examined during anoxic acclimation, with the greatest in vivo H2-production rates observed at 7.0% NaCl. These Tetraselmis strains maintain robust hydrogenase activity even after 24 h of anoxic acclimation and show increased hydrogenase activity relative to C. reinhardtii after extended anoxia. Transcriptional analysis of Tetraselmis GSL1 enabled sequencing of the cDNA encoding the FeFe-hydrogenase structural enzyme (HYDA) and its maturation proteins (HYDE, HYDEF and HYDG). In contrast to freshwater Chlorophyceae, the halophilic Tetraselmis GSL1 strain likely encodes a single HYDA and two copies of HYDE, one of which is fused to HYDF. Phylogenetic analyses of HYDA and concatenated HYDA, HYDE, HYDF and HYDG in Tetraselmis GSL1 fill existing knowledge gaps in the evolution of algal hydrogenases and indicate that the algal hydrogenases sequenced to date are derived from a common ancestor. This is consistent with recent hypotheses that suggest fermentative metabolism in the majority of eukaryotes is derived from a common base set of enzymes that emerged early in eukaryotic evolution with subsequent losses in some organisms. PMID

  5. Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

    PubMed Central

    2017-01-01

    Catalysis of H2 production and oxidation reactions is critical in renewable energy systems based around H2 as a clean fuel, but the present reliance on platinum-based catalysts is not sustainable. In nature, H2 is oxidized at minimal overpotential and high turnover frequencies at [NiFe] catalytic sites in hydrogenase enzymes. Although an outline mechanism has been established for the [NiFe] hydrogenases involving heterolytic cleavage of H2 followed by a first and then second transfer of a proton and electron away from the active site, details remain vague concerning how the proton transfers are facilitated by the protein environment close to the active site. Furthermore, although [NiFe] hydrogenases from different organisms or cellular environments share a common active site, they exhibit a broad range of catalytic characteristics indicating the importance of subtle changes in the surrounding protein in controlling their behavior. Here we review recent time-resolved infrared (IR) spectroscopic studies and IR spectroelectrochemical studies carried out in situ during electrocatalytic turnover. Additionally, we re-evaluate the significant body of IR spectroscopic data on hydrogenase active site states determined through more conventional solution studies, in order to highlight mechanistic steps that seem to apply generally across the [NiFe] hydrogenases, as well as steps which so far seem limited to specific groups of these enzymes. This analysis is intended to help focus attention on the key open questions where further work is needed to assess important aspects of proton and electron transfer in the mechanism of [NiFe] hydrogenases.

  6. Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii.

    PubMed Central

    Pihl, T D; Maier, R J

    1991-01-01

    Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value. Images PMID:1900502

  7. Vibrational spectroscopic characterization of the phosphate mineral barbosalite FeFe23+()2( - Implications for the molecular structure

    NASA Astrophysics Data System (ADS)

    Frost, Ray L.; Xi, Yunfei; López, Andrés; Scholz, Ricardo; Lana, Cristiano de Carvalho; Souza, Bárbara Firmino e.

    2013-11-01

    Natural single-crystal specimens of barbosalite from Brazil, with general formula FeFe23+()2( were investigated by Raman and infrared spectroscopy. The mineral occurs as secondary products in granitic pegmatites. The Raman spectrum of barbosalite is characterized by bands at 1020, 1033 and 1044 cm-1 cm-1, assigned to ν1 symmetric stretching mode of the HOPO33- and PO43- units. Raman bands at around 1067, 1083 and 1138 cm-1 are attributed to both the HOP and PO antisymmetric stretching vibrations. The set of Raman bands observed at 575, 589 and 606 cm-1 are assigned to the ν4 out of plane bending modes of the PO4 and H2PO4 units. Raman bands at 439, 461, 475 and 503 cm-1 are attributed to the ν2 PO4 and H2PO4 bending modes. Strong Raman bands observed at 312, 346 cm-1 with shoulder bands at 361, 381 and 398 cm-1 are assigned to FeO stretching vibrations. No bands which are attributable to water vibrations were found. Vibrational spectroscopy enables aspects of the molecular structure of barbosalite to be assessed.

  8. Dinuclear iron(II)-cyanocarbonyl complexes linked by two/three bridging ethylthiolates: relevance to the active site of [Fe] hydrogenases.

    PubMed

    Liaw, Wen-Feng; Tsai, Wen-Ting; Gau, Hung-Bin; Lee, Chien-Ming; Chou, Shin-Yuan; Chen, Wen-Yuan; Lee, Gene-Hsiang

    2003-04-21

    Dinuclear iron(II)-cyanocarbonyl complex [PPN](2)[Fe(CN)(2)(CO)(2)(mu-SEt)](2) (1) was prepared by the reaction of [PPN][FeBr(CN)(2)(CO)(3)] and [Na][SEt] in THF at ambient temperature. Reaction of complex 1 with [PPN][SEt] produced the triply thiolate-bridged dinuclear Fe(II) complex [PPN][(CN)(CO)(2)Fe(mu-SEt)(3)Fe(CO)(2)(CN)] (2) with the torsion angle of two CN(-) groups (C(5)N(2) and C(3)N(1)) being 126.9 degrees. The extrusion of two sigma-donor CN(-) ligands from Fe(II)Fe(II) centers of complex 1 as a result of the reaction of complex 1 and [PPN][SEt] reflects the electron-rich character of the dinuclear iron(II) when ligated by the third bridging ethylthiolate. The Fe-S distances (2.338(2) and 2.320(3) A for complexes 1 and 2, respectively) do not change significantly, but the Fe(II)-Fe(II) distance contracts from 3.505 A in complex 1 to 3.073 A in complex 2. The considerably longer Fe(II)-Fe(II) distance of 3.073 A in complex 2, compared to the reported Fe-Fe distances of 2.6/2.62 A in DdHase and CpHase, was attributed to the presence of the third bridging ethylthiolate, instead of pi-accepting CO-bridged ligand as observed in [Fe] hydrogenases. Additionally, in a compound of unusual composition ([Na.(5)/(2)H(2)O][(CN)(CO)(2)Fe(mu-SEt)(3)Fe(CO)(2)(CN)])(n)((1)/(2)O(Et)(2))(n) (3), the Na(+) cations and H(2)O molecules combining with dinuclear [(CN)(CO)(2)Fe(mu-SEt)(3)Fe(CO)(2)(CN)](-) anions create a polymeric framework wherein two CN(-) ligands are coordinated via CN(-)-Na(+)/CN(-)-(Na(+))(2) linkages, respectively.

  9. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    SciTech Connect

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  10. Determination of the hydrogenase status of individual legume nodules by a methylene blue reduction assay.

    PubMed

    Lambert, G R; Hanus, F J; Russell, S A; Evans, H J

    1985-08-01

    We adapted a method for the rapid screening of colonies of free-living Rhizobium japonicum for hydrogenase activity to determine the hydrogenase status of individual soybean nodules. Crude bacteroid suspensions from nodules containing strains known to be hydrogen uptake positive (Hup) caused a localized decolorization of filter paper disks, whereas suspensions from nodules arising from inoculation with hydrogen uptake-negative (Hup) mutants or strains did not decolorize the disks. The reliability of the method was demonstrated by its successful application to 29 slow-growing rhizobia. The Hup phenotype on methylene blue filters agreed with that determined amperometrically with either methylene blue or oxygen as the electron acceptor.

  11. Improved O2-tolerance in variants of a H2-evolving [NiFe]-hydrogenase from Klebsiella oxytoca HP1.

    PubMed

    Huang, Gang-Feng; Wu, Xiao-Bing; Bai, Li-Ping; Liu, Ke; Jiang, Li-Jing; Long, Min-Nan; Chen, Qing-Xi

    2015-04-02

    In this study, we investigated the mechanism of O2 tolerance of Klebsiella oxytoca HP1 H2-evolving hydrogenase 3 (KHyd3) by mutational analysis and three-dimensional structure modeling. Results revealed that certain surface amino acid residues of KHyd3 large subunit, in particular those at the outer entrance of the gas channel, have a visible effect on its oxygen tolerance. Additionally, solution pH, immobilization and O2 partial pressure also affect KHyd3 O2-tolerance to some extent. We propose that the extent of KHyd3 O2-tolerance is determined by a balance between the rate of O2 access to the active center through gas channels and the deoxidation rate of the oxidized active center. Based on our findings, two higher O2-tolerant KHyd3 mutations G300E and G300M were developed.

  12. Analysis of Redox Responses During TNT Transformation by Clostridium acetobutylicum ATCC 824 and Mutants Exhibiting Altered Metabolism

    DTIC Science & Technology

    2012-01-01

    2006) Functional studies of [ FeFe ] hydrogenase maturation in an Escherichia coli biosynthetic system. J Bacteriol 188(6):2163–2172 Kutty R, Bennett...activation of [ FeFe ] hydrogenase: new insights into hydrogenase maturation. J Biol Inorg Chem 12(4):443–447 Mermelstein LD, Papoutsakis ET (1993) In

  13. Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes.

    PubMed

    Pi, Jian; Jawed, Muhammad; Wang, Jun; Xu, Li; Yan, Yunjun

    2016-01-01

    In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.

  14. Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg.

    PubMed Central

    Braks, I J; Hoppert, M; Roge, S; Mayer, F

    1994-01-01

    The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme. Images PMID:8002593

  15. Regulation and genetic organization of hydrogenase: Final progress report for the period June 1, 1985--July 31, 1988

    SciTech Connect

    Krasna, A.I.

    1988-10-01

    Hydrogenase is an enzyme which plays an important role in the anaerobic metabolism of many bacteria. The objectives of the research were to elucidate the regulation and genetic organization of hydrogenase in microorganisms. A mutation in the E. coli hydE gene leads to loss of all hydrogenase activities and isoenzymes as well as all formate-related activities. A 0.9 kb DNA fragment has been cloned from an E. coli genomic DNA library which restored all hydrogenase and formate activities to a hydE mutant strain. This gene coded for a polypeptide of subunit mw 36 kDa which is required for hydrogenase synthesis. It is involved in incorporation of nickel into hydrogenase. A mutation in the E coli hupB gene leads to the loss of the uptake of H/sub 2/ by dyes and the ability to grow on fumarate plus H/sub 2/, but expresses normal levels of hydrogenase when assayed by deuterium exchange. This mutation also leads to loss of all formate-related activities. The mutation mapped near minute 17 and a single mutation was responsible for loss of both activities. A 1.4 kb DNA fragment was isolated which restored the hydrogen uptake activities and coded for a polypeptide of subunit mw 30 kDa. Dna fragments have been isolated from Chromatium vinosum and Proteus vulgaris which restored hydrogenase activities to E. coli strains with mutations in the hydA or hydB regulatory genes and which lack all hydrogenase activities. 6 refs., 12 figs.

  16. hypD as a marker for [NiFe]-hydrogenases in microbial communities of surface waters.

    PubMed

    Beimgraben, Christian; Gutekunst, Kirstin; Opitz, Friederike; Appel, Jens

    2014-06-01

    Hydrogen is an important trace gas in the atmosphere. Soil microorganisms are known to be an important part of the biogeochemical H2 cycle, contributing 80 to 90% of the annual hydrogen uptake. Different aquatic ecosystems act as either sources or sinks of hydrogen, but the contribution of their microbial communities is unknown. [NiFe]-hydrogenases are the best candidates for hydrogen turnover in these environments since they are able to cope with oxygen. As they lack sufficiently conserved sequence motifs, reliable markers for these enzymes are missing, and consequently, little is known about their environmental distribution. We analyzed the essential maturation genes of [NiFe]-hydrogenases, including their frequency of horizontal gene transfer, and found hypD to be an applicable marker for the detection of the different known hydrogenase groups. Investigation of two freshwater lakes showed that [NiFe]-hydrogenases occur in many prokaryotic orders. We found that the respective hypD genes cooccur with oxygen-tolerant [NiFe]-hydrogenases (groups 1 and 5) mainly of Actinobacteria, Acidobacteria, and Burkholderiales; cyanobacterial uptake hydrogenases (group 2a) of cyanobacteria; H2-sensing hydrogenases (group 2b) of Burkholderiales, Rhizobiales, and Rhodobacterales; and two groups of multimeric soluble hydrogenases (groups 3b and 3d) of Legionellales and cyanobacteria. These findings support and expand a previous analysis of metagenomic data (M. Barz et al., PLoS One 5:e13846, 2010, http://dx.doi.org/10.1371/journal.pone.0013846) and further identify [NiFe]-hydrogenases that could be involved in hydrogen cycling in aquatic surface waters.

  17. hypD as a Marker for [NiFe]-Hydrogenases in Microbial Communities of Surface Waters

    PubMed Central

    Beimgraben, Christian; Gutekunst, Kirstin; Opitz, Friederike

    2014-01-01

    Hydrogen is an important trace gas in the atmosphere. Soil microorganisms are known to be an important part of the biogeochemical H2 cycle, contributing 80 to 90% of the annual hydrogen uptake. Different aquatic ecosystems act as either sources or sinks of hydrogen, but the contribution of their microbial communities is unknown. [NiFe]-hydrogenases are the best candidates for hydrogen turnover in these environments since they are able to cope with oxygen. As they lack sufficiently conserved sequence motifs, reliable markers for these enzymes are missing, and consequently, little is known about their environmental distribution. We analyzed the essential maturation genes of [NiFe]-hydrogenases, including their frequency of horizontal gene transfer, and found hypD to be an applicable marker for the detection of the different known hydrogenase groups. Investigation of two freshwater lakes showed that [NiFe]-hydrogenases occur in many prokaryotic orders. We found that the respective hypD genes cooccur with oxygen-tolerant [NiFe]-hydrogenases (groups 1 and 5) mainly of Actinobacteria, Acidobacteria, and Burkholderiales; cyanobacterial uptake hydrogenases (group 2a) of cyanobacteria; H2-sensing hydrogenases (group 2b) of Burkholderiales, Rhizobiales, and Rhodobacterales; and two groups of multimeric soluble hydrogenases (groups 3b and 3d) of Legionellales and cyanobacteria. These findings support and expand a previous analysis of metagenomic data (M. Barz et al., PLoS One 5:e13846, 2010, http://dx.doi.org/10.1371/journal.pone.0013846) and further identify [NiFe]-hydrogenases that could be involved in hydrogen cycling in aquatic surface waters. PMID:24727276

  18. The uptake hydrogenase in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain PCC 7822 protects nitrogenase from oxygen toxicity.

    PubMed

    Zhang, Xiaohui; Sherman, Debra M; Sherman, Louis A

    2014-02-01

    Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H2 produced by the nitrogenase.

  19. The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

    PubMed Central

    Zhang, Xiaohui; Sherman, Debra M.

    2014-01-01

    Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H2 produced by the nitrogenase. PMID:24317398

  20. Flexible plastic bioreactors for photobiological hydrogen production by hydrogenase-deficient cyanobacteria.

    PubMed

    Kitashima, Masaharu; Masukawa, Hajime; Sakurai, Hidehiro; Inoue, Kazuhito

    2012-01-01

    Uptake hydrogenase mutant cells of the cyanobacterium Nostoc sp. PCC 7422 photobiologically produced H(2) catalyzed by nitrogenase for several days in H(2)-barrier transparent plastic bags, and accumulated H(2) in the presence of O(2) evolved by photosynthesis. Their H(2) production activity was higher in the sealed flexible bags than in stoppered serum bottles of fixed gas volume.

  1. Hydrogenase activity of mineral-associated and suspended populations of Desulfovibrio Desulfuricans Essex 6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate...

  2. Lyophilization protects [FeFe]-hydrogenases against O2-induced H-cluster degradation

    PubMed Central

    Noth, Jens; Kositzki, Ramona; Klein, Kathrin; Winkler, Martin; Haumann, Michael; Happe, Thomas

    2015-01-01

    Nature has developed an impressive repertoire of metal-based enzymes that perform complex chemical reactions under moderate conditions. Catalysts that produce molecular hydrogen (H2) are particularly promising for renewable energy applications. Unfortunately, natural and chemical H2-catalysts are often irreversibly degraded by molecular oxygen (O2). Here we present a straightforward procedure based on freeze-drying (lyophilization), that turns [FeFe]-hydrogenases, which are excellent H2-producers, but typically extremely O2-sensitive in solution, into enzymes that are fully resistant against O2. Complete dryness protects and conserves both, the [FeFe]-hydrogenase proteins and their inorganic active-site cofactor (H-cluster), when exposed to 100% O2 for days. The full H2-formation capacity is restored after solvation of the lyophilized enzymes. However, even minimal moisturizing re-establishes O2-sensitivity. The dry [FeFe]-hydrogenase material is superior also for advanced spectroscopic investigations on the H-cluster reaction mechanism. Our method provides a convenient way for long-term storage and impacts on potential biotechnological hydrogen production applications of hydrogenase enzymes. PMID:26364994

  3. Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants.

    PubMed Central

    Merberg, D; Maier, R J

    1984-01-01

    In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity. PMID:6384199

  4. Role of the NiFe Hydrogenase Hya in Oxidative Stress Defense in Geobacter sulfurreducens

    PubMed Central

    Lovley, Derek R.

    2012-01-01

    Geobacter sulfurreducens, an Fe(III)-reducing deltaproteobacterium found in anoxic subsurface environments, contains 4 NiFe hydrogenases. Hyb, a periplasmically oriented membrane-bound NiFe hydrogenase, is essential for hydrogen-dependent growth. The functions of the three other hydrogenases are unknown. We show here that the other periplasmically oriented membrane-bound NiFe hydrogenase, Hya, is necessary for growth after exposure to oxidative stress when hydrogen or a highly limiting concentration of acetate is the electron source. The beneficial impact of Hya on growth was dependent on the presence of H2 in the atmosphere. Moreover, the Hya-deficient strain was more sensitive to the presence of superoxide or hydrogen peroxide. Hya was also required to safeguard Hyb hydrogen oxidation activity after exposure to O2. Overexpression studies demonstrated that Hya was more resistant to oxidative stress than Hyb. Overexpression of Hya also resulted in the creation of a recombinant strain better fitted for exposure to oxidative stress than wild-type G. sulfurreducens. These results demonstrate that one of the physiological roles of the O2-resistant Hya is to participate in the oxidative stress defense of G. sulfurreducens. PMID:22366414

  5. Protonation State of MnFe and FeFe Cofactors in a Ligand-Binding Oxidase Revealed by X-ray Absorption, Emission, and Vibrational Spectroscopy and QM/MM Calculations.

    PubMed

    Kositzki, Ramona; Mebs, Stefan; Marx, Jennifer; Griese, Julia J; Schuth, Nils; Högbom, Martin; Schünemann, Volker; Haumann, Michael

    2016-10-03

    Enzymes with a dimetal-carboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O2) activation. Recently described proteins bind a manganese-iron cofactor (MnFe) instead of the classical diiron cofactor (FeFe). Determination of atomic-level differences of homo- versus hetero-bimetallic cofactors is crucial to understand their diverse redox reactions. We studied a ligand-binding oxidase from the bacterium Geobacillus kaustophilus (R2lox) loaded with a FeFe or MnFe cofactor, which catalyzes O2 reduction and an unusual tyrosine-valine ether cross-link formation, as revealed by X-ray crystallography. Advanced X-ray absorption, emission, and vibrational spectroscopy methods and quantum chemical and molecular mechanics calculations provided relative Mn/Fe contents, X-ray photoreduction kinetics, metal-ligand bond lengths, metal-metal distances, metal oxidation states, spin configurations, valence-level degeneracy, molecular orbital composition, nuclear quadrupole splitting energies, and vibrational normal modes for both cofactors. A protonation state with an axial water (H2O) ligand at Mn or Fe in binding site 1 and a metal-bridging hydroxo group (μOH) in a hydrogen-bonded network is assigned. Our comprehensive picture of the molecular, electronic, and dynamic properties of the cofactors highlights reorientation of the unique axis along the Mn-OH2 bond for the Mn1(III) Jahn-Teller ion but along the Fe-μOH bond for the octahedral Fe1(III). This likely corresponds to a more positive redox potential of the Mn(III)Fe(III) cofactor and higher proton affinity of its μOH group. Refined model structures for the Mn(III)Fe(III) and Fe(III)Fe(III) cofactors are presented. Implications of our findings for the site-specific metalation of R2lox and performance of the O2 reduction and cross-link formation reactions are discussed.

  6. Theoretical 57Fe Mössbauer spectroscopy: isomer shifts of [Fe]-hydrogenase intermediates.

    PubMed

    Hedegård, Erik Donovan; Knecht, Stefan; Ryde, Ulf; Kongsted, Jacob; Saue, Trond

    2014-03-14

    Mössbauer spectroscopy is an indispensable spectroscopic technique and analytical tool in iron coordination chemistry. The linear correlation between the electron density at the nucleus ("contact density") and experimental isomer shifts has been used to link calculated contact densities to experimental isomer shifts. Here we have investigated relativistic methods of systematically increasing sophistication, including the eXact 2-Component (X2C) Hamiltonian and a finite-nucleus model, for the calculation of isomer shifts of iron compounds. While being of similar accuracy as the full four-component treatment, X2C calculations are far more efficient. We find that effects of spin-orbit coupling can safely be neglected, leading to further speedup. Linear correlation plots using effective densities rather than contact densities versus experimental isomer shift lead to a correlation constant a = -0.294 a0(-3) mm s(-1) (PBE functional) which is close to an experimentally derived value. Isomer shifts of similar quality can thus be obtained both with and without fitting, which is not the case if one pursues a priori a non-relativistic model approach. As an application for a biologically relevant system, we have studied three recently proposed [Fe]-hydrogenase intermediates. The structures of these intermediates were extracted from QM/MM calculations using large QM regions surrounded by the full enzyme and a solvation shell of water molecules. We show that a comparison between calculated and experimentally observed isomer shifts can be used to discriminate between different intermediates, whereas calculated atomic charges do not necessarily correlate with Mössbauer isomer shifts. Detailed analysis reveals that the difference in isomer shifts between two intermediates is due to an overlap effect.

  7. Novel H2-oxidizing [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F.

    PubMed

    Nonaka, Kyoshiro; Nguyen, Nga T; Yoon, Ki-Seok; Ogo, Seiji

    2013-04-01

    [NiFeSe]hydrogenases are promising biocatalysts in H2-based technology due to their high catalytic activity and O2-stability. Here, we report purification and characterization of a new membrane-associated [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F ([NiFeSe]DvMF). The [NiFeSe]DvMF was composed of two subunits, corresponding to a large subunit of 58.3 kDa and a small subunit of 29.3 kDa determined by SDS-PAGE. Unlike conventional [NiFeSe]hydrogenases having catalytic bias toward H2-production, the [NiFeSe]DvMF showed 11-fold higher specific activity of H2-oxidation (2444 U/mg) than that of H2-production (217 U/mg). At the optimal reaction temperature of the enzyme (65°C), the specific activity of H2-oxidation could reach up to 21,553 U/mg. Amperometric assays of the [NiFeSe]DvMF clearly indicated that the enzyme had a remarkable O2-stability. According to the amino acid sequence alignment, the conserved cysteine residue at position 281 in medial cluster of other [NiFeSe]hydrogenases was specifically replaced by a serine residue (Ser281) in the [NiFeSe]DvMF. These results indicate that the [NiFeSe]DvMF can play as a new H2-oxidizing and O2-stable biocatalyst, along with providing helpful insights into the structure-function relationship of [NiFeSe]hydrogenases.

  8. The Hyb Hydrogenase Permits Hydrogen-Dependent Respiratory Growth of Salmonella enterica Serovar Typhimurium

    PubMed Central

    Lamichhane-Khadka, Reena; Kwiatkowski, Andrea; Maier, Robert J.

    2010-01-01

    Salmonella enterica serovar Typhimurium contains three distinct respiratory hydrogenases, all of which contribute to virulence. Addition of H2 significantly enhanced the growth rate and yield of S. Typhimurium in an amino acid-containing medium; this occurred with three different terminal respiratory electron acceptors. Based on studies with site-specific double-hydrogenase mutant strains, most of this H2-dependent growth increase was attributed to the Hyb hydrogenase, rather than to the Hya or Hyd respiratory H2-oxidizing enzymes. The wild type strain with H2 had 4.0-fold greater uptake of 14C-labeled amino acids over a period of minutes than did cells incubated without H2. The double-uptake hydrogenase mutant containing only the Hyb hydrogenase transported amino acids H2 dependently like the wild type. The Hyb-only-containing strain produced a membrane potential comparable to that of the wild type. The H2-stimulated amino acid uptake of the wild type and the Hyb-only strain was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but was less affected by the ATP synthase inhibitor sodium orthovanadate. In the wild type, proteins TonB and ExbD, which are known to couple proton motive force (PMF) to transport processes, were induced by H2 exposure, as were the genes corresponding to these periplasmic PMF-coupling factors. However, studies on tonB and exbD single mutant strains could not confirm a major role for these proteins in amino acid transport. The results link H2 oxidation via the Hyb enzyme to growth, amino acid transport, and expression of periplasmic proteins that facilitate PMF-mediated transport across the outer membrane. PMID:21157514

  9. Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum.

    PubMed Central

    Baron, S F; Ferry, J G

    1989-01-01

    The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any amino acid) was conserved in the N-termini of the alpha subunits of the F420-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F420-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F420. Maximal coenzyme F420-reducing activity was obtained at 55 degrees C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H2 production at a rate threefold lower than that for H2 uptake and reduced coenzyme F420, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited the coenzyme F420-dependent but not the methyl viologen-dependent activity of the purified enzyme. Images PMID:2738024

  10. Human Performance and Biosystems

    DTIC Science & Technology

    2013-03-08

    M., Grossman, A.R., and Posewitz. M.C. (2012) Genetic disruption of both Chlamydomonas reinhardtii [ FeFe ]-hydrogenases: insight into the role of...structure of HydF scaffold protein provides insights into [ FeFe ]-hydrogenase maturation. Journal of Biological Chemistry 286, 43944-43950. 8...variabilis NC64A [ FeFe ]-hydrogenase. Planta 234, 829-843. CSM Publications citing AFOSR funding during the last year DISTRIBUTION A: Approved for

  11. A redox hydrogel protects the O2 -sensitive [FeFe]-hydrogenase from Chlamydomonas reinhardtii from oxidative damage.

    PubMed

    Oughli, Alaa Alsheikh; Conzuelo, Felipe; Winkler, Martin; Happe, Thomas; Lubitz, Wolfgang; Schuhmann, Wolfgang; Rüdiger, Olaf; Plumeré, Nicolas

    2015-10-12

    The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2 . [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2 . Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2 , demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2 /O2 mixed feed.

  12. [FeFe]-hydrogenase in Yellowstone National Park: evidence for dispersal limitation and phylogenetic niche conservatism.

    PubMed

    Boyd, Eric S; Hamilton, Trinity L; Spear, John R; Lavin, Matthew; Peters, John W

    2010-12-01

    Hydrogen (H₂) has an important role in the anaerobic degradation of organic carbon and is the basis for many syntrophic interactions that commonly occur in microbial communities. Little is known, however, with regard to the biotic and/or abiotic factors that control the distribution and phylogenetic diversity of organisms which produce H₂ in microbial communities. In this study, we examined the [FeFe]-hydrogenase gene (hydA) as a proxy for fermentative bacterial H₂ production along physical and chemical gradients in various geothermal springs in Yellowstone National Park (YNP), WY, USA. The distribution of hydA in YNP geothermal springs was constrained by pH to environments co-inhabited by oxygenic phototrophs and to environments predicted to have low inputs of abiotic H₂. The individual HydA asssemblages from YNP springs were more closely related when compared with randomly assembled communities, which suggests ecological filtering. Model selection approaches revealed that geographic distance was the best explanatory variable to predict the phylogenetic relatedness of HydA communities. This evinces the dispersal limitation imposed by the geothermal spring environment on HydA phylogenetic diversity even at small spatial scales. pH differences between sites is the second highest ranked explanatory variable of HydA phylogenetic relatedness, which suggests that the ecology related to pH imposes strong phylogenetic niche conservatism. Collectively, these results indicate that pH has imposed strong niche conservatism on fermentative bacteria and that, within a narrow pH realm, YNP springs are dispersal limited with respect to fermentative bacterial communities.

  13. Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen.

    PubMed Central

    Kim, H; Yu, C; Maier, R J

    1991-01-01

    Bradyrhizobium japonicum expresses hydrogenase in microaerophilic free-living conditions in the presence of nickel. Plasmid-borne hup-lacZ transcriptional fusion constructs were used to study the regulation of the hydrogenase gene. The hydrogenase gene was transcriptionally induced under microaerobic conditions (0.1 to 3.0% partial pressure O2). The hydrogenase gene was not transcribed or was poorly transcribed in strictly anaerobic conditions or conditions above 3.0% O2. Hydrogen gas at levels as low as 0.1% partial pressure induced hydrogenase transcription, and a high level of transcription was maintained up to at least 10% H2 concentration. No transcription was observed in the absence of H2. Hydrogenase was regulated by H2, O2, and Ni when the 5'-upstream sequence was pared down to include base number -168. However, when the upstream sequence was pared down to base number -118, the regulatory response to O2, H2, and Ni levels was negated. Thus, a common cis-acting regulatory region localized within 50 bp is critical for the regulation of hydrogenase by hydrogen, oxygen, and nickel. As a control, the B. japonicum hemA gene which codes for delta-aminolevulinic acid synthase was also fused to the promoterless lacZ gene, and its regulation was tested in the presence of various concentrations of O2 and H2. hemA-lacZ transcription was not dependent on levels of Ni, O2, or H2. Two different hup-lacZ fusions were tested in a Hup- background, strain JH47; these hup-lacZ constructs in JH47 demonstrated dependency on nickel, O2, and H2, indicating that the hydrogenase protein itself is not a sensor for regulation by O2, H2, or nickel. PMID:2061281

  14. Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

    PubMed Central

    Jenney, Francis E.; McTernan, Patrick M.; Adams, Michael W. W.

    2010-01-01

    Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins. PMID:20463892

  15. Light-driven hydrogen production by a hybrid complex of a [NiFe]-hydrogenase and the cyanobacterial photosystem I.

    PubMed

    Ihara, Masaki; Nishihara, Hirofumi; Yoon, Ki-Seok; Lenz, Oliver; Friedrich, Bärbel; Nakamoto, Hitoshi; Kojima, Kouji; Honma, Daisuke; Kamachi, Toshiaki; Okura, Ichiro

    2006-01-01

    In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a 'hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the beta-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 mumol H(2).mg chlorophyll(-1).h(-1). The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.

  16. Impact of the chemicals, essential for the purification process of strict Fe-hydrogenase, on the corrosion of mild steel.

    PubMed

    Rouvre, Ingrid; Gauquelin, Charles; Meynial-Salles, Isabelle; Basseguy, Régine

    2016-06-01

    The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.1 M pH7 Tris-HCl medium with or without pure hydrogenase. The results showed that hydrogenase and the additional molecules were in competition, and the electrochemical response could not be attributed solely to hydrogenase. Then, solutions with additional chemicals of different compositions were studied electrochemically. DTT polluted the electrochemical signal by increasing the Eoc by 35 mV 24 h after the injection of 300 μL of control solutions with DTT, whereas it drastically decreased the corrosion rate by increasing the charge transfer resistance (Rct 10 times the initial value). Thus, DTT was shown to have a strong antagonistic effect on corrosion and was removed from the purification process. An optimal composition of the medium was selected (0.5 mM dithionite, 7.5 mM desthiobiotin) that simultaneously allowed a high activity of hydrogenase and a lower impact on the electrochemical response for corrosion tests.

  17. Expression of a clostridial [FeFe]-hydrogenase in Chlamydomonas reinhardtii prolongs photo-production of hydrogen from water splitting

    DOE PAGES

    Noone, Seth; Ratcliff, Kathleen; Davis, ReAnna; ...

    2016-12-24

    The high oxygen (O2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1–hydA2–) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H2 photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active formmore » that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O2 inactivation of CaI led to more sustained H2 photoproduction when cultures were challenged with O2 or kept under prolonged illumination at solar intensities. Lastly, these results provide new insights into the requisites for attaining photobiological H2 production from water splitting using a more O2-tolerant hydrogenase.« less

  18. A Core-Shell Fe/Fe2 O3 Nanowire as a High-Performance Anode Material for Lithium-Ion Batteries.

    PubMed

    Na, Zhaolin; Huang, Gang; Liang, Fei; Yin, Dongming; Wang, Limin

    2016-08-16

    The preparation of novel one-dimensional core-shell Fe/Fe2 O3 nanowires as anodes for high-performance lithium-ion batteries (LIBs) is reported. The nanowires are prepared in a facile synthetic process in aqueous solution under ambient conditions with subsequent annealing treatment that could tune the capacity for lithium storage. When this hybrid is used as an anode material for LIBs, the outer Fe2 O3 shell can act as an electrochemically active material to store and release lithium ions, whereas the highly conductive and inactive Fe core functions as nothing more than an efficient electrical conducting pathway and a remarkable buffer to tolerate volume changes of the electrode materials during the insertion and extraction of lithium ions. The core-shell Fe/Fe2 O3 nanowire maintains an excellent reversible capacity of over 767 mA h g(-1) at 500 mA g(-1) after 200 cycles with a high average Coulombic efficiency of 98.6 %. Even at 2000 mA g(-1) , a stable capacity as high as 538 mA h g(-1) could be obtained. The unique composition and nanostructure of this electrode material contribute to this enhanced electrochemical performance. Due to the ease of large-scale fabrication and superior electrochemical performance, these hybrid nanowires are promising anode materials for the next generation of high-performance LIBs.

