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1

Organometallic Complexes that Model the Active Sites of the [FeFe]- and [Fe]-Hydrogenases  

E-print Network

observed mono- and bis-O-atom adducts at sulfur. The FeII(mu-H)FeII diiron model, (mu-pdt)(mu-H)[Fe(CO)2PMe3]2 (IV-5), for which the HOMO is largely of sulfur character, exclusively yields S-oxygenation. Deoxygenation with reclamation of the mu-pdt parent...

Liu, Tianbiao

2011-02-22

2

[FeFe] hydrogenases and their evolution: a genomic perspective  

Microsoft Academic Search

.  Most hydrogenases (H2ases), the enzymes that produce or oxidize dihydrogen, possess dimetallic active sites and belong to either one of two phylogenetically\\u000a distinct classes, the [NiFe] and the [FeFe] H2ases. These families of H2ases share a number of similarities regarding active site structure and reaction mechanism, as a result of convergent evolution.\\u000a They are otherwise alien to each other, in

J. Meyer

2007-01-01

3

Silicon-Heteroaromatic [FeFe] Hydrogenase Model Complexes: Insight into Protonation, Electrochemical Properties, and Molecular Structures.  

PubMed

To learn from Nature how to create an efficient hydrogen-producing catalyst, much attention has been paid to the investigation of structural and functional biomimics of the active site of [FeFe]-hydrogenase. To understand their catalytic activities, the ?-S atoms of the dithiolate bridge have been considered as possible basic sites during the catalytic processes. For this reason, a series of [FeFe]-H2 ase mimics have been synthesized and characterized. Different [FeFe]-hydrogenase model complexes containing bulky Si-heteroaromatic systems or fluorene directly attached to the dithiolate moiety as well as their mono-PPh3 -substituted derivatives have been prepared and investigated in detail by spectroscopic, electrochemical, X-ray diffraction, and computational methods. The assembly of the herein reported series of complexes shows that the ?-S atoms can be a favored basic site in the catalytic process. Small changes in the (hetero)-aromatic system of the dithiolate moiety are responsible for large differences in their structures. This was elucidated in detail by DFT calculations, which were consistent with the experimental results. PMID:25689265

Goy, Roman; Bertini, Luca; Görls, Helmar; De Gioia, Luca; Talarmin, Jean; Zampella, Giuseppe; Schollhammer, Philippe; Weigand, Wolfgang

2015-03-23

4

In Vitro Activation of [FeFe] Hydrogenase: New Insights into Hydrogenase Maturation  

SciTech Connect

The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with addition of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation.

McGlynn, S. E.; Ruebush, S. S.; Naumov, A.; Nagy, L. E.; Dubini, A.; King, P. W.; Broderick, J. B.; Posewitz, M. C.; Peters, J. W.

2007-01-01

5

Models for the active site in [FeFe] hydrogenase with iron-bound ligands derived from bis-, tris-, and tetrakis(mercaptomethyl)silanes.  

PubMed

A series of multifunctional (mercaptomethyl)silanes of the general formula type R(n)Si(CH(2)SH)(4-n) (n = 0-2; R = organyl) was synthesized, starting from the corresponding (chloromethyl)silanes. They were used as multidentate ligands for the conversion of dodecacarbonyltriiron, Fe(3)(CO)(12), into iron carbonyl complexes in which the deprotonated (mercaptomethyl)silanes act as ?-bridging ligands. These complexes can be regarded as models for the [FeFe] hydrogenase. They were characterized by elemental analyses (C, H, S), NMR spectroscopic studies ((1)H, (13)C, (29)Si), and single-crystal X-ray diffraction. Their electrochemical properties were investigated by cyclic voltammetry to disclose a new mechanism for the formation of dihydrogen catalyzed by these compounds, whereby one sulfur atom was protonated in the catalytic cycle. The reaction of the tridentate ligand MeSi(CH(2)SH)(3) with Fe(3)(CO)(12) yielded a tetranuclear cluster compound. A detailed investigation by X-ray diffraction, electrochemical, Raman, Mo?ssbauer, and susceptibility techniques indicates that for this compound initially [Fe(2){?-MeSi(CH(2)S)(2)CH(2)SH}(CO)(6)] is formed. This dinuclear complex, however, is slowly transformed into the tetranuclear species [Fe(4){?-MeSi(CH(2)S)(3)}(2)(CO)(8)]. PMID:20873759

Apfel, Ulf-Peter; Troegel, Dennis; Halpin, Yvonne; Tschierlei, Stefanie; Uhlemann, Ute; Görls, Helmar; Schmitt, Michael; Popp, Jürgen; Dunne, Peter; Venkatesan, Munuswamy; Coey, Michael; Rudolph, Manfred; Vos, Johannes G; Tacke, Reinhold; Weigand, Wolfgang

2010-11-01

6

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

DOEpatents

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.

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

2014-09-16

7

Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation  

PubMed Central

Background [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H2. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated. Principal Findings We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation. Significance The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives. PMID:19855833

Kuchenreuther, Jon M.; Stapleton, James A.; Swartz, James R.

2009-01-01

8

Proton transport in Clostridium pasteurianum [FeFe] hydrogenase I: a computational study.  

PubMed

To better understand the proton transport through the H2 production catalysts, the [FeFe] hydrogenases, we have undertaken a modeling and simulation study of the proton transfer processes mediated by amino acid side-chain residues in hydrogenase I from Clostridium pasteurianum. Free-energy calculation studies show that the side chains of two conserved glutamate residues, Glu-279 and Glu-282, each possess two stable conformations with energies that are sensitive to protonation state. Coordinated conformational changes of these residues can form a proton shuttle between the surface Glu-282 and Cys-299, which is the penultimate proton donor to the catalytic H-cluster. Calculated acid dissociation constants are consistent with a proton relay connecting the H-cluster to the bulk solution. The complete proton-transport process from the surface-disposed Glu-282 to Cys-299 is studied using coupled semiempirical quantum-mechanical/classical-mechanical dynamics. Two-dimensional free-energy maps show the mechanisms of proton transport, which involve Glu-279, Ser-319, and a short internal water relay to connect functionally Glu-282 with the H-cluster. The findings of conformational bistability, PT event coupling with pKa mismatch, and water participation have implications in the design of artificial water reduction or general electrocatalytic H2-production catalysts. PMID:24405487

Long, Hai; King, Paul W; Chang, Christopher H

2014-01-30

9

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

PubMed Central

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

Stapleton, James A.; Swartz, James R.

2010-01-01

10

The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.  