  19. Effect of H2 binding on the nonadiabatic transition probability between singlet and triplet states of the [NiFe]-hydrogenase active site.

    PubMed

    Kaliakin, Danil S; Zaari, Ryan R; Varganov, Sergey A

    2015-02-12

    We investigate the effect of H2 binding on the spin-forbidden nonadiabatic transition probability between the lowest energy singlet and triplet electronic states of [NiFe]-hydrogenase active site model, using a velocity averaged Landau-Zener theory. Density functional and multireference perturbation theories were used to provide parameters for the Landau-Zener calculations. It was found that variation of the torsion angle between the terminal thiolate ligands around the Ni center induces an intersystem crossing between the lowest energy singlet and triplet electronic states in the bare active site and in the active site with bound H2. Potential energy curves between the singlet and triplet minima along the torsion angle and H2 binding energies to the two spin states were calculated. Upon H2 binding to the active site, there is a decrease in the torsion angle at the minimum energy crossing point between the singlet and triplet states. The probability of nonadiabatic transitions at temperatures between 270 and 370 K ranges from 35% to 32% for the active site with bound H2 and from 42% to 38% for the bare active site, thus indicating the importance of spin-forbidden nonadiabatic pathways for H2 binding on the [NiFe]-hydrogenase active site.

  20. From hydrogenases to noble metal-free catalytic nanomaterials for H2 production and uptake.

    PubMed

    Le Goff, Alan; Artero, Vincent; Jousselme, Bruno; Tran, Phong Dinh; Guillet, Nicolas; Métayé, Romain; Fihri, Aziz; Palacin, Serge; Fontecave, Marc

    2009-12-04

    Interconversion of water and hydrogen in unitized regenerative fuel cells is a promising energy storage framework for smoothing out the temporal fluctuations of solar and wind power. However, replacement of presently available platinum catalysts by lower-cost and more abundant materials is a requisite for this technology to become economically viable. Here, we show that the covalent attachment of a nickel bisdiphosphine-based mimic of the active site of hydrogenase enzymes onto multiwalled carbon nanotubes results in a high-surface area cathode material with high catalytic activity under the strongly acidic conditions required in proton exchange membrane technology. Hydrogen evolves from aqueous sulfuric acid solution with very low overvoltages (20 millivolts), and the catalyst exhibits exceptional stability (more than 100,000 turnovers). The same catalyst is also very efficient for hydrogen oxidation in this environment, exhibiting current densities similar to those observed for hydrogenase-based materials.

  1. Artificial hydrogenases: biohybrid and supramolecular systems for catalytic hydrogen production or uptake.

    PubMed

    Caserta, Giorgio; Roy, Souvik; Atta, Mohamed; Artero, Vincent; Fontecave, Marc

    2015-04-01

    There is an urgent need for cheap, abundant and efficient catalysts as an alternative to platinum for hydrogen production and oxidation in (photo)electrolyzers and fuel cells. Hydrogenases are attractive solutions. These enzymes use exclusively nickel and iron in their active sites and function with high catalytic rates at the thermodynamic equilibrium. As an alternative, a number of biomimetic and bioinspired catalysts for H2 production and/or uptake, based on Ni, Fe and Co, have been developed and shown to display encouraging performances. In this review we discuss specifically recent approaches aiming at incorporating these compounds within oligomeric and polymeric hosts. The latter are most often biological compounds (peptides, proteins, polysaccharides, etc.) but we also discuss non-biological scaffolds (synthetic polymers, Metal-organic-Frameworks, etc.) which can provide the appropriate environment to tune the activity and stability of the synthetic catalysts. These supramolecular catalytic systems thus define a class of original compounds so-called artificial hydrogenases.

  2. Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

    PubMed Central

    Flanagan, Lindsey A.; Parkin, Alison

    2016-01-01

    Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

  3. Isolation, purification and characterization of the hydrogen evolution promoting factor of hydrogenase of Spirulina platensis

    NASA Astrophysics Data System (ADS)

    Gu, Tian-Qing; Zhang, Hui-Miao; Sun, Shi-Hua

    1996-03-01

    A component (s-factor) with obvious promoting effect on hydrogen evolution of hydrogenase has been isolated and extracted from a cell-free preparation of Spirulina platensis. The effect of the s-factor in the reaction system is similar to that of Na2S2O4, but is coupled with light. The s-factor has the maximum absorption peak at 620 nm in the oxidized state, at 590 nm in the reduced state. The partially purified s-factor showed two bands by SDS-PAGE and is distinctly different from phycocyanin, which has no change of oxidized state and reduced state absorption spectra, and also has no promoting effect on hydrogenase of Spirulina platensis under the light.

  4. Pathways of H2 toward the Active Site of [NiFe]-Hydrogenase

    PubMed Central

    Teixeira, Vitor H.; Baptista, António M.; Soares, Cláudio M.

    2006-01-01

    Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2), but little is known about the diffusion of H2 toward the active site. Here we analyze pathways for H2 permeation using molecular dynamics (MD) simulations in explicit solvent. Various MD simulation replicates were done, to improve the sampling of the system states. H2 easily permeates hydrogenase in every simulation and it moves preferentially in channels. All H2 molecules that reach the active site made their approach from the side of the Ni ion. H2 is able to reach distances of <4 Å from the active site, although after 6 Å permeation is difficult. In this region we mutated Val-67 into alanine and perform new MD simulations. These simulations show an increase of H2 inside the protein and at lower distances from the active site. This valine can be a control point in the H2 access to the active center. PMID:16731562

  5. A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.

    PubMed Central

    Reeve, J N; Beckler, G S; Cram, D S; Hamilton, P T; Brown, J W; Krzycki, J A; Kolodziej, A F; Alex, L; Orme-Johnson, W H; Walsh, C T

    1989-01-01

    The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters. PMID:2654933

  6. Alcaligenes Eutrophus as a Source of Hydrogenase: An Evaluation of Techniques for Its Large Scale Production

    DTIC Science & Technology

    1990-08-01

    block number) -A Alcaligenes eutroplus H16 (AICC 17699), an aerobic H, oxidizing bacteria, has been selected as the most sutable enzyme source for a NAD...laboratory animals (Wistar rats). 2. SELECTION OF Alcaligenes eutrophus AS THE HYDROGENASE SOURCE. 2.1. Hydrogen-oxidizing bacteria: Historical...among strains, and therefore 2 the indMdual species have been placed later under preexisting genera of heterotrophic bacteria - such as Alcaligenes

  7. Proton-coupled electron transfer dynamics in the catalytic mechanism of a [NiFe]-hydrogenase.

    PubMed

    Greene, Brandon L; Wu, Chang-Hao; McTernan, Patrick M; Adams, Michael W W; Dyer, R Brian

    2015-04-08

    The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

  8. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    PubMed

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes.

  9. Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis.

    PubMed

    Lohner, Svenja T; Deutzmann, Jörg S; Logan, Bruce E; Leigh, John; Spormann, Alfred M

    2014-08-01

    Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<-414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.

  10. Proton Reduction Using a Hydrogenase-Modified Nanoporous Black Silicon Photoelectrode

    SciTech Connect

    Zhao, Yixin; Anderson, Nicholas C.; Ratzloff, Michael W.; Mulder, David W.; Zhu, Kai; Turner, John A.; Neale, Nathan R.; King, Paul W.; Branz, Howard M.

    2016-06-15

    Metalloenzymes featuring earth-abundant metal-based cores exhibit rates for catalytic processes such as hydrogen evolution comparable to those of noble metals. Realizing these superb catalytic properties in artificial systems is challenging owing to the difficulty of effectively interfacing metalloenzymes with an electrode surface in a manner that supports efficient charge-transfer. Here, we demonstrate that a nanoporous 'black' silicon (b-Si) photocathode provides a unique interface for binding an adsorbed [FeFe]-hydrogenase enzyme ([FeFe]-H2ase). The resulting [FeFe]-H2ase/b-Si photoelectrode displays a 280 mV more positive onset potential for hydrogen generation than bare b-Si without hydrogenase, similar to that observed for a b-Si/Pt photoelectrode at the same light intensity. Additionally, we show that this H2ase/b-Si electrode exhibits a turnover frequency of >/=1300 s-1 and a turnover number above 107 and sustains current densities of at least 1 mA/cm2 based on the actual surface area of the electrode (not the smaller projected geometric area), orders of magnitude greater than that observed for previous enzyme-catalyzed electrodes. While the long-term stability of hydrogenase on the b-Si surface remains too low for practical applications, this work extends the proof-of-concept that biologically derived metalloenzymes can be interfaced with inorganic substrates to support technologically relevant current densities.

  11. The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation.

    PubMed Central

    Lenz, O.; Schwartz, E.; Dernedde, J.; Eitinger, M.; Friedrich, B.

    1994-01-01

    Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis. Images PMID:8021224

  12. Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis

    PubMed Central

    Lohner, Svenja T; Deutzmann, Jörg S; Logan, Bruce E; Leigh, John; Spormann, Alfred M

    2014-01-01

    Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism. PMID:24844759

  13. Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions.

    PubMed

    Fernandez, V M; Rua, M L; Reyes, P; Cammack, R; Hatchikian, E C

    1989-11-06

    The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(II) and Ni(II) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(II) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the 'unready' enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(II) also inactivated the enzyme irreversible in the 'unready' state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe-4S] signal greater than H2-uptake activity greater than Ni-A signal. Similar results were obtained when the protein was treated with Hg(II). The results demonstrate that the [3Fe-4S] cluster is not essential for H2-uptake activity with methyl viologen, but the integrity of [4Fe-4S] clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D. gigas hydrogenase was found to be highly resistant to digestion by proteases.

  14. Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans.

    PubMed

    Hatchikian, C E; Traore, A S; Fernandez, V M; Cammack, R

    1990-02-14

    The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.

  15. Halotolerant and Resistant to High pH Hydrogenase from Haloalkaliphilic Sulfate-Reducing Bacterium Desulfonatronum thiodismutans

    NASA Technical Reports Server (NTRS)

    Detkova, Ekaterina N.; Pikuta, Elena V.; Hoover, Richard B.

    2004-01-01

    Hydrogenase is the key enzyme of energetic metabolism in cells, it catalyzing the converse reaction of hydrogen oxidation and responsible for consumption and excretion of hydrogen in bacteria. Hydrogenases are proteins containing either Nickel and Iron, or the only Iron in theirs active center. Hydrogenases have been found in many microorganisms, such as Methanogenic, acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria that could utilize the hydrogen as energy source or use it as electron sink. Hydrogenases are subject for wide physiological, biochemical, physicochemical and genetic studies due to theirs abilities produce the molecular hydrogen as alternative source of pure energy. Notwithstanding on enough large quantity of works that deal with intracellular and extrasellular enzymes of halophilic bacteria, the data about hydrogenases and theirs functions of salts practically are absent. The study of hydrogenase in cell-free extracts of extremely halophilic eubacterium Acetohalobium mabaticum showed dramatic increasing activity of the enzyme at high concentrations of NaCl and KCI (close to saturated solution). Here we present the data of free-cells extracted hydrogenase from new haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans, which grow on highly miniralized carbonate-bicarbonate medium in salinity range 1 to 7 % and at pH 7.8 - 10.5. Studied enzyme was active in Concentration range from 0 to 4.3 M NaCl with optimum at 1.0 M NaCl. At 1.0 M NaCl the enzyme activity was increased on 20 %, but with changing concentration from 2.1 M to 3.4 M the activity decreased and was kept on constant level. NaHCO3 inhibited hydrogenase activity on more then 30 %. The maximum of enzyme activity was observed at pH 9.5 with limits 7.5 and 11.5 that practically equal to pH optimum of bacterial growth. Therefore the hydrogenase of Desulfanatronum thiodismutans is tolerant to high concentrations of sodium salts and it also resistant to

  16. Hydrogen-dependent growth of Escherichia coli in anaerobic respiration and the presence of hydrogenases with different functions.

    PubMed

    Yamamoto, I; Ishimoto, M

    1978-09-01

    E. coli K10 was found to grow anaerobically on molecular hydrogen by reducing nitrate, fumarate, and trimethylamine N-oxide when peptone was added to the culture medium. Molar growth yields based on consumed hydrogen estimated from the amounts of reduction products were all 7.8 g cells/mol, suggesting that 1 mol of ATP was produced in the oxidation of 1 mol of hydrogen. Hydrogenase activity measured in terms of hydrogen evolution was several times higher in cells grown on glucose than in cells grown on hydrogen in the presence of fumarate and trimethylamine N-oxide, while hydrogenase activity measured in terms of hydrogen uptake was unchanged in both cases. The ratio of hydrogenase activities measured in terms of hydrogen uptake and evolution was also high in the extract and centrifugal fractions from cells grown in hydrogen. The soluble fraction and trypsin digest of the precipitate at 100,000 X g were subjected to polyacrylamide disc gel electrophoresis and hydrogenase bands were stained by reduction of benzyl viologen with hydrogen and by oxidation of reduced methyl viologen. The resulting patterns suggest that multiple forms of hydrogenase are present and that the amounts of forms functioning in hydrogen evolution were greatly decresed in cells grown on hydrogen in the presence of acceptors.

  17. Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli.

    PubMed

    Maier, Johannes A H; Ragozin, Sergey; Jeltsch, Albert

    2015-04-10

    Hydrogen (H2) is a potential alternative energy carrier which only produces water and heat upon combustion. Today, industrial hydrogen production mainly uses thermochemical processes based on fossil fuels or electrolysis of water. Therefore, biotechnological approaches to produce H2 from biomass are an interesting alternative. We introduce here a novel direct hydrogen measurement system using a semiconducting device specific for hydrogen detection. Using this device, a bacterium producing considerable amounts of hydrogen under aerobic cultivation was isolated and identified by 16S ribosomal DNA sequencing as Citrobacter sp. The enzyme responsible for the observed hydrogenase activity was partially purified by 3 chromatographic purification steps and could be identified by peptide mass fingerprinting to be a type 2 [NiFe]-hydrogenase. Expression of the [NiFe]-hydrogenase 2 containing operon from Citrobacter sp. SG in Escherichia coli allowed recombinant hydrogen production. The [NiFe]-hydrogenase 2 identified here may be useful for biotechnological hydrogen production. We speculate that the expression of the hydrogenase in Citrobacter may be an adaptation to growth in acidic conditions.

  18. Synthesis, structure and geometrically frustrated magnetism of the layered oxide-stannide compounds Fe(Fe3-xMnx)Si2Sn7O16.

    PubMed

    Allison, M C; Avdeev, M; Schmid, S; Liu, S; Söhnel, T; Ling, C D

    2016-06-21

    Fe4Si2Sn7O16 has a unique crystal structure that contains alternating layers of Fe(2+) ions octahedrally coordinated by O (oxide layer) and Sn (stannide layer), bridged by SiO4 tetrahedra. The formula can be written as FeFe3Si2Sn7O16 to emphasise the distinction between the layers. Here, we report the changes in structure and properties as iron is selectively replaced by manganese in the oxide layer. Solid-state synthesis was used to produce polycrystalline samples of Fe(Fe3-xMnx)Si2Sn7O16 for x≤ 2.55, the structures of which were characterised using high-resolution synchrotron X-ray and neutron powder diffraction. Single-crystal samples were also grown at x = 0.35, 0.95, 2.60 and characterised by single crystal X-ray diffraction. We show that manganese is doped exclusively into the oxide layer, and that this layer contains exclusively magnetically active high-spin M(2+) transition metal cations; while the stannide layer only accommodates non-magnetic low-spin Fe(2+). All samples show clear evidence of geometrically frustrated magnetism, which we associate with the fact that the topology of the high-spin M(2+) ions in the oxide layer describes a perfect kagomé lattice. Despite this frustration, the x = 0 and x = 2.55 samples undergo long-range antiferromagnetic ordering transitions at 3.0 K and 2.5 K, respectively.

  19. The Mössbauer Parameters of the Proximal Cluster of Membrane-Bound Hydrogenase Revisited: A Density Functional Theory Study

    PubMed Central

    2015-01-01

    An unprecedented [4Fe-3S] cluster proximal to the regular [NiFe] active site has recently been found to be responsible for the ability of membrane-bound hydrogenases (MBHs) to oxidize dihydrogen in the presence of ambient levels of oxygen. Starting from proximal cluster models of a recent DFT study on the redox-dependent structural transformation of the [4Fe-3S] cluster, 57Fe Mössbauer parameters (electric field gradients, isomer shifts, and nuclear hyperfine couplings) were calculated using DFT. Our results revise the previously reported correspondence of Mössbauer signals and iron centers in the [4Fe-3S]3+ reduced-state proximal cluster. Similar conflicting assignments are also resolved for the [4Fe-3S]5+ superoxidized state with particular regard to spin-coupling in the broken-symmetry DFT calculations. Calculated 57Fe hyperfine coupling (HFC) tensors expose discrepancies in the experimental set of HFC tensors and substantiate the need for additional experimental work on the magnetic properties of the MBH proximal cluster in its reduced and superoxidized redox states. PMID:26598030

  20. Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival.

    PubMed

    Greening, Chris; Biswas, Ambarish; Carere, Carlo R; Jackson, Colin J; Taylor, Matthew C; Stott, Matthew B; Cook, Gregory M; Morales, Sergio E

    2016-03-01

    Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H2) is a niche process. To gain a broader insight into the importance of microbial H2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H2. The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H2-based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H2-sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content (pO2) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H2

  1. Novel [NiFe]- and [FeFe]-hydrogenase gene transcripts indicative of active facultative aerobes and obligate anaerobes in earthworm gut contents.

    PubMed

    Schmidt, Oliver; Wüst, Pia K; Hellmuth, Susanne; Borst, Katharina; Horn, Marcus A; Drake, Harold L

    2011-09-01

    The concomitant occurrence of molecular hydrogen (H(2)) and organic acids along the alimentary canal of the earthworm is indicative of ongoing fermentation during gut passage. Fermentative H(2) production is catalyzed by [FeFe]-hydrogenases and group 4 [NiFe]-hydrogenases in obligate anaerobes (e.g., Clostridiales) and facultative aerobes (e.g., Enterobacteriaceae), respectively, functional groups that might respond differently to contrasting redox conditions. Thus, the objectives of this study were to assess the redox potentials of the alimentary canal of Lumbricus terrestris and analyze the hydrogenase transcript diversities of H(2) producers in glucose-supplemented gut content microcosms. Although redox potentials in the core of the alimentary canal were variable on an individual worm basis, average redox potentials were similar. The lowest redox potentials occurred in the foregut and midgut regions, averaging 40 and 110 mV, respectively. Correlation plots between hydrogenase amino acid sequences and 16S rRNA gene sequences indicated that closely related hydrogenases belonged to closely related taxa, whereas distantly related hydrogenases did not necessarily belong to distantly related taxa. Of 178 [FeFe]-hydrogenase gene transcripts, 177 clustered in 12 Clostridiales-affiliated operational taxonomic units, the majority of which were indicative of heretofore unknown hydrogenases. Of 86 group 4 [NiFe]-hydrogenase gene transcripts, 79% and 21% were affiliated with organisms in the Enterobacteriaceae and Aeromonadaceae, respectively. The collective results (i) suggest that fermenters must cope with variable and moderately oxidative redox conditions along the alimentary canal, (ii) demonstrate that heretofore undetected hydrogenases are present in the earthworm gut, and (iii) corroborate previous findings implicating Clostridiaceae and Enterobacteriaceae as active fermentative taxa in earthworm gut content.

  2. High-performance hydrogen production and oxidation electrodes with hydrogenase supported on metallic single-wall carbon nanotube networks.

    PubMed

    Svedružić, Draženka; Blackburn, Jeffrey L; Tenent, Robert C; Rocha, John-David R; Vinzant, Todd B; Heben, Michael J; King, Paul W

    2011-03-30

    We studied the electrocatalytic activity of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaH2ase) immobilized on single-wall carbon nanotube (SWNT) networks. SWNT networks were prepared on carbon cloth by ultrasonic spraying of suspensions with predetermined ratios of metallic and semiconducting nanotubes. Current densities for both proton reduction and hydrogen oxidation electrocatalytic activities were at least 1 order of magnitude higher when hydrogenase was immobilized onto SWNT networks with high metallic tube (m-SWNT) content in comparison to hydrogenase supported on networks with low metallic tube content or when SWNTs were absent. We conclude that the increase in electrocatalytic activities in the presence of SWNTs was mainly due to the m-SWNT fraction and can be attributed to (i) substantial increases in the active electrode surface area, and (ii) improved electronic coupling between CaH2ase redox-active sites and the electrode surface.

  3. Effects of anaerobic regulatory mutations and catabolite repression on regulation of hydrogen metabolism and hydrogenase isoenzyme composition in Salmonella typhimurium.

    PubMed

    Jamieson, D J; Sawers, R G; Rugman, P A; Boxer, D H; Higgins, C F

    1986-10-01

    Hydrogen metabolism in Salmonella typhimurium is differentially regulated by mutations in the two anaerobic regulatory pathways, defined by the fnr (oxrA) and oxrC genes, and is controlled by catabolite repression. The synthesis of the individual hydrogenase isoenzymes is also specifically influenced by fnr and oxrC mutations and by catabolite repression in a manner entirely consistent with the proposed role for each isoenzyme in hydrogen metabolism. Synthesis of hydrogenase isoenzyme 2 was found to be fnr dependent and oxrC independent, consistent with a role in respiration-linked hydrogen uptake which was shown to be similarly regulated. Also in keeping with such a respiratory role was the finding that both hydrogen uptake and the expression of isoenzyme 2 are under catabolite repression. In contrast, formate hydrogenlyase-dependent hydrogen evolution, characteristic of fermentative growth, was reduced in oxrC strains but not in fnr strains. Hydrogenase 3 activity was similarly regulated, consistent with a role in hydrogen evolution. Unlike the expression of hydrogenases 2 and 3, hydrogenase 1 expression was both fnr and oxrC dependent. Hydrogen uptake during fermentative growth was also both fnr and oxrC dependent. This provided good evidence for a distinction between hydrogen uptake during fermentation- and respiration-dependent growth and for a hydrogen-recycling process. The pattern of anaerobic control of hydrogenase activities illustrated the functional diversity of the isoenzymes and, in addition, the physiological distinction between the two anaerobic regulatory pathways, anaerobic respiratory genes being fnr dependent and enzymes required during fermentative growth being oxrC dependent.

  4. Density functional study of the catalytic cycle of nickel-iron [NiFe] hydrogenases and the involvement of high-spin nickel(II).

    PubMed

    Pardo, Alejandro; De Lacey, Antonio L; Fernández, Víctor M; Fan, Hua-Jun; Fan, Yubo; Hall, Michael B

    2006-04-01

    In light of recent experiments suggesting high-spin (HS) Ni(II) species in the catalytic cycle of [NiFe] hydrogenase, a series of models of the Ni(II) forms Ni-SI(I,II), SI-CO and Ni-R(I,II,III) were examined in their high-spin states via density functional calculations. Because of its importance in the catalytic cycle, the Ni-C form was also included in this study. Unlike the Ni(II) forms in previous studies, in which a low-spin (LS) state was assumed and a square-planar structure found, the optimized geometries of these HS Ni(II) forms resemble those observed in the crystal structures: a distorted tetrahedral to distorted pyramidal coordination for the NiS4. This resemblance is particularly significant because the LS state is 20-30 kcal/mol less stable than the HS state for the geometry of the crystal structure. If these Ni(II) forms in the enzyme are not high spin, a large change in geometry at the active site is required during the catalytic cycle. Furthermore, only the HS state for the CO-inhibited form SI-CO has CO stretching frequencies that match the experimental results. As in the previous work, these new results show that the heterolytic cleavage reaction of dihydrogen (where H2 is cleaved with the metal acting as a hydride acceptor and a cysteine as the proton acceptor) has a lower energy barrier and is more exothermic when the active site is oxidized to Ni(III). The enzyme models described here are supported by a calibrated correlation of the calculated and measured CO stretching frequencies of the forms of the enzyme. The correlation coefficient for the final set of models of the forms of [NiFe] hydrogenase is 0.8.

  5. Requirements for construction of a functional hybrid complex of photosystem I and [NiFe]-hydrogenase.

    PubMed

    Schwarze, Alexander; Kopczak, Marta J; Rögner, Matthias; Lenz, Oliver

    2010-04-01

    The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H(2) sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O(2)-tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH(PsaE)) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H(2) production capacity of the purified MBH(PsaE) fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH(PsaE) with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH(PsaE)-PS I complex formation and its light-driven H(2) production capacity by means of spectroelectrochemistry.

  6. A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF.

    PubMed

    Shepard, Eric M; Byer, Amanda S; Betz, Jeremiah N; Peters, John W; Broderick, Joan B

    2016-06-28

    [FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.

  7. A Transmissible Plant Shoot Factor Promotes Uptake Hydrogenase Activity in Rhizobium Symbionts 1

    PubMed Central

    Bedmar, Eulogio J.; Phillips, Donald A.

    1984-01-01

    Shoot/root grafting studies showed organ and host cultivar effects on net H2 evolution from Pisum sativum L. root nodules. Net H2 evolution from those nodules represents the sum of H2 formed by Rhizobium nitrogenase and H2 oxidized by any uptake hydrogenase present in the bacteria. Grafts between pea cultivars `JI1205' or `Alaska' and `Feltham First' in symbioses with R. leguminosarum 128C53 showed that shoots of both JI1205 and Alaska increased H2 uptake significantly (P ≤ 0.05) in Feltham First root nodules. The same plants also had less net H2 evolution at similar rates of C2H2 reduction than plants formed by grafting Feltham First shoots on Feltham First roots. Although JI1205 and Alaska shoots increased H2-uptake activity of Feltham First root nodules 28 days after the graft was made, intermediate to high levels of H2 uptake activity were still present in nodules on roots of both JI1205 and Alaska grafted to Feltham First shoots. These results indicate the presence of a transmissible shoot factor(s) which can increase uptake hydrogenase activity in a Rhizobium symbiont and show that root genotype also can influence that parameter. Parallel grafting experiments using the same pea cultivars in symbioses with R. leguminosarum strain 300, which lacks uptake hydrogenase activity, suggested that a transmissible shoot factor(s) altered H2 formation from nitrogenase by changing the electron allocation coefficient of that enzyme complex. The root and shoot factor(s) detected in this study had no permanent effect on strain 128C53. Bacterial cells isolated from Feltham First nodules with low H2 uptake activity formed root nodules on JI1205 and Alaska with high H2 uptake activity. Bacteroids isolated from nodules on intact JI1205, Alaska, or Feltham First plants with high, medium, or low H2 uptake activity, respectively, maintained those phenotypes during in vitro assays. PMID:16663677

  8. A transmissible plant shoot factor promotes uptake hydrogenase activity in Rhizobium symbionts.

    PubMed

    Bedmar, E J; Phillips, D A

    1984-07-01

    Shoot/root grafting studies showed organ and host cultivar effects on net H(2) evolution from Pisum sativum L. root nodules. Net H(2) evolution from those nodules represents the sum of H(2) formed by Rhizobium nitrogenase and H(2) oxidized by any uptake hydrogenase present in the bacteria. Grafts between pea cultivars ;JI1205' or ;Alaska' and ;Feltham First' in symbioses with R. leguminosarum 128C53 showed that shoots of both JI1205 and Alaska increased H(2) uptake significantly (P hydrogenase activity in a Rhizobium symbiont and show that root genotype also can influence that parameter.Parallel grafting experiments using the same pea cultivars in symbioses with R. leguminosarum strain 300, which lacks uptake hydrogenase activity, suggested that a transmissible shoot factor(s) altered H(2) formation from nitrogenase by changing the electron allocation coefficient of that enzyme complex.The root and shoot factor(s) detected in this study had no permanent effect on strain 128C53. Bacterial cells isolated from Feltham First nodules with low H(2) uptake activity formed root nodules on JI1205 and Alaska with high H(2) uptake activity. Bacteroids isolated from nodules on intact JI1205, Alaska, or Feltham First plants with high, medium, or low H(2) uptake activity, respectively, maintained those phenotypes during in vitro assays.

  9. Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases.

    PubMed

    Pandey, Kavita; Islam, Shams T A; Happe, Thomas; Armstrong, Fraser A

    2017-04-11

    The kinetics of hydrogen oxidation and evolution by [FeFe]-hydrogenases have been investigated by electrochemical impedance spectroscopy-resolving factors that determine the exceptional activity of these enzymes, and introducing an unusual and powerful way of analyzing their catalytic electron transport properties. Attached to an electrode, hydrogenases display reversible electrocatalytic behavior close to the 2H(+)/H2 potential, making them paradigms for efficiency: the electrocatalytic "exchange" rate (measured around zero driving force) is therefore an unusual parameter with theoretical and practical significance. Experiments were carried out on two [FeFe]-hydrogenases, CrHydA1 from the green alga Chlamydomonas reinhardtii, which contains only the active-site "H cluster," and CpI from the fermentative anaerobe Clostridium pasteurianum, which contains four low-potential FeS clusters that serve as an electron relay in addition to the H cluster. Data analysis yields catalytic exchange rates (at the formal 2H(+)/H2 potential, at 0 °C) of 157 electrons (78 molecules H2) per second for CpI and 25 electrons (12 molecules H2) per second for CrHydA1. The experiments show how the potential dependence of catalytic electron flow comprises frequency-dependent and frequency-independent terms that reflect the proficiencies of the catalytic site and the electron transfer pathway in each enzyme. The results highlight the "wire-like" behavior of the Fe-S electron relay in CpI and a low reorganization energy for electron transfer on/off the H cluster.

  10. Three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina.

    PubMed Central

    Sherman, M B; Orlova, E V; Smirnova, E A; Hovmöller, S; Zorin, N A

    1991-01-01

    The three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina has been determined at a resolution of 2 nm in the plane and 4 nm in the vertical direction by electron microscopy and computerized image processing on microcrystals of the enzyme. The enzyme forms a large ring-shaped complex containing six each of the large (62-kDa) and small (26-kDa) subunits. The complex is very open, with six well-separated dumbbell-shaped masses surrounding a large cylindrical hole. Each dumbbell is interpreted as consisting of one large and one small subunit. Images PMID:1901570

  11. Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.

    PubMed Central

    Colbeau, A; Vignais, P M

    1992-01-01

    The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis. PMID:1624420

  12. HupO, a Novel Regulator Involved in Thiosulfate-Responsive Control of HupSL [NiFe]-Hydrogenase Synthesis in Thiocapsa roseopersicina

    PubMed Central

    Nagy, Ildikó K.; Kovács, Kornél L.

    2016-01-01

    [NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacterium Thiocapsa roseopersicina harbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2 in Rhodobacter capsulatus and Cupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional in T. roseopersicina and that thiosulfate is a regulating factor of hup expression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase in T. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in that hup expression was decreased significantly in the hox1 mutant. This reduction in Hup synthesis could be reversed by mutation of hupO, which resulted in strongly elevated hup expression, as well as Hup protein levels, and concomitant in vivo hydrogen uptake activity in the hox1 mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependent hup expression was shown in the hupO mutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network in T. roseopersicina. PMID:26801573

  13. The AbrB2 autorepressor, expressed from an atypical promoter, represses the hydrogenase operon to regulate hydrogen production in Synechocystis strain PCC6803.

    PubMed

    Dutheil, Jérémy; Saenkham, Panatda; Sakr, Samer; Leplat, Christophe; Ortega-Ramos, Marcia; Bottin, Hervé; Cournac, Laurent; Cassier-Chauvat, Corinne; Chauvat, Franck

    2012-10-01

    We have thoroughly investigated the abrB2 gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium Synechocystis strain PCC6803. We report that abrB2 is expressed from an active but atypical promoter that possesses an extended -10 element (TGTAATAT) that compensates for the absence of a -35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended -10 promoter box and the absence of a -35 element are two well-conserved features in abrB2 genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the hox operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the hox operon is weakly expressed even though it possesses the two sequences resembling canonical -10 and -35 promoter boxes. In both the AbrB2-repressed promoters of the abrB2 gene and the hox operon, we found a repeated DNA motif [TT-(N(5))-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the abrB2 promoter. We think that our abrB2-deleted mutant with increased expression of the hox operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the abrB2 gene and the hox operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in Synechocystis.

  14. Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobium japonicum uptake hydrogenase.

    PubMed Central

    Sayavedra-Soto, L A; Powell, G K; Evans, H J; Morris, R O

    1988-01-01

    An indispensable part of the hydrogen-recycling system in Bradyrhizobium japonicum is the uptake hydrogenase, which is composed of 34.5- and 65.9-kDa subunits. The gene encoding the large subunit is located on a 5.9-kilobase fragment of the H2-uptake-complementing cosmid pHU52 [Zuber, M., Harker, A.R., Sultana, M.A. & Evans, H.J. (1986) Proc. Natl. Acad. Sci. USA 83, 7668-7672]. We have now determined that the structural genes for both subunits are present on this fragment. Two open reading frames are present that correspond in size and deduced amino acid sequence to the hydrogenase subunits, except that the small-subunit coding region contains a leader peptide of 46 amino acids. The two genes are separated by a 32-nucleotide intergenic region and likely constitute an operon. Comparison of the deduced amino acid sequences of the B. japonicum genes with those from Desulfovibrio gigas, Desulfovibrio baculatus, and Rhodobacter capsulatus indicates significant sequence identity. Images PMID:3054886

  15. Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

    PubMed Central

    Petrenko, Alexander; Stein, Matthias

    2017-01-01

    Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron–sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from Density Functional Theory (DFT) calculations. The distal cubane FeS-cluster has a three-cysteine and one-histidine coordination [Fe4S4](His)(Cys)3 first ligation sphere. The reorganization energy (inner- and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and an all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe4S4](Cys)4 case. The fast and efficient electron transfer from the distal iron–sulfur cluster is realized by a fine-tuned protein environment, which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering. PMID:28067774

  16. Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

    DOE PAGES

    Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; ...

    2015-08-10

    The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique ‘wagging’ mode involving H- motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate amore » low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H- binding Ni more tightly than Fe. Lastly, the present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.« less

  17. Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

    SciTech Connect

    Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; Schilter, David; Pelmenschikov, Vladimir; van Gastel, Maurice; Neese, Frank; Rauchfuss, Thomas B.; Gee, Leland B.; Scott, Aubrey D.; Yoda, Yoshitaka; Lubitz, Wolfgang; Cramer, Stephen P.

    2015-08-10

    The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique ‘wagging’ mode involving H- motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H- binding Ni more tightly than Fe. Lastly, the present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.

  18. Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

    PubMed Central

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Wittkamp, Florian; Apfel, Ulf-Peter; Heberle, Joachim; Haumann, Michael; Stripp, Sven Timo

    2016-01-01

    The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN−) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of 13CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN− at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle. PMID:27432985

  19. Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

    NASA Astrophysics Data System (ADS)

    Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; Schilter, David; Pelmenschikov, Vladimir; van Gastel, Maurice; Neese, Frank; Rauchfuss, Thomas B.; Gee, Leland B.; Scott, Aubrey D.; Yoda, Yoshitaka; Tanaka, Yoshihito; Lubitz, Wolfgang; Cramer, Stephen P.

    2015-08-01

    The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique `wagging' mode involving H- motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe-CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H- binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe-H moieties in other important natural and synthetic catalysts.

  20. Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

    PubMed Central

    Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; Schilter, David; Pelmenschikov, Vladimir; van Gastel, Maurice; Neese, Frank; Rauchfuss, Thomas B.; Gee, Leland B.; Scott, Aubrey D.; Yoda, Yoshitaka; Tanaka, Yoshihito; Lubitz, Wolfgang; Cramer, Stephen P.

    2015-01-01

    The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique ‘wagging' mode involving H− motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H− binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts. PMID:26259066

  1. Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase

    SciTech Connect

    Sode, Olaseni; Voth, Gregory A.

    2014-12-14

    Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A{sup 2−}, a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A{sup 2-} by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.

  2. THE USE OF A HYDROGENASE-METHYLENE BLUE SYSTEM IN A BIOCHEMICAL FUEL CELL (AN ANODE REACTION)

    DTIC Science & Technology

    An electron carrier system similar to one found in living cells has been studied in vitro at the anode of a biochemical fuel cell . The action of an...the anode of a biochemical fuel cell . A current of 0.16 mA/(sq. cm.) has been shown using a hydrogenase solution obtained from E. coli. It gave

  3. Expression of Shewanella oneidensis MR-1 [FeFe]-Hydrogenase Genes in Anabaena sp. Strain PCC 7120

    PubMed Central

    Gärtner, Katrin; Lechno-Yossef, Sigal; Cornish, Adam J.; Wolk, C. Peter

    2012-01-01

    H2 generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H2 from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O2 sensitive. Certain filamentous cyanobacteria protect nitrogenase from O2 by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter PhetN. Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O2. PMID:23023750

  4. Structure of an Actinobacterial-Type [NiFe]-Hydrogenase Reveals Insight into O2-Tolerant H2 Oxidation.

    PubMed

    Schäfer, Caspar; Bommer, Martin; Hennig, Sandra E; Jeoung, Jae-Hun; Dobbek, Holger; Lenz, Oliver

    2016-02-02

    A novel group of bacterial [NiFe]-hydrogenases is responsible for high-affinity H2 uptake from the troposphere, and is therefore thought to play an important role in the global H2 cycle. Here we present the first crystal structure at 2.85-Å resolution of such an actinobacterial-type hydrogenase (AH), which was isolated from the dihydrogen oxidizing bacterium, Ralstonia eutropha. The enzyme has a dimeric structure carrying two active [NiFe] sites that are interconnected by six [4Fe4S] clusters over a range of approximately 90 Å. Unlike most other [NiFe]-hydrogenases, the [4Fe4S] cluster proximal to the [NiFe] site is coordinated by three cysteines and one aspartate. Mutagenesis experiments revealed that this aspartate residue is related to the apparent O2 insensitivity of the AH. Our data provide first structural insight into specialized hydrogenases that are supposed to consume atmospheric H2 under challenging conditions, i.e. at high O2 concentration and wide temperature and pH ranges.

  5. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG).

    PubMed

    Mulder, David W; Boyd, Eric S; Sarma, Ranjana; Lange, Rachel K; Endrizzi, James A; Broderick, Joan B; Peters, John W

    2010-05-13

    Complex enzymes containing Fe-S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear. Here we show the structure of HydA(DeltaEFG) (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe-4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

  6. Phase relations in the Fe-Fe3C-Fe3N system at 7.8 GPa and 1350 °C: Implications for carbon and nitrogen hosts in Fe0-saturated upper mantle

    NASA Astrophysics Data System (ADS)

    Sokol, Alexander G.; Kruk, Alexey N.; Seryotkin, Yurii V.; Korablin, Alexander A.; Palyanov, Yury N.

    2017-04-01

    Phase relations in the Fe-Fe3C-Fe3N system are studied in high-pressure experiments at 7.8 GPa and 1350 °C using a split-sphere multi-anvil apparatus. The starting mixtures consisting of Fe, Fe3C and Fe3N are loaded into ceramic or graphite capsules. Contamination with trace amounts of oxygen leads to the appearance of wüstite in the system retaining oxygen fugacity (fO2) near the iron-wüstite (IW) buffer. The metal melt rich in carbon and nitrogen has a large stability field in the central part of the phase diagram, and this field at 1350 °C is tangent to the Fe-Fe3C side of the Fe-Fe3C-Fe3N triangle at the point of the Fe-Fe3C eutectics. Iron nitride ε-Fe3N (space group P6322 or P63/mmc) contains variable amounts of C and N: up to 2.0-2.5 wt% C and 6.0-7.3 wt% N in equilibrium with a C- and N-rich melt and as little as 1.0 wt% C and 3.2 wt% N in equilibrium with γ- Fe. The limit C and N contents in γ-Fe equilibrated with the C- and N-rich melt is about 1.0 wt%, while the N solubility in cementite (Fe3C) does not exceed 0.5 wt%. The obtained data make basis for the isothermal section of the Fe-Fe3C-Fe3N system. The metal melt phase is inferred to be the main host of carbon and nitrogen in the Fe0-saturated (0.1 wt%) mantle at a depth of ∼250 km. In particular, C- and N-bearing austenite (γ-Fe) and metal melts host carbon and nitrogen in the mantle depleted in volatiles (20 ppm C and 1 ppm N), whereas carbon and nitrogen in the mantle with high concentrations of volatiles (250 ppm C and 100 ppm N) reside in C- and N-rich melts with a minor amount of iron carbide (Fe3C). The presence of nickel and sulphur in metal are expected to inhibit the formation of iron carbide and increases the melt phase stability. Redox freezing of N-rich carbonate melts from subduction slabs in Fe0-saturated mantle may produce iron melts supersaturated with nitrogen and stable ε-Fe3N.

  7. Vibrational cooling dynamics of a [FeFe]-hydrogenase mimic probed by time-resolved infrared spectroscopy.

    PubMed

    Caplins, Benjamin W; Lomont, Justin P; Nguyen, Son C; Harris, Charles B

    2014-12-11

    Picosecond time-resolved infrared spectroscopy (TRIR) was performed for the first time on a dithiolate bridged binuclear iron(I) hexacarbonyl complex ([Fe₂(μ-bdt)(CO)₆], bdt = benzene-1,2-dithiolate) which is a structural mimic of the active site of the [FeFe]-hydrogenase enzyme. As these model active sites are increasingly being studied for their potential in photocatalytic systems for hydrogen production, understanding their excited and ground state dynamics is critical. In n-heptane, absorption of 400 nm light causes carbonyl loss with low quantum yield (<10%), while the majority (ca. 90%) of the parent complex is regenerated with biexponential kinetics (τ₁ = 21 ps and τ₂ = 134 ps). In order to understand the mechanism of picosecond bleach recovery, a series of UV-pump TRIR experiments were performed in different solvents. The long time decay (τ₂) of the transient spectra is seen to change substantially as a function of solvent, from 95 ps in THF to 262 ps in CCl₄. Broadband IR-pump TRIR experiments were performed for comparison. The measured vibrational lifetimes (T₁(avg)) of the carbonyl stretches were found to be in excellent correspondence to the observed τ₂ decays in the UV-pump experiments, signifying that vibrationally excited carbonyl stretches are responsible for the observed longtime decays. The fast spectral evolution (τ₁) was determined to be due to vibrational cooling of low frequency modes anharmonically coupled to the carbonyl stretches that were excited after electronic internal conversion. The results show that cooling of both low and high frequency vibrational modes on the electronic ground state give rise to the observed picosecond TRIR transient spectra of this compound, without the need to invoke electronically excited states.

  8. Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning.

    PubMed

    La Favre, J S; Focht, D D

    1983-08-01

    Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing (15)N-enriched organic matter. Seasonal N(2) fixation activity was determined by periodically assaying plants for reduction of C(2)H(2). N(2) fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N(2) fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of (15)N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N(2) fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.Gas samples were taken from soil columns several times during the growth cycle of the plants. H(2) was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H(2) consumption by soil bacteria. Estimation of N(2) fixation by acetylene reduction activity was closest to the direct (15)N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct (15)N fixation rates ranged

  9. The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic, Nitrate-limiting Conditions*

    PubMed Central

    Gutekunst, Kirstin; Chen, Xi; Schreiber, Karoline; Kaspar, Ursula; Makam, Srinivas; Appel, Jens

    2014-01-01

    Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO2-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase. PMID:24311779

  10. Single crystal EPR study of the Ni center of NiFe hydrogenase

    NASA Astrophysics Data System (ADS)

    Geßner, Ch.; Trofanchuk, O.; Kawagoe, K.; Higuchi, Y.; Yasuoka, N.; Lubitz, W.

    1996-07-01

    EPR spectra of single crystals of NiFe hydrogenase from Desulfovibrio vulgaris Miyazaki F were evaluated and yielded the g-tensors of the Ni center for two different states of enzyme. The g-values associated with these states are identical to those measured in frozen solutions for the ready (NiB) and the unready (NiA) form of the Ni center. Directions of the g-tensor axes were determined relative to the crystal symmetry axes. The obtained changes of g-values and tensor axes orientations between NiA and NiB can be explained by a structural difference involving modification of a cysteine sulfur ligand.

  11. [Conditions for effective hydrogen photoevolution by chloroplasts in the presence of bacterial hydrogenase].

    PubMed

    Krasnovskiĭ, A A; Chan-van-Ni; Nikandrov, V V; Brin, G P

    1980-01-01

    The hydrogen photoevolution was studied to compare the efficiency of chloroplasts or solubilized chlorophyll in the presence of hydrogenase from Clostridium butyricum and methylviologen which links the electron transfer from photosystems to the exogenous enzyme. The hydrogen evolution by chloroplasts in the absence of exogeneous electron donors (or in the presence of irreversibly oxidized dithiotreitol or cysteine) is probably limited by cyclic electron flow shot-circuiting the photosystem 1. Efficiency of hydrogen photoproduction when ascorbate or NADP.H are used as electron donors is probably limited by reverse reaction of photoreduced methylviologen with the oxidized electron donor. The combination of both dithiotreitol and ascorbate prevents the shot-circuiting of photosystem 1 by methylviologen; in this case the maximum efficiency of hydrogen photoevolution was achieved up to 400 mumol H2 per 1 mg chlorophyll per hour.

  12. Photocatalytic Hydrogen Production using Polymeric Carbon Nitride with a Hydrogenase and a Bioinspired Synthetic Ni Catalyst**

    PubMed Central

    Caputo, Christine A; Gross, Manuela A; Lau, Vincent W; Cavazza, Christine; Lotsch, Bettina V; Reisner, Erwin

    2014-01-01

    Solar-light-driven H2 production in water with a [NiFeSe]-hydrogenase (H2ase) and a bioinspired synthetic nickel catalyst (NiP) in combination with a heptazine carbon nitride polymer, melon (CNx), is reported. The semibiological and purely synthetic systems show catalytic activity during solar light irradiation with turnover numbers (TONs) of more than 50 000 mol H2 (mol H2ase)−1 and approximately 155 mol H2 (mol NiP)−1 in redox-mediator-free aqueous solution at pH 6 and 4.5, respectively. Both systems maintained a reduced photoactivity under UV-free solar light irradiation (λ>420 nm). PMID:26300567

  13. Photocatalytic Hydrogen Production using Polymeric Carbon Nitride with a Hydrogenase and a Bioinspired Synthetic Ni Catalyst**

    PubMed Central

    Caputo, Christine A; Gross, Manuela A; Lau, Vincent W; Cavazza, Christine; Lotsch, Bettina V; Reisner, Erwin

    2014-01-01

    Solar-light-driven H2 production in water with a [NiFeSe]-hydrogenase (H2ase) and a bioinspired synthetic nickel catalyst (NiP) in combination with a heptazine carbon nitride polymer, melon (CNx), is reported. The semibiological and purely synthetic systems show catalytic activity during solar light irradiation with turnover numbers (TONs) of more than 50 000 mol H2 (mol H2ase)−1 and approximately 155 mol H2 (mol NiP)−1 in redox-mediator-free aqueous solution at pH 6 and 4.5, respectively. Both systems maintained a reduced photoactivity under UV-free solar light irradiation (λ>420 nm). PMID:25205168

  14. Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes †

    PubMed Central

    Lambert, Grant R.; Harker, Alan R.; Cantrell, Michael A.; Hanus, F. Joe; Russell, Sterling A.; Haugland, Richard A.; Evans, Harold J.

    1987-01-01

    The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup+ phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules. Images PMID:16347291

  15. The NiFe Hydrogenases of the Tetrachloroethene-Respiring Epsilonproteobacterium Sulfurospirillum multivorans: Biochemical Studies and Transcription Analysis

    PubMed Central

    Kruse, Stefan; Goris, Tobias; Wolf, Maria; Wei, Xi; Diekert, Gabriele

    2017-01-01

    The organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans is able to grow with hydrogen as electron donor and with tetrachloroethene (PCE) as electron acceptor; PCE is reductively dechlorinated to cis-1,2-dichloroethene. Recently, a genomic survey revealed the presence of four gene clusters encoding NiFe hydrogenases in its genome, one of which is presumably periplasmic and membrane-bound (MBH), whereas the remaining three are cytoplasmic. To explore the role and regulation of the four hydrogenases, quantitative real-time PCR and biochemical studies were performed with S. multivorans cells grown under different growth conditions. The large subunit genes of the MBH and of a cytoplasmic group 4 hydrogenase, which is assumed to be membrane-associated, show high transcript levels under nearly all growth conditions tested, pointing toward a constitutive expression in S. multivorans. The gene transcripts encoding the large subunits of the other two hydrogenases were either not detected at all or only present at very low amounts. The presence of MBH under all growth conditions tested, even with oxygen as electron acceptor under microoxic conditions, indicates that MBH gene transcription is not regulated in contrast to other facultative hydrogen-oxidizing bacteria. The MBH showed quinone-reactivity and a characteristic UV/VIS spectrum implying a cytochrome b as membrane-integral subunit. Cell extracts of S. multivorans were subjected to native polyacrylamide gel electrophoresis (PAGE) and hydrogen oxidizing activity was tested by native staining. Only one band was detected at about 270 kDa in the particulate fraction of the extracts, indicating that there is only one hydrogen-oxidizing enzyme present in S. multivorans. An enrichment of this enzyme and SDS PAGE revealed a subunit composition corresponding to that of the MBH. From these findings we conclude that the MBH is the electron-donating enzyme system in the PCE respiratory chain. The roles for

  16. Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16.

    PubMed Central

    Tran-Betcke, A; Warnecke, U; Böcker, C; Zaborosch, C; Friedrich, B

    1990-01-01

    The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide. Images PMID:2188945

  17. Visible light-driven H(2) production by hydrogenases attached to dye-sensitized TiO(2) nanoparticles.

    PubMed

    Reisner, Erwin; Powell, Daniel J; Cavazza, Christine; Fontecilla-Camps, Juan C; Armstrong, Fraser A

    2009-12-30

    A study of hybrid, enzyme-modified nanoparticles able to produce H(2) using visible light as the energy source has been carried out to establish per-site performance standards for H(2) production catalysts able to operate under ambient conditions. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) is identified as a particularly proficient catalyst. The optimized system consisting of Db [NiFeSe]-H attached to Ru dye-sensitized TiO(2), with triethanolamine as a sacrificial electron donor, produces H(2) at a turnover frequency of approximately 50 (mol H(2)) s(-1) (mol total hydrogenase)(-1) at pH 7 and 25 degrees C, even under the typical solar irradiation of a northern European sky. The system shows high electrocatalytic stability not only under anaerobic conditions but also after prolonged exposure to air, thus making it sufficiently robust for benchtop applications.

  18. Photosensitivity of the Ni-A state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F with visible light

    SciTech Connect

    Osuka, Hisao; Shomura, Yasuhito; Komori, Hirofumi; Shibata, Naoki; Nagao, Satoshi; Higuchi, Yoshiki; Hirota, Shun

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Ni-A state of [NiFe] hydrogenase showed light sensitivity. Black-Right-Pointing-Pointer New FT-IR bands were observed with light irradiation of the Ni-A state. Black-Right-Pointing-Pointer EPR g-values of the Ni-A state shifted upon light irradiation. Black-Right-Pointing-Pointer The light-induced state converted back to the Ni-A state under the dark condition. -- Abstract: [NiFe] hydrogenase catalyzes reversible oxidation of molecular hydrogen. Its active site is constructed of a hetero dinuclear Ni-Fe complex, and the oxidation state of the Ni ion changes according to the redox state of the enzyme. We found that the Ni-A state (an inactive unready, oxidized state) of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) is light sensitive and forms a new state (Ni-AL) with irradiation of visible light. The Fourier transform infrared (FT-IR) bands at 1956, 2084 and 2094 cm{sup -1} of the Ni-A state shifted to 1971, 2086 and 2098 cm{sup -1} in the Ni-AL state. The g-values of g{sub x} = 2.30, g{sub y} = 2.23 and g{sub z} = 2.01 for the signals in the electron paramagnetic resonance (EPR) spectrum of the Ni-A state at room temperature varied for -0.009, +0.012 and +0.010, respectively, upon light irradiation. The light-induced Ni-AL state converted back immediately to the Ni-A state under dark condition at room temperature. These results show that the coordination structure of the Fe site of the Ni-A state of [NiFe] hydrogenase is perturbed significantly by light irradiation with relatively small coordination change at the Ni site.

  19. Salmonella Typhimurium Strain ATCC14028 Requires H2-Hydrogenases for Growth in the Gut, but Not at Systemic Sites

    PubMed Central

    Maier, Lisa; Barthel, Manja; Stecher, Bärbel; Maier, Robert J.; Gunn, John S.; Hardt, Wolf-Dietrich

    2014-01-01

    Salmonella enterica is a common cause of diarrhea. For eliciting disease, the pathogen has to colonize the gut lumen, a site colonized by the microbiota. This process/initial stage is incompletely understood. Recent work established that one particular strain, Salmonella enterica subspecies 1 serovar Typhimurium strain SL1344, employs the hyb H2-hydrogenase for consuming microbiota-derived H2 to support gut luminal pathogen growth: Protons from the H2-splitting reaction contribute to the proton gradient across the outer bacterial membrane which can be harvested for ATP production or for import of carbon sources. However, it remained unclear, if other Salmonella strains would use the same strategy. In particular, earlier work had left unanswered if strain ATCC14028 might use H2 for growth at systemic sites. To clarify the role of the hydrogenases, it seems important to establish if H2 is used at systemic sites or in the gut and if Salmonella strains may differ with respect to the host sites where they require H2 in vivo. In order to resolve this, we constructed a strain lacking all three H2-hydrogenases of ATCC14028 (14028hyd3) and performed competitive infection experiments. Upon intragastric inoculation, 14028hyd3 was present at 100-fold lower numbers than 14028WT in the stool and at systemic sites. In contrast, i.v. inoculation led to equivalent systemic loads of 14028hyd3 and the wild type strain. However, the pathogen population spreading to the gut lumen featured again up to 100-fold attenuation of 14028hyd3. Therefore, ATCC14028 requires H2-hydrogenases for growth in the gut lumen and not at systemic sites. This extends previous work on ATCC14028 and supports the notion that H2-utilization might be a general feature of S. Typhimurium gut colonization. PMID:25303479

  20. Photosensitivity of the Ni-A state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F with visible light.

    PubMed

    Osuka, Hisao; Shomura, Yasuhito; Komori, Hirofumi; Shibata, Naoki; Nagao, Satoshi; Higuchi, Yoshiki; Hirota, Shun

    2013-01-04

    [NiFe] hydrogenase catalyzes reversible oxidation of molecular hydrogen. Its active site is constructed of a hetero dinuclear Ni-Fe complex, and the oxidation state of the Ni ion changes according to the redox state of the enzyme. We found that the Ni-A state (an inactive unready, oxidized state) of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) is light sensitive and forms a new state (Ni-AL) with irradiation of visible light. The Fourier transform infrared (FT-IR) bands at 1956, 2084 and 2094 cm(-1) of the Ni-A state shifted to 1971, 2086 and 2098 cm(-1) in the Ni-AL state. The g-values of g(x)=2.30, g(y)=2.23 and g(z)=2.01 for the signals in the electron paramagnetic resonance (EPR) spectrum of the Ni-A state at room temperature varied for -0.009, +0.012 and +0.010, respectively, upon light irradiation. The light-induced Ni-AL state converted back immediately to the Ni-A state under dark condition at room temperature. These results show that the coordination structure of the Fe site of the Ni-A state of [NiFe] hydrogenase is perturbed significantly by light irradiation with relatively small coordination change at the Ni site.

  1. Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2‐Protected Silicon Electrode

    PubMed Central

    Lee, Chong‐Yong; Park, Hyun S.; Fontecilla‐Camps, Juan C.

    2016-01-01

    Abstract The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron‐transfer processes at highly active and well‐defined catalytic sites on a light‐harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO2‐coated p‐Si photocathode for the photo‐reduction of protons to H2. The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO2 layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme. The p‐Si|TiO2|hydrogenase photocathode displays visible‐light driven production of H2 at an energy‐storing, positive electrochemical potential and an essentially quantitative faradaic efficiency. We have thus established a widely applicable platform to wire redox enzymes in an active configuration on a p‐type semiconductor photocathode through the engineering of the enzyme–materials interface. PMID:27570301

  2. Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2‐Protected Silicon Electrode

    PubMed Central

    Lee, Chong‐Yong; Park, Hyun S.; Fontecilla‐Camps, Juan C.

    2016-01-01

    Abstract The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron‐transfer processes at highly active and well‐defined catalytic sites on a light‐harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO2‐coated p‐Si photocathode for the photo‐reduction of protons to H2. The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO2 layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme. The p‐Si|TiO2|hydrogenase photocathode displays visible‐light driven production of H2 at an energy‐storing, positive electrochemical potential and an essentially quantitative faradaic efficiency. We have thus established a widely applicable platform to wire redox enzymes in an active configuration on a p‐type semiconductor photocathode through the engineering of the enzyme–materials interface. PMID:27061334

  3. Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12.

    PubMed Central

    Stoker, K; Reijnders, W N; Oltmann, L F; Stouthamer, A H

    1989-01-01

    To isolate genes from Escherichia coli which regulate the labile hydrogenase activity, a plasmid library was used to transform hydL mutants lacking the labile hydrogenase. A single type of gene, designated hydG, was isolated. This gene also partially restored the hydrogenase activity in hydF mutants (which are defective in all hydrogenase isoenzymes), although the low hydrogenase 1 and 2 levels were not induced. Therefore, hydG apparently regulates, specifically, the labile hydrogenase activity. Restoration of this latter activity in hydF mutants was accompanied by a proportional increase of the H2 uptake activity, suggesting a functional relationship. H2:fumarate oxidoreductase activity was not restored in complemented hydL mutants. These latter strains may therefore lack, in addition to the labile hydrogenase, a second component (provisionally designated component R), possibly an electron carrier coupling H2 oxidation to the anerobic respiratory chain. Sequence analysis showed an open reading frame of 1,314 base pairs for hydG. It was preceded by a ribosome-binding site but apparently lacked a promoter. Minicell experiments revealed a single polypeptide of approximately 50 kilodaltons. Comparison of the predicted amino acid sequence with a protein sequence data base revealed strong homology to NtrC from Klebsiella pneumoniae, a DNA-binding transcriptional activator. The 411 base pairs upstream from pHG40 contained a second open reading frame overlapping hydG by four bases. The deduced amino acid sequence showed considerable homology with the C-terminal part of NtrB. This sequence was therefore assumed to be part of a second gene, encoding the NtrB-like component, and was designated hydH. The labile hydrogenase activity in E. coli is apparently regulated by a multicomponent system analogous to the NtrB-NtrC system. This conclusion is in agreement with the results of Birkmann et al. (A. Birkmann, R. G. Sawers, and A. Böck, Mol. Gen. Genet. 210:535-542, 1987), who

  4. Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment1[OPEN

    PubMed Central

    Liran, Oded; Milrad, Yuval; Eilenberg, Haviva; Weiner, Iddo

    2016-01-01

    Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, 18O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells. PMID:27443604

  5. Genetic diversity of Desulfovibrio spp. in environmental samples analyzed by denaturing gradient gel electrophoresis of [NiFe] hydrogenase gene fragments.

    PubMed Central

    Wawer, C; Muyzer, G

    1995-01-01

    The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors. PMID:7793940

  6. H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase.

    PubMed

    Lonsdale, Thomas H; Lauterbach, Lars; Honda Malca, Sumire; Nestl, Bettina M; Hauer, Bernhard; Lenz, Oliver

    2015-11-21

    An in vivo biotransformation system is presented that affords the hydroxylation of n-octane to 1-octanol on the basis of NADH-dependent CYP153A monooxygenase and NAD(+)-reducing hydrogenase heterologously synthesized in a bacterial host. The hydrogenase sustains H2-driven NADH cofactor regeneration even in the presence of O2, the co-substrate of monooxygenase.

  7. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

    SciTech Connect

    Cornish, Adam J.; Ginovska, Bojana; Thelen, Adam; da Silva, Julio C. S.; Soares, Thereza A.; Raugei, Simone; Dupuis, Michel; Shaw, Wendy J.; Hegg, Eric L.

    2016-06-07

    The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a modeled water molecule. Recently, a computational analysis of this pathway revealed that the solvent-exposed residue of the pathway (Glu282) could form hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implicating that these residues could function in regulating proton transfer. Substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in a 2.5-fold enhancement in H2 production activity, suggesting that Arg286 serves an important role in controlling the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate approximately 5-fold, implying that it either acts as a member of the pathway or influences Glu282 to enable proton transfer. Interestingly, QM/MM and molecular dynamics calculations indicate that Ser320 does not play an electronic or structural role. QM calculations also estimate that including Ser320 in the pathway does not significantly change the barrier to proton movement, providing further support for its role as a member of the proton pathway. While further studies are needed to quantify the role of Ser320, collectively, these data provide evidence that the enzyme scaffold plays a significant role in modulating the activity of the enzyme, demonstrating that the rate of intraprotein proton transfer can be accelerated, particularly in a non-biological context. This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science, DE-FC02-07ER64494). In addition, support from the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences (WJS, BGP, SR) is gratefully acknowledged. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of

  8. Basic studies of hydrogen evolution by Escherichia coli containing a cloned Citrobacter freundii hydrogenase gene.

    PubMed

    Kanayama, H; Sode, K; Karube, I

    1987-08-01

    Citrobacter freundii genes that complemented Escherichia coli hyd-(hydrogenase activity) mutation were cloned in plasmids pCBH4 (6.2 kb) and pCBH6(5.7 kb). Hydrogen evolution by the transformant E. coli HK-8(pCBH4 or pCBH6) was investigated. The optimum culture temperature of recombinant E. coli cells for hydrogen evolution from glucose was in the neighborhood of 18 degrees C. The recombinant E. coli cells cultured at this condition showed a several-fold increase of hydrogen evolution, as compared with that of the wild-type cells. The plasmid-retention stability of this recombinant E. coli was extremely high, especially plasmid pCBH4, which was completely retained during 2 wk without any restriction. Hydrogen production by immobilized recombinant E. coli was then investigated using cells cultured at 18 degrees C. The hydrogen evolution rate from glucose and Lennox-broth were about twofold higher than that of E. coli C600, and this high hydrogen evolution rate was maintained for more than 1 mo.

  9. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  10. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster.

  11. Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello.

    PubMed Central

    Voordouw, G; Strang, J D; Wilson, F R

    1989-01-01

    The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of alpha and beta subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) alpha subunit precedes that of the 13.5-kDa beta subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both alpha subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed beta subunits (124 and 123 amino acid residues, respectively) share 71% sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was found approximately 90 base pairs upstream from the translational start of the hydA gene. The recently discovered hydC gene, which may code for a 65.8-kDa fusion protein (gamma) of the alpha and beta subunits and is present immediately downstream from the hydAB genes in the Hildenborough strain, was found to be absent from the Monticello strain. The implication of this result for the possible function of the hydC gene product in Desulfovibrio species is discussed. Images PMID:2661538

  12. Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

    PubMed Central

    Eberz, G; Eitinger, T; Friedrich, B

    1989-01-01

    Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium. PMID:2646280

  13. Expression of a clostridial [FeFe]-hydrogenase in Chlamydomonas reinhardtii prolongs photo-production of hydrogen from water splitting

    SciTech Connect

    Noone, Seth; Ratcliff, Kathleen; Davis, ReAnna; Subramanian, Venkataramanan; Meuser, Jonathan; Posewitz, Matthew C.; King, Paul W.; Ghirardi, Maria L.

    2016-12-24

    The high oxygen (O2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1hydA2) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H2 photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O2 inactivation of CaI led to more sustained H2 photoproduction when cultures were challenged with O2 or kept under prolonged illumination at solar intensities. Lastly, these results provide new insights into the requisites for attaining photobiological H2 production from water splitting using a more O2-tolerant hydrogenase.

  14. Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus

    SciTech Connect

    Menon, N.K.; Peck, H.D. Jr.; Le Gall, J.; Przybyla, A.E.

    1987-12-01

    The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.

  15. Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5.

    PubMed

    Lindberg, Pia; Lindblad, Peter; Cournac, Laurent

    2004-04-01

    Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.

  16. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.

    PubMed

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw(-1) h(-1) was observed at a dilution rate of 0.31 h(-1). We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h(-1), the SHPR was 36.2 mmol H2 g-dcw(-1) h(-1), corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h(-1) or 1.07 h(-1) resulted in a SHPR of 120 mmol H2 g-dcw(-1) h(-1), which is one of the highest production rates observed in microbial fermentation.

  17. Fractionation of sulfur isotopes by Desulfovibrio vulgaris mutants lacking hydrogenases or type I tetraheme cytochrome c 3.

    PubMed

    Sim, Min Sub; Wang, David T; Zane, Grant M; Wall, Judy D; Bosak, Tanja; Ono, Shuhei

    2013-01-01

    The sulfur isotope effect produced by sulfate reducing microbes is commonly used to trace biogeochemical cycles of sulfur and carbon in aquatic and sedimentary environments. To test the contribution of intracellular coupling between carbon and sulfur metabolisms to the overall magnitude of the sulfur isotope effect, this study compared sulfur isotope fractionations by mutants of Desulfovibrio vulgaris Hildenborough. We tested mutant strains lacking one or two periplasmic (Hyd, Hyn-1, Hyn-2, and Hys) or cytoplasmic hydrogenases (Ech and CooL), and a mutant lacking type I tetraheme cytochrome (TpI-c 3). In batch culture, wild-type D. vulgaris and its hydrogenase mutants had comparable growth kinetics and produced the same sulfur isotope effects. This is consistent with the reported redundancy of hydrogenases in D. vulgaris. However, the TpI-c 3 mutant (ΔcycA) exhibited slower growth and sulfate reduction rates in batch culture, and produced more H2 and an approximately 50% larger sulfur isotope effect, compared to the wild type. The magnitude of sulfur isotope fractionation in the CycA deletion strain, thus, increased due to the disrupted coupling of the carbon oxidation and sulfate reduction pathways. In continuous culture, wild-type D. vulgaris and the CycA mutant produced similar sulfur isotope effects, underscoring the influence of environmental conditions on the relative contribution of hydrogen cycling to the electron transport. The large sulfur isotope effects associated with the non-ideal stoichiometry of sulfate reduction in this study imply that simultaneous fermentation and sulfate reduction may be responsible for some of the large naturally-occurring sulfur isotope effects. Overall, mutant strains provide a powerful tool to test the effect of specific redox proteins and pathways on sulfur isotope fractionation.