PubMed

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation. PMID:24651202

Fourmond, Vincent; Greco, Claudio; Sybirna, Kateryna; Baffert, Carole; Wang, Po-Hung; Ezanno, Pierre; Montefiori, Marco; Bruschi, Maurizio; Meynial-Salles, Isabelle; Soucaille, Philippe; Blumberger, Jochen; Bottin, Hervé; De Gioia, Luca; Léger, Christophe

2014-04-01

11

The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster  

NASA Astrophysics Data System (ADS)

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

Fourmond, Vincent; Greco, Claudio; Sybirna, Kateryna; Baffert, Carole; Wang, Po-Hung; Ezanno, Pierre; Montefiori, Marco; Bruschi, Maurizio; Meynial-Salles, Isabelle; Soucaille, Philippe; Blumberger, Jochen; Bottin, Hervé; de Gioia, Luca; Léger, Christophe

2014-04-01

12

Analysis of extensive [FeFe] hydrogenase gene diversity within the gut microbiota of insects representing five families of Dictyoptera.  

PubMed

We have designed and utilized degenerate primers in the phylogenetic analysis of [FeFe] hydrogenase gene diversity in the gut ecosystems of roaches and lower termites. 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 primers designed target with specificity the largest group of enzymatic H domain proteins previously identified in a termite gut metagenome. "Family 3" hydrogenase sequences were amplified from the guts of lower termites, Incisitermes minor, Zootermopsis nevadensis, and Reticulitermes hesperus, and two roaches, Cryptocercus punctulatus and Periplaneta americana. Subsequent analyses revealed that all termite and Cryptocercus sequences were phylogenetically distinct from non-termite-associated hydrogenases available from public databases. The abundance of unique sequence operational taxonomic units (as many as 21 from each species) underscores the previously demonstrated physiological importance of H(2) to the gut ecosystems of these wood-feeding insects. The diversity of sequences observed might be reflective of multiple niches that the enzymes have been evolved to accommodate. Sequences cloned from Cryptocercus and the lower termite samples, all of which are wood feeding insects, clustered closely with one another in phylogenetic analyses to the exclusion of alleles from P. americana, an omnivorous cockroach, also cloned during this study. We present primers targeting a family of termite gut [FeFe] hydrogenases and provide results that are consistent with a pivotal role for hydrogen in the termite gut ecosystem and point toward unique evolutionary adaptations to the gut ecosystem. PMID:21935609

Ballor, Nicholas R; Leadbetter, Jared R

2012-04-01

13

Computational chemical analysis of [FeFe] hydrogenase H-cluster analogues to discern catalytically relevant features of the natural diatomic ligand configuration.  

PubMed

Density functional theoretical models of the electronic structure of several configurational isomers and analogues of the [2Fe](H) H-cluster in [FeFe] hydrogenase were analyzed to identify distinguishing features of the canonical cofactor structure potentially relevant to catalysis. Collective analysis of geometric changes over models of oxidized and reduced [2Fe] clusters highlighted movement of the bridging carbonyl and anticorrelation of the proximal and distal Fe-C(terminal) bonds as key explanatory factors for variance over the considered models. Charge and bond order analysis suggest that as the bridging carbonyl favors the distal iron upon reduction, bonding simultaneously becomes more ionic in nature, raising the possibility of simple electrostatic stabilization as a factor in charge accumulation prior to ultimate H(2) creation and release. Frontier orbital energies show cis and trans arrangements of cyanide on the Fe-Fe core to have distinctive energies from the other models, which may be important for redox poise. Altogether, few factors qualitatively distinguish the cis- from the trans-cyano configurations, which may in fact enhance catalytic robustness under conditions leading to exchange of the bridging and terminal carbonyl ligands. However, the naturally occurring trans configuration possesses two distinct donor-metal-acceptor S-Fe-C(O) interactions, which might play a role in enforcing a low-spin ground state for the hydridic mechanism of H(2) production. PMID:21682274

Chang, Christopher H

2011-08-11

14

Immobilization of FeFe hydrogenase mimics onto carbon and gold electrodes by controlled aryldiazonium salt reduction: an electrochemical, XPS and ATR-IR study  

Microsoft Academic Search

A dithiolate-bridged hexacarbonyldiiron complex was synthesized from the reaction of the N-hydroxysuccinimide (NHS) ester of lipoic acid with Fe3(CO)12 in toluene. This mimic of the active site of FeFe hydrogenases could be covalently attached, using an NHS ester route, to carbon or gold electrode first decorated with amino functions. Once grafted this complex catalyzes hydrogen electro-evolution under strongly acidic conditions

Alan Le Goff; Vincent Artero; Romain Metayé; Fabrice Moggia; Bruno Jousselme; Mathieu Razavet; Phong D. Tran; Serge Palacin; Marc Fontecave

2010-01-01

15

Hydrogen-producing microflora and Fe-Fe hydrogenase diversities in seaweed bed associated with marine hot springs of Kalianda, Indonesia.  

PubMed

Microbial fermentation is a promising technology for hydrogen (H(2)) production. H(2) producers in marine geothermal environments are thermophilic and halotolerant. However, no one has surveyed an environment specifically for thermophilic bacteria that produce H(2) through Fe-Fe hydrogenases (H(2)ase). Using heterotrophic medium, several microflora from a seaweed bed associated with marine hot springs were enriched and analyzed for H(2) production. A H(2)-producing microflora was obtained from Sargassum sp., 16S rRNA genes and Fe-Fe H(2)ase diversities of this enrichment were also analyzed. Based on 16S rRNA genes analysis, 10 phylotypes were found in the H(2)-producing microflora showing 90.0-99.5 % identities to known species, and belonged to Clostridia, Gammaproteobacteria, and Bacillales. Clostridia were the most abundant group, and three Clostridia phylotypes were most related to known H(2) producers such as Anaerovorax odorimutans (94.0 % identity), Clostridium papyrosolvens (98.4 % identity), and Clostridium tepidiprofundi (93.1 % identity). For Fe-Fe H(2)ases, seven phylotypes were obtained, showing 63-97 % identities to known Fe-Fe H(2)ases, and fell into four distinct clusters. Phylotypes HW55-3 and HM55-1 belonged to thermophilic and salt-tolerant H(2)-producing Clostridia, Halothermothrix orenii-like Fe-Fe H(2)ases (80 % identity), and cellulolytic H(2)-producing Clostridia, C. papyrosolvens-like Fe-Fe H(2)ases (97 % identity), respectively. The results of both 16S rRNA genes and Fe-Fe H(2)ases surveys suggested that the thermophilic and halotolerant H(2)-producing microflora in seaweed bed of hot spring area represented previously unknown H(2) producers, and have potential application for H(2) production. PMID:23325032

Xu, Shou-Ying; He, Pei-Qing; Dewi, Seswita-Zilda; Zhang, Xue-Lei; Ekowati, Chasanah; Liu, Tong-Jun; Huang, Xiao-Hang

2013-05-01

16

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

PubMed

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. PMID:22895256

Roy, Anindya; Madden, Christopher; Ghirlanda, Giovanna

2012-10-11

17

A radical intermediate in tyrosine scission to the CO and CN- ligands of FeFe hydrogenase.  