  18. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production

    PubMed Central

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw−1 h−1 was observed at a dilution rate of 0.31 h−1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h−1, the SHPR was 36.2 mmol H2 g-dcw−1 h−1, corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h−1 or 1.07 h−1 resulted in a SHPR of 120 mmol H2 g-dcw−1 h−1, which is one of the highest production rates observed in microbial fermentation. PMID:26379632

  19. Distribution of Hydrogenases in Cyanobacteria: A Phylum-Wide Genomic Survey.

    PubMed

    Puggioni, Vincenzo; Tempel, Sébastien; Latifi, Amel

    2016-01-01

    Microbial Molecular hydrogen (H2) cycling plays an important role in several ecological niches. Hydrogenases (H2ases), enzymes involved in H2 metabolism, are of great interest for investigating microbial communities, and producing BioH2. To obtain an overall picture of the genetic ability of Cyanobacteria to produce H2ases, we conducted a phylum wide analysis of the distribution of the genes encoding these enzymes in 130 cyanobacterial genomes. The concomitant presence of the H2ase and genes involved in the maturation process, and that of well-conserved catalytic sites in the enzymes were the three minimal criteria used to classify a strain as being able to produce a functional H2ase. The [NiFe] H2ases were found to be the only enzymes present in this phylum. Fifty-five strains were found to be potentially able produce the bidirectional Hox enzyme and 33 to produce the uptake (Hup) enzyme. H2 metabolism in Cyanobacteria has a broad ecological distribution, since only the genomes of strains collected from the open ocean do not possess hox genes. In addition, the presence of H2ase was found to increase in the late branching clades of the phylogenetic tree of the species. Surprisingly, five cyanobacterial genomes were found to possess homologs of oxygen tolerant H2ases belonging to groups 1, 3b, and 3d. Overall, these data show that H2ases are widely distributed, and are therefore probably of great functional importance in Cyanobacteria. The present finding that homologs to oxygen-tolerant H2ases are present in this phylum opens new perspectives for applying the process of photosynthesis in the field of H2 production.

  20. Proton Management as a Design Principle for Hydrogenase-inspired Catalysts

    SciTech Connect

    Muckerman, J.T.; Small, Y.A.; DuBois, D.L.; Fujita, E.

    2011-08-01

    The properties of the hydrogenase-inspired [Ni(PNP){sub 2}]{sup 2+} (PNP = Et{sub 2}PCH{sub 2}NMeCH{sub 2}PEt{sub 2}) catalyst for homogeneous hydrogen oxidation in acetonitrile solution are explored from a theoretical perspective for hydrogen production. The defining characteristic of this catalyst is the presence of pendent bases in the second coordination sphere that function as proton relays between the solution and the metal center. DFT calculations of the possible intermediates along proposed catalytic pathways are carried out and used to construct coupled Pourbaix diagrams of the redox processes and free-energy profiles along the reaction pathways. Analysis of the coupled Pourbaix diagrams reveals insights into the intermediate species and the mechanisms favored at different pH values of the solution. Consideration of the acid-base behavior of the metal hydride and H{sub 2} adduct species imposes additional constraints on the reaction mechanism, which can involve intramolecular as well as intermolecular proton-coupled electron-transfer steps. The efficacy of the catalyst is shown to depend critically on the pK{sub a} values of these potential intermediates, as they control both the species in solution at a given pH and the free-energy profile of reaction pathways. Optimal relationships among these pK{sub a} values can be identified, and it is demonstrated that 'proton management', i.e., the manipulation of these pK{sub a} values (e.g., through choice of metal or substituents on ligands), can serve as a design principle for improved catalytic behavior.

  1. Distribution of Hydrogenases in Cyanobacteria: A Phylum-Wide Genomic Survey

    PubMed Central

    Puggioni, Vincenzo; Tempel, Sébastien; Latifi, Amel

    2016-01-01

    Microbial Molecular hydrogen (H2) cycling plays an important role in several ecological niches. Hydrogenases (H2ases), enzymes involved in H2 metabolism, are of great interest for investigating microbial communities, and producing BioH2. To obtain an overall picture of the genetic ability of Cyanobacteria to produce H2ases, we conducted a phylum wide analysis of the distribution of the genes encoding these enzymes in 130 cyanobacterial genomes. The concomitant presence of the H2ase and genes involved in the maturation process, and that of well-conserved catalytic sites in the enzymes were the three minimal criteria used to classify a strain as being able to produce a functional H2ase. The [NiFe] H2ases were found to be the only enzymes present in this phylum. Fifty-five strains were found to be potentially able produce the bidirectional Hox enzyme and 33 to produce the uptake (Hup) enzyme. H2 metabolism in Cyanobacteria has a broad ecological distribution, since only the genomes of strains collected from the open ocean do not possess hox genes. In addition, the presence of H2ase was found to increase in the late branching clades of the phylogenetic tree of the species. Surprisingly, five cyanobacterial genomes were found to possess homologs of oxygen tolerant H2ases belonging to groups 1, 3b, and 3d. Overall, these data show that H2ases are widely distributed, and are therefore probably of great functional importance in Cyanobacteria. The present finding that homologs to oxygen-tolerant H2ases are present in this phylum opens new perspectives for applying the process of photosynthesis in the field of H2 production. PMID:28083017

  2. Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation.

    PubMed

    Trchounian, Karen; Blbulyan, Syuzanna; Trchounian, Armen

    2013-06-01

    Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical-the membrane potential (∆φ) and chemical-the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pHex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pHex at pH 7.5 and 6.5, respectively; and it was higher than pHex at pH 5.5. At high pHex, the increase of ∆φ (-130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pHex ∆φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp might be due to distinguished mechanisms for H(+) transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F0F1-ATPase. H(+) efflux was determined to depend on pHex; overall and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibitory H(+) efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pHex, and a role of Hyd enzymes in its generation is suggested.

  3. Unification of [FeFe]-hydrogenases into three structural and functional groups

    DOE PAGES

    Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; ...

    2016-05-27

    [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatorymore » proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggests that they are post-translationally modified by phosphorylation. In conclusion, these results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.« less

  4. Unification of [FeFe]-hydrogenases into three structural and functional groups

    SciTech Connect

    Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Refai, Mohammed; Schut, Gerrit J.; King, Paul W.; Maness, Pin-Ching; Adams, Michael W. W.; Peters, John W.; Bothner, Brian; Boyd, Eric S.

    2016-05-27

    [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggests that they are post-translationally modified by phosphorylation. In conclusion, these results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.

  5. Theoretical insights into [NiFe]-hydrogenases oxidation resulting in a slowly reactivating inactive state.

    PubMed

    Breglia, Raffaella; Ruiz-Rodriguez, Manuel Antonio; Vitriolo, Alessandro; Gonzàlez-Laredo, Rubén Francisco; De Gioia, Luca; Greco, Claudio; Bruschi, Maurizio

    2017-01-01

    [NiFe]-hydrogenases catalyse the relevant H2 → 2H(+) + 2e(-) reaction. Aerobic oxidation or anaerobic oxidation of this enzyme yields two inactive states called Ni-A and Ni-B. These states differ for the reactivation kinetics which are slower for Ni-A than Ni-B. While there is a general consensus on the structure of Ni-B, the nature of Ni-A is still controversial. Indeed, several crystallographic structures assigned to the Ni-A state have been proposed, which, however, differ for the nature of the bridging ligand and for the presence of modified cysteine residues. The spectroscopic characterization of Ni-A has been of little help due to small differences of calculated spectroscopic parameters, which does not allow to discriminate among the various forms proposed for Ni-A. Here, we report a DFT investigation on the nature of the Ni-A state, based on systematic explorations of conformational and configurational space relying on accurate energy calculations, and on comparisons of theoretical geometries with the X-ray structures currently available. The results presented in this work show that, among all plausible isomers featuring various protonation patterns and oxygenic ligands, the one corresponding to the crystallographic structure recently reported by Volbeda et al. (J Biol Inorg Chem 20:11-22, 19)-featuring a bridging hydroxide ligand and the sulphur atom of Cys64 oxidized to bridging sulfenate-is the most stable. However, isomers with cysteine residues oxidized to terminal sulfenate are very close in energy, and modifications in the network of H-bond with neighbouring residues may alter the stability order of such species.

  6. Improved purification, crystallization and crystallographic study of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77.

    PubMed

    Muhd Noor, Noor Dina; Nishikawa, Koji; Nishihara, Hirofumi; Yoon, Ki Seok; Ogo, Seiji; Higuchi, Yoshiki

    2016-01-01

    The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P21, with unit-cell parameters a = 63.90, b = 118.89, c = 96.70 Å, β = 100.61° for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63 Å, β = 102.29° for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60 Å resolution, which is considerably better than the 2.00 Å resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O2, indicating that it has clear O2 tolerance.

  7. Radioassay for Hydrogenase Activity in Viable Cells and Documentation of Aerobic Hydrogen-Consuming Bacteria Living in Extreme Environments

    PubMed Central

    Schink, Bernhard; Lupton, F. S.; Zeikus, J. G.

    1983-01-01

    An isotopic tracer assay based on the hydrogenase-dependent formation of tritiated water from tritium gas was developed for in life analysis of microbial hydrogen transformation. This method allowed detection of bacterial hydrogen metabolism in pure cultures or in natural samples obtained from aquatic ecosystems. A differentiation between chemical-biological and aerobic-anaerobic hydrogen metabolism was established by variation of the experimental incubation temperature or by addition of selective inhibitors. Hydrogenase activity was shown to be proportional to the consumption or production of hydrogen by cultures of Desulfovibrio vulgaris, Clostridium pasteurianum, and Methanosarcina barkeri. This method was applied, in connection with measurements of free hydrogen and most-probable-number enumerations, in aerobic natural source waters to establish the activity and document the ecology of hydrogen-consuming bacteria in extreme acid, thermal, or saline environments. The utility of the assay is based in part on the ability to quantify bacterial hydrogen transformation at natural hydrogen partial pressures, without the use of artificial electron acceptors. PMID:16346288

  8. [FeFe]-hydrogenase abundance and diversity along a vertical redox gradient in Great Salt Lake, USA.

    PubMed

    Boyd, Eric S; Hamilton, Trinity L; Swanson, Kevin D; Howells, Alta E; Baxter, Bonnie K; Meuser, Jonathan E; Posewitz, Matthew C; Peters, John W

    2014-11-28

    The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications.

  9. Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.

    PubMed Central

    Menon, N K; Robbins, J; Peck, H D; Chatelus, C Y; Choi, E S; Przybyla, A E

    1990-01-01

    DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown. Images FIG. 2 FIG. 3 PMID:2180913

  10. Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F(0)F(1)-ATPase.

    PubMed

    Trchounian, Karen; Poladyan, Anna; Vassilian, Anait; Trchounian, Armen

    2012-01-01

    Molecular hydrogen (H(2)) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H(2)-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH](out)) and the presence of formate. FHL is related with the F(0)F(1)-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H(2)-oxidizing enzymes during glucose fermentation at neutral and low [pH](out). They operate in a reverse, H(2)-producing mode during glycerol fermentation at neutral [pH](out). Hyd-1 and Hyd-2 activity depends on F(0)F(1). Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H(2) production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H(2) production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H(2) production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.

  11. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

    PubMed

    Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

    2016-04-25

    [NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

  12. Sequence analysis and interposon mutagenesis of the hupT gene, which encodes a sensor protein involved in repression of hydrogenase synthesis in Rhodobacter capsulatus.

    PubMed Central

    Elsen, S; Richaud, P; Colbeau, A; Vignais, P M

    1993-01-01

    The hupT gene, which represses hydrogenase gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus, has been identified and sequenced. The nucleotide sequence of hupT and of the contiguous downstream open reading frame, hupU, is reported. The HupT protein of 456 amino acids (48,414 Da) has sequence similarity with the FixL, DctB, NtrB, and ArcB proteins and is predicted to be a soluble sensor kinase. Insertional inactivation of the hupT gene led to deregulation of transcriptional control, so that the hydrogenase structural operon hupSLC became overexpressed in cells grown anaerobically or aerobically. The HupT- mutants were complemented in trans by a plasmid containing an intact copy of the hupT gene. The hupU open reading frame, capable of encoding a protein of 84,879 Da, shared identity with [NiFe]hydrogenase subunits; the strongest similarity was observed with the periplasmic hydrogenase of Desulfovibrio baculatus. Images PMID:8226687

  13. Iron Acyl Thiolato Carbonyls: Structural Models for the Active Site of the [Fe]-Hydrogenase (Hmd)

    PubMed Central

    Royer, Aaron M.; Salomone-Stagni, Marco

    2012-01-01

    Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls via the reaction of phosphine thioesters and sources of Fe(0). The reaction generates both Fe(SPh)(Ph2PC6H4CO)(CO)3 (1) and the diferrous diacyl Fe2(SPh)2(CO)3(Ph2PC6H4CO)2, which carbonylates to give 1. For the extremely bulky arylthioester Ph2PC6H4C(O)SC6H4-2,6-(2,4,6-trimethylphenyl)2, oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained. Complex 1 reacts with cyanide to give Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2] (Et4N[2]). 13C and 31P NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2]− in CH2Cl2 solution very closely matches that for HmdCN. XANES and EXAFS measurements also indicate close structural and electronic similarity of Et4N[2] to the active site of wild-type Hmd. Complex 1 also stereospecifically forms a derivative with TsCH2NC, but the adduct is more labile than Et4N[2]. Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd. PMID:21062066

  14. Mixed-Valence Nickel-Iron Dithiolate Models of the [NiFe]-Hydrogenase Active Site

    PubMed Central

    Schilter, David; Nilges, Mark J.; Chakrabarti, Mrinmoy; Lindahl, Paul A.; Rauchfuss, Thomas B.; Stein, Matthias

    2012-01-01

    A series of mixed-valence iron-nickel dithiolates is described. Oxidation of (diphosphine)Ni(dithiolate)Fe(CO)3 complexes 1, 2, and 3 with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)Ni(pdt)Fe(CO)3]+ ([1]+), [(dppe)Ni(edt)Fe(CO)3]+ ([2]+) and [(dcpe)Ni(pdt)Fe(CO)3]+ ([3]+), respectively, where dppe = Ph2PCH2CH2PPh2, dcpe = Cy2PCH2CH2PCy2, pdtH2 = HSCH2CH2CH2SH and edtH2 = HSCH2CH2SH. The cation [2]+ proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as Cs-symmetric species. Crystallographic characterization of [3]BF4 shows that Ni is square planar. Interaction of [1]BF4 with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)Ni(pdt)Fe(CO)2L]BF4 for L = P(OPh)3 ([4a]BF4), P(p-C6H4Cl)3 ([4b]BF4), PPh2(2-py) ([4c]BF4), PPh2(OEt) ([4d]BF4), PPh3 ([4e]BF4), PPh2(o-C6H4OMe) ([4f]BF4), PPh2(o-C6H4OCH2OMe) ([4g]BF4), P(p-tol)3 ([4h]BF4), P(p-C6H4OMe)3 ([4i]BF4), PMePh2 ([4j]BF4). EPR analysis indicates that ethanedithiolate [2]+ exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of 1 and oxidized S = ½ [4e]BF4 are both consistent with a low-spin Fe(i) state. The hyperfine coupling tensor of [4e]BF4 has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in a Fe(i)-centered d(z2) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [1]+ with CN- and compact basic phosphines results in complex reactions. With dppe, [1]+ undergoes quasi-disproportionation to give 1 and the diamagnetic complex [(dppe)Ni(pdt)Fe(CO)2(dppe)]2+ ([5]2+), which features square-planar Ni linked to an octahedral Fe center. PMID:22304696

  15. Protonation of Nickel–Iron Hydrogenase Models Proceeds after Isomerization at Nickel

    PubMed Central

    2015-01-01

    Theory and experiment indicate that the protonation of reduced NiFe dithiolates proceeds via a previously undetected isomer with enhanced basicity. In particular, it is proposed that protonation of (OC)3Fe(pdt)Ni(dppe) (1; pdt2– = –S(CH2)3S–; dppe = Ph2P(CH2)2PPh2) occurs at the Fe site of the two-electron mixed-valence Fe(0)Ni(II) species, not the Fe(I)-Ni(I) bond for the homovalence isomer of 1. The new pathway, which may have implications for protonation of other complexes and clusters, was uncovered through studies on the homologous series L(OC)2Fe(pdt)M(dppe), where M = Ni, Pd (2), and Pt (3) and L = CO, PCy3. Similar to 1, complexes 2 and 3 undergo both protonation and 1e– oxidation to afford well-characterized hydrides ([2H]+ and [3H]+) and mixed-valence derivatives ([2]+ and [3]+), respectively. Whereas the Pd site is tetrahedral in 2, the Pt site is square-planar in 3, indicating that this complex is best described as Fe(0)Pt(II). In view of the results on 2 and 3, the potential energy surface of 1 was reinvestigated with density functional theory. These calculations revealed the existence of an energetically accessible and more basic Fe(0)Ni(II) isomer with a square-planar Ni site. PMID:25094041

  16. Purification and characterization of the [NiFe]-hydrogenase of Shewanella oneidensis MR-1.

    PubMed

    Shi, Liang; Belchik, Sara M; Plymale, Andrew E; Heald, Steve; Dohnalkova, Alice C; Sybirna, Kateryna; Bottin, Hervé; Squier, Thomas C; Zachara, John M; Fredrickson, James K

    2011-08-15

    Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.

  17. Molecular Biomarkers for Detecting, Monitoring and Quantifying Reductive Microbial Processes

    DTIC Science & Technology

    2013-07-01

    Termite  Gut  Metagenome,  IMG/M  ID  2005608198   5.6%   [FeFe]  Hydrogenase  from   Termite  Gut  Metagenome,  IMG/M  ID...2005622586   5.0%   [FeFe]  Hydrogenase  from   Termite  Gut  Metagenome,  IMG/M  ID  2005599188   4.2%   [FeFe]  Hydrogenase...Dde_0474,  Desulfovibrio  alaskensis  G20   2.3%   [FeFe]  Hydrogenase  from   Termite

  18. Accumulation of Ferrous Iron in Chlamydomonas reinhardtii. Influence of CO2 and Anaerobic Induction of the Reversible Hydrogenase1

    PubMed Central

    Semin, Boris K.; Davletshina, Lira N.; Novakova, Alla A.; Kiseleva, Tat'yana Y.; Lanchinskaya, Victoriya Y.; Aleksandrov, Anatolii Y.; Seifulina, Nora; Ivanov, Il'ya I.; Seibert, Michael; Rubin, Andrei B.

    2003-01-01

    The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H2 via ferredoxin and the reversible [Fe]hydrogenase enzyme under anaerobic conditions. Recently, a novel approach for sustained H2 gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A. Melis, L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert [2000] Plant Physiol 122: 127–135). The close relationship between S and Fe in the H2-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase. In this study, we used Mössbauer spectroscopy to examine both the uptake of Fe by the alga at different CO2 concentrations during growth and the influence of anaerobiosis on the accumulation of Fe. Algal cells grown in media with 57Fe(III) at elevated (3%, v/v) CO2 concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with Mössbauer parameters of quadrupole splitting = 0.65 mm s−1 and isomeric shift = 0.46 mm s−1 and (b) Fe(II) with quadrupole splitting = 3.1 mm s−1 and isomeric shift = 1.36 mm s−1. Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell. The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis. Growing the algae under atmospheric CO2 (limiting) conditions, compared with 3% (v/v) CO2, resulted in a decrease in the intracellular Fe(II) content by a factor of 3. Incubating C. reinhardtii cells, grown at atmospheric CO2 for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO2. This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO2 concentration, and anaerobiosis. A

  19. Accumulation of ferrous iron in Chlamydomonas reinhardtii. Influence of CO2 and anaerobic induction of the reversible hydrogenase.

    PubMed

    Semin, Boris K; Davletshina, Lira N; Novakova, Alla A; Kiseleva, Tat'yana Y; Lanchinskaya, Victoriya Y; Aleksandrov, Anatolii Y; Seifulina, Nora; Ivanov, Il'ya I; Seibert, Michael; Rubin, Andrei B

    2003-04-01

    The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H(2) via ferredoxin and the reversible [Fe]hydrogenase enzyme under anaerobic conditions. Recently, a novel approach for sustained H(2) gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A. Melis, L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert [2000] Plant Physiol 122: 127-135). The close relationship between S and Fe in the H(2)-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase. In this study, we used Mössbauer spectroscopy to examine both the uptake of Fe by the alga at different CO(2) concentrations during growth and the influence of anaerobiosis on the accumulation of Fe. Algal cells grown in media with (57)Fe(III) at elevated (3%, v/v) CO(2) concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with Mössbauer parameters of quadrupole splitting = 0.65 mm s(-1) and isomeric shift = 0.46 mm s(-1) and (b) Fe(II) with quadrupole splitting = 3.1 mm s(-1) and isomeric shift = 1.36 mm s(-1). Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell. The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis. Growing the algae under atmospheric CO(2) (limiting) conditions, compared with 3% (v/v) CO(2), resulted in a decrease in the intracellular Fe(II) content by a factor of 3. Incubating C. reinhardtii cells, grown at atmospheric CO(2) for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO(2). This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO(2) concentration, and

  20. Dependence on the F0F1-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in Escherichia coli.

    PubMed

    Trchounian, Karen; Pinske, Constanze; Sawers, R Gary; Trchounian, Armen

    2011-12-01

    Escherichia coli has four [NiFe]-hydrogenases (Hyd); three of these, Hyd-1, Hyd-2 and Hyd-3 have been characterized well. In this study the requirement for the F(0)F(1)-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases Hyd-1 and Hyd-2 was examined. During fermentative growth on glucose at pH 7.5 an E. coli F(0)F(1)-ATP synthase mutant (DK8) lacked hydrogenase activity. At pH 5.5 hydrogenase activity was only 20% that of the wild type. Using in-gel activity staining, it could be demonstrated that both Hyd-1 and Hyd-2 were essentially inactive at these pHs, indicating that the residual activity at pH 5.5 was due to the hydrogen-evolving Hyd-3 enzyme. During fermentative growth in the presence of glycerol, hydrogenase activity in the mutant was highest at pH 7.5 attaining a value of 0.76 U/mg, or ~50% of wild type activity, and Hyd-2 was only partially active at this pH, while Hyd-1 was inactive. Essentially no hydrogenase activity was measured at pH 5.5 during growth with glycerol. At this pH the mutant had a hydrogenase activity that was maximally only ~10% of wild type activity with either carbon substrate but a weak activity of both Hyd-1 and Hyd-2 could be detected. Taken together, these results demonstrate for the first time that the activity of the hydrogen-oxidizing hydrogenases in E. coli depends on an active F(0)F(1)-ATP synthase during growth at high and low pH.

  1. [NiFe]Hydrogenase from Citrobacter sp. S-77 surpasses platinum as an electrode for H2 oxidation reaction.

    PubMed

    Matsumoto, Takahiro; Eguchi, Shigenobu; Nakai, Hidetaka; Hibino, Takashi; Yoon, Ki-Seok; Ogo, Seiji

    2014-08-18

    Reported herein is an electrode for dihydrogen (H2) oxidation, and it is based on [NiFe]Hydrogenase from Citrobacter sp. S-77 ([NiFe]S77). It has a 637 times higher mass activity than Pt (calculated based on 1 mg of [NiFe]S77 or Pt) at 50 mV in a hydrogen half-cell. The [NiFe]S77 electrode is also stable in air and, unlike Pt, can be recovered 100 % after poisoning by carbon monoxide. Following characterization of the [NiFe]S77 electrode, a fuel cell comprising a [NiFe]S77 anode and Pt cathode was constructed and shown to have a a higher power density than that achievable by Pt.

  2. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

    PubMed

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

  3. Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

    SciTech Connect

    Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

    2014-09-30

    Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

  4. Evidence for selenocysteine coordination to the active site nickel in the [NiFeSe]hydrogenases from Desulfovibrio baculatus.

    PubMed Central

    Eidsness, M K; Scott, R A; Prickril, B C; DerVartanian, D V; Legall, J; Moura, I; Moura, J J; Peck, H D

    1989-01-01

    Ni and Se x-ray absorption spectroscopic studies of the [NiFeSe]hydrogenases from Desulfovibrio baculatus are described. The Ni site geometry is pseudo-octahedral with a coordinating ligand composition of 3-4 (N,O) at 2.06 A, 1-2 (S,Cl) at 2.17 A, and 1 Se at 2.44 A. The Se coordination environment consists of 1 C at 2.0 A and a heavy scatterer M (M = Ni or Fe) at approximately 2.4 A. These results are interpreted in terms of a selenocysteine residue coordinated to the Ni site. The possible role of the Ni-Se site in the catalytic activation of H2 is discussed. PMID:2521386

  5. The weak, fluctuating, dipole moment of membrane-bound hydrogenase from Aquifex aeolicus accounts for its adaptability to charged electrodes.

    PubMed

    Oteri, Francesco; Ciaccafava, Alexandre; de Poulpiquet, Anne; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

    2014-06-21

    [NiFe] hydrogenases from Aquifex aeolicus (AaHase) and Desulfovibrio fructosovorans (DfHase) have been mainly studied to characterize physiological electron transfer processes, or to develop biotechnological devices such as biofuel cells. In this context, it remains difficult to control the orientation of AaHases on electrodes to achieve a fast interfacial electron transfer. Here, we study the electrostatic properties of these two proteins based on microsecond-long molecular dynamics simulations that we compare to voltammetry experiments. Our calculations show weak values and large fluctuations of the dipole direction in AaHase compared to DfHase, enabling the AaHase to absorb on both negatively and positively charged electrodes, with an orientation distribution that induces a spread in electron transfer rates. Moreover, we discuss the role of the transmembrane helix of AaHase and show that it does not substantially impact the general features of the dipole moment.

  6. [FeFe]-Hydrogenase Abundance and Diversity along a Vertical Redox Gradient in Great Salt Lake, USA

    PubMed Central

    Boyd, Eric S.; Hamilton, Trinity L.; Swanson, Kevin D.; Howells, Alta E.; Baxter, Bonnie K.; Meuser, Jonathan E.; Posewitz, Matthew C.; Peters, John W.

    2014-01-01

    The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications. PMID:25464382

  7. Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation

    PubMed Central

    Tominaga, Taiga; Watanabe, Satoshi; Matsumi, Rie; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2013-01-01

    Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN)2CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53- and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively. PMID:24297906

  8. Occurrence of H2-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus1

    PubMed Central

    Murillo, Jesús; Villa, Ana; Chamber, Manuel; Ruiz-Argüeso, Tomás

    1989-01-01

    Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H2 uptake by bacteroids and the relative efficiency of N2 fixation (RE = 1 - [H2 evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H2-uptake activity in symbiosis with O. compressus. The inability of these putative Hup+ strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization

  9. Immobilization of the [FeFe]-hydrogenase CrHydA1 on a gold electrode: design of a catalytic surface for the production of molecular hydrogen.

    PubMed

    Krassen, Henning; Stripp, Sven; von Abendroth, Gregory; Ataka, Kenichi; Happe, Thomas; Heberle, Joachim

    2009-06-01

    Hydrogenase-modified electrodes are a promising catalytic surface for the electrolysis of water with an overpotential close to zero. The [FeFe]-hydrogenase CrHydA1 from the photosynthetic green alga Chlamydomonas reinhardtii is the smallest [FeFe]-hydrogenase known and exhibits an extraordinary high hydrogen evolution activity. For the first time, we immobilized CrHydA1 on a gold surface which was modified by different carboxy-terminated self-assembled monolayers. The immobilization was in situ monitored by surface-enhanced infrared spectroscopy. In the presence of the electron mediator methyl viologen the electron transfer from the electrode to the hydrogenase was detected by cyclic voltammetry. The hydrogen evolution potential (-290 mV vs NHE, pH 6.8) of this protein modified electrode is close to the value for bare platinum (-270 mV vs NHE). The surface coverage by CrHydA1 was determined to 2.25 ng mm(-2) by surface plasmon resonance, which is consistent with the formation of a protein monolayer. Hydrogen evolution was quantified by gas chromatography and the specific hydrogen evolution activity of surface-bound CrHydA1 was calculated to 1.3 micromol H(2)min(-1)mg(-1) (or 85 mol H(2)min(-1)mol(-1)). In conclusion, a viable hydrogen-evolving surface was developed that may be employed in combination with immobilized photosystems to provide a platform for hydrogen production from water and solar energy with enzymes as catalysts.

  10. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

    DOE PAGES

    Lauterbach, Lars; Wang, Hongxin; Horch, Marius; ...

    2014-10-30

    Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, whichmore » is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.« less

  11. Oxygen-tolerant [NiFe]-hydrogenases: the individual and collective importance of supernumerary cysteines at the proximal Fe-S cluster.

    PubMed

    Lukey, Michael J; Roessler, Maxie M; Parkin, Alison; Evans, Rhiannon M; Davies, Rosalind A; Lenz, Oliver; Friedrich, Baerbel; Sargent, Frank; Armstrong, Fraser A

    2011-10-26

    An important clue to the mechanism for O(2) tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron-sulfur cluster that is proximal to the active site. The O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron-sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O(2).

  12. Exposure studies of core-shell Fe/Fe(3)O(4) and Cu/CuO NPs to lettuce (Lactuca sativa) plants: Are they a potential physiological and nutritional hazard?

    PubMed

    Trujillo-Reyes, J; Majumdar, S; Botez, C E; Peralta-Videa, J R; Gardea-Torresdey, J L

    2014-02-28

    Iron and copper nanomaterials are widely used in environmental remediation and agriculture. However, their effects on physiological parameters and nutritional quality of terrestrial plants such as lettuce (Lactuca sativa) are still unknown. In this research, 18-day-old hydroponically grown lettuce seedlings were treated for 15 days with core-shell nanoscale materials (Fe/Fe(3)O(4), Cu/CuO) at 10 and 20mg/L, and FeSO(4)·7H(2)O and CuSO(4)·5H(2)O at 10mg/L. At harvest, Fe, Cu, micro and macronutrients were determined by ICP-OES. Also, we evaluated chlorophyll content, plant growth, and catalase (CAT) and ascorbate peroxidase (APX) activities. Our results showed that iron ions/NPs did not affect the physiological parameters with respect to water control. Conversely, Cu ions/NPs reduced water content, root length, and dry biomass of the lettuce plants. ICP-OES results showed that nano-Cu/CuO treatments produced significant accumulation of Cu in roots compared to the CuSO(4)·5H(2)O treatment. In roots, all Cu treatments increased CAT activity but decreased APX activity. In addition, relative to the control, nano-Cu/CuO altered the nutritional quality of lettuce, since the treated plants had significantly more Cu, Al and S but less Mn, P, Ca, and Mg.

  13. Fractionation of Sulfur Isotopes by Desulfovibrio vulgaris Mutants Lacking Periplasmic Hydrogenases or the Type I Tetraheme Cytochrome c3

    NASA Astrophysics Data System (ADS)

    Sim, M.; Ono, S.; Bosak, T.

    2012-12-01

    A large fraction of anaerobic mineralization of organic compounds relies on microbial sulfate reduction. Sulfur isotope fractionation by these microbes has been widely used to trace the biogeochemical cycling of sulfur and carbon, but intracellular mechanisms behind the wide range of fractionations observed in nature and cultures are not fully understood. In this study, we investigated the influence of electron transport chain components on the fractionation of sulfur isotopes by culturing Desulfovibrio vulgaris Hildenborough mutants lacking hydrogenases or type I tetraheme cytochrome c3 (Tp1-c3). The mutants were grown both in batch and continuous cultures. All tested mutants grew on lactate or pyruvate as the sole carbon and energy sources, generating sulfide. Mutants lacking cytoplasmic and periplasmic hydrogenases exhibited similar growth physiologies and sulfur isotope fractionations to their parent strains. On the other hand, a mutant lacking Tp1-c3 (ΔcycA) fractionated the 34S/32S ratio more than the wild type, evolving H2 in the headspace and exhibiting a lower specific respiration rate. In the presence of high concentrations of pyruvate, the growth of ΔcycA relied largely on fermentation rather than sulfate reduction, even when sulfate was abundant, producing the largest sulfur isotope effect observed in this study. Differences between sulfur isotope fractionation by ΔcycA and the wild type highlight the effect of electron transfer chains on the magnitude of sulfur isotope fractionation. Because Tp1-c3 is known to exclusively shuttle electrons from periplasmic hydrogenases to transmembrane complexes, electron transfers in the absence of Tp1-c3 should bypass the periplasmic hydrogen cycling, and the loss of reducing equivalents in the form of H2 can impair the flow of electrons from organic acids to sulfur, increasing isotope fractionation. Larger fractionation by ΔcycA can inform interpretations of sulfur isotope data at an environmental scale as well

  14. In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected

    PubMed Central

    Sayavedra-Soto, L A; Arp, D J

    1993-01-01

    The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected]. Images PMID:8501046

  15. A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase*

    PubMed Central

    Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L.; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

    2015-01-01

    The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. PMID:25666617

  16. An iron-iron hydrogenase mimic with appended electron reservoir for efficient proton reduction in aqueous media.

    PubMed

    Becker, René; Amirjalayer, Saeed; Li, Ping; Woutersen, Sander; Reek, Joost N H

    2016-01-01

    The transition from a fossil-based economy to a hydrogen-based economy requires cheap and abundant, yet stable and efficient, hydrogen production catalysts. Nature shows the potential of iron-based catalysts such as the iron-iron hydrogenase (H2ase) enzyme, which catalyzes hydrogen evolution at rates similar to platinum with low overpotential. However, existing synthetic H2ase mimics generally suffer from low efficiency and oxygen sensitivity and generally operate in organic solvents. We report on a synthetic H2ase mimic that contains a redox-active phosphole ligand as an electron reservoir, a feature that is also crucial for the working of the natural enzyme. Using a combination of (spectro)electrochemistry and time-resolved infrared spectroscopy, we elucidate the unique redox behavior of the catalyst. We find that the electron reservoir actively partakes in the reduction of protons and that its electron-rich redox states are stabilized through ligand protonation. In dilute sulfuric acid, the catalyst has a turnover frequency of 7.0 × 10(4) s(-1) at an overpotential of 0.66 V. This catalyst is tolerant to the presence of oxygen, thereby paving the way for a new generation of synthetic H2ase mimics that combine the benefits of the enzyme with synthetic versatility and improved stability.

  17. An iron-iron hydrogenase mimic with appended electron reservoir for efficient proton reduction in aqueous media

    PubMed Central

    Becker, René; Amirjalayer, Saeed; Li, Ping; Woutersen, Sander; Reek, Joost N. H.

    2016-01-01

    The transition from a fossil-based economy to a hydrogen-based economy requires cheap and abundant, yet stable and efficient, hydrogen production catalysts. Nature shows the potential of iron-based catalysts such as the iron-iron hydrogenase (H2ase) enzyme, which catalyzes hydrogen evolution at rates similar to platinum with low overpotential. However, existing synthetic H2ase mimics generally suffer from low efficiency and oxygen sensitivity and generally operate in organic solvents. We report on a synthetic H2ase mimic that contains a redox-active phosphole ligand as an electron reservoir, a feature that is also crucial for the working of the natural enzyme. Using a combination of (spectro)electrochemistry and time-resolved infrared spectroscopy, we elucidate the unique redox behavior of the catalyst. We find that the electron reservoir actively partakes in the reduction of protons and that its electron-rich redox states are stabilized through ligand protonation. In dilute sulfuric acid, the catalyst has a turnover frequency of 7.0 × 104 s−1 at an overpotential of 0.66 V. This catalyst is tolerant to the presence of oxygen, thereby paving the way for a new generation of synthetic H2ase mimics that combine the benefits of the enzyme with synthetic versatility and improved stability. PMID:26844297

  18. Immobilization of hydrogenase on carbon nanotube polyelectrolytes as heterogeneous catalysts for electrocatalytic interconversion of protons and hydrogen

    NASA Astrophysics Data System (ADS)

    Liu, Jiang; Wu, Wen-Jie; Fang, Fang; Zorin, Nikolay A.; Chen, Meng; Qian, Dong-Jin

    2016-08-01

    Immobilization of active enzymes on the surfaces of electrodes and nanomaterials is important in the fields of bioscience, and biotechnology. In this study, we investigated electrocatalytic properties of the interconversion of protons and hydrogen by means of hydrogenase (H2ase)-functionalized carbon nanotube polyelectrolyte composites. Multiwalled carbon nanotube polyelectrolytes (MWNT-PEs) were synthesized through a diazonium and an addition reaction with poly(4-vinylpyridine) (P4VP), followed by another addition reaction with either methyl iodide (CH3I) or N-methyl- N'-benzyl bromide bipyridinium (VBenBr) to produce MWNT-P4VPMe or MWNT-P4VPBenV polyelectrolytes, respectively. The MWNT-PE@H2ase bio-nanocomposites were then prepared by means of MWNT-PEs as substrates to bind with H2ase. The redox current density of the MWNT-PE@H2ase-modified electrodes increased with a decrease in pH values of the Ar-saturated electrolyte solution owing to the catalytic reduction of protons (H2 production); further, it increased with the increasing pH values of the H2-saturated solution owing to the catalytic oxidation of hydrogen. The reversible color change between blue-colored and colorless viologen (catalyzed by the MWNT-PE@H2ase bio-nanocomposites) suggested that they may be developed as nano-biosensors for molecular H2. The as-synthesized bio-nanocomposites showed strong long-term stability and high bioactivity.

  19. Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

    PubMed Central

    Cunningham, Scott D.; Kapulnik, Yoram; Brewin, Nicholas J.; Phillips, Donald A.

    1985-01-01

    Six mutants of Rhizobium leguminosarum 3855 lacking uptake hydrogenase activity (Hup− phenotype) as a result of Tn5-mob mutagenesis of the hup-containing plasmid pRL6JI were tested for symbiotic performance on Pisum sativum L. and Vicia benghalensis L. Three pea cultivars and one vetch line, which induce four different levels of Hup activity in strain 3855, were grown to flowering under microbiologically controlled conditions in the absence of combined N. Direct Kjeldahl N measurements showed that in every case at least one Hup− mutant fixed as much N2 as the isogenic Hup+ strain. Measures of C2H2 reduction, H2 evolution, 3H2 incorporation, and plant dry weight were consistent with the interpretation that the oxidation of H2 produced by the nitrogenase enzyme complex was not necessarily associated with increased N2 fixation in these symbiotic associations. Tests with a smaller subset of the Hup− strains under four different root environments ranging from pH 5.0 to 8.2 likewise showed no significant advantage for the isogenic Hup+ strain. It was concluded that the improvements in symbiotic N2 fixation produced by pRL6JI are associated with some trait other than the Hup+ phenotype. PMID:16346912

  20. A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.

    PubMed

    Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

    2015-03-27

    The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

  1. Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

    PubMed Central

    Manyani, Hamid; Rey, Luis; Palacios, José M.; Imperial, Juan; Ruiz-Argüeso, Tomás

    2005-01-01

    In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (ΔhupG, ΔhupH, ΔhupI, and ΔhupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The ΔhupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the ΔhupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from ΔhupH, ΔhupI, and ΔhupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the ΔhupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit. PMID:16199572

  2. Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution.

    PubMed Central

    McTavish, H; Sayavedra-Soto, L A; Arp, D J

    1995-01-01

    Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H2 oxidation-to-H2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H2 oxidation activity, while having less effect on evolution. The K64,65C-->S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H2 evolution and H2 oxidation activities with whole cells and membranes of the K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C-->S or K297C-->S hydrogenase were uninhibited by O2 in H2 oxidation and uninhibited by H2 in H2 evolution. Wild-type membranes and membranes with K64,65C-->S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O2 or H2. PMID:7608067

  3. Impact of membrane-associated hydrogenases on the F₀F₁-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N'-dicyclohexylcarbodiimide in the mutants lacking hydrogenases.

    PubMed

    Blbulyan, Syuzanna; Trchounian, Armen

    2015-08-01

    Escherichia coli is able to ferment glycerol and to produce molecular hydrogen (H2) by four membrane-associated hydrogenases (Hyd) changing activity in response to different conditions. In this study, overall ATPase activity of glycerol alone and mixed carbon sources (glucose and glycerol) fermented E. coli wild type and different Hyd mutants and its inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) were first investigated. ATPase activity was higher in glycerol fermented wild type cells at pH 7.5 compared to pH 6.5 and pH 5.5; DCCD inhibited markedly ATPase activity at pH 7.5. The ATPase activity at pH 7.5, compared with wild type, was lower in selC and less in hypF single mutants, suppressed in hyaB hybC selC triple mutant. Moreover, total ATPase activity of mixed carbon fermented wild type cells was maximal at pH 7.5 and lowered at pH 5.5. The ATPase activities of hypF and hyaB hybC selC mutants were higher at pH 5.5, compared with wild type; DCCD inhibited markedly ATPase activity of hypF mutant. These results demonstrate that in E. coli during glycerol fermentation the membrane proton-translocating FOF1-ATPase has major input in overall ATPase activity and alkaline pH is more optimal for the FOF1-ATPase operation. Hyd-1 and Hyd-2 are required for the FOF1-ATPase activity upon anaerobic fermentation of glycerol. The impact of Hyd-1 and Hyd-2 on the FOF1-ATPase is more obvious during mixed carbon fermentation at slightly acidic pH.

  4. Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16.

    PubMed

    Buhrke, Thorsten; Löscher, Simone; Lenz, Oliver; Schlodder, Eberhard; Zebger, Ingo; Andersen, Lars K; Hildebrandt, Peter; Meyer-Klaucke, Wolfram; Dau, Holger; Friedrich, Bärbel; Haumann, Michael

    2005-05-20

    The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

  5. Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

    PubMed Central

    Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A.; Lenz, Oliver

    2011-01-01

    The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with KI values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. PMID:22016788

  6. Contribution of hydrogenase 2 to stationary phase H2 production by Escherichia coli during fermentation of glycerol.

    PubMed

    Trchounian, Karen; Soboh, Basem; Sawers, R Gary; Trchounian, Armen

    2013-05-01

    Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H2 production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H2 production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H2 production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)(-1) and Hyd-1 activity was 0.1 U (mg protein)(-1). As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)(-1), and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)(-1) but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H2 production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.

  7. Synthesis and Photophysical Study of a [NiFe] Hydrogenase Biomimetic Compound Covalently Linked to a Re-diimine Photosensitizer

    PubMed Central

    2015-01-01

    The synthesis, photophysics, and photochemistry of a linked dyad ([Re]-[NiFe2]) containing an analogue ([NiFe2]) of the active site of [NiFe] hydrogenase, covalently bound to a Re-diimine photosensitizer ([Re]), are described. Following excitation, the mechanisms of electron transfer involving the [Re] and [NiFe2] centers and the resulting decomposition were investigated. Excitation of the [Re] center results in the population of a diimine-based metal-to-ligand charge transfer excited state. Reductive quenching by NEt3 produces the radically reduced form of [Re], [Re]− (kq = 1.4 ± 0.1 × 107 M–1 s–1). Once formed, [Re]− reduces the [NiFe2] center to [NiFe2]−, and this reduction was followed using time-resolved infrared spectroscopy. The concentration dependence of the electron transfer rate constants suggests that both inter- and intramolecular electron transfer pathways are involved, and the rate constants for these processes have been estimated (kinter = 5.9 ± 0.7 × 108 M–1 s–1, kintra = 1.5 ± 0.1 × 105 s–1). For the analogous bimolecular system, only intermolecular electron transfer could be observed (kinter = 3.8 ± 0.5 × 109 M–1 s–1). Fourier transform infrared spectroscopic studies confirms that decomposition of the dyad occurs upon prolonged photolysis, and this appears to be a major factor for the low activity of the system toward H2 production in acidic conditions. PMID:26605700

  8. Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells.

    PubMed

    Monsalve, Karen; Roger, Magali; Gutierrez-Sanchez, Cristina; Ilbert, Marianne; Nitsche, Serge; Byrne-Kodjabachian, Deborah; Marchi, Valérie; Lojou, Elisabeth

    2015-12-01

    For the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV-Vis spectroscopy. Two sizes around 20.0±5.3 nm and 37.2±4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidation over a large range of temperatures. A high current density up to 1.85±0.15 mA·cm(-2) was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was especially studied as a function of the AuNP size and amount, and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power density of 0.25 mW·cm(-2) was recorded.

  9. Direct Comparison of the Performance of a Bio-inspired Synthetic Nickel Catalyst and a [NiFe]-Hydrogenase, Both Covalently Attached to Electrodes

    SciTech Connect

    Rodriguez-Macia, Patricia; Dutta, Arnab; Lubitz, Wolfgang; Shaw, Wendy J.; Rudiger, Olaf

    2015-10-12

    The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we’ve come in mimicking the impressive enzymatic performance. Here we directly compare the catalytic properties of the [Ni(PCy2NGly2)2]2+ complex with the [NiFe]-hydrogenase from Desulfobivrio vulgaris Miyazaki F (DvMF) immobilized to a functionalized electrode under identical conditions. At pH=7, the enzyme has higher performance in both activity and overpotential, and is more stable, while at low pH, the molecular catalyst outperforms the enzyme in all respects. The Ni complex also has increased tolerance to CO. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bioinspired complexes in fuel cells. AD and WJS acknowledge the Office of Science Early Career Research Program through the US Department of Energy (US DOE), Office of Science, Office of Basic Energy Sciences (BES), and Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the US DOE.

  10. Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

    PubMed

    Liot, Quentin; Constant, Philippe

    2016-02-01

    The Streptomyces avermitilis genome encodes a putative high-affinity [NiFe]-hydrogenase conferring the ability to oxidize tropospheric H2 in mature spores. Here, we used a combination of transcriptomic and mutagenesis approaches to shed light on the potential ecophysiological role of the enzyme. First, S. avermitilis was either exposed to low or hydrogenase-saturating levels of H2 to investigate the impact of H2 on spore transcriptome. In total, 1293 genes were differentially expressed, with 1127 and 166 showing lower and higher expression under elevated H2 concentration, respectively. High H2 exposure lowered the expression of the Sec protein secretion pathway and ATP-binding cassette-transporters, with increased expression of genes encoding proteins directing carbon metabolism toward sugar anabolism and lower expression of NADH dehydrogenase in the respiratory chain. Overall, the expression of relA responsible for the synthesis of the pleiotropic alarmone ppGpp decreased upon elevated H2 exposure, which likely explained the reduced expression of antibiotic synthesis and stress response genes. Finally, deletion of hhySL genes resulted in a loss of H2 uptake activity and a dramatic loss of viability in spores. We propose that H2 is restricted to support the seed bank of Streptomyces under a unique survival-mixotrophic energy mode and discuss important ecological implications of this finding.

  11. A strenuous experimental journey searching for spectroscopic evidence of a bridging nickel–iron–hydride in [NiFe] hydrogenase

    PubMed Central

    Wang, Hongxin; Yoda, Yoshitaka; Ogata, Hideaki; Tanaka, Yoshihito; Lubitz, Wolfgang

    2015-01-01

    Direct spectroscopic evidence for a hydride bridge in the Ni–R form of [NiFe] hydrogenase has been obtained using iron-specific nuclear resonance vibrational spectroscopy (NRVS). The Ni–H–Fe wag mode at 675 cm−1 is the first spectroscopic evidence for a bridging hydride in Ni–R as well as the first iron-hydride-related NRVS feature observed for a biological system. Although density function theory (DFT) calculation assisted the determination of the Ni–R structure, it did not predict the Ni–H–Fe wag mode at ∼675 cm−1 before NRVS. Instead, the observed Ni–H–Fe mode provided a critical reference for the DFT calculations. While the overall science about Ni–R is presented and discussed elsewhere, this article focuses on the long and strenuous experimental journey to search for and experimentally identify the Ni–H–Fe wag mode in a Ni–R sample. As a methodology, the results presented here will go beyond Ni–R and hydrogenase research and will also be of interest to other scientists who use synchrotron radiation for measuring dilute samples or weak spectroscopic features. PMID:26524296

  12. Analysis and comparison of nucleotide sequences encoding the genes for [NiFe] and [NiFeSe] hydrogenases from Desulfovibrio gigas and Desulfovibrio baculatus.

    PubMed Central

    Voordouw, G; Menon, N K; LeGall, J; Choi, E S; Peck, H D; Przybyla, A E

    1989-01-01

    The nucleotide sequences encoding the [NiFe] hydrogenase from Desulfovibrio gigas and the [NiFeSe] hydrogenase from Desulfovibrio baculatus (N.K. Menon, H.D. Peck, Jr., J. LeGall, and A.E. Przybyla, J. Bacteriol. 169:5401-5407, 1987; C. Li, H.D. Peck, Jr., J. LeGall, and A.E. Przybyla, DNA 6:539-551, 1987) were analyzed by the codon usage method of Staden and McLachlan. The reported reading frames were found to contain regions of low codon probability which are matched by more probable sequences in other frames. Renewed nucleotide sequencing showed the probable frames to be correct. The corrected sequences of the two small and large subunits share a significant degree of sequence homology. The small subunit, which contains 10 conserved cysteine residues, is likely to coordinate at least 2 iron-sulfur clusters, while the finding of a selenocysteine codon (TGA) near the 3' end of the [NiFeSe] large-subunit gene matched by a regular cysteine codon (TGC) in the [NiFe] large-subunit gene indicates the presence of some of the ligands to the active-site nickel in the large subunit. PMID:2651421

  13. [Nitrogenase, hydrogenase and nitrate reductase activities, oxygen consumption, and ATP content in nodules formed by strains of Rhizobium leguminosarum 128C53 and 300 in symbiosis with pea plants].

    PubMed

    Bedmar, E J; Olivares, J

    1986-10-01

    The nitrogenase activity, nitrate reductase activity and oxygen uptake as well as the hydrogen incorporation and ATP content were examined in the root nodules and bacteroids, respectively, formed by Rhizobium leguminosarum strains 128C53 (hydrogenase positive) and 300 (hydrogenase negative) in symbiosis with Pisum sativum plants grown in the presence of 2 mM KNO3. The strain 128C53 showed the greatest values for all parameters analyzed, except for the nitrate reductase activity, which was higher for the strain 300. Similarly, nodule nitrate reductase activity in strain 300 was greater than that in strain 128C53 when plants grew in the absence of combined nitrogen. In general, the highest values were obtained when determinations were made after 7 hours of plant illumination. However, the hydrogenase activity of strain 128C53 and the nitrate reductase activities of both strains increased with the light period, reaching a maximum after 14 hours of illumination. These results suggest that the benefits derived from the superior symbiotic properties and from the presence of hydrogenase activity in strain 128C53 could be counteracted by the higher rates of the nodule nitrate reductase activity in strain 300.

  14. Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H2

    SciTech Connect

    Kreuzer, Helen W.; Hill, Eric A.; Moran, James J.; Bartholomew, Rachel A.; Hui, Yang; Hegg, Eric L.

    2014-03-01

    Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data is consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organism, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

  15. Genome data mining and soil survey for the novel group 5 [NiFe]-hydrogenase to explore the diversity and ecological importance of presumptive high-affinity H(2)-oxidizing bacteria.

    PubMed

    Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

    2011-09-01

    Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.

  16. The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound [NiFe] Hydrogenase in Ralstonia eutropha H16 ▿ †

    PubMed Central

    Fritsch, Johannes; Lenz, Oliver; Friedrich, Bärbel

    2011-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O2 and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O2 levels. MBH-driven growth on H2 is inhibited or retarded at high O2 partial pressure (pO2) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO2. Lack of hoxR and hoxT can phenotypically be restored by providing O2-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions. PMID:21441514

  17. Designing interfaces of hydrogenase-nanomaterial hybrids for efficient solar conversion.

    PubMed

    King, Paul W

    2013-01-01

    The direct conversion of sunlight into biofuels is an intriguing alternative to a continued reliance on fossil fuels. Natural photosynthesis has long been investigated both as a potential solution, and as a model for utilizing solar energy to drive a water-to-fuel cycle. The molecules and organizational structure provide a template to inspire the design of efficient molecular systems for photocatalysis. A clear design strategy is the coordination of molecular interactions that match kinetic rates and energetic levels to control the direction and flow of energy from light harvesting to catalysis. Energy transduction and electron-transfer reactions occur through interfaces formed between complexes of donor-acceptor molecules. Although the structures of several of the key biological complexes have been solved, detailed descriptions of many electron-transfer complexes are lacking, which presents a challenge to designing and engineering biomolecular systems for solar conversion. Alternatively, it is possible to couple the catalytic power of biological enzymes to light harvesting by semiconductor nanomaterials. In these molecules, surface chemistry and structure can be designed using ligands. The passivation effect of the ligand can also dramatically affect the photophysical properties of the semiconductor, and energetics of external charge-transfer. The length, degree of bond saturation (aromaticity), and solvent exposed functional groups of ligands can be manipulated to further tune the interface to control molecular assembly, and complex stability in photocatalytic hybrids. The results of this research show how ligand selection is critical to designing molecular interfaces that promote efficient self-assembly, charge-transfer and photocatalysis. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

  18. A hydrogenase model system based on the sequence of cytochrome c: photochemical hydrogen evolution in aqueous media.

    PubMed

    Sano, Yohei; Onoda, Akira; Hayashi, Takashi

    2011-08-07

    The diiron carbonyl cluster is held by a native CXXC motif, which includes Cys14 and Cys17, in the cytochrome c sequence. It is found that the diiron carbonyl complex works well as a catalyst for H(2) evolution. It has a TON of ∼80 over 2 h at pH 4.7 in the presence of a Ru-photosensitizer and ascorbate as a sacrificial reagent in aqueous media.

  19. Photoinduced hydrogen evolution in supramolecular devices with a rhenium photosensitizer linked to FeFe-hydrogenase model complexes.

    PubMed

    Liu, Jianhui; Jiang, Weina

    2012-08-28

    Coordination of the pyridyl-attached diiron azadithiolate hexacarbonyl complexes (2 and 3) through the pyridyl nitrogen to the Re on 10-phenanthroline rhenium (5a) and 2,9-diphenyl-1,10-phenanthroline rhenium (5b) forms novel [Re-Fe] complexes 7a, 7b and 8 respectively. Under visible light illumination using triethylamine as a sacrificial electron donor and [Re-Fe] type complexes (7a, 7b or 8) as catalysts, remarkably increased efficiency was observed for photoinduced hydrogen production with a turnover number reaching 11.8 from complex 7a and 8.75 from 7b. To the best of our knowledge, these are the best values compared to other [Re-Fe] photocatalysts reported so far. In contrast to the parent molecules, the turnover number by the intermolecular combination of complexes 6a and 2 showed a value of 5.23, and that from 6b and 2 is 3.8, while no H(2) was detected from 8a and 3 under the same experimental conditions. Obviously, the intramolecular combination of rhenium(I) and [2Fe2S] as a catalyst is promising for efficient H(2) evolution, and it is better than the intermolecular multi-component system.

  20. Synthetic Models for the [FeFe]-Hydrogenase: Catalytic Proton Reduction and the Structure of the Doubly Protonated Intermediate

    PubMed Central

    Carroll, Maria E.; Barton, Bryan E.; Rauchfuss, Thomas B.; Carroll, Patrick J.

    2012-01-01

    This report compares biomimetic HER catalysts with and without the amine cofactor (adtNH): Fe2(adtNH)(CO)2(dppv)2 (1NH) and Fe2(pdt)(CO)2(dppv)2 (2; (adtNH)2− = (HN(CH2S)22−, pdt2− = 1,3-(CH2)3S22−). These compounds are spectroscopically, structurally, and stereodynamically very similar but exhibit very different catalytic properties. Protonation of 1NH and 2 each give three isomeric hydrides beginning with the kinetically favored terminal hydride, which converts sequentially to sym and unsym isomers of the bridging hydrides. In the case of the amine, the corresponding ammonium-hydrides are also observed. In the case of the terminal amine hydride [t-H1NH]BF4, the ammonium/amine-hydride equilibrium is sensitive to counteranions and solvent. The species [t-H1NH2](BF4)2 represents the first example of a crystallographically characterized terminal hydride produced by protonation. The NH--HFe distance of 1.88(7) Å indicates dihydrogen bonding. The bridging hydrides [µ-H1NH]+ and [µ-H2]+ reduce near −1.8 V, about 150 mV more negative than the reductions of the terminal hydride [t-H1NH]+ and [t-H2]+ at −1.65 V. Reductions of the amine hydrides [t-H1NH]+ and [t-H1NH2]2+ are irreversible. For the pdt analog, the [t-H2]+/0 couple is unaffected by weak acids (pKaMeCN 15.3) but exhibits catalysis with HBF4•Et2O, albeit with a TOF around 4 s−1 and an overpotential greater than 1 V. The voltammetry of [t-H1NH]+ is strongly affected by relatively weak acids and proceeds at 5000 s−1 with an overpotential of 0.7 V. The ammonium-hydride [t-H1NH2]2+ is a faster catalyst with an estimated TOF of 58,000 s−1 and an overpotential of 0.5 V. PMID:23126330

  1. Active-Site Models for the Nickel-Iron Hydrogenases: Effects of Ligands on Reactivity and Catalytic Properties

    PubMed Central

    Carroll, Maria E.; Barton, Bryan E.; Gray, Danielle L.; Mack, Amanda E.; Rauchfuss, Thomas B.

    2011-01-01

    Described are new derivatives of the type [HNiFe(SR)2(diphosphine)(CO)3]+, which feature a Ni(diphosphine) group linked to a Fe(CO)3 group via two bridging thiolate ligands. Previous work had described [HNiFe(pdt)(dppe)(CO)3]+ ([1H]+) and its activity as a catalyst for the reduction of protons. Work described in this paper focused on the effects of the diphosphine attached to nickel as well as the dithiolate bridge, 1,3-propanedithiolate (pdt) vs 1,2-ethanedithiolate (edt). A new synthetic route to these Ni-Fe dithiolates is described, involving reaction of Ni(SR)2(diphosphine) with FeI2(CO)4 followed by in situ reduction with cobaltocene. Evidence is presented that this route proceeds via metastable μ-iodo derivatives. Attempted isolation of such species led to the crystallization of NiFe(Me2pdt)(dppe)I2, which features tetrahedral Fe(II) and square planar Ni(II) centers (Me2pdt = 2,2-dimethylpropanedithiol). The new tricarbonyls prepared in this work are NiFe(pdt)(dcpe)(CO)3 (2, dcpe = 1,2-bis(dicyclohexylphosphino)ethane), NiFe(edt)(dppe)(CO)3 (3), and NiFe(edt)(dcpe)(CO)3 (4). Attempted preparation of a phenylthiolate-bridged complex via the FeI2(CO)4 + Ni(SPh)2(dppe) route gave the tetrametallic species [(CO)2Fe(SPh)2Ni(CO)]2(μ-dppe)2. Crystallographic analysis of the edt-dcpe compund [2H]BF4 and the edt-dppe compound [3H]BF4 verified their close resemblance. Each features pseudo-octahedral Fe and square pyramidal Ni centers. Starting from [4H]BF4 we prepared the PPh3 derivative [HNiFe(edt)(dppe)(PPh3)(CO)2]BF4 ([5H]BF4), which was obtained as a ~2:1 mixture of unsymmetrical and symmetrical isomers. Acid-base measurements indicate that changing from Ni(dppe) to Ni(dcpe) decreases the acidity of the cationic hydride complexes by 2.5 pKaMeCN units, from ~11 to ~13.5 (previous work showed that substitution at Fe leads to more dramatic effects). The redox potentials are more strongly affected by the change from dppe to dcpe, for example the [2]0/+ couple occurs at E1/2 = −820 for [2]0/+ vs −574 mV (vs Fc+/0) for [1]0/+. Changes in the dithiolate do not affect the acidity or the reduction potentials of the hydrides. The acid-independent rate of reduction of CH2ClCO2H by [2H]+ is ca. 50 s−1 (25 °C), twice that of [1H]+. The edt-dppe complex [2H]+ proved to be the most active catalyst, with an acid-independent rate of 300 s−1. PMID:21866886

  2. Mechanism of H2 Production by Models for the [NiFe]-Hydrogenases: Role of Reduced Hydrides.

    PubMed

    Ulloa, Olbelina A; Huynh, Mioy T; Richers, Casseday P; Bertke, Jeffery A; Nilges, Mark J; Hammes-Schiffer, Sharon; Rauchfuss, Thomas B

    2016-07-27

    The intermediacy of a reduced nickel-iron hydride in hydrogen evolution catalyzed by Ni-Fe complexes was verified experimentally and computationally. In addition to catalyzing hydrogen evolution, the highly basic and bulky (dppv)Ni(μ-pdt)Fe(CO)(dppv) ([1](0); dppv = cis-C2H2(PPh2)2) and its hydride derivatives have yielded to detailed characterization in terms of spectroscopy, bonding, and reactivity. The protonation of [1](0) initially produces unsym-[H1](+), which converts by a first-order pathway to sym-[H1](+). These species have C1 (unsym) and Cs (sym) symmetries, respectively, depending on the stereochemistry of the octahedral Fe site. Both experimental and computational studies show that [H1](+) protonates at sulfur. The S = 1/2 hydride [H1](0) was generated by reduction of [H1](+) with Cp*2Co. Density functional theory (DFT) calculations indicate that [H1](0) is best described as a Ni(I)-Fe(II) derivative with significant spin density on Ni and some delocalization on S and Fe. EPR spectroscopy reveals both kinetic and thermodynamic isomers of [H1](0). Whereas [H1](+) does not evolve H2 upon protonation, treatment of [H1](0) with acids gives H2. The redox state of the "remote" metal (Ni) modulates the hydridic character of the Fe(II)-H center. As supported by DFT calculations, H2 evolution proceeds either directly from [H1](0) and external acid or from protonation of the Fe-H bond in [H1](0) to give a labile dihydrogen complex. Stoichiometric tests indicate that protonation-induced hydrogen evolution from [H1](0) initially produces [1](+), which is reduced by [H1](0). Our results reconcile the required reductive activation of a metal hydride and the resistance of metal hydrides toward reduction. This dichotomy is resolved by reduction of the remote (non-hydride) metal of the bimetallic unit.

  3. The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster.

    PubMed

    Suess, Daniel L M; Pham, Cindy C; Bürstel, Ingmar; Swartz, James R; Cramer, Stephen P; Britt, R David

    2016-02-03

    Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key intermediate in H-cluster maturation.

  4. Transcriptional regulation of genes encoding the selenium-free [NiFe]-hydrogenases in the archaeon Methanococcus voltae involves positive and negative control elements.

    PubMed Central

    Noll, I; Müller, S; Klein, A

    1999-01-01

    Methanococcus voltae harbors genetic information for two pairs of homologous [NiFe]-hydrogenases. Two of the enzymes contain selenocysteine, while the other two gene groups encode apparent isoenzymes that carry cysteinyl residues in the homologous positions. The genes coding for the selenium-free enzymes, frc and vhc, are expressed only under selenium limitation. They are transcribed out of a common intergenic region. A series of deletions made in the intergenic region localized a common negative regulatory element for the vhc and frc promoters as well as two activator elements that are specific for each of the two transcription units. Repeated sequences, partially overlapping the frc promoter, were also detected. Mutations in these repeated heptanucleotide sequences led to a weak induction of a reporter gene under the control of the frc promoters in the presence of selenium. This result suggests that the heptamer repeats contribute to the negative regulation of the frc transcription unit. PMID:10430564

  5. The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster

    PubMed Central

    Suess, Daniel L. M.; Pham, Cindy C.; Bürstel, Ingmar; Swartz, James R.; Cramer, Stephen P.; Britt, R. David

    2016-01-01

    Three maturase enzymes—HydE, HydF, and HydG—synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)]− species is a key intermediate in H-cluster maturation. PMID:26764535

  6. Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB*

    PubMed Central

    Sydor, Andrew M.; Lebrette, Hugo; Ariyakumaran, Rishikesh; Cavazza, Christine; Zamble, Deborah B.

    2014-01-01

    The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination. PMID:24338018

  7. Bis[1,2-bis-(eth-oxy-carbon-yl)ethene-1,2-dithiol-ato-κ(2) S,S']bis-(η(5)-penta-methyl-cyclo-penta-dien-yl)tetra-μ3-sulfido-diiron(IV)diiron(III)(3 Fe-Fe).

    PubMed

    Ito, Shohei; Hisamichi, Nozomu; Takase, Tsugiko; Inomata, Shinji

    2013-04-01

    The title compound, [Fe4(C10H15)2(C8H10O4S2)2S4], contains a twisted Fe4S4 cubane-like core. A twofold rotation axis passes through the Fe4S4 core, completing the coordination of the four Fe atoms with two penta-methyl-cyclo-penta-dienyl ligands and two chelating dithiol-ate ligands. There are three short Fe-Fe and three long Fe⋯Fe contacts in the Fe4S4 core, suggesting bonding and non-bonding inter-actions, respectively. The Fe-S bonds in the Fe4S4 core range from 2.1523 (5) to 2.2667 (6) Å and are somewhat longer than the Fe-S bonds involving the dithiol-ate ligand.

  8. Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase.

    PubMed

    Wang, Rui-Yan; Shi, Zhen-Yu; Chen, Jin-Chun; Wu, Qiong; Chen, Guo-Qiang

    2012-09-01

    Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153 mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source.

  9. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

    SciTech Connect

    Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B.; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P.

    2014-10-30

    Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

  10. Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus.

    PubMed Central

    Richaud, P; Colbeau, A; Toussaint, B; Vignais, P M

    1991-01-01

    The hupR1 gene from Rhodobacter capsulatus was cloned and sequenced. It can encode a protein of 53,843 Da which shares significant similarity with several transcriptional regulators and activates transcription of the structural hupSL genes of [NiFe]hydrogenase, as shown by the use of a translational fusion of lacZ with the hupSL promoter. A Hup- mutant having a point mutation in the hupR1 gene is described. PMID:1885559

  11. RAPD analysis and sequencing of ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase as tools for genetic classification of potentially pathogenic isolates of Trichomonas gallinae.

    PubMed

    Sansano-Maestre, José; Martínez-Herrero, María Del Carmen; Garijo-Toledo, María Magdalena; Gómez-Muñoz, María Teresa

    2016-08-01

    Trichomonas gallinae is a worldwide parasite that causes oropharyngeal avian trichomonosis. During eight years, 60 axenic isolates were obtained from different bird species and characterized by three molecular methods: RAPD analysis and PCR-sequencing of ITS1/5.8S rRNA/ITS2 fragment and Fe-hydrogenase gene. We have found two genotypes of ITS1/5.8S rRNA/ITS2 widely distributed among bird populations, a new variant and also two sequences with mixed pattern. Genotype ITS-OBT-Tg-1 was associated with the presence of gross lesions in birds. We have found eight genotypes of the Fe-hydrogenase (A1, A2, C2, C2.1, C4, C5, C6 and C7), three of them are new reports (C5, C6 and C7), and also three sequences with mixed pattern. Subtype A1 of the Fe-hydrogenase was also related with the presence of lesions. RAPD analyses included most of the strains isolated from animals with lesions in one of the sub-clusters. Potentially pathogenic isolates of T. gallinae obtained in this study fulfill the following criteria with one exception: isolated from lesions+ITS-OBT-Tg-1 genotype+FeHyd A1+RAPD sub-cluster I2.