PubMed

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the C?-C? bond forms a transient 4-oxidobenzyl (4OB(•)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(•) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN(-), a key intermediate in the assembly of the 2Fe subunit of the H cluster. PMID:24159045

Kuchenreuther, Jon M; Myers, William K; Stich, Troy A; George, Simon J; Nejatyjahromy, Yaser; Swartz, James R; Britt, R David

2013-10-25

18

Inhibition of biocatalysis in [Fe-Fe] hydrogenase by oxygen: molecular dynamics and density functional theory calculations.  

PubMed

Designing O(2)-tolerant hydrogenases is a major challenge in applying [Fe-Fe]H(2)ases for H(2) production. The inhibition involves transport of oxygen through the enzyme to the H-cluster, followed by binding and subsequent deactivation of the active site. To explore the nature of the oxygen diffusion channel for the hydrogenases from Desulfovibrio desulfuricans (Dd) and Clostridium pasteurianum (Cp), empirical molecular dynamics simulations were performed. The dynamic nature of the oxygen pathways in Dd and Cp was elucidated, and insight is provided, in part, into the experimental observation on the difference of oxygen inhibition in Dd and the hydrogenase from Clostridium acetobutylicum (Ca, assumed homologous to Cp). Further, to gain an understanding of the mechanism of oxygen inhibition of the [Fe-Fe]H(2)ase, density functional theory calculations of model compounds composed of the H-cluster and proximate amino acids are reported. Confirmation of the experimentally based suppositions on inactivation by oxygen at the [2Fe](H) domain is provided, validating the model compounds used and oxidation state assumptions, further explaining the mode of damage. This unified approach provides insight into oxygen diffusion in the enzyme, followed by deactivation at the H-cluster. PMID:22563793

Hong, Gongyi; Pachter, Ruth

2012-07-20

19

Artificially maturated [FeFe] hydrogenase from Chlamydomonas reinhardtii: a HYSCORE and ENDOR study of a non-natural H-cluster.  

PubMed

Hydrogenases are enzymes that catalyze the oxidation of H2 as well as the reduction of protons to form H2. The active site of [FeFe] hydrogenase is referred to as the "H-cluster" and consists of a "classical" [4Fe-4S] cluster connected via a bridging cysteine thiol group to a unique [2Fe]H sub-cluster, containing CN(-) and CO ligands as well as a bidentate azadithiolate ligand. It has been recently shown that the biomimetic [Fe2(adt)(CO)4(CN)2](2-) (adt(2-) = azadithiolate) complex resembling the diiron sub-cluster can be inserted in vitro into the apo-protein of [FeFe] hydrogenase, which contains only the [4Fe-4S] part of the H-cluster, resulting in a fully active enzyme. This synthetic tool allows convenient incorporation of a variety of diiron mimics, thus generating hydrogenases with artificial active sites. [FeFe] hydrogenase from Chlamydomonas reinhardtii maturated with the biomimetic complex [Fe2(pdt)(CO)4(CN)2](2-) (pdt(2-) = propanedithiolate), in which the bridging adt(2-) ligand is replaced by pdt(2-), can be stabilized in a state strongly resembling the active oxidized (Hox) state of the native protein. This state is EPR active and the signal originates from the mixed valence Fe(I)Fe(II) state of the diiron sub-cluster. Taking advantage of the variant with (15)N and (13)C isotope labeled CN(-) ligands we performed HYSCORE and ENDOR studies on this hybrid protein. The (13)C hyperfine couplings originating from both CN(-) ligands were determined and assigned. Only the (15)N coupling from the CN(-) ligand bound to the terminal iron was observed. Detailed orientation selective ENDOR and HYSCORE experiments at multiple field positions enabled the extraction of accurate data for the relative orientations of the nitrogen and carbon hyperfine tensors. These data are consistent with the crystal structure assuming a g-tensor orientation following the local symmetry of the binuclear sub-cluster. PMID:25613229

Adamska-Venkatesh, Agnieszka; Simmons, Trevor R; Siebel, Judith F; Artero, Vincent; Fontecave, Marc; Reijerse, Edward; Lubitz, Wolfgang

2015-02-21

20

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

SciTech Connect

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.

Kania, Rafal; Hunt, Neil T. [Department of Physics, University of Strathclyde, SUPA, Glasgow G4 0NG (United Kingdom); Frederix, Pim W. J. M. [Department of Physics, University of Strathclyde, SUPA, Glasgow G4 0NG (United Kingdom); WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL (United Kingdom); Wright, Joseph A.; Pickett, Christopher J. [Energy Materials Laboratory, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ (United Kingdom); Ulijn, Rein V. [WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL (United Kingdom)

2012-01-28

21

Vibrational Analysis of the Model Complex (?-edt)[Fe(CO)3]2 and Comparison to Iron-only Hydrogenase: The Activation Scale of Hydrogenase Model Systems  

PubMed Central

Research on simple [FeFe] hydrogenase model systems of type (?-S2R)[Fe(CO)3]2 (R = ethane, propane) which have been shown to function as robust electrocatalysts for proton reduction, provides a reference to understand the electronic and vibrational properties of the active site of [FeFe] hydrogenases and of more sophisticated model systems. In this study, the solution and solid Raman spectra of (?-S2R)[Fe(CO)3]2 (R = ethane) and of the corresponding 13CO-labeled complex are presented and analyzed in detail, with focus on the ?(C=O) and ?(Fe-CO)/?(Fe-C=O) vibrational regions. These regions are specifically important as vibrations involving CO ligands serve as probes for the ‘electron richness’ of low-valent transition metal centers and the geometric structures of the complexes. The obtained vibrational spectra have been completely assigned in terms of the ?(C=O), ?(Fe-CO) and ?(Fe-C=O) modes, and the force constants of the important C=O and Fe-CO bonds have been determined using our Quantum Chemistry Centered Normal Coordinate Analysis (QCC-NCA). In the 400–650 cm?1 region, 15 mixed ?(Fe-CO)/?(Fe-C=O) modes have been identified. The most prominent Raman peaks at 454, 456 and 483 cm?1 correspond to a combination of ?(Fe-CO) stretching and ?(Fe-C=O) linear bending modes. The less intense peaks at 416 cm?1 and 419 cm?1 correspond to pure ?(Fe-C=O) linear bends. In the ?(C=O) region, the ?(C=O) normal modes at lower energy (1968 and 1964 cm?1) are almost pure equatorial (eq) ?(C=O)eq stretching vibrations, whereas the remaining four ?(C=O) normal modes show dominant (C=O)eq (2070 and 1961 cm?1) and (C=O)ax (2005 and 1979 cm?1; ax = axial) contributions. Importantly, an inverse correlation between the f(C=O)ax/eq and f(Fe-CO)ax/eq force constants is obtained, in agreement with the idea that the Fe(I)-CO bond in these types of complexes is dominated by ? backdonation. Compared to the reduced form of [FeFe] hydrogenase (Hred), the ?(C=O) vibrational frequencies of (?-edt)[Fe(CO)3]2 are higher in energy, indicating that the dinuclear iron core in (?-edt)[Fe(CO)3]2 is less electron rich compared to Hred in the actual enzyme. Finally, quantum yields for the photodecomposition of (?-edt)[Fe(CO)3]2 have been determined. PMID:20225804

Galinato, Mary Grace I.; Whaley, C. Matthew; Lehnert, Nicolai

2010-01-01

22

Spin distribution of the H-cluster in the H(ox)-CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of (14)N and (13)C nuclear interactions.  

PubMed

Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the "H-cluster." It consists of the redox-active binuclear subcluster ([2Fe](H)) coordinated by CN(-) and CO ligands and the cubane-like [4Fe-4S](H) subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe](H) subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H(ox)-CO state the open coordination site at the [2Fe](H) subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with (13)CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three (13)C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to (13)CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No (13)CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, (14)N nuclear spin interactions were measured. On the basis of the observed (14)N hyperfine couplings, which result from the CN(-) ligands of the [2Fe](H) subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster. PMID:19011912

Silakov, Alexey; Wenk, Brian; Reijerse, Eduard; Albracht, Simon P J; Lubitz, Wolfgang

2009-02-01

23

EPR/ENDOR, Mössbauer, and Quantum Chemical Investigation of Di-iron Complexes Mimicking the Active Oxidized State of [FeFe] Hydrogenase  

PubMed Central

Understanding the catalytic process of the heterolytic splitting and formation of molecular hydrogen is one of the key topics for the development of a future hydrogen economy. With an interest in elucidating the enzymatic mechanism of the [Fe2(S2C2H4NH)(CN)2(CO)2(µ-CO)] active center uniquely found in the [FeFe]-hydrogenases, we present a detailed spectroscopic and theoretical analysis of its inorganic model [Fe2(S2X)(CO)3(dppv)(PMe3)]+ in two forms with S2X = ethanedithiolate (1edt) and azadithiolate (1adt) (dppv =cis-1,2-bis(diphenylphosphino)ethylene). These complexes represent models for the oxidized mixed-valent Fe(I)Fe(II) state analogous to the active oxidized "Hox" state of the native H-cluster. For both complexes, the 31P hyperfine (HF) interactions were determined by pulse EPR and ENDOR methods. For 1edt, the 57Fe parameters were measured by ESEEM and Mössbauer spectroscopy, while for 1adt 14N and selected 1H couplings could be obtained by ENDOR and HYSCORE. The spin density was found to be predominantly localized on the Fe(dppv) site. This spin distribution is different from the H-cluster where both the spin and charge density are delocalized over the two Fe centers. This difference is attributed to the influence of “native” cubane subcluster that is lacking in the inorganic models. The degree and character of the unpaired spin delocalization was found to vary from 1edt, with an abiological dithiolate, to 1adt, which features the authentic cofactor. For 1adt, we find two 14N signals, which are indicative for two possible isomers of the azadithiolate, demonstrating its high flexibility. All interaction parameters were also evaluated through density functional theory calculations at various levels. PMID:22800196

Olsen, Matthew T.; Sproules, Stephen; Reijerse, Eduard J.; Rauchfuss, Thomas B.

2012-01-01

24

Artificial Hydrogenases  

PubMed Central

Decades of biophysical study on the hydrogenase (H2ase) enzymes have yielded sufficient information to guide the synthesis of analogues of their active sites. Three families of enzymes serve as inspiration for this work: the [FeFe]-, [NiFe]-, and [Fe]-H2ases, all of which feature iron centers bound to both CO and thiolate. Artificial H2ases effect the oxidation of H2 of H2 and the reverse reaction, the reduction of protons. These reactions occur via the intermediacy of metal hydrides. The inclusion of amine bases within the catalysts is an important design feature that is emulated in related bioinspired catalysts. Continuing challenges are the low reactivity of H2 towards biomimetic H2ases. PMID:20356731

Barton, Bryan E.; Olsen, Matthew T.; Rauchfuss, Thomas B.

2010-01-01

25

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

PubMed

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)?Ĺ between the protic N-H(?+) and hydridic Fe-H(?-) 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. PMID:24757087

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

2014-05-19

26

Explorations of iron-iron hydrogenase active site models by experiment and theory  

E-print Network

This dissertation describes computational and experimental studies of synthetic complexes that model the active site of the iron-iron hydrogenase [FeFe]H 2ase enzyme. Simple dinuclear iron dithiolate complexes act as functional models of the ironiron...