  12. An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

    PubMed Central

    Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A. A.; Baumann, Marcus E. M.; Selmer, Thorsten

    2013-01-01

    Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the

  13. Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

    PubMed

    Graentzdoerffer, Andrea; Rauh, David; Pich, Andreas; Andreesen, Jan R

    2003-01-01

    Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine

  14. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water.

    PubMed

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-07-15

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA(-)) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts.

  15. Hydrogen evolution of Enterobacter aerogenes depending on culture pH: mechanism of hydrogen evolution from NADH by means of membrane-bound hydrogenase.

    PubMed

    Tanisho, S; Kamiya, N; Wakao, N

    1989-01-26

    The pH dependency of cell mass productivity, the hydrogen evolution rate and the yield of hydrogen from glucose was measured by controlling the pH of the culture automatically. The cell mass productivity of Enterobacter aerogenes increased in a linear fashion up to a pH value of approx. 7.0. In contrast, both the evolution rate and the yield of hydrogen showed convex relationships up to a pH value of 7.0, both having maximum values at a pH of approx. 5.8. The maximum evolution rate was approx. 11.3 mmol H2 per g dry cell per h at 38 degrees C. A hypothetical mechanism for hydrogen evolution was proposed by taking our results and other research work into consideration. The proposed mechanism of hydrogen evolution was that NADH was oxidized on the inside surface of the cell membrane and protons were reduced on the outside surface by means of membrane-bound hydrogenase. This mechanism explains in a thermodynamic context the relation between the activity of the hydrogen evolution and the pH of the culture.

  16. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts.

    PubMed

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-09-29

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

  17. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water

    PubMed Central

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-01-01

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA−) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts. PMID:27417065

  18. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts

    PubMed Central

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-01-01

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil. PMID:27319579

  19. Investigating the Role of the Outer-Coordination Sphere in [Ni(PPh2NPh-R2)2]2+ Hydrogenase Mimics

    SciTech Connect

    Jain, Avijita; Reback, Matthew L.; Lindstrom, Mary L.; Thogerson, Colleen E.; Helm, Monte L.; Appel, Aaron M.; Shaw, Wendy J.

    2012-06-18

    A series of dipeptide nickel complexes with the general formula, [Ni(PPh2NNNA-amino acid/ester2)2](BF4)2, have been synthesized and characterized (P2N2= 1,5-diaza-3,7-diphosphacyclooctane, amino acid/esters = glutamic acid, alanine, lysine, and aspartic acid). Each of these complexes is an efficient electrocatalyst for H2 production. The contribution of the outer-coordination sphere, specifically the impact of sterics, the ability to protonate and the pKa of amino acid side chain on the hydrogen production activity of these complexes, was investigated. The rates of all of the catalysts ranged over an order of magnitude. The amino acid containing complexes display 2-3 times higher rates of hydrogen production than the corresponding ester complexes, suggesting the significance of protonated species (side chains/backbone of amino acids) in the outer-coordination sphere. The largest had the fastest rates suggesting that catalytic activity is not hindered by sterics. However, the shapes of catalytic waves are indicative of hindered electron transfer and may suggest a competing mechanism for catalysis than that observed for the unsubstituted parent complex. These studies demonstrate the significant contribution that the outer-coordination sphere can have in tuning the catalytic activity of small molecule hydrogenase mimics.

  20. Escherichia coli multiple [Ni-Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs.

    PubMed

    Trchounian, Karen; Trchounian, Armen

    2013-11-01

    Escherichia coli evolves H2 via multiple [Ni-Fe]-hydrogenases (Hyd). This activity under hyper- and hypo-osmotic stress was investigated with mutants lacking different Hyd enzymes during glycerol fermentation. Inhibitory effects of hypo-stress on H2 production was stronger at pH 6.5 in wild type and mutants except fhlA, which encodes a transcriptional activator for Hyd-3, compared with the effects of N,N'-dicyclohexylcarbodiimide. These results indicate that Hyd-3 and Hyd-4 are osmosensitive at pH 7.5. Hyd-4 and FhlA are implicated in osmotic stress response at pH 6.5. Hyd-1 and FhlA might be osmosensitive at pH 5.5. Thus, osmosensitivity of Hyd enzymes is a novel property that depends on pH. This is significant for mechanisms of cell osmoregulation and H2 production biotechnology when glycerol is used as a fermentation substrate.

  1. Polarized potential and electrode materials implication on electro-fermentative di-hydrogen production: Microbial assemblages and hydrogenase gene copy variation.

    PubMed

    Arunasri, Kotakonda; Annie Modestra, J; Yeruva, Dileep Kumar; Vamshi Krishna, K; Venkata Mohan, S

    2016-01-01

    This study examined the changes in microbial diversity in response to different electrode materials viz., stainless steel mesh (SS) and graphite plate as anodes in two microbial electrolysis cell (MEC) each poised at 0.2V, 0.4V, 0.6V and 0.8V. Changes in microbiota prior to and after pretreatment along with microbiota enriched in response to various poised potentials with SS and graphite are monitored by 16S rRNA gene based DGGE profiling. Significant shifts in microbial community were noticed at all these experimental conditions. Correspondingly, the level of hydrogenase belonging to genera Bacillus, Pseudomonas, Rhodopseudomonas and Clostridium was studied by quantitative real time PCR (RT-PCR) at various applied potentials. DGGE based 16S rRNA gene profiling revealed enriched members belonging to phylum Firmicutes predominantly present at 0.8V in both MECs contributing to high hydrogen production. This study first time explored the growth behavior of mixed consortia in response to poised potentials and electrode materials.

  2. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water

    NASA Astrophysics Data System (ADS)

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-07-01

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA‑) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts.

  3. Molecular recognition and self-assembly special feature: Self-assembled biomimetic [2Fe2S]-hydrogenase-based photocatalyst for molecular hydrogen evolution.

    PubMed

    Kluwer, A M; Kapre, R; Hartl, F; Lutz, M; Spek, A L; Brouwer, A M; van Leeuwen, P W N M; Reek, J N H

    2009-06-30

    The large-scale production of clean energy is one of the major challenges society is currently facing. Molecular hydrogen is envisaged as a key green fuel for the future, but it becomes a sustainable alternative for classical fuels only if it is also produced in a clean fashion. Here, we report a supramolecular biomimetic approach to form a catalyst that produces molecular hydrogen using light as the energy source. It is composed of an assembly of chromophores to a bis(thiolate)-bridged diiron ([2Fe2S]) based hydrogenase catalyst. The supramolecular building block approach introduced in this article enabled the easy formation of a series of complexes, which are all thoroughly characterized, revealing that the photoactivity of the catalyst assembly strongly depends on its nature. The active species, formed from different complexes, appears to be the [Fe(2)(micro-pdt)(CO)(4){PPh(2)(4-py)}(2)] (3) with 2 different types of porphyrins (5a and 5b) coordinated to it. The modular supramolecular approach was important in this study as with a limited number of building blocks several different complexes were generated.

  4. FT-IR Characterization of the Light-Induced Ni-L2 and Ni-L3 States of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F.

    PubMed

    Tai, Hulin; Nishikawa, Koji; Inoue, Seiya; Higuchi, Yoshiki; Hirota, Shun

    2015-10-29

    Different light-induced Ni-L states of [NiFe] hydrogenase from its Ni-C state have previously been observed by EPR spectroscopy. Herein, we succeeded in detecting simultaneously two Ni-L states of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by FT-IR spectroscopy. A new light-induced νCO band at 1890 cm(-1) and νCN bands at 2034 and 2047 cm(-1) were detected in the FT-IR spectra of the H2-activated enzyme under N2 atmosphere at basic conditions, in addition to the 1910 cm(-1) νCO band and 2047 and 2061 cm(-1) νCN bands of the Ni-L2 state. The new bands were attributed to the Ni-L3 state by comparison of the FT-IR and EPR spectra. The νCO and νCN frequencies of the Ni-L3 state are the lowest frequencies observed among the corresponding frequencies of standard-type [NiFe] hydrogenases in various redox states. These results indicate that a residue, presumably Ni-coordinating Cys546, is protonated and deprotonated in the Ni-L2 and Ni-L3 states, respectively. Relatively small ΔH (6.4 ± 0.8 kJ mol(-1)) and ΔS (25.5 ± 10.3 J mol(-1) K(-1)) values were obtained for the conversion from the Ni-L2 to Ni-L3 state, which was in agreement with the previous proposals that deprotonation of Cys546 is important for the catalytic reaction of the enzyme.

  5. Bis[1,2-bis-(meth-oxy-carbon-yl)ethene-1,2-dithiol-ato-κ(2) S,S']bis-(η(5)-penta-methyl-cyclo-penta-dien-yl)tetra-μ3-sulfido-tetra-iron(4 Fe-Fe) hexa-fluoridophosphate.

    PubMed

    Inomata, Shinji; Ito, Shohei; Takase, Tsugiko

    2013-04-01

    The asymmetric unit of the title compound, [Fe4(C6H6O4S2)2(C10H15)2S4]PF6, contains two different complex cations and two PF6 (-) anions. The two complex cations have similar conformations with the butterfly-like Fe4S4 core surrounded by two penta-methyl-cyclo-penta-dienyl ligands and the S atoms of two dithiol-ate ligands. In each Fe4S4 core, there are four short Fe-Fe and two long Fe⋯Fe contacts, suggesting bonding and non-bonding inter-actions, respectively. The Fe-S distances range from 2.1287 (13) to 2.2706 (16) Å for one and from 2.1233 (13) to 2.2650 (16) Å for the other Fe4S4 core. The Fe-S distances involving the dithiol-ate ligands are in a more narrow range [2.1764 (16)-2.1874 (13) Å for one and 2.1743 (14)-2.1779 (16) Å for the other cation]. There are no significant inter-actions between cations and anions.

  6. Electronic Structure and Chemistry of Iron-Based Metal Oxide Nanostructured Materials: A NEXAFS Investigation of BiFeO3, Bi2Fe4O9, α-Fe2O3, γ-Fe2O3, and Fe/Fe3O4

    SciTech Connect

    Park,T.; Sambasivan, S.; Fischer, D.; Yoon, W.; Misewich, J.; Wong, S.

    2008-01-01

    We present a systematic and detailed near edge X-ray absorption fine structure (NEXAFS) experimental investigation of the electronic structure and chemistry of iron-based metal oxide nanostructured (FeMONS) materials including BiFeO3, Bi2Fe4O9, a-Fe2O3, ?-Fe2O3, and Fe/Fe3O4. Correlations of the electronic structure and structural chemistry of these intriguing nanomaterials are presented, ranging from the nano to the bulk scale. In this work, variations in the shape, position, and intensity of the O K-edge and Fe L-edge NEXAFS spectra have been analyzed in terms of electronic structure and surface chemistry of the FeMONS materials as compared with that of the bulk. We hypothesize that surface imperfection and surface strain anisotropies in nanoparticles induce distortion and site inequivalency of the oxygen Oh sites around the Fe ion located close to the surface, resulting in an increase in the degree of multiplicity as well as in nonstoichiometric effects in FeMONS materials.

  7. Moments of Inertia and the Chandlerian Period for Two- and Three-Layer Models of the Galilean Satellite Io

    NASA Astrophysics Data System (ADS)

    Zharkov, V. N.; Sobisevich, A. L.

    2005-03-01

    We consider two-layer (Fe-FeS core+silicate mantle) and three-layer (Fe-FeS core+silicate mantle+crust) models of the Galilean satellite Io. Two parameters are known from observations for the equilibrium figure of the satellite, the mean density ρ 0 and the Love number k 2. Previously, the Radau-Darwin formula was used to determine the mean moment of inertia. Using formulas of the Figure Theory, we calculated the principal moments of inertia A, B, and C and the mean moment of inertia I for the two-and three-layer models of Io using ρ 0 and k 2 as the boundary conditions. We concluded that when modeling the internal structure of Io, it is better to use the observed value of k 2 than the moment of inertia I derived from k 2 using the Radau-Darwin formula. For the models under consideration, we calculated the Chandlerian wobble periods of Io. For the three-layer model, this period is approximately 460 days.

  8. Escherichia coli K-12 survives anaerobic exposure at pH 2 without RpoS, Gad, or hydrogenases, but shows sensitivity to autoclaved broth products.

    PubMed

    Riggins, Daniel P; Narvaez, Maria J; Martinez, Keith A; Harden, Mark M; Slonczewski, Joan L

    2013-01-01

    Escherichia coli and other enteric bacteria survive exposure to extreme acid (pH 2 or lower) in gastric fluid. Aerated cultures survive via regulons expressing glutamate decarboxylase (Gad, activated by RpoS), cyclopropane fatty acid synthase (Cfa) and others. But extreme-acid survival is rarely tested under low oxygen, a condition found in the stomach and the intestinal tract. We observed survival of E. coli K-12 W3110 at pH 1.2-pH 2.0, conducting all manipulations (overnight culture at pH 5.5, extreme-acid exposure, dilution and plating) in a glove box excluding oxygen (10% H2, 5% CO2, balance N2). With dissolved O2 concentrations maintained below 6 µM, survival at pH 2 required Cfa but did not require GadC, RpoS, or hydrogenases. Extreme-acid survival in broth (containing tryptone and yeast extract) was diminished in media that had been autoclaved compared to media that had been filtered. The effect of autoclaved media on extreme-acid survival was most pronounced when oxygen was excluded. Exposure to H2O2 during extreme-acid treatment increased the death rate slightly for W3110 and to a greater extent for the rpoS deletion strain. Survival at pH 2 was increased in strains lacking the anaerobic regulator fnr. During anaerobic growth at pH 5.5, strains deleted for fnr showed enhanced transcription of acid-survival genes gadB, cfa, and hdeA, as well as catalase (katE). We show that E. coli cultured under oxygen exclusion (<6 µM O2) requires mechanisms different from those of aerated cultures. Extreme acid survival is more sensitive to autoclave products under oxygen exclusion.

  9. The Relationship between H2 Evolution and Acetylene Reduction in Pisum sativum-Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity 1

    PubMed Central

    Mahon, John D.; Nelson, Louise M.

    1986-01-01

    Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H2 evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H2 or 3H2 in the presence of acetylene. In all cases, the rate of H2 evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H2 evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup− strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H2 evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup− strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H2 evolution by Hup+ strains were similar in magnitude to their H2-saturated rates of Hup activity. The curvilinear relationship between H2 evolution and acetylene reduction for a representative Hup+ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity. PMID:16664984

  10. How Formaldehyde Inhibits Hydrogen Evolution by [FeFe]-Hydrogenases: Determination by ¹³C ENDOR of Direct Fe-C Coordination and Order of Electron and Proton Transfers.

    PubMed

    Bachmeier, Andreas; Esselborn, Julian; Hexter, Suzannah V; Krämer, Tobias; Klein, Kathrin; Happe, Thomas; McGrady, John E; Myers, William K; Armstrong, Fraser A

    2015-04-29

    Formaldehyde (HCHO), a strong electrophile and a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases, is used to identify the point in the catalytic cycle at which a highly reactive metal-hydrido species is formed. Investigations of the reaction of Chlamydomonas reinhardtii [FeFe]-hydrogenase with formaldehyde using pulsed-EPR techniques including electron-nuclear double resonance spectroscopy establish that formaldehyde binds close to the active site. Density functional theory calculations support an inhibited super-reduced state having a short Fe-(13)C bond in the 2Fe subsite. The adduct forms when HCHO is available to compete with H(+) transfer to a vacant, nucleophilic Fe site: had H(+) transfer already occurred, the reaction of HCHO with the Fe-hydrido species would lead to methanol, release of which is not detected. Instead, Fe-bound formaldehyde is a metal-hydrido mimic, a locked, inhibited form analogous to that in which two electrons and only one proton have transferred to the H-cluster. The results provide strong support for a mechanism in which the fastest pathway for H2 evolution involves two consecutive proton transfer steps to the H-cluster following transfer of a second electron to the active site.

  11. Metagenomic and PCR-Based Diversity Surveys of [FeFe]-Hydrogenases Combined with Isolation of Alkaliphilic Hydrogen-Producing Bacteria from the Serpentinite-Hosted Prony Hydrothermal Field, New Caledonia

    PubMed Central

    Mei, Nan; Postec, Anne; Monnin, Christophe; Pelletier, Bernard; Payri, Claude E.; Ménez, Bénédicte; Frouin, Eléonore; Ollivier, Bernard; Erauso, Gaël; Quéméneur, Marianne

    2016-01-01

    High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (~11), low-temperature (< 40°C), and low-salinity fluids are discharged in both intertidal and shallow submarine environments. In this study, we investigated the diversity and distribution of potentially hydrogen-producing bacteria in Prony hyperalkaline springs by using metagenomic analyses and different PCR-amplified DNA sequencing methods. The retrieved sequences of hydA genes, encoding the catalytic subunit of [FeFe]-hydrogenases and, used as a molecular marker of hydrogen-producing bacteria, were mainly related to those of Firmicutes and clustered into two distinct groups depending on sampling locations. Intertidal samples were dominated by new hydA sequences related to uncultured Firmicutes retrieved from paddy soils, while submarine samples were dominated by diverse hydA sequences affiliated with anaerobic and/or thermophilic submarine Firmicutes pertaining to the orders Thermoanaerobacterales or Clostridiales. The novelty and diversity of these [FeFe]-hydrogenases may reflect the unique environmental conditions prevailing in the PHF (i.e., high-pH, low-salt, mesothermic fluids). In addition, novel alkaliphilic hydrogen-producing Firmicutes (Clostridiales and Bacillales) were successfully isolated from both intertidal and submarine PHF chimney samples. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce hydrogen by fermentation, potentially contributing to the molecular hydrogen balance in situ. PMID:27625634

  12. Role of different Escherichia coli hydrogenases in H+ efflux and F₁F(o)-ATPase activity during glycerol fermentation at different pH values.

    PubMed

    Blbulyan, Syuzanna; Avagyan, Arev; Poladyan, Anna; Trchounian, Armen

    2011-06-01

    Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild-type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

  13. Precursors to [FeFe]-Hydrogenase Models: Syntheses of Fe2(SR)2(CO)6 from CO-Free Iron Sources

    PubMed Central

    Chen, Jinzhu; Boyke, Christine; Rauchfuss, Thomas B.; Volkers, Phillip I.; Whaley, C. Matthew; Wilson, Scott R.; Yao, Haijun

    2008-01-01

    This report describes routes to iron dithiolato carbonyls that do not require preformed iron carbonyls. The reaction of FeCl2, Zn, and Q2S2CnH2n (Q+ = Na+, Et3NH+) under an atmosphere of CO affords Fe2(S2CnH2n)(CO)6 (n = 2, 3) in yields >70%. The method was employed to prepare Fe2(S2C2H4)(13CO)6. Treatment of these carbonylated mixtures with tertiary phosphines gave the ferrous species Fe3(S2C3H6)3(CO)4(PR3)2, for R = Et, Bu, and Ph. Like the related complex Fe3(SPh)6(CO)6, these compounds consist of a linear arrangement of three conjoined face-shared octahedral centers. Omitting the phosphine but with an excess of dithiolate, we obtained the related mixed-valence triiron species [Fe3(S2CnH2n)4(CO)4]−. The highly reducing all-ferrous species [Fe3(S2CnH2n)4(CO)4]2− is implicated as an intermediate in this transformation. Reactive forms of iron, prepared by the method of Rieke, also combined with dithiols under a CO atmosphere to give Fe2(S2CnH2n)(CO)6 in modest yields under mild conditions. Studies on the order of addition indicate that ferrous thiolates are formed prior to the onset of carbonylation. Crystallographic characterization demonstrated that the complexes Fe3(S2C3H6)3(CO)4(PEt3)2 and PBnPh3[Fe3(S2C3H6)4(CO)4] feature high spin ferrous and low spin ferric as the central metal, respectively. PMID:18610969

  14. Coordination chemistry of [HFe(CN)(2)(CO)(3)](-) and its derivatives: toward a model for the iron subsite of the [NiFe]-hydrogenases.

    PubMed

    Whaley, C Matthew; Rauchfuss, Thomas B; Wilson, Scott R

    2009-05-18

    The photoreaction of Fe(CO)(5) and cyanide salts in MeCN solution affords the dianion [Fe(CN)(2)(CO)(3)](2-), conveniently isolated as [K(18-crown-6)](2)[Fe(CN)(2)(CO)(3)]. Solutions of [Fe(CN)(2)(CO)(3)](2-) oxidize irreversibly at -600 mV (vs Ag/AgCl) to give primarily [Fe(CN)(3)(CO)(3)](-). Protonation of the dianion affords the hydride [K(18-crown-6)][HFe(CN)(2)(CO)(3)] with a pK(a) approximately 17 (MeCN). The ferrous hydride exhibits enhanced electrophilicity vs its dianionic precursor, which resists substitution. Treatment of [K(18-crown-6)][Fe(CN)(2)(CO)(3)] with tertiary phosphines and phosphites gives isomeric mixtures of [HFe(CN)(2)(CO)(2)L](-) (L = P(OPh)(3) and PPh(3)). Carbonyl substitution on [1H(CO)(2)](-) by P(OPh)(3) is first-order in both the phosphite and iron (k = 0.18 M(-1) s(-1) at 22 degrees C) with DeltaH(double dagger) = 51.6 kJ mol(-1) and DeltaS(double dagger) = -83.0 J K(-1) mol(-1). These ligands are displaced under an atmosphere of CO. With cis-Ph(2)PCH=CHPPh(2) (dppv), we obtained the monocarbonyl, [HFe(CN)(2)(CO)(dppv)](-), a highly basic hydride (pK(a) > 23.3) that rearranges in solution to a single isomer. Treatment of [K(18-crown-6)][HFe(CN)(2)(CO)(3)] with Et(4)NCN resulted in rapid deprotonation to give [Fe(CN)(2)(CO)(3)](2-) and HCN. The tricyano hydride [HFe(CN)(3)(CO)(2)](2-) is prepared by the reaction of [HFe(CN)(2)(CO)(2)(PPh(3))](-) and [K(18-crown-6)]CN. Similar to the phosphine and phosphite derivatives, [HFe(CN)(3)(CO)(2)](2-) exists as a mixture of all three possible isomers. Protonation of the hydrides [HFe(CN)(2)(CO)(dppv)](-) and [HFe(CN)(3)(CO)(2)](-) in acetonitrile solutions releases H(2) and gives the corresponding acetonitrile complexes [K(18-crown-6)][Fe(CN)(3)(NCMe)(CO)(2)] and Fe(CN)(2)(NCMe)(CO)(dppv). Alkylation of [K(18-crown-6)](2)[Fe(CN)(2)(CO)(3)] with MeOTf gives the thermally unstable [MeFe(CN)(2)(CO)(3)](-), which was characterized spectroscopically at -40 degrees C. Reaction of dppv with [MeFe(CN)(2)(CO)(3)](-) gives the acetyl complex, [Fe(CN)(2)(COMe)(CO)(dppv)](-). Whereas [Fe(CN)(2)(CO)(3)](2-) undergoes protonation and methylation at Fe, acid chlorides give the iron(0) N-acylisocyanides [Fe(CN)(CO)(3)(CNCOR)](-) (R = Ph, CH(3)). The solid state structures of [K(18-crown-6)][HFe(CN)(2)(CO)(dppv)], Fe(CN)(2)(NCMe)(CO)(dppv), and [K(18-crown-6)](2)[HFe(CN)(3)(CO)(2)] were confirmed crystallographically. In all three cases, the cyanide ligands are cis to the hydride or acetonitrile ligands.

  15. 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

    2017-03-28

    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.The ISME Journal advance online publication, 28 March 2017; doi:10.1038/ismej.2017.39.

  16. Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation

    SciTech Connect

    Morra, Simone; Maurelli, Sara; Chiesa, Mario; Mulder, David W.; Ratzloff, Michael W.; Giamello, Elio; King, Paul W.; Gilardi, Gianfranco; Valettia, Francesca

    2016-01-01

    A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid caused significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.

  17. Hierarchical Modeling of Ferromagnetic SMAs and Composites

    DTIC Science & Technology

    2006-01-20

    1(33σ (49) oFeo A FeFe σRAσσ)KI)(SE(Iσ ⋅=⇒⋅⋅−⋅−= f (50) where [ ] ; )KI)(SE(I RA ; 0 0 0 1 0 0V AFeTo ⋅−⋅−== f (51...loading of NiTi. 35 The total volume averaged stress in domain D becomes [ ] [ ] f f FeFe f TFeoFe f **Feoo f **oFeo f oFeo D σ σWI)(SEVI)(SEI σεWI...SEIR FeFe The volume averaged strain is computed as:- ( ) ( ) ( )( ) ToFe D T**oToo D εIWσVEε εεε)εεε(εεε)ε ⋅−+⋅+=⇒ −+=−++++−= − )( 1 1 ff fff

  18. Protein environmental effects on iron-sulfur clusters: A set of rules for constructing computational models for inner and outer coordination spheres.

    PubMed

    Harris, Travis V; Szilagyi, Robert K

    2016-07-05

    The structural properties and reactivity of iron-sulfur proteins are greatly affected by interactions between the prosthetic groups and the surrounding amino acid residues. Thus, quantum chemical investigations of the structure and properties of protein-bound iron-sulfur clusters can be severely limited by truncation of computational models. The aim of this study was to identify, a priori, significant interactions that must be included in a quantum chemical model. Using the [2Fe-2S] accessory cluster of the FeFe-hydrogenase as a demonstrative example with rich electronic structural features, the electrostatic and covalent effects of the surrounding side chains, charged groups, and backbone moieties were systematically mapped through density functional theoretical calculations. Electron affinities, spin density differences, and delocalization indexes from the quantum theory of atoms in molecules were used to evaluate the importance of each interaction. Case studies for hydrogen bonding and charged side-chain interactions were used to develop selection rules regarding the significance of a given protein environmental effect. A set of general rules is proposed for constructing quantum chemical models for iron-sulfur active sites that capture all significant interactions from the protein environment. This methodology was applied to our previously used models in galactose oxidase and the 6Fe-cluster of FeFe-hydrogenase. © 2016 Wiley Periodicals, Inc.

  19. A Compilation of Static Stability and Fin Loads Data for Slender Body Missile Models With and Without Tail Fins and Wings. Volume 1

    DTIC Science & Technology

    1976-03-01

    0 0 0 0 ^^m^^^m 5^g fefe H3D ^^r’" -8 -y o 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 h. CLL versus a Figure 45. Continued. to 00 00...NSRDC TEST 7 DELI DEL2 0EL3 DEL4 PHI 0 0 0 0 0 0 -10 0 -10 0 0 -20 0 -20 0 0 -30 0 -30 0 3J U1 M U1 A- .... ragS^ fefeS ^ M

  20. Modelling the Thermal History of Asteroid 4 Vesta

    NASA Technical Reports Server (NTRS)

    Solano, James M.; Kiefer, W. S.; Mittlefehldt, D. W.

    2012-01-01

    The asteroid 4 Vesta is widely thought to be the source of the HED (Howardite, Eucrite and Diogenite) meteorites, with this link supported by spectroscopic and dynamical studies. The availability of the HED meteorites for study and the new data being gained from the Dawn mission provides an excellent opportunity to investigate Vesta s history. In this study, modelling of Vesta has been undertaken to investigate its evolution from an unconsolidated chondritic body to a differentiated body with an iron core. In contrast to previous modelling, both heat and mass transfer are considered as coupled processes. This work draws on models of melt segregation in terrestrial environments to inform the evolution of Vesta into the differentiated body observed today. In order for a core to form in this body, a separation of the metallic iron from the silicates must take place. Temperatures in excess of the solidus temperatures for the Fe-FeS system and the silicates are therefore required. Thermal modelling has shown accretion before 2Myr leads to temperatures in excess of the silicate solidus.

  1. Contributing factors in the improvement of cellulosic H2 production in Clostridium thermocellum/Thermoanaerobacterium co-cultures.

    PubMed

    Wang, Mingyu; Zhao, Qi; Li, Ling; Niu, Kangle; Li, Yi; Wang, Fangzhong; Jiang, Baojie; Liu, Kuimei; Jiang, Yi; Fang, Xu

    2016-10-01

    Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium.

  2. Integrated analysis of transcriptomic and proteomic data of Desulfovibrio vulgaris: Zero-Inflated Poisson regression models to predict abundance of undetected proteins

    SciTech Connect

    Nie, Lei; Wu, Gang; Brockman, Fred J.; Zhang, Weiwen

    2006-05-04

    Abstract Advances in DNA microarray and proteomics technologies have enabled high-throughput measurement of mRNA expression and protein abundance. Parallel profiling of mRNA and protein on a global scale and integrative analysis of these two data types could provide additional insight into the metabolic mechanisms underlying complex biological systems. However, because protein abundance and mRNA expression are affected by many cellular and physical processes, there have been conflicting results on the correlation of these two measurements. In addition, as current proteomic methods can detect only a small fraction of proteins present in cells, no correlation study of these two data types has been done thus far at the whole-genome level. In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), Zero-inflated Poisson models were used to define the correlation pattern of mRNA and protein abundance. The models assumed that there is a probability mass at zero representing some of the undetected proteins because of technical limitations. The models thus use abundance measurements of transcripts and proteins experimentally detected as input to generate predictions of protein abundances as output for all genes in the genome. We demonstrated the statistical models by comparatively analyzing D. vulgaris grown on lactate-based versus formate-based media. The increased expressions of Ech hydrogenase and alcohol dehydrogenase (Adh)-periplasmic Fe-only hydrogenase (Hyd) pathway for ATP synthesis were predicted for D. vulgaris grown on formate.

  3. Growth of thin Fe/Fe2O3 films on the Cu(110) surface

    NASA Astrophysics Data System (ADS)

    Pflitsch, Christian; David, Rudolf; Verheij, Laurens K.; Franchy, René

    2001-08-01

    The growth of Fe/Fe-oxide double-layers on Cu(110) was studied with thermal energy atom scattering (TEAS), Auger electron spectroscopy, and low-energy electron diffraction (LEED). An iron film with a thickness of about 0.6 nm was evaporated at low temperature (130 K) on a smooth, well-ordered thin film of Fe2O3 prepared on Cu(110). This Fe film is disordered. Ordering of the film was observed at temperatures between 400 and 600 K. At 530 K, a structure corresponding to that of a well-ordered α-Fe(001) surface was observed with TEAS and LEED. Clear evidence was found for a mixing of the Fe and Fe2O3 layers at the interface, already beginning at the deposition temperature of 130 K. With increasing temperature, the mixing of the Fe and Fe2O3 layers became gradually more effective until, at around 600 K, it was essentially completed. Upon annealing the sample to 1000 K the structure of the film changes and a very thin (less than 2 ML) FeO film on top of the Cu(110) surface is obtained.

  4. Cluster molecular orbital description of the electronic structures of mixed-valence iron oxides and silicates

    USGS Publications Warehouse

    Sherman, David M.

    1986-01-01

    A molecular orbital description, based on spin-unrestricted X??-scattered wave calculations, is given for the electronic structures of mixed valence iron oxides and silicates. The cluster calculations show that electron hopping and optical intervalence charge-transger result from weak FeFe bonding across shared edges of FeO6 coordination polyhedra. In agreement with Zener's double exchange model, FeFe bonding is found to stabilize ferromagnetic coupling between Fe2+ and Fe3+ cations. ?? 1986.

  5. First-principles computation of electron transfer and reaction rate at a perovskite cathode for hydrogen production.

    PubMed

    Liu, C T; Chu, J F; Lin, C K; Hong, C W

    2017-03-22

    The focus of this research is on the electron transfer and its reaction rate at the perovskite cathode of a photoelectrochemical cell for hydrogen production. By employing the density functional theory (DFT), the electron density, projected density of states (PDOS), electron distribution and electron transfer path between [Fe-Fe] hydrogenase and the perovskite cathode can be obtained. Simulation results show that the perovskite cathode is better than traditional cathodes for hydrogen production. Before transmission to the [Fe-Fe] hydrogenase, electron clouds mainly aggregate at the periphery of amine molecules. Simulations also show that the key to hydrogen production at the perovskite structure lies in the organic molecules. Electrons are transferred to the hydrocarbon structural chain before reaching the Fe atoms. The Rice, Ramsperger, Kassel and Marcus (RRKM) theory was used to predict the reaction rates at different temperatures. It was found that the reaction rates are in good agreement with the experimental results. This research provides more physical insight into the electron transfer mechanism during the hydrogen production process.

  6. Sodium Stimulation of Uptake Hydrogenase Activity In Symbiotic Rhizobium1

    PubMed Central

    Kapulnik, Yoram; Phillips, Donald A.

    1986-01-01

    Initial observations showed a 100% increase in H2-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H2 evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na+ (29 versus 9 milligrams per kilogram) and less K+ (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na+ concentration of clay-grown plants with a much smaller reduction in K+ concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na+ strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na+ concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na+ increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.). PMID:16665057

  7. Survey of Hydrogenase Activity in Algae: Final Report

    SciTech Connect

    Brand, J. J.