Tye, Jesse Wayne

2009-05-15

27

Ultrafast Photodriven Intramolecular Electron Transfer from a Zinc Porphyrin to a Readily Reduced Diiron Hydrogenase Model Complex  

SciTech Connect

Diiron complexes modeled on the active site of the [FeFe] hydrogenases having the general formula [Fe{sub 2}(?-R)(CO){sub 6?n}(L){sub n}], where commonly R = alkyl or aryl dithiolate and L = CO, CN{sup ?}, or PR{sub 3}, are a promising class of catalysts for use in photodriven H{sub 2} production. However, many of these catalysts are difficult to photoreduce using chromophores that absorb visible light. Here we report the synthesis and spectroscopic characterization of a naphthalene-4,5-dicarboximide-1,8-dithiolate diiron complex [NMI-Fe{sub 2}S{sub 2}(CO){sub 6}, 1] and a covalently linked, fixed-distance zinc 5,10,15-tri-n-pentyl-20-phenylporphyrin-NMI-Fe{sub 2}S{sub 2}(CO){sub 6} donor?acceptor dyad (2). The electron-withdrawing nature of the NMI group makes the diiron complex among the most easily reduced hydrogenase mimics reported to date (?0.74 V vs SCE). In the presence of triflic acid, the cyclic voltammogram of 1 showed an increase in current at the first reduction wave at ?0.78 V and a new reduction wave at ?1.4 V. As the acid concentration was increased, the current at ?0.78 V remained constant while the current at ?1.4 V increased significantly, which is consistent with a catalytic proton reduction process. Selective photoexcitation of the Zn porphyrin in 2 with 553 nm, 110 fs laser pulses in both toluene and CH{sub 2}Cl{sub 2} yielded transient absorption spectra showing a distinct peak at 616 nm, which has been assigned to [NMI-Fe{sub 2}S{sub 2}(CO){sub 6}]{sup ?•} on the basis of spectroelectrochemical measurements on 1. The 616 nm peak was used to monitor the charge separation (CS) and charge recombination (CR) dynamics of 2, which yielded ?{sub CS} = 12 ± 1 ps and ?{sub CR} = 3.0 ± 0.2 ns in toluene and ?{sub CS} = 24 ± 1 ps and ?{sub CR} = 57 ± 1 ps in CH{sub 2}Cl{sub 2}. Photoexcitation of the disulfide precursor to 2 in both toluene and CH{sub 2}Cl{sub 2} produced only the singlet and triplet excited states of the Zn porphyrin, showing that electron transfer is favorable only when the diiron complex is present. Photoexcitation of 2 in the presence of trifluoroacetic acid was shown to generate H{sub 2}.

Samuel, Amanda P.S.; Co, Dick T.; Stern, C. L.; Wasielewski, Michael R.

2010-01-01

28

Microbial communities responsible for fixation of CO2 revealed by using mcrA, cbbM, cbbL, fthfs, fefe-hydrogenase genes as molecular biomarkers in petroleum reservoirs of different temperatures  

NASA Astrophysics Data System (ADS)

Sequestration of CO2 in oil reservoir is one of the feasible options for mitigating atmospheric CO2 building up. The in situ bioconversion of sequestrated CO2 to methane by microorganisms inhabiting oil reservoirs is feasible. To evaluate the potential of in situ microbial fixation and conversion of CO2 into CH4 in oil reservoirs, a comprehensive molecular survey was performed to reveal microbial communities inhabiting four oil reservoirs with different temperatures by analysis of functional genes involved in the biochemical pathways of CO2 fixation and CH4 synthesis (cbbM, cbbL, fthfs, [FeFe]-hydrogenase encoding gene, and mcrA). A rich diversity of these functional genes was found in all the samples with both high and low temperatures and they were affiliated to members of the Proteobacteria (cbbL and cbbM, fthfs), Firmicutes and Actinobacteria (fthfs), uncultured bacteria ([FeFe]-hydrogenase), and Methanomirobiales, Methanobacteriales and Methanosarcinales (mcrA). The predominant methanogens were all identified to be hydrogenotrophic CO2-reducing physiological types. These results showed that functional microbial communities capable of microbial fixation and bioconversion of CO2 into methane inhabit widely in oil reservoirs, which is helpful to microbial recycling of sequestrated CO2 to further new energy in oil reservoirs.

Liu, J.-F.; Mbadinga, S. M.; Sun, X.-B.; Yang, G.-C.; Yang, S.-Z.; Gu, J.-D.; Mu, B.-Z.

2015-01-01

29

The organometallic active site of [Fe]hydrogenase: Models and entatic states  

PubMed Central

The simple organometallic, (?-S2)Fe2(CO)6, serves as a precursor to synthetic analogues of the chemically rudimentary iron-only hydrogenase enzyme active site. The fundamental properties of the (?-SCH2CH2CH2S)[Fe(CO)3]2 compound, including structural mobility and regioselectivity in cyanide/carbon monoxide substitution reactions, relate to the enzyme active site in the form of transition-state structures along reaction paths rather than ground-state structures. Even in the absence of protein-based active-site organization, the ground-state structural model complexes are shown to serve as hydrogenase enzyme reaction models, H2 uptake and H2 production, with the input of photo- or electrochemical energy, respectively. PMID:12642671

Darensbourg, Marcetta Y.; Lyon, Erica J.; Zhao, Xuan; Georgakaki, Irene P.

2003-01-01

30

Synthesis, structure and reactivity of Ni site models of [NiFeSe] hydrogenases.  

PubMed

A series of structural models of the Ni centre in [NiFeSe] hydrogenases has been developed which exhibits key structural features of the Ni site in the H2 cycling enzyme. Specifically, two complexes with a hydrogenase-analogous four-coordinate 'NiS3Se' primary coordination sphere and complexes with a 'NiS2Se2' and a 'NiS4' core are reported. The reactivity of the complexes towards oxygen and protons shows some relevance to the chemistry of [NiFeSe] hydrogenases. Exposure of a 'NiS3Se' complex to atmospheric oxygen results in the oxidation of the selenolate group in the complex to a diselenide, which is released from the nickel site. Oxidation of the selenolate ligand on Ni occurs approximately four times faster than oxidation with the analogous sulfur complex. Reaction of the complexes with one equivalent of HBF4 results in protonation of the monodentate chalcogenolate and the release of this ligand from the metal centre as a thiol or selenol. Unrelated to their biomimetic nature, the complexes serve also as molecular precursors to modify electrodes with Ni-S-Se containing particles by electrochemical deposition. The activated electrodes evolve H2 in pH neutral water with an electrocatalytic onset potential of -0.6 V and a current density of 15 ?A cm(-2) at -0.75 V vs. NHE. PMID:24366040

Wombwell, Claire; Reisner, Erwin

2014-03-21

31

Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity  

SciTech Connect

The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

Dubini, A.; Mus, F.; Seibert, M.; Grossman, A. R.; Posewitz, M. C.

2009-03-13

32

Functional model for the [Fe] hydrogenase inspired by the frustrated Lewis pair concept.  