    1982-04-01

    The capacity for hydrogen gas production was examined in nearly 100 strains of Eukaryotic algae. Each strain was assessed for rate of H2 production in darkness, at compensating light intensity and at saturating Tight intensity. Maximum H2 yield on illumination and sensitivity to molecular oxygen were also measured.

  8. Hydrogen Production Using Hydrogenase-Containing Oxygenic Photosynthetic Organisms

    DOEpatents

    Melis, A.; Zhang, L.; Benemann, J. R.; Forestier, M.; Ghirardi, M.; Seibert, M.

    2006-01-24

    A reversible physiological process provides for the temporal separation of oxygen evolution and hydrogen production in a microorganism, which includes the steps of growing a culture of the microorganism in medium under illuminated conditions to accumulate an endogenous substrate, depleting from the medium a nutrient selected from the group consisting of sulfur, iron, and/or manganese, sealing the culture from atmospheric oxygen, incubating the culture in light whereby a rate of light-induced oxygen production is equal to or less than a rate of respiration, and collecting an evolved gas. The process is particularly useful to accomplish a sustained photobiological hydrogen gas production in cultures of microorganisms, such as Chlamydomonas reinhardtii.

  9. Hydrogen production using hydrogenase-containing oxygenic photosynthetic organisms

    DOEpatents

    Melis, Anastasios; Zhang, Liping; Benemann, John R.; Forestier, Marc; Ghirardi, Maria; Seibert, Michael

    2006-01-24

    A reversible physiological process provides for the temporal separation of oxygen evolution and hydrogen production in a microorganism, which includes the steps of growing a culture of the microorganism in medium under illuminated conditions to accumulate an endogenous substrate, depleting from the medium a nutrient selected from the group consisting of sulfur, iron, and/or manganese, sealing the culture from atmospheric oxygen, incubating the culture in light whereby a rate of light-induced oxygen production is equal to or less than a rate of respiration, and collecting an evolved gas. The process is particularly useful to accomplish a sustained photobiological hydrogen gas production in cultures of microorganisms, such as Chlamydomonas reinhardtii.

  10. Geochemical modeling of low melt-fraction anatexis in a peraluminous system: The Pena Negra complex (central Spain)

    SciTech Connect

    Bea, F. )

    1991-07-01

    A study was made of the chemical fractionation associated with four cases of anatectic segregation of low melt-fraction cordieritic granites from migmatized meta-greywackes. The aims of the study were to (1) reveal the fractionation patterns of major and trace elements, (2) compare the major element chemistry of leucogranites and the quantitative behavior of source minerals during anatexis - inferred by mass-balance adjustment - with available experimental data for peraluminous systems, and (3) discuss the behavior of trace elements in crustal melting by comparing the chemically determined composition of leucogranites with the results of three fractionation models. Two of these assume a perfect diffusive behavior of trace elements within residual solids, but they use a different set of distribution coefficients. The third assumes a perfect nondiffusive behavior. In relation to their source rocks, the leucogranites are strongly depleted in Li, Transition Elements, and Light Rare Earth Elements, but enriched in K{sub 2}O, SiO{sub 2}, and Ba. Mass balance analysis using the Anatexis Mixing Model shows that the chemistry of cordierite leucogranites is compatible with its having originated by closed-system, water-undersaturated anatexis on previously migmatized meta-greywackes, leaving a residue enriched in cordierite plus biotite and exhausted in K-feldspar. Biotite melts congruently unless important amounts of sillimanite were also present in the source. Compared with experimental metals obtained from sources with the same chemical composition but with a different femic mineralogy (biotite + sillimanite, instead of cordierite + biotite), the Pena Negra leucogranites are richer in K{sub 2}O and MgO with a lower Fe/(Fe + Mg) ratio. The differences in magnesium are believed to result from the changes in the mineral assemblage of the source rocks.

  11. First-principles study of point defects in an fcc Fe-10Ni-20Cr model alloy

    NASA Astrophysics Data System (ADS)

    Piochaud, J. B.; Klaver, T. P. C.; Adjanor, G.; Olsson, P.; Domain, C.; Becquart, C. S.

    2014-01-01

    The influence of the local environment on vacancy and self-interstitial formation energies has been investigated in a face-centered-cubic (fcc) Fe-10Ni-20Cr model alloy by analyzing an extensive set of first-principle calculations based on density functional theory. Chemical disorder has been considered by designing special quasirandom structures and four different collinear magnetic structures have been investigated in order to determine a relevant reference state to perform point defect calculations at 0 K. Two different convergence methods have also been used to characterize the importance of the method on the results. Although our fcc Fe-10Ni-20Cr would be better represented in terms of applications by the paramagnetic state, we found that the antiferromagnetic single-layer magnetic structure was the most stable at 0 K and we chose it as a reference state to determine the point defect properties. Point defects have been introduced in this reference state, i.e., vacancies and Fe-Fe, Fe-Ni, Fe-Cr, Cr-Cr, Ni-Ni, and Ni-Cr dumbbell interstitials oriented either parallel or perpendicular to the single layer antiferromagnetic planes. Each point defect studied was introduced at different lattice sites to consider a sufficient variety of local environments and analyze its influence on the formation energy values. We have estimated the point defect formation energies with linear regressions using variables which describe the local environment surrounding the point defects. The number and the position of Ni and Cr first nearest neighbors to the point defects were found to drive the evolution of the formation energies. In particular, Ni is found to decrease and Cr to increase the vacancy formation energy of the model alloy, while the opposite trends are found for the dumbbell interstitials. This study suggested that, to a first approximation, the first nearest atoms to point defects can provide reliable estimates of point defect formation energies.

  12. The bonding of FeN2, FeCO, and Fe2N2 - Model systems for side-on bonding of CO and N2

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Pettersson, Lars G. M.; Siegbahn, Per E. M.

    1987-01-01

    Qualitative calculations are performed to elucidate the nature of the side-on interaction of both N2 and CO with a single Fe atom. The systems are found to be quite similar, with bonding leading to an increase in the CO or N2 bond length and a decrease in the vibrational frequency. The CO or N2 stretching modes lead to a large dipole derivative along the metal-ligand bond axis. The populations show an almost identical, large donation from the Fe 3d orbitals into the CO or N2 Pi-asterisk. The larger system Fe2N2 is then considered, with the N2 bridging the Fe2, both parallel and perpendicular to the Fe2 bond axis for two different Fe-Fe distances. For FeN2, the shift in the observed N2 frequency is smaller than observed for the alpha state of N2/Fe(111). The shift in the N2 vibrational frequency increases when the N2 interacts with two Fe atoms, either at the Fe-Fe nearest neighbor distance or at the first layer Fe-Fe distance, when the side-on N2 axis is oriented perpendicular to an Fe-Fe bond.

  13. An electron-flow model can predict complex redox reactions in mixed-culture fermentative bioH2: microbial ecology evidence.

    PubMed

    Lee, Hyung-Sool; Krajmalinik-Brown, Rosa; Zhang, Husen; Rittmann, Bruce E

    2009-11-01

    We developed the first model for predicting community structure in mixed-culture fermentative biohydrogen production using electron flows and NADH2 balances. A key assumption of the model is that H2 is produced only via the pyruvate decarboxylation-ferredoxin-hydrogenase pathway, which is commonly the case for fermentation by Clostridium and Ethanoligenens species. We experimentally tested the model using clone libraries to gauge community structures with mixed cultures in which we did not pre-select for specific bacterial groups, such as spore-formers. For experiments having final pHs 3.5 and 4.0, where H2 yield and soluble end-product distribution were distinctly different, we established stoichiometric reactions for each condition by using experimentally determined electron equivalent balances. The error in electron balancing was only 3% at final pH 3.5, in which butyrate and acetate were dominant organic products and the H2 yield was 2.1 mol H2/mol glucose. Clone-library analysis showed that clones affiliated with Clostridium sp. BL-22 and Clostridium sp. HPB-16 were dominant at final pH 3.5. For final pH 4.0, the H2 yield was 0.9 mol H2/mol glucose, ethanol, and acetate were the dominant organic products, and the electron balance error was 13%. The significant error indicates that a second pathway for H2 generation was active. The most abundant clones were affiliated with Klebsiella pneumoniae, which uses the formate-cleavage pathway for H2 production. Thus, the clone-library analyses confirmed that the model predictions for when the pyruvate decarboxylation-ferredoxin-hydrogenase pathway was (final pH 3.5) or was not (final pH 4.0) dominant. With the electron-flow model, we can easily assess the main mechanisms for H2 formation and the dominant H2-producing bacteria in mixed-culture fermentative bioH2.

  14. A Tale of Two Gases: Isotope Effects Associated with the Enzymatic Production of H2 and N2O

    NASA Astrophysics Data System (ADS)

    Yang, H.; Gandhi, H.; Kreuzer, H. W.; Moran, J.; Hill, E. A.; McQuarters, A.; Lehnert, N.; Ostrom, N. E.; Hegg, E. L.

    2014-12-01

    Stable isotopes can provide considerable insight into enzymatic mechanisms and fluxes in various biological processes. In our studies, we used stable isotopes to characterize both enzyme-catalyzed H2 and N2O production. H2 is a potential alternative clean energy source and also a key metabolite in many microbial communities. Biological H2 production is generally catalyzed by hydrogenases, enzymes that combine protons and electrons to produce H2 under anaerobic conditions. In our study, H isotopes and fractionation factors (α) were used to characterize two types of hydrogenases: [FeFe]- and [NiFe]-hydrogenases. Due to differences in the active site, the α associated with H2 production for [FeFe]- and [NiFe]-hydrogenases separated into two distinct clusters (αFeFe > αNiFe). The calculated kinetic isotope effects indicate that hydrogenase-catalyzed H2 production has a preference for light isotopes, consistent with the relative bond strengths of O-H and H-H bonds. Interestingly, the isotope effects associated with H2 consumption and H2-H2O exchange reactions were also characterized, but in this case no specific difference was observed between the different enzymes. N2O is a potent greenhouse gas with a global warming potential 300 times that of CO2, and the concentration of N2O is currently increasing at a rate of ~0.25% per year. Thus far, bacterial and fungal denitrification processes have been identified as two of the major sources of biologically generated N2O. In this study, we measured the δ15N, δ18O, δ15Nα (central N atom in N2O), and δ15Nβ (terminal N atom in N2O) of N2O generated by purified fungal P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum. We observed normal isotope effects for δ18O and δ15Nα, and inverse isotope effects for bulk δ15N (the average of Nα and Nβ) and δ15Nβ. The observed isotope effects have been used in conjunction with DFT calculations to provide important insight into the mechanism of P450nor. Similar

  15. Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri

    SciTech Connect

    Feist, Adam; Scholten, Johannes C.; Palsson, Bernard O.; Brockman, Fred J.; Ideker, Trey

    2006-01-31

    We present a genome-scale metabolic reconstruction for the archaeal methanogen Methanosarcina barkeri. This reconstruction represents the first large-scale, predictive model of a methanogen and an archael species. We characterize this reconstruction and compare it to those from the prokaryotic, eukaryotic, and archael domains. We further apply constraint-based methods to stimulate the metabolic fluxes and resulting phenotypes under different environmental and genetic conditions. These results are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for mutants of the methanogenic pathway were found to have a high level of agreement with experimental findings. The active reactions and pathways under selected growth conditions are presented and characterized. We also examined the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an in silico analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation, and further uncover the metabolic characteristics of methanogenesis.

  16. Ion irradiation of Fe-Fe oxide core-shell nanocluster films: Effect of interface on stability of magnetic properties

    NASA Astrophysics Data System (ADS)

    McCloy, John S.; Jiang, Weilin; Droubay, Timothy C.; Varga, Tamas; Kovarik, Libor; Sundararajan, Jennifer A.; Kaur, Maninder; Qiang, You; Burks, Edward C.; Liu, Kai

    2013-08-01

    A cluster deposition method was used to produce films of loosely aggregated nanoclusters (NCs) of Fe core-Fe3O4 shell or fully oxidized Fe3O4. Films of these NC on Si(100) or MgO(100)/Fe3O4(100) were irradiated to 1016 Si2+/cm2 near room temperature using an ion accelerator. Ion irradiation creates structural change in the NC film with corresponding chemical and magnetic changes which depend on the initial oxidation state of the cluster. Films were characterized using magnetometry (hysteresis, first order reversal curves), microscopy (transmission electron, helium ion), and x-ray diffraction. In all cases, the particle sizes increased due to ion irradiation, and when a core of Fe is present, irradiation reduces the oxide shells to lower valent Fe species. These results show that ion irradiated behavior of the NC films depends strongly on the initial nanostructure and chemistry, but in general saturation magnetization decreases slightly.

  17. Toward a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Chhabra, S.R.; Joachimiak, M.P.; Petzold, C.J.; Zane, G.M.; Price, M.N.; Gaucher, S.; Reveco, S.A.; Fok, V.; Johanson, A.R.; Batth, T.S.; Singer, M.; Chandonia, J.M.; Joyner, D.; Hazen, T.C.; Arkin, A.P.; Wall, J.D.; Singh, A.K.; Keasling, J.D.

    2011-05-01

    Protein–protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study E. coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio 5 vulgaris Hildenborough, a model anaerobe and sulfate reducer. In this paper we present the first attempt to identify protein-protein interactions in an obligate anaerobic bacterium. We used suicide vector-assisted chromosomal modification of 12 open reading frames encoded by this sulfate reducer to append an eight amino acid affinity tag to the carboxy-terminus of the chosen proteins. Three biological replicates of the 10 ‘pulled-down’ proteins were separated and analyzed using liquid chromatography-mass spectrometry. Replicate agreement ranged between 35% and 69%. An interaction network among 12 bait and 90 prey proteins was reconstructed based on 134 bait-prey interactions computationally identified to be of high confidence. We discuss the biological significance of several unique metabolic features of D. vulgaris revealed by this protein-protein interaction data 15 and protein modifications that were observed. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.

  18. Towards a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough.

    PubMed

    Chhabra, Swapnil R; Joachimiak, Marcin P; Petzold, Christopher J; Zane, Grant M; Price, Morgan N; Reveco, Sonia A; Fok, Veronica; Johanson, Alyssa R; Batth, Tanveer S; Singer, Mary; Chandonia, John-Marc; Joyner, Dominique; Hazen, Terry C; Arkin, Adam P; Wall, Judy D; Singh, Anup K; Keasling, Jay D

    2011-01-01

    Protein-protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study Escherichia coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio vulgaris Hildenborough, a model obligate anaerobe and sulfate reducer and the subject of this study. Here we carried out affinity purification followed by mass spectrometry to reconstruct an interaction network among 12 chromosomally encoded bait and 90 prey proteins based on 134 bait-prey interactions identified to be of high confidence. Protein-protein interaction data are often plagued by the lack of adequate controls and replication analyses necessary to assess confidence in the results, including identification of potential false positives. We addressed these issues through the use of biological replication, exponentially modified protein abundance indices, results from an experimental negative control, and a statistical test to assign confidence to each putative interacting pair applicable to small interaction data studies. We discuss the biological significance of metabolic features of D. vulgaris revealed by these protein-protein interaction data and the observed protein modifications. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.

  19. Towards a Rigorous Network of Protein-Protein Interactions of the Model Sulfate Reducer Desulfovibrio vulgaris Hildenborough

    PubMed Central

    Petzold, Christopher J.; Zane, Grant M.; Price, Morgan N.; Reveco, Sonia A.; Fok, Veronica; Johanson, Alyssa R.; Batth, Tanveer S.; Singer, Mary; Chandonia, John-Marc; Joyner, Dominique; Hazen, Terry C.; Arkin, Adam P.; Wall, Judy D.; Singh, Anup K.; Keasling, Jay D.

    2011-01-01

    Protein–protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study Escherichia coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio vulgaris Hildenborough, a model obligate anaerobe and sulfate reducer and the subject of this study. Here we carried out affinity purification followed by mass spectrometry to reconstruct an interaction network among 12 chromosomally encoded bait and 90 prey proteins based on 134 bait-prey interactions identified to be of high confidence. Protein-protein interaction data are often plagued by the lack of adequate controls and replication analyses necessary to assess confidence in the results, including identification of potential false positives. We addressed these issues through the use of biological replication, exponentially modified protein abundance indices, results from an experimental negative control, and a statistical test to assign confidence to each putative interacting pair applicable to small interaction data studies. We discuss the biological significance of metabolic features of D. vulgaris revealed by these protein-protein interaction data and the observed protein modifications. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction. PMID:21738675

  20. Hydrogen Production by Water Biophotolysis

    SciTech Connect

    Ghirardi, Maria L.; King, Paul W.; Mulder, David W.; Eckert, Carrie; Dubini, Alexandra; Maness, Pin-Ching; Yu, Jianping

    2014-01-22

    The use of microalgae for production of hydrogen gas from water photolysis has been studied for many years, but its commercialization is still limited by multiple challenges. Most of the barriers to commercialization are attributed to the existence of biological regulatory mechanisms that, under anaerobic conditions, quench the absorbed light energy, down-regulate linear electron transfer, inactivate the H2-producing enzyme, and compete for electrons with the hydrogenase. Consequently, the conversion efficiency of absorbed photons into H2 is significantly lower than its estimated potential of 12–13 %. However, extensive research continues towards addressing these barriers by either trying to understand and circumvent intracellular regulatory mechanisms at the enzyme and metabolic level or by developing biological systems that achieve prolonged H2 production albeit under lower than 12–13 % solar conversion efficiency. This chapter describes the metabolic pathways involved in biological H2 photoproduction from water photolysis, the attributes of the two hydrogenases, [FeFe] and [NiFe], that catalyze biological H2 production, and highlights research related to addressing the barriers described above. These highlights include: (a) recent advances in improving our understanding of the O2 inactivation mechanism in different classes of hydrogenases; (b) progress made in preventing competitive pathways from diverting electrons from H2 photoproduction; and (c) new developments in bypassing the non-dissipated proton gradient from down-regulating photosynthetic electron transfer. As an example of a major success story, we mention the generation of truncated-antenna mutants in Chlamydomonas and Synechocystis that address the inherent low-light saturation of photosynthesis. In addition, we highlight the rationale and progress towards coupling biological hydrogenases to non-biological, photochemical charge-separation as a means to bypass the barriers of photobiological

  1. Bioinspired Hydrogenase Models: The Mixed-Valence Triiron Complex [Fe3(CO)7(μ-edt)2] and Phosphine Derivatives [Fe3(CO)7-x (PPh3) x (μ-edt)2] (x = 1, 2) and [Fe3(CO)5(κ(2)-diphosphine)(μ-edt)2] as Proton Reduction Catalysts.

    PubMed

    Rahaman, Ahibur; Ghosh, Shishir; Unwin, David G; Basak-Modi, Sucharita; Holt, Katherine B; Kabir, Shariff E; Nordlander, Ebbe; Richmond, Michael G; Hogarth, Graeme

    2014-03-24

    The mixed-valence triiron complexes [Fe3(CO)7-x (PPh3) x (μ-edt)2] (x = 0-2; edt = SCH2CH2S) and [Fe3(CO)5(κ(2)-diphosphine)(μ-edt)2] (diphosphine = dppv, dppe, dppb, dppn) have been prepared and structurally characterized. All adopt an anti arrangement of the dithiolate bridges, and PPh3 substitution occurs at the apical positions of the outer iron atoms, while the diphosphine complexes exist only in the dibasal form in both the solid state and solution. The carbonyl on the central iron atom is semibridging, and this leads to a rotated structure between the bridged diiron center. IR studies reveal that all complexes are inert to protonation by HBF4·Et2O, but addition of acid to the pentacarbonyl complexes results in one-electron oxidation to yield the moderately stable cations [Fe3(CO)5(PPh3)2(μ-edt)2](+) and [Fe3(CO)5(κ(2)-diphosphine)(μ-edt)2](+), species which also result upon oxidation by [Cp2Fe][PF6]. The electrochemistry of the formally Fe(I)-Fe(II)-Fe(I) complexes has been investigated. Each undergoes a quasi-reversible oxidation, the potential of which is sensitive to phosphine substitution, generally occurring between 0.15 and 0.50 V, although [Fe3(CO)5(PPh3)2(μ-edt)2] is oxidized at -0.05 V. Reduction of all complexes is irreversible and is again sensitive to phosphine substitution, varying between -1.47 V for [Fe3(CO)7(μ-edt)2] and around -1.7 V for phosphine-substituted complexes. In their one-electron-reduced states, all complexes are catalysts for the reduction of protons to hydrogen, the catalytic overpotential being increased upon successive phosphine substitution. In comparison to the diiron complex [Fe2(CO)6(μ-edt)], [Fe3(CO)7(μ-edt)2] catalyzes proton reduction at 0.36 V less negative potentials. Electronic structure calculations have been carried out in order to fully elucidate the nature of the oxidation and reduction processes. In all complexes, the HOMO comprises an iron-iron bonding orbital localized between the two iron atoms not ligated by the semibridging carbonyl, while the LUMO is highly delocalized in nature and is antibonding between both pairs of iron atoms but also contains an antibonding dithiolate interaction.

  2. Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus.

    PubMed

    Morais-Silva, Fabio O; Rezende, Antonio Mauro; Pimentel, Catarina; Santos, Catia I; Clemente, Carla; Varela-Raposo, Ana; Resende, Daniela M; da Silva, Sofia M; de Oliveira, Luciana Márcia; Matos, Marcia; Costa, Daniela A; Flores, Orfeu; Ruiz, Jerónimo C; Rodrigues-Pousada, Claudina

    2014-08-01

    Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.

  3. Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus*

    PubMed Central

    Morais-Silva, Fabio O; Rezende, Antonio Mauro; Pimentel, Catarina; Santos, Catia I; Clemente, Carla; Varela–Raposo, Ana; Resende, Daniela M; da Silva, Sofia M; de Oliveira, Luciana Márcia; Matos, Marcia; Costa, Daniela A; Flores, Orfeu; Ruiz, Jerónimo C; Rodrigues-Pousada, Claudina

    2014-01-01

    Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh. PMID:25055974

  4. Regioselectivity of H Cluster Oxidation

    PubMed Central

    2011-01-01

    The H2-evolving potential of [FeFe] hydrogenases is severely limited by the oxygen sensitivity of this class of enzymes. Recent experimental studies on hydrogenase from C. reinhardtii point to O2-induced structural changes in the [Fe4S4] subsite of the H cluster. Here, we investigate the mechanistic basis of this observation by means of density functional theory. Unexpectedly, we find that the isolated H cluster shows a pathological catalytic activity for the formation of reactive oxygen species such as O2– and HO2–. After protonation of O2–, an OOH radical may coordinate to the Fe atoms of the cubane, whereas H2O2 specifically reacts with the S atoms of the cubane-coordinating cysteine residues. Both pathways are accompanied by significant structural distortions that compromise cluster integrity and thus catalytic activity. These results explain the experimental observation that O2-induced inhibition is accompanied by distortions of the [Fe4S4] moiety and account for the irreversibility of this process. PMID:22106822

  5. A Synthetic Nickel Electrocatalyst With a Turnover Frequency Above 100,000 s-1 for H2 Production

    SciTech Connect

    Helm, Monte L.; Stewart, Michael P.; Bullock, R. Morris; Rakowski DuBois, Mary; DuBois, Daniel L.

    2011-08-12

    Increased worldwide energy demand will require greater use of carbon-neutral sustainable energy sources. The intermittent nature of solar and wind power requires storage of energy, so electrocatalysts that convert electrical energy to chemical bonds in fuels are needed. Platinum is an excellent catalyst, but it is of low abundance and high cost. Hydrogenase enzymes in Nature catalyze the evolution of H2 and use earth-abundant metals such as nickel and iron. We report that a synthetic nickel catalyst, [Ni(7PPh2NPh)2](BF4)2, (7PPh2NPh = 1,3,6-triphenyl-1-aza-3,6-diphosphacycloheptane) catalyzes the production of H2 using [(DMF)H]+OTf as the proton source, with turnover frequencies of 31,000 s-1 in dry acetonitrile and 108,000 s-1 in the presence of H2O (1.2 M), at a potential of -1.13 V (vs. the ferrocenium/ferrocene couple). These turnover frequencies exceed those reported for the [FeFe] hydrogenase enzyme by more than an order of magnitude, and are the fastest reported for any molecular catalyst for H2 production. This material is based upon work supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.

  6. Genome-Guided Analysis and Whole Transcriptome Profiling of the Mesophilic Syntrophic Acetate Oxidising Bacterium Syntrophaceticus schinkii

    PubMed Central

    Manzoor, Shahid; Bongcam-Rudloff, Erik; Schnürer, Anna; Müller, Bettina

    2016-01-01

    Syntrophaceticus schinkii is a mesophilic, anaerobic bacterium capable of oxidising acetate to CO2 and H2 in intimate association with a methanogenic partner, a syntrophic relationship which operates close to the energetic limits of microbial life. Syntrophaceticus schinkii has been identified as a key organism in engineered methane-producing processes relying on syntrophic acetate oxidation as the main methane-producing pathway. However, due to strict cultivation requirements and difficulties in reconstituting the thermodynamically unfavourable acetate oxidation, the physiology of this functional group is poorly understood. Genome-guided and whole transcriptome analyses performed in the present study provide new insights into habitat adaptation, syntrophic acetate oxidation and energy conservation. The working draft genome of Syntrophaceticus schinkii indicates limited metabolic capacities, with lack of organic nutrient uptake systems, chemotactic machineries, carbon catabolite repression and incomplete biosynthesis pathways. Ech hydrogenase, [FeFe] hydrogenases, [NiFe] hydrogenases, F1F0-ATP synthase and membrane-bound and cytoplasmic formate dehydrogenases were found clearly expressed, whereas Rnf and a predicted oxidoreductase/heterodisulphide reductase complex, both found encoded in the genome, were not expressed under syntrophic growth condition. A transporter sharing similarities to the high-affinity acetate transporters of aceticlastic methanogens was also found expressed, suggesting that Syntrophaceticus schinkii can potentially compete with methanogens for acetate. Acetate oxidation seems to proceed via the Wood-Ljungdahl pathway as all genes involved in this pathway were highly expressed. This study shows that Syntrophaceticus schinkii is a highly specialised, habitat-adapted organism relying on syntrophic acetate oxidation rather than metabolic versatility. By expanding its complement of respiratory complexes, it might overcome limiting bioenergetic

  7. Metabolic network reconstruction and genome-scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052

    PubMed Central

    2011-01-01

    hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed. Conclusions Microbial production of butanol by C. beijerinckii offers a promising, sustainable, method for generation of this important chemical and potential biofuel. iCM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production. PMID:21846360

  8. Experimental partitioning studies near the Fe-FeS eutectic, with an emphasis on elements important to iron meteorite chronologies (Pb, Ag, Pd, and Tl)

    NASA Astrophysics Data System (ADS)

    Jones, J. H.; Hart, S. R.; Benjamin, T. M.

    1993-01-01

    Partitioning coefficients for metal/sulfide liquid, troilite/sulfide liquid, and schreibersite/sulfide liquid were determined for Ag, Au, Mo, Ni, Pd, and Tl (using EMPA and proton-induced X-ray microprobe and ion microprobe analyses) in order to understand the chronometer systems of iron meteorites. In general, the obtained schreibersite/metal and troilite/metal partition coefficients for 'compatible' elements were quite similar to those inferred from natural assemblages, reinforcing an earlier made conclusion that there is a class of elements for which experimental troilite/metal and schreibersite/metal partition coefficients approximate those inferred from natural samples. The consistency between experimental and natural assemblages, however, was not observed for Ag, Pb, and Tl, indicating that the abundances of these elements determined in 'metal' and 'troilite' separates from iron meteorites are influenced by trace minerals that concentrate incompatible elements.

  9. Comprehensive structural and chemical (CO2, Fe/Fe Mg, H2O) investigations of Mg-Fe cordierite with micro Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Haefeker, U.; Kaindl, R.; Tropper, P.

    2012-04-01

    The Mg-Fe silicate cordierite with the idealized formula (Fe, Mg)2Al4Si5O18 occurs as a hexagonal and an orthorhombic polymorph with disordered/ordered Al-Si distribution on the tetrahedral sites. Most of the natural cordierites are fully ordered. Six-membered rings of (Si,Al)O4 are piled in the direction of the crystallographic c-axis and form channels, laterally and vertically linked by additional (Al, Si) tetrahedrons. Mg and Fe in varying fractions occupy the octahedrally coordinated M-sites. CO2 and H2O (and other volatiles) can be incorporated into the structural channels, thus cordierite can be used for paleofluid reconstruction. The vibration energies of incorporated volatiles, their interaction with the lattice and variations of certain lattice-vibration energies caused by the Mg-Fe exchange can be determined with Raman spectroscopy, allowing chemical quantifications and structural investigations. A method for the semi-quantitative determination of CO2-contents of natural cordierites by Kaindl et al. (2006) was optimized and enhanced by Haefeker et al. (2007). CO2 contents can be measured in single crystals and thin sections with an error of ± 0.05 - 0.09 wt.-%. Based on the Mg-Fe exchange with garnet, cordierite can be used as a geothermobarometer. Recent investigations of synthetic Mg-Fe cordierites with XFe = 0 - 1 have shown a linear downshift of six selected lattice peaks between 100 and 1250 cm-1 with the Mg-Fe contents. Correlation diagrams allow an estimation of the Mg-Fe contents in synthetic and natural samples. The experimental data are in good agreement with the results of quantum-mechanical calculations of the Raman spectra of Mg- and Fe cordierite (Kaindl et al., 2011) allowing the assignment of the peaks to specific vibrations of tetrahedral and octahedral sites. Natural Mg-Fe cordierites are mainly orthorhombic with a fully ordered Al/Si distribution on the tetrahedral sites. However, the disordered hexagonal polymorph is observed in many experiments. Raman spectroscopy allows easy distinguishing between the two polymorphs by the splitting of a characteristic peak at ~569 cm-1. Crystallographic and Raman spectroscopic data of the Fe endmember polymorphs are rare in literature, therefore, Raman and single-crystal x-ray data of synthetic samples were collected and compared with the well-known Mg and Mg-Fe cordierites. First compositional Raman maps show a relation between the degree of ordering of Fe cordierite and the amount of water incorporated into the channels. The effects of water incorporation on the Raman spectra of Mg cordierites is currently being evaluated. Preliminary investigations indicate a downshift of the peak at ~1186 cm-1with increasing water contents. Literature: Kaindl, R., Tropper P., Deibl, I. (2006) A semi-quantitative technique for determination of CO2in cordierite by Raman spectroscopy in thin sections. Eur. J. Mineral, 18, 331-335 Haefeker, U. (2007) Verbesserte semiquantitative Analyse von CO2 in natürlichem Cordierit mit Hilfe der Mikro-Raman-Spektroskopie. Unpublished master thesis. University of Innsbruck, 86p Kaindl, R., Többens, D. M., Haefeker, U. (2011) Quantum-mechanical calculations of the Raman spectra of Mg- and Fe-cordierite. American Mineralogist, 96, 1568-1574

  10. Models and role models.

    PubMed

    ten Cate, Jacob M

    2015-01-01

    Developing experimental models to understand dental caries has been the theme in our research group. Our first, the pH-cycling model, was developed to investigate the chemical reactions in enamel or dentine, which lead to dental caries. It aimed to leverage our understanding of the fluoride mode of action and was also utilized for the formulation of oral care products. In addition, we made use of intra-oral (in situ) models to study other features of the oral environment that drive the de/remineralization balance in individual patients. This model addressed basic questions, such as how enamel and dentine are affected by challenges in the oral cavity, as well as practical issues related to fluoride toothpaste efficacy. The observation that perhaps fluoride is not sufficiently potent to reduce dental caries in the present-day society triggered us to expand our knowledge in the bacterial aetiology of dental caries. For this we developed the Amsterdam Active Attachment biofilm model. Different from studies on planktonic ('single') bacteria, this biofilm model captures bacteria in a habitat similar to dental plaque. With data from the combination of these models, it should be possible to study separate processes which together may lead to dental caries. Also products and novel agents could be evaluated that interfere with either of the processes. Having these separate models in place, a suggestion is made to design computer models to encompass the available information. Models but also role models are of the utmost importance in bringing and guiding research and researchers.

  11. Reengineering cyt b562 for hydrogen production: A facile route to artificial hydrogenases.

    PubMed

    Sommer, Dayn Joseph; Vaughn, Michael David; Clark, Brett Colby; Tomlin, John; Roy, Anindya; Ghirlanda, Giovanna

    2016-05-01

    Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. 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.