PubMed

[Fe] hydrogenase (Hmd) catalyzes the heterolytic splitting of H2 by using, in its active site, a unique organometallic iron-guanylylpyridinol (FeGP) cofactor and, as a hydride acceptor, the substrate methenyltetrahydromethanopterin (methenyl-H4MPT(+)). The combination FeGP/methenyl-H4MPT(+) and its reactivity bear resemblance to the concept of frustrated Lewis pairs (FLPs), some of which have been shown to heterolytically activate H2. The present work exploits this interpretation of Hmd reactivity by using the combination of Lewis basic ruthenium metalates, namely K[CpRu(CO)2] (KRp) and a related polymeric Cp/Ru/CO compound (Rs), with the new imidazolinium salt 1,3-bis(2,6-difluorophenyl)-2-(4-tolyl)imidazolinium bromide ([(Tol)Im(F4)](+)Br(-)) that was designed to emulate the hydride acceptor properties of methenyl-H4MPT(+). Solid-state structures of [(Tol)Im(F4)](+)Br(-) and the corresponding imidazolidine H(Tol)Im(F4) reveal that the heterocycle undergoes similar structural changes as in the biological substrate. DFT calculations indicate that heterolytic splitting of dihydrogen by the FLP Rp(-)/[(Tol)Im(F4)](+) is exothermic, but the formation of the initial Lewis pair should be unfavorable in polar solvents. Consequently the combination Rp(-)/[(Tol)Im(F4)](+) does not react with H2 but leads instead to side products from nucleophilic substitution (k = 4 × 10(-2) L mol (-1) s(-1) at room temperature). In contrast, the heterogeneous combination Rs/[(Tol)Im(F4)](+) does split H2 heterolytically to give H(Tol)Im(F4) and HRuCp(CO)2 (HRp) or D(Tol)Im(F4) and DRp when using D2. The reaction has been followed by (1)H/(2)H and (19)F NMR spectroscopy as well as by IR spectroscopy and reaches 96% conversion after 1 d. Formation of H(Tol)Im(F4) under these conditions demonstrates that superelectrophilic activation by protonation, which has been proposed for methenyl-H4MPT(+) to increase its carbocationic character, is not necessarily required for an imidazolinium ion to serve as a hydride acceptor. This unprecedented functional model for the [Fe] hydrogenase, using a Lewis acidic imidazolinium salt as a biomimetic hydride acceptor in combination with an organometallic Lewis base, may provide new inspiration for biomimetic H2 activation. PMID:25353322

Kalz, Kai F; Brinkmeier, Alexander; Dechert, Sebastian; Mata, Ricardo A; Meyer, Franc

2014-11-26

33

Fundamental Studies of Recombinant Hydrogenases  

SciTech Connect

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.

Adams, Michael W

2014-01-25

34

Aza- and Oxadithiolates Are Proton Relays in Functional Models for the [FeFe]-Hydrogenases  

PubMed Central

The dithiolate cofactor for the [FeFe]-hydrogenase models, Fe2(xdt)(CO)2(dppv)2 (where xdt = 1,3-propanedithiolate (pdt), azadithiolate (adt), (SCH2)2NH, and oxadithiolate (odt), (SCH2)2O; dppv = cis-1,2-bis-(diphenylphosphino)ethylene) have been probed for their functionality as proton relays enabling formation and deprotonation of terminal hydrides. Compared to the propanedithiolate derivative, the azadithiolate and oxaditiholate show enhanced rates of proton transfer between solution and the terminal site on one Fe center. The results are consistent with the heteroatom of the dithiolate serving a gating role for both protonation and deprotonation. The pKa of the transiently formed ammonium (pK CD2Cl2 5.7–8.2) or oxonium (pK CD2Cl2 ?4.7–1.6) regulates the proton transfer. As consequence, only the azadithiolate is capable of yielding the terminal hydride from weak acids. The aza- and oxadithiolates manifested the advantages of proton relays: the odt derivative proved to be a faster catalyst for hydrogen evolution than the pdt derivative as indicated from cyclic voltammetry plots of ic/ip vs. [H+]. The adt derivative was capable of proton reduction from the weak acid [HPMe2Ph]BF4 (pK CD2Cl2 = 5.7). The proton relay function does not apply to the isomeric bridged-hydrides [Fe2(xdt)(?-H)(CO)2(dppv)2]+, where the hydride is too distant and too basic to interact to be affected by the heteroatomic relay site. None of these ?-H species can be deprotonated. PMID:19053433

Barton, Bryan E.; Olsen, Matthew T.; Rauchfuss, Thomas B.

2009-01-01

35

Hydrogen Activation by Biomimetic [NiFe]-Hydrogenase Model Containing Protected Cyanide Cofactors  

PubMed Central

Described are experiments that allow incorporation of cyanide cofactors and hydride substrate into active site models [NiFe]-hydrogenases (H2ases). Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe), (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BArF3) to give the adducts (CO)2(CNBArF3)2Fe(pdt)Ni(dxpe), (1(BArF3)2, 2(BArF3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives Et4N[(CO)(CNBArF3)2Fe(H)(pdt)Ni(dxpe)], (dxpe = dppe, Et4N[H3(BArF3)2]; dxpe = dcpe, Et4N[H4(BArF3)2]). Crystallographic analysis shows that Et4N[H3(BArF3)2] generally resembles the active site of the enzyme in the reduced, hydride-containing states (Ni-C/R). The Fe-H…Ni center is unsymmetrical with rFe-H = 1.51(3) and rNi-H = 1.71(3) Ĺ. Both crystallographic and 19F NMR analysis show that the CNBArF3? ligands occupy basal and apical sites. Unlike cationic Ni-Fe hydrides, [H3(BArF3)2]? and [H4(BArF3)2]? oxidize at mild potentials, near the Fc+/0 couple. Electrochemical measurements indicate that in the presence of base, [H3(BArF3)2]? catalyzes the oxidation of H2. NMR evidence indicates dihydrogen bonding between these anionic hydrides and ammonium salts, which is relevant to the mechanism of hydrogenogenesis. In the case of Et4N[H3(BArF3)2], strong acids such as HCl induce H2 release to give the chloride Et4N[(CO)(CNBArF3)2Fe(pdt)(Cl)Ni(dppe)]. PMID:23899049

Manor, Brian C.; Rauchfuss, Thomas B.

2013-01-01

36

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

PubMed Central

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 Ĺ 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, ?GH• and ?GH? are 57 and 79 kcal/mol for [1H]+ and [1]2+, respectively. PMID:20925337

Barton, Bryan E.; Rauchfuss, Thomas B.

2010-01-01

37

Accurate calculations of geometries and singlet-triplet energy differences for active-site models of [NiFe] hydrogenase.  