  12. Mechanism of Hydrogen Production in [Fe-Fe]-Hydrogenase: A Density Functional Theory Study (Preprint)

    DTIC Science & Technology

    2007-03-01

    SCHUMACHER, Chief Survivability and Sensor Materials Division This report is published in the interest of scientific and...Journal Article Preprint 5a. CONTRACT NUMBER In-house 5b. GRANT NUMBER 4. TITLE AND SUBTITLE MECHANISM OF HYDROGEN PRODUCTION IN [Fe-Fe...distribution unlimited. 13. SUPPLEMENTARY NOTES Journal article submitted to Energy and Fuels. The U.S. Government is joint author of this work

  13. Mechanism of Hydrogen Production in [Fe-Fe]-Hydrogenase: A Density Functional Theory Study (Postprint)

    DTIC Science & Technology

    2007-03-01

    scientific and technical information exchange, and its publication does not constitute the Government’s approval or disapproval of its ideas or...March 2007 Journal Article Postprint 5a. CONTRACT NUMBER In-house 5b. GRANT NUMBER 4. TITLE AND SUBTITLE MECHANISM OF HYDROGEN PRODUCTION IN...public release; distribution unlimited. 13. SUPPLEMENTARY NOTES Journal article published in Energy & Fuels (2007) Vol. 21. © 2007 American

  14. Cobalt complexes as artificial hydrogenases for the reductive side of water splitting.

    PubMed

    Eckenhoff, William T; McNamara, William R; Du, Pingwu; Eisenberg, Richard

    2013-01-01

    The generation of H2 from protons and electrons by complexes of cobalt has an extensive history. During the past decade, interest in this subject has increased as a result of developments in hydrogen generation that are driven electrochemically or photochemically. This article reviews the subject of hydrogen generation using Co complexes as catalysts and discusses the mechanistic implications of the systems studied for making H2. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

  15. Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus

    SciTech Connect

    Wawrousek, Karen; Noble, Scott; Korlach, Jonas; Eckert, Carrie; Yu, Jianping; Maness, Pin -Ching

    2014-12-05

    In this article, we report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO2 and H2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H2 in support of cell growth, and a regulatory hydrogenase that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Finally and collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth.

  16. Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus

    DOE PAGES

    Wawrousek, Karen; Noble, Scott; Korlach, Jonas; ...

    2014-12-05

    In this article, we report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO2 and H2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzymemore » complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H2 in support of cell growth, and a regulatory hydrogenase that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Finally and collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth.« less

  17. Models, Fiction, and Fictional Models

    NASA Astrophysics Data System (ADS)

    Liu, Chuang

    2014-03-01

    The following sections are included: * Introduction * Why Most Models in Science Are Not Fictional * Typically Fictional Models in Science * Modeling the Unobservable * Fictional Models for the Unobservable? * References

  18. Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes

    PubMed Central

    2015-01-01

    Conspectus A [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5′-deoxyadenosyl radical (5′-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5′-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5′-dA• to the product radical l-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set

  19. Mental Models, Conceptual Models, and Modelling.

    ERIC Educational Resources Information Center

    Greca, Ileana Maria; Moreira, Marco Antonio

    2000-01-01

    Reviews science education research into representations constructed by students in their interactions with the world, its phenomena, and artefacts. Features discussions of mental models, conceptual models, and the activity of modeling. (Contains 30 references.) (Author/WRM)

  20. Hydrogen Metabolism in Helicobacter pylori Plays a Role in Gastric Carcinogenesis through Facilitating CagA Translocation

    PubMed Central

    Wang, Ge; Romero-Gallo, Judith; Benoit, Stéphane L.; Piazuelo, M. Blanca; Dominguez, Ricardo L.; Morgan, Douglas R.; Peek, Richard M.

    2016-01-01

    ABSTRACT A known virulence factor of Helicobacter pylori that augments gastric cancer risk is the CagA cytotoxin. A carcinogenic derivative strain, 7.13, that has a greater ability to translocate CagA exhibits much higher hydrogenase activity than its parent noncarcinogenic strain, B128. A Δhyd mutant strain with deletion of hydrogenase genes was ineffective in CagA translocation into human gastric epithelial AGS cells, while no significant attenuation of cell adhesion was observed. The quinone reductase inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) was used to specifically inhibit the H2-utilizing respiratory chain of outer membrane-permeabilized bacterial cells; that level of inhibitor also greatly attenuated CagA translocation into AGS cells, indicating the H2-generated transmembrane potential is a contributor to toxin translocation. The Δhyd strain showed a decreased frequency of DNA transformation, suggesting that H. pylori hydrogenase is also involved in energizing the DNA uptake apparatus. In a gerbil model of infection, the ability of the Δhyd strain to induce inflammation was significantly attenuated (at 12 weeks postinoculation), while all of the gerbils infected with the parent strain (7.13) exhibited a high level of inflammation. Gastric cancer developed in 50% of gerbils infected with the wild-type strain 7.13 but in none of the animals infected with the Δhyd strain. By examining the hydrogenase activities from well-defined clinical H. pylori isolates, we observed that strains isolated from cancer patients (n = 6) have a significantly higher hydrogenase (H2/O2) activity than the strains isolated from gastritis patients (n = 6), further supporting an association between H. pylori hydrogenase activity and gastric carcinogenesis in humans. PMID:27531909

  1. Formate-derived H2 , a driver of hydrogenotrophic processes in the root-zone of a methane-emitting fen.

    PubMed

    Hunger, Sindy; Schmidt, Oliver; Gößner, Anita S; Drake, Harold L

    2016-09-01

    Wetlands are important sources of globally emitted methane. Plants mediate much of that emission by releasing root-derived organic carbon, including formate, a direct precursor of methane. Thus, the objective of this study was to resolve formate-driven processes potentially linked to methanogenesis in the fen root-zone. Although, formate was anticipated to directly trigger methanogenesis, the rapid anaerobic consumption of formate by Carex roots unexpectedly yielded H2 and CO2 via enzymes such as formate-H2 -lyase (FHL), and likewise appeared to enhance the utilization of organic carbon. Collectively, 57 [FeFe]- and [NiFe]-hydrogenase-containing family level phylotypes potentially linked to FHL activity were detected. Under anoxic conditions, root-derived fermentative Citrobacter and Hafnia isolates produced H2 from formate via FHL. Formate-derived H2 fueled methanogenesis and acetogenesis, and methanogenic (Methanoregula, Methanobacterium, Methanocella) and acetogenic (Acetonema, Clostridum, Sporomusa) genera potentially linked to these hydrogenotrophic activities were identified. The findings (i) provide novel insights on highly diverse root-associated FHL-containing taxa that can augment secondary hydrogenotrophic processes via the production of formate-derived H2 , (ii) demonstrate that formate can have a 'priming' effect on the utilization of organic carbon, and (iii) raise questions regarding the fate of formate-derived H2 when it diffuses away from the root-zone.

  2. Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes.

    PubMed

    Stechmann, Alexandra; Hamblin, Karleigh; Pérez-Brocal, Vicente; Gaston, Daniel; Richmond, Gregory S; van der Giezen, Mark; Clark, C Graham; Roger, Andrew J

    2008-04-22

    Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.

  3. A continuous system for biocatalytic hydrogenation of CO2 to formate.

    PubMed

    Mourato, Cláudia; Martins, Mónica; da Silva, Sofia M; Pereira, Inês A C

    2017-03-20

    In this work a novel bioprocess for hydrogenation of CO2 to formate was developed, using whole cell catalysis by a sulfate-reducing bacterium. Three Desulfovibrio species were tested (D. vulgaris Hildenborough, D. alaskensis G20, and D. desulfuricans ATCC 27774), of which D. desulfuricans showed the highest activity, producing 12mM of formate in batch, with a production rate of 0.09mMh(-1). Gene expression analysis indicated that among the three formate dehydrogenases and five hydrogenases, the cytoplasmic FdhAB and the periplasmic [FeFe] HydAB are the main enzymes expressed in D. desulfuricans in these conditions. The new bioprocess for continuous formate production by D. desulfuricans had a maximum specific formate production rate of 14mMgdcw(-1)h(-1), and more than 45mM of formate were obtained with a production rate of 0.40mMh(-1). This is the first report of a continuous process for biocatalytic formate production.

  4. Genome-Guided Analysis of Physiological Capacities of Tepidanaerobacter acetatoxydans Provides Insights into Environmental Adaptations and Syntrophic Acetate Oxidation

    PubMed Central

    Niazi, Adnan; Bongcam-Rudloff, Erik; Schnürer, Anna

    2015-01-01

    This paper describes the genome-based analysis of Tepidanaerobacter acetatoxydans strain Re1, a syntrophic acetate-oxidising bacterium (SAOB). Principal issues such as environmental adaptations, metabolic capacities, and energy conserving systems have been investigated and the potential consequences for syntrophic acetate oxidation discussed. Briefly, in pure culture, T. acetatoxydans grows with different organic compounds and produces acetate as the main product. In a syntrophic consortium with a hydrogenotrophic methanogen, it can also reverse its metabolism and instead convert acetate to formate/H2 and CO2. It can only proceed if the product formed is continuously removed. This process generates a very small amount of energy that is scarcely enough for growth, which makes this particular syntrophy of special interest. As a crucial member of the biogas-producing community in ammonium-rich engineered AD processes, genomic features conferring ammonium resistance, bacterial defense, oxygen and temperature tolerance were found, as well as attributes related to biofilm formation and flocculation. It is likely that T. acetatoxydans can form an electrochemical gradient by putative electron-bifurcating Rnf complex and [Fe-Fe] hydrogenases, as observed in other acetogens. However, genomic deficiencies related to acetogenic metabolism and anaerobic respiration were discovered, such as the lack of formate dehydrogenase and F1F0 ATP synthase. This has potential consequences for the metabolic pathways used under SAO and non-SAO conditions. The two complete sets of bacteriophage genomes, which were found to be encoded in the genome, are also worthy of mention. PMID:25811859

  5. Supermatrix models

    SciTech Connect

    Yost, S.A.

    1991-05-01

    Radom matrix models based on an integral over supermatrices are proposed as a natural extension of bosonic matrix models. The subtle nature of superspace integration allows these models to have very different properties from the analogous bosonic models. Two choices of integration slice are investigated. One leads to a perturbative structure which is reminiscent of, and perhaps identical to, the usual Hermitian matrix models. Another leads to an eigenvalue reduction which can be described by a two component plasma in one dimension. A stationary point of the model is described.

  6. ENTRAINMENT MODELS

    EPA Science Inventory

    This presentation presented information on entrainment models. Entrainment models use entrainment hypotheses to express the continuity equation. The advantage is that plume boundaries are known. A major disadvantage is that the problems that can be solved are rather simple. The ...

  7. Radiation Models

    ERIC Educational Resources Information Center

    James, W. G. G.

    1970-01-01

    Discusses the historical development of both the wave and the corpuscular photon model of light. Suggests that students should be informed that the two models are complementary and that each model successfully describes a wide range of radiation phenomena. Cites 19 references which might be of interest to physics teachers and students. (LC)

  8. Cover Image, Volume 114, Number 2, February 2017.

    PubMed

    Wecker, Matt S A; Beaton, Stephen E; Chado, Robert A; Ghirardi, Maria L

    2017-02-01

    The cover image, by Maria L. Ghirardi et al., is based on the Article Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production, DOI: 10.1002/bit.26076.

  9. Synthesis, Purification, and Characterization of a [mu]-(1,3-Propanedithiolato)-Hexacarbonyldiiron

    ERIC Educational Resources Information Center

    Works, Carmen F.

    2007-01-01

    A project which exposes students to biologically important transition-metal chemistry is illustrated by taking an example of the iron-carbonyl compound, [mu]-(1,3-Propanedithiolaro)-hexa-carbonyldiiron as a structural model for an iron-only hydro-genase. The project provides the students with experience of Schlenk line techniques, purification,…

  10. Hydrological models are mediating models

    NASA Astrophysics Data System (ADS)

    Babel, L. V.; Karssenberg, D.

    2013-08-01

    Despite the increasing role of models in hydrological research and decision-making processes, only few accounts of the nature and function of models exist in hydrology. Earlier considerations have traditionally been conducted while making a clear distinction between physically-based and conceptual models. A new philosophical account, primarily based on the fields of physics and economics, transcends classes of models and scientific disciplines by considering models as "mediators" between theory and observations. The core of this approach lies in identifying models as (1) being only partially dependent on theory and observations, (2) integrating non-deductive elements in their construction, and (3) carrying the role of instruments of scientific enquiry about both theory and the world. The applicability of this approach to hydrology is evaluated in the present article. Three widely used hydrological models, each showing a different degree of apparent physicality, are confronted to the main characteristics of the "mediating models" concept. We argue that irrespective of their kind, hydrological models depend on both theory and observations, rather than merely on one of these two domains. Their construction is additionally involving a large number of miscellaneous, external ingredients, such as past experiences, model objectives, knowledge and preferences of the modeller, as well as hardware and software resources. We show that hydrological models convey the role of instruments in scientific practice by mediating between theory and the world. It results from these considerations that the traditional distinction between physically-based and conceptual models is necessarily too simplistic and refers at best to the stage at which theory and observations are steering model construction. The large variety of ingredients involved in model construction would deserve closer attention, for being rarely explicitly presented in peer-reviewed literature. We believe that devoting

  11. Model Experiments and Model Descriptions

    NASA Technical Reports Server (NTRS)

    Jackman, Charles H.; Ko, Malcolm K. W.; Weisenstein, Debra; Scott, Courtney J.; Shia, Run-Lie; Rodriguez, Jose; Sze, N. D.; Vohralik, Peter; Randeniya, Lakshman; Plumb, Ian

    1999-01-01

    The Second Workshop on Stratospheric Models and Measurements Workshop (M&M II) is the continuation of the effort previously started in the first Workshop (M&M I, Prather and Remsberg [1993]) held in 1992. As originally stated, the aim of M&M is to provide a foundation for establishing the credibility of stratospheric models used in environmental assessments of the ozone response to chlorofluorocarbons, aircraft emissions, and other climate-chemistry interactions. To accomplish this, a set of measurements of the present day atmosphere was selected. The intent was that successful simulations of the set of measurements should become the prerequisite for the acceptance of these models as having a reliable prediction for future ozone behavior. This section is divided into two: model experiment and model descriptions. In the model experiment, participant were given the charge to design a number of experiments that would use observations to test whether models are using the correct mechanisms to simulate the distributions of ozone and other trace gases in the atmosphere. The purpose is closely tied to the needs to reduce the uncertainties in the model predicted responses of stratospheric ozone to perturbations. The specifications for the experiments were sent out to the modeling community in June 1997. Twenty eight modeling groups responded to the requests for input. The first part of this section discusses the different modeling group, along with the experiments performed. Part two of this section, gives brief descriptions of each model as provided by the individual modeling groups.

  12. Climate Models

    NASA Technical Reports Server (NTRS)

    Druyan, Leonard M.

    2012-01-01

    Climate models is a very broad topic, so a single volume can only offer a small sampling of relevant research activities. This volume of 14 chapters includes descriptions of a variety of modeling studies for a variety of geographic regions by an international roster of authors. The climate research community generally uses the rubric climate models to refer to organized sets of computer instructions that produce simulations of climate evolution. The code is based on physical relationships that describe the shared variability of meteorological parameters such as temperature, humidity, precipitation rate, circulation, radiation fluxes, etc. Three-dimensional climate models are integrated over time in order to compute the temporal and spatial variations of these parameters. Model domains can be global or regional and the horizontal and vertical resolutions of the computational grid vary from model to model. Considering the entire climate system requires accounting for interactions between solar insolation, atmospheric, oceanic and continental processes, the latter including land hydrology and vegetation. Model simulations may concentrate on one or more of these components, but the most sophisticated models will estimate the mutual interactions of all of these environments. Advances in computer technology have prompted investments in more complex model configurations that consider more phenomena interactions than were possible with yesterday s computers. However, not every attempt to add to the computational layers is rewarded by better model performance. Extensive research is required to test and document any advantages gained by greater sophistication in model formulation. One purpose for publishing climate model research results is to present purported advances for evaluation by the scientific community.

  13. Phenomenological models

    SciTech Connect

    Braby, L.A.

    1990-09-01

    The biological effects of ionizing radiation exposure are the result of a complex sequence of physical, chemical, biochemical, and physiological interactions. One way to begin a search for an understanding of health effects of radiation is through the development of phenomenological models of the response. Many models have been presented and tested in the slowly evolving process of characterizing cellular response. A range of models covering different endpoints and phenomena has developed in parallel. Many of these models employ similar assumptions about some underlying processes while differing about the nature of others. An attempt is made to organize many of the models into groups with similar features and to compare the consequences of those features with the actual experimental observations. It is assumed that by showing that some assumptions are inconsistent with experimental observations, the job of devising and testing mechanistic models can be simplified. 43 refs., 13 figs.

  14. Cloud Modeling

    NASA Technical Reports Server (NTRS)

    Tao, Wei-Kuo; Moncrieff, Mitchell; Einaud, Franco (Technical Monitor)

    2001-01-01

    Numerical cloud models have been developed and applied extensively to study cloud-scale and mesoscale processes during the past four decades. The distinctive aspect of these cloud models is their ability to treat explicitly (or resolve) cloud-scale dynamics. This requires the cloud models to be formulated from the non-hydrostatic equations of motion that explicitly include the vertical acceleration terms since the vertical and horizontal scales of convection are similar. Such models are also necessary in order to allow gravity waves, such as those triggered by clouds, to be resolved explicitly. In contrast, the hydrostatic approximation, usually applied in global or regional models, does allow the presence of gravity waves. In addition, the availability of exponentially increasing computer capabilities has resulted in time integrations increasing from hours to days, domain grids boxes (points) increasing from less than 2000 to more than 2,500,000 grid points with 500 to 1000 m resolution, and 3-D models becoming increasingly prevalent. The cloud resolving model is now at a stage where it can provide reasonably accurate statistical information of the sub-grid, cloud-resolving processes poorly parameterized in climate models and numerical prediction models.

  15. Ventilation Model

    SciTech Connect

    V. Chipman

    2002-10-05

    The purpose of the Ventilation Model is to simulate the heat transfer processes in and around waste emplacement drifts during periods of forced ventilation. The model evaluates the effects of emplacement drift ventilation on the thermal conditions in the emplacement drifts and surrounding rock mass, and calculates the heat removal by ventilation as a measure of the viability of ventilation to delay the onset of peak repository temperature and reduce its magnitude. The heat removal by ventilation is temporally and spatially dependent, and is expressed as the fraction of heat carried away by the ventilation air compared to the fraction of heat produced by radionuclide decay. One minus the heat removal is called the wall heat fraction, or the remaining amount of heat that is transferred via conduction to the surrounding rock mass. Downstream models, such as the ''Multiscale Thermohydrologic Model'' (BSC 2001), use the wall heat fractions as outputted from the Ventilation Model to initialize their post-closure analyses. The Ventilation Model report was initially developed to analyze the effects of preclosure continuous ventilation in the Engineered Barrier System (EBS) emplacement drifts, and to provide heat removal data to support EBS design. Revision 00 of the Ventilation Model included documentation of the modeling results from the ANSYS-based heat transfer model. The purposes of Revision 01 of the Ventilation Model are: (1) To validate the conceptual model for preclosure ventilation of emplacement drifts and verify its numerical application in accordance with new procedural requirements as outlined in AP-SIII-10Q, Models (Section 7.0). (2) To satisfy technical issues posed in KTI agreement RDTME 3.14 (Reamer and Williams 2001a). Specifically to demonstrate, with respect to the ANSYS ventilation model, the adequacy of the discretization (Section 6.2.3.1), and the downstream applicability of the model results (i.e. wall heat fractions) to initialize post

  16. Model Selection for Geostatistical Models

    SciTech Connect

    Hoeting, Jennifer A.; Davis, Richard A.; Merton, Andrew A.; Thompson, Sandra E.

    2006-02-01

    We consider the problem of model selection for geospatial data. Spatial correlation is typically ignored in the selection of explanatory variables and this can influence model selection results. For example, the inclusion or exclusion of particular explanatory variables may not be apparent when spatial correlation is ignored. To address this problem, we consider the Akaike Information Criterion (AIC) as applied to a geostatistical model. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical model is superior to the often used approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a model for lizard abundance. We also employ the principle of minimum description length (MDL) to variable selection for the geostatistical model. The effect of sampling design on the selection of explanatory covariates is also explored.

  17. Modeling Sunspots

    ERIC Educational Resources Information Center

    Oh, Phil Seok; Oh, Sung Jin

    2013-01-01

    Modeling in science has been studied by education researchers for decades and is now being applied broadly in school. It is among the scientific practices featured in the "Next Generation Science Standards" ("NGSS") (Achieve Inc. 2013). This article describes modeling activities in an extracurricular science club in a high…

  18. Dispersion Modeling.

    ERIC Educational Resources Information Center

    Budiansky, Stephen

    1980-01-01

    This article discusses the need for more accurate and complete input data and field verification of the various models of air pollutant dispension. Consideration should be given to changing the form of air quality standards based on enhanced dispersion modeling techniques. (Author/RE)

  19. Qualitative modeling.

    PubMed

    Forbus, Kenneth D

    2011-07-01

    Qualitative modeling concerns the representations and reasoning that people use to understand continuous aspects of the world. Qualitative models formalize everyday notions of causality and provide accounts of how to ground symbolic, relational representations in perceptual processes. This article surveys the basic ideas of qualitative modeling and their applications from a cognitive science perspective. It describes the basic principles of qualitative modeling, and a variety of qualitative representations that have been developed for quantities and for relationships between them, providing a kind of qualitative mathematics. Three ontological frameworks for organizing modeling knowledge (processes, components, and field) are summarized, along with research on automatically assembling models for particular tasks from such knowledge. Qualitative simulation and how it carves up time into meaningful units is discussed. We discuss several accounts of causal reasoning about dynamical systems, based on different choices of qualitative mathematics and ontology. Qualitative spatial reasoning is explored, both in terms of relational systems and visual reasoning. Applications of qualitative models of particular interest to cognitive scientists are described, including how they have been used to capture the expertise of scientists and engineers and how they have been used in education. Open questions and frontiers are also discussed, focusing on relationships between ideas developed in the qualitative modeling community and other areas of cognitive science. WIREs Cogni Sci 2011 2 374-391 DOI: 10.1002/wcs.115 For further resources related to this article, please visit the WIREs website.

  20. Turbulence modeling

    NASA Technical Reports Server (NTRS)

    Bardina, Jorge E.

    1995-01-01

    The objective of this work is to develop, verify, and incorporate the baseline two-equation turbulence models which account for the effects of compressibility into the three-dimensional Reynolds averaged Navier-Stokes (RANS) code and to provide documented descriptions of the models and their numerical procedures so that they can be implemented into 3-D CFD codes for engineering applications.

  1. Climate models and model evaluation

    SciTech Connect

    Gates, W.L.

    1994-12-31

    This brief overview addresses aspects of the nature, uses, evaluation and limitations of climate models. A comprehensive global modeling capability has been achieved only for the physical climate system, which is characterized by processes that serve to transport and exchange momentum, heat and moisture within and between the atmosphere, ocean and land surface. The fundamental aim of climate modeling, and the justification for the use of climate models, is the need to achieve a quantitative understanding of the operation of the climate system and to exploit any potential predictability that may exist.

  2. OSPREY Model

    SciTech Connect

    Veronica J. Rutledge

    2013-01-01

    The absence of industrial scale nuclear fuel reprocessing in the U.S. has precluded the necessary driver for developing the advanced simulation capability now prevalent in so many other countries. Thus, it is essential to model complex series of unit operations to simulate, understand, and predict inherent transient behavior and feedback loops. A capability of accurately simulating the dynamic behavior of advanced fuel cycle separation processes will provide substantial cost savings and many technical benefits. The specific fuel cycle separation process discussed in this report is the off-gas treatment system. The off-gas separation consists of a series of scrubbers and adsorption beds to capture constituents of interest. Dynamic models are being developed to simulate each unit operation involved so each unit operation can be used as a stand-alone model and in series with multiple others. Currently, an adsorption model has been developed within Multi-physics Object Oriented Simulation Environment (MOOSE) developed at the Idaho National Laboratory (INL). Off-gas Separation and REcoverY (OSPREY) models the adsorption of off-gas constituents for dispersed plug flow in a packed bed under non-isothermal and non-isobaric conditions. Inputs to the model include gas, sorbent, and column properties, equilibrium and kinetic data, and inlet conditions. The simulation outputs component concentrations along the column length as a function of time from which breakthrough data is obtained. The breakthrough data can be used to determine bed capacity, which in turn can be used to size columns. It also outputs temperature along the column length as a function of time and pressure drop along the column length. Experimental data and parameters were input into the adsorption model to develop models specific for krypton adsorption. The same can be done for iodine, xenon, and tritium. The model will be validated with experimental breakthrough curves. Customers will be given access to

  3. Model hydrographs

    USGS Publications Warehouse

    Mitchell, W.D.

    1972-01-01

    Model hydrographs are composed of pairs of dimensionless ratios, arrayed in tabular form, which, when modified by the appropriate values of rainfall exceed and by the time and areal characteristics of the drainage basin, satisfactorily represent the flood hydrograph for the basin. Model bydrographs are developed from a dimensionless translation hydrograph, having a time base of T hours and appropriately modified for storm duration by routing through reservoir storage, S=kOx. Models fall into two distinct classes: (1) those for which the value of x is unity and which have all the characteristics of true unit hydrographs and (2) those for which the value of x is other than unity and to which the unit-hydrograph principles of proportionality and superposition do not apply. Twenty-six families of linear models and eight families of nonlinear models in tabular form from the principal subject of this report. Supplemental discussions describe the development of the models and illustrate their application. Other sections of the report, supplemental to the tables, describe methods of determining the hydrograph characteristics, T, k, and x, both from observed hydrograph and from the physical characteristics of the drainage basin. Five illustrative examples of use show that the models, when properly converted to incorporate actual rainfall excess and the time and areal characteristics of the drainage basins, do indeed satisfactorily represent the observed flood hydrographs for the basins.

  4. Stereometric Modelling

    NASA Astrophysics Data System (ADS)

    Grimaldi, P.

    2012-07-01

    These mandatory guidelines are provided for preparation of papers accepted for publication in the series of Volumes of The The stereometric modelling means modelling achieved with : - the use of a pair of virtual cameras, with parallel axes and positioned at a mutual distance average of 1/10 of the distance camera-object (in practice the realization and use of a stereometric camera in the modeling program); - the shot visualization in two distinct windows - the stereoscopic viewing of the shot while modelling. Since the definition of "3D vision" is inaccurately referred to as the simple perspective of an object, it is required to add the word stereo so that "3D stereo vision " shall stand for "three-dimensional view" and ,therefore, measure the width, height and depth of the surveyed image. Thanks to the development of a stereo metric model , either real or virtual, through the "materialization", either real or virtual, of the optical-stereo metric model made visible with a stereoscope. It is feasible a continuous on line updating of the cultural heritage with the help of photogrammetry and stereometric modelling. The catalogue of the Architectonic Photogrammetry Laboratory of Politecnico di Bari is available on line at: http://rappresentazione.stereofot.it:591/StereoFot/FMPro?-db=StereoFot.fp5&-lay=Scheda&-format=cerca.htm&-view

  5. A Model for Math Modeling

    ERIC Educational Resources Information Center

    Lin, Tony; Erfan, Sasan

    2016-01-01

    Mathematical modeling is an open-ended research subject where no definite answers exist for any problem. Math modeling enables thinking outside the box to connect different fields of studies together including statistics, algebra, calculus, matrices, programming and scientific writing. As an integral part of society, it is the foundation for many…

  6. Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium.

    PubMed

    Phillips, D A; Kapulnik, Y; Bedmar, E J; Joseph, C M

    1990-04-01

    Some Rhizobium bacteria have H(2)-uptake (Hup) systems that oxidize H(2) evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars ;JI1205' and ;Alaska' produce high Hup (Hup(++)) and moderate Hup (Hup(+)) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup(++) and Hup(+) phenotypes in R. leguminosarum 128C53 were identified in 173 F(2)-derived F(6) families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P

  7. Sodium Ions Affect the pH behavior of the Soluble Hydrogenase of Alcaligenes Eutrophus H16

    DTIC Science & Technology

    1993-01-01

    were relatively ineffective as inhibitors. Similar results were observed with MgCI2, 5 CaC2, and BaCl2 , possibly suggesting a common binding site on...Inhibition at pH 8.5 at pH 7.0 100 mM LiCI 50.2 4.7 300 mM KCI 50.1 4.4 30 mM MgCl2 43.8 0 30 mM CaCI2 54.6 3.4 40 mM BaCQ2 65.3 7.3 10 FIGURE LEGENDS

  8. Systems Level Approaches to Understanding and Manipulating Heterocyst Differentiation in Nostoc Punctiforme: Sites of Hydrogenase and Nitrogenase Synthesis and Activity

    SciTech Connect

    Meeks, John C.

    2015-04-02

    Heterocysts are specialized cells that establish a physiologically low oxygen concentration; they function as the sites of oxygen-sensitive nitrogen fixation and hydrogen metabolism in certain filamentous cyanobacteria. They are present at a frequency of less than 10% of the cells and singly in a nonrandom spacing pattern in the filaments. The extent of differential gene expression during heterocyst differentiation was defined by DNA microarray analysis in wild type and mutant cultures of Nostoc punctiforme. The results in wild-type cultures identified two groups of genes; approximately 440 that are unique to heterocyst formation and function, and 500 that respond positively and negatively to the transient stress of nitrogen starvation. Nitrogen fixation is initiated within 24 h after induction, but the cultures require another 24 h before growth is reinitiated. Microarray analyses were conducted on strains with altered expression of three genes that regulate the presence and spacing of heterocysts in the filaments; loss of function or over expression of these genes increases the heterocyst frequency 2 to 3 fold compared to the wild-type. Mutations in the genes hetR and hetF result in the inability to differentiate heterocysts, whereas over expression of each gene individually yields multiple contiguous heterocysts at sites in the filaments; they are positive regulatory elements. Mutation of the gene patN results in an increase in heterocysts frequency, but, in this case, the heterocysts are singly spaced in the filaments with a decrease in the number of vegetative cells in the interval between heterocysts; this is a negative regulatory element. However, over expression of patN resulted in the wild-type heterocyst frequency and spacing pattern. Microarray results indicated HetR and HetF influence the transcription of a common set of about 395 genes, as well as about 350 genes unique to each protein. HetR is known to be a transcriptional regulator and HetF is predicted to be a protease, perhaps operating thorough stability of HetR; thus, the influence of HetF on transcription of a unique set of genes was unanticipated. These two proteins are also found in non-heterocyst-forming filamentous cyanobacteria and the results have implications on their other physiological role(s). The PatN protein is unique to heterocyst-forming cyanobacteria. Cytological analysis indicated PatN is present in only one of the two daughter cells following division, but is present in both cell less than 8 h after division. Microarray analysis indicated only five genes were differentially transcribed in the patN mutant compared to the wild type; three up-regulated genes that are known to influence heterocyst differentiation and two down-regulated genes that have an unassigned function. Mutational analyses indicted the two down-regulated genes do not have a distinct role in heterocyst differentiation. Thus, PatN only indirectly impacts transcription. These databases provide lists of differentially transcribed genes involved in nitrogen starvation and cellular differentiation that can be mined for detailed genetic analysis of the regulation of heterocyst formation and function for subsequent photo-biohydrogen production.

  9. Anchor Modeling

    NASA Astrophysics Data System (ADS)

    Regardt, Olle; Rönnbäck, Lars; Bergholtz, Maria; Johannesson, Paul; Wohed, Petia

    Maintaining and evolving data warehouses is a complex, error prone, and time consuming activity. The main reason for this state of affairs is that the environment of a data warehouse is in constant change, while the warehouse itself needs to provide a stable and consistent interface to information spanning extended periods of time. In this paper, we propose a modeling technique for data warehousing, called anchor modeling, that offers non-destructive extensibility mechanisms, thereby enabling robust and flexible management of changes in source systems. A key benefit of anchor modeling is that changes in a data warehouse environment only require extensions, not modifications, to the data warehouse. This ensures that existing data warehouse applications will remain unaffected by the evolution of the data warehouse, i.e. existing views and functions will not have to be modified as a result of changes in the warehouse model.

  10. Programming models

    SciTech Connect

    Daniel, David J; Mc Pherson, Allen; Thorp, John R; Barrett, Richard; Clay, Robert; De Supinski, Bronis; Dube, Evi; Heroux, Mike; Janssen, Curtis; Langer, Steve; Laros, Jim

    2011-01-14

    A programming model is a set of software technologies that support the expression of algorithms and provide applications with an abstract representation of the capabilities of the underlying hardware architecture. The primary goals are productivity, portability and performance.

  11. Model Lungs.

    ERIC Educational Resources Information Center

    Taylor, Emma

    1991-01-01

    A cheap and simple model that can be made and used by pupils to study the human breathing mechanism is presented. A list of needed materials, procedures for construction, possible refinements, and method of use are included. (KR)

  12. Micromolecular modeling

    NASA Technical Reports Server (NTRS)

    Guillet, J. E.

    1984-01-01

    A reaction kinetics based model of the photodegradation process, which measures all important rate constants, and a computerized model capable of predicting the photodegradation rate and failure modes of a 30 year period, were developed. It is shown that the computerized photodegradation model for polyethylene correctly predicts failure of ELVAX 15 and cross linked ELVAX 150 on outdoor exposure. It is indicated that cross linking ethylene vinyl acetate (EVA) does not significantly change its degradation rate. It is shown that the effect of the stabilizer package is approximately equivalent on both polymers. The computerized model indicates that peroxide decomposers and UV absorbers are the most effective stabilizers. It is found that a combination of UV absorbers and a hindered amine light stabilizer (HALS) is the most effective stabilizer system.

  13. Environmental Modeling

    EPA Pesticide Factsheets

    EPA's modeling community is working to gain insights into certain parts of a physical, biological, economic, or social system by conducting environmental assessments for Agency decision making to complex environmental issues.

  14. Energy Models

    EPA Science Inventory

    Energy models characterize the energy system, its evolution, and its interactions with the broader economy. The energy system consists of primary resources, including both fossil fu