PubMed

We have studied the geometry and singlet-triplet energy difference of two mono-nuclear Ni(2+) models related to the active site in [NiFe] hydrogenase. Multiconfigurational second-order perturbation theory based on a complete active-space wavefunction with an active space of 12 electrons in 12 orbitals, CASPT2(12,12), reproduces experimental bond lengths to within 1 pm. Calculated singlet-triplet energy differences agree with those obtained from coupled-cluster calculations with single, double and (perturbatively treated) triple excitations (CCSD(T)) to within 12 kJ mol(-1). For a bimetallic model of the active site of [NiFe] hydrogenase, the CASPT2(12,12) results were compared with the results obtained with an extended active space of 22 electrons in 22 orbitals. This is so large that we need to use restricted active-space theory (RASPT2). The calculations predict that the singlet state is 48-57 kJ mol(-1) more stable than the triplet state for this model of the Ni-SIa state. However, in the [NiFe] hydrogenase protein, the structure around the Ni ion is far from the square-planar structure preferred by the singlet state. This destabilises the singlet state so that it is only ?24 kJ mol(-1) more stable than the triplet state. Finally, we have studied how various density functional theory methods compare to the experimental, CCSD(T), CASPT2, and RASPT2 results. Semi-local functionals predict the best singlet-triplet energy differences, with BP86, TPSS, and PBE giving mean unsigned errors of 12-13 kJ mol(-1) (maximum errors of 25-31 kJ mol(-1)) compared to CCSD(T). For bond lengths, several methods give good results, e.g. TPSS, BP86, and M06, with mean unsigned errors of 2 pm for the bond lengths if relativistic effects are considered. PMID:24647807

Delcey, Mickaël G; Pierloot, Kristine; Phung, Quan M; Vancoillie, Steven; Lindh, Roland; Ryde, Ulf

2014-05-01

38

Role of the Azadithiolate Cofactor in Models for the [FeFe]-Hydrogenase: Novel Structures and Catalytic Implications  

PubMed Central

The report summarizes studies on the redox behavior of synthetic models for the [FeFe]-hydrogenases, consisting of diiron dithiolato carbonyl complexes bearing the amine cofactor and its N-benzyl derivative. Of specific interest are the causes of the low reactivity of oxidized models toward H2, which contrasts with the high activity of these enzymes for H2 oxidation. The redox and acid-base properties of the model complexes [Fe2[(SCH2)2NR](CO)3(dppv)(PMe3)]+ ([2]+ for R = H and [2?]+ for R = CH2C6H5, dppv = cis-1,2-bis(diphenylphosphino)ethylene)) indicate that addition of H2 and followed by deprotonation are (i) endothermic for the mixed valence (FeIIFeI) state and (ii) exothermic for the diferrous (FeIIFeII) state. The diferrous state is shown to be unstable with respect to coordination of the amine to Fe, a derivative of which was characterized crystallographically. The redox and acid-base properties for the mixed valence models differ strongly for those containing the amine cofactor versus those derived from propanedithiolate. Protonation of [2?]+ induces disproportionation to a 1:1 mixture of the ammonium-FeIFeI and the dication [2?]2+ (FeIIFeII). This effect is consistent with substantial enhancement of the basicity of the amine in the FeIFeI state vs the FeIIFeI state. The FeIFeI ammonium compounds are rapid and efficient H-atom donors toward the nitroxyl compound TEMPO. The atom transfer is proposed to proceed via the hydride, as indicated by the reaction of [HFe2[(SCH2)2NH](CO)2(dppv)2]+ with TEMPO. Collectively, the results suggest that proton-coupled electron-transfer pathways should be considered for H2 activation by the [FeFe]-hydrogenases. PMID:21114298

Olsen, Matthew T.; Rauchfuss, Thomas B.; Wilson, Scott R.

2010-01-01

39

Cobaloximes as functional models for hydrogenases. 2. Proton electroreduction catalyzed by difluoroborylbis(dimethylglyoximato)cobalt(II) complexes in organic media.  

PubMed

Cobaloximes are effective electrocatalysts for hydrogen evolution and thus functional models for hydrogenases. Among them, difluoroboryl-bridged complexes appear both to mediate proton electroreduction with low overpotentials and to be quite stable in acidic conditions. We report here a mechanistic study of [Co(dmgBF2)2L] (dmg2- = dimethylglyoximato dianion; L = CH3CN or N,N-dimethylformamide) catalyzed proton electroreduction in organic solvents. Depending on the applied potential and the strength of the acid used, three different pathways for hydrogen production were identified and a unified mechanistic scheme involving cobalt(II) or cobalt(III) hydride species is proposed. As far as working potential and turnover frequency are concerned, [Co(dmgBF2)2(CH3CN)2], in the presence of p-cyanoanilinium cation in acetonitrile, is one of the best synthetic catalysts of the first-row transition-metal series for hydrogen evolution. PMID:17269760

Baffert, Carole; Artero, Vincent; Fontecave, Marc

2007-03-01

40

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

SciTech Connect

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.

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

2008-01-01

41

Diiron Dithiolato Carbonyls Related to the HoxCO State of [FeFe]-Hydrogenase  

PubMed Central

Oxidation of the electron-rich (E1/2 = ?175 vs Ag/AgCl) ethanedithiolato complex Fe2(S2C2H4)-(CO)2(dppv)2 (1) under a CO atmosphere yielded [Fe2(S2C2H4)(?-CO)(CO)2(dppv)2]+ ([1(CO)]+), a model for the HoxCO state of the [FeFe]-hydrogenases. This complex exists as two isomers: a kinetically favored unsymmetrical derivative, unsym-[1(CO)]+, and a thermodynamically favored isomer, sym-[1(CO)]+, wherein both diphosphines span apical and basal sites. Crystallographic characterization of sym-[1(CO)]+ confirmed a C2-symmetric structure with a bridging CO ligand and an elongated Fe–Fe bond of 2.7012(14) Ĺ, as predicted previously. Oxidation of sym-[1(CO)]+ and unsym-[1(CO)]+ again by 1e? oxidation afforded the respective diamagnetic diferrous derivatives where the relative stabilities of the sym and unsym isomers are reversed. DFT calculations indicate that the stabilities of sym and unsym isomers are affected differently by the oxidation state of the diiron unit: the mutually trans CO ligands in the sym isomer are more destabilizing in the mixed-valence state than in the diferrous state. EPR analysis of mixed-valence complexes revealed that, for [1]+, the unpaired spin is localized on a single iron center, whereas for unsym/sym-[1(CO)]+, the unpaired spin was delocalized over both iron centers, as indicated by the magnitude of the hyperfine coupling to the phosphine ligands trans to the Fe–Fe vector. Oxidation of 1 by 2 equiv of acetylferrocenium afforded the dication [1]2+, which, on the basis of low-temperature IR spectrum, is structurally similar to [1]+. Treatment of [1]2+ with CO gives unsym-[1(CO)]2+. PMID:18341276

Justice, Aaron K.; Nilges, Mark J.; Rauchfuss, Thomas B.; Wilson, Scott R.; De Gioia, Luca; Zampella, Giuseppe

2008-01-01

42

Studies on hydrogenase  

PubMed Central

Hydrogenases are microbial enzymes which catalyze uptake and production of H2. Hydrogenases are classified into 10 classes based on the electron carrier specificity, or into 3 families, [NiFe]-family (including [NiFeSe]-subfamily), [FeFe]-family and [Fe]-family, based on the metal composition of the active site. H2 is heterolytically cleaved on the enzyme (E) to produce EHaHb, where Ha and Hb have different rate constants for exchange with the medium hydron. X-ray crystallography unveiled the three-dimensional structures of hydrogenases. The simplest [NiFe]-hydrogenase is a heterodimer, in which the large subunit bears the Ni-Fe center buried deep in the protein, and the small subunit bears iron-sulfur clusters, which mediate electron transfer between the Ni-Fe center and the protein surface. Some hydrogenases have additional subunit(s) for interaction with their electron carriers. Various redox states of the enzyme were characterized by EPR, FTIR, etc. Based on the kinetic, structural and spectroscopic studies, the catalytic mechanism of [NiFe]-hydrogenase was proposed to explain H2-uptake, H2-production and isotopic exchange reactions. PMID:23318679

YAGI, Tatsuhiko; HIGUCHI, Yoshiki

2013-01-01

43

Multiple Forms of Bacterial Hydrogenases  

PubMed Central

Ackrell, B. A. C. (University of Hawaii, Honolulu), R. N. Asato, and H. F. Mower. Multiple forms of bacterial hydrogenases. J. Bacteriol. 92:828–838. 1966.—Extracts of certain bacterial species have been shown by disc electrophoresis on polyacrylamide gel to contain multiple hydrogenase systems. The hydrogenase enzymes comprising these systems have different electrophoretic mobilities and produce a band pattern that is unique for each bacterial species. Of 20 bacterial species known to possess hydrogenase activity and which were examined by this technique, only the activities of Clostridium tetanomorphum and C. thermosaccharolyticum could be attributed, at pH 8.3, to a single hydrogenase enzyme. This multiplicity of hydrogenase forms was found both in bacteria which contain mostly soluble hydrogenases and in those where the hydrogenase is predominantly associated with particulate material. When solubilization of this particulate material could be effected, at least two solubilized hydrogenases were released, and, of these, one would have the same electrophoretic properties (i.e., RF) as one of the soluble hydrogenases already present in small amounts within the cell. Different growth conditions for various types of bacteria, such as the nitrogen source, the degree of aeration, and photosynthetic versus aerobic growth in the dark, as well as the conditions under which the cells were stored, markedly affected the hydrogenase activity of the cells, but not their hydrogenase band pattern. The disc electrophoresis technique proved to be 10 times more sensitive than the manometric technique in detecting hydrogenase activity. PMID:5926752

Ackrell, B. A. C.; Asato, R. N.; Mower, H. F.

1966-01-01

44

Hydrogenases and Hydrogen Photoproduction in Oxygenic Photosynthetic Organisms  

SciTech Connect

The photobiological production of H{sub 2} gas, using water as the only electron donor, is a property of two types of photosynthetic microorganisms: green algae and cyanobacteria. In these organisms, photosynthetic water splitting is functionally linked to H{sub 2} production by the activity of hydrogenase enzymes. Interestingly, each of these organisms contains only one of two major types of hydrogenases, [FeFe] or [NiFe] enzymes, which are phylogenetically distinct but perform the same catalytic reaction, suggesting convergent evolution. This idea is supported by the observation that each of the two classes of hydrogenases has a different metallo-cluster, is encoded by entirely different sets of genes (apparently under the control of different promoter elements), and exhibits different maturation pathways. The genetics, biosynthesis, structure, function, and O{sub 2} sensitivity of these enzymes have been the focus of extensive research in recent years. Some of this effort is clearly driven by the potential for using these enzymes in future biological or biohybrid systems to produce renewable fuel or in fuel cell applications.

Ghirardi, M. L.; Posewitz, M. C.; Maness, P. C.; Dubini, A.; Yu, J.; Seibert, M.

2007-01-01

45

Combining acid-base, redox and substrate binding functionalities to give a complete model for the [FeFe]-hydrogenase  

PubMed Central

Some enzymes function by coupling substrate turnover with electron transfer from a redox cofactor such as ferredoxin. In the [FeFe]-hydrogenases, nature’s fastest catalysts for the production and oxidation of H2, the one-electron redox by a ferredoxin complements the one-electron redox by the diiron active site. In this Article, we replicate the function of the ferredoxins with the redox-active ligand Cp*Fe(C5Me4CH2PEt2) (FcP*). FcP* oxidizes at mild potentials, in contrast to most ferrocene-based ligands, which suggests that it might be a useful mimic of ferredoxin cofactors. The specific model is Fe2[(SCH2)2NBn](CO)3(FcP*)(dppv) (1), which contains the three functional components of the active site: a reactive diiron centre, an amine as a proton relay and, for the first time, a one-electron redox module. By virtue of the synthetic redox cofactor, [1]2+ exhibits unique reactivity towards hydrogen and CO. In the presence of excess oxidant and base, H2 oxidation by [1]2+ is catalytic. PMID:22169868

Camara, James M.; Rauchfuss, Thomas B.

2012-01-01

46

Time resolved infrared spectroscopy: kinetic studies of weakly binding ligands in an iron-iron hydrogenase model compound.  

PubMed

Solution photochemistry of (?-pdt)[Fe(CO)(3)](2) (pdt = ?(2)-S(CH(2))(3)S), a precursor model of the 2-Fe subsite of the H-cluster of the hydrogenase enzyme, has been studied using time-resolved infrared spectroscopy. Following the loss of CO, solvation of the Fe center by the weakly binding ligands cyclohexene, 3-hexyne, THF, and 2,3-dihydrofuran (DHF) occurred. Subsequent ligand substitution of these weakly bound ligands by pyridine or cyclooctene to afford a more stable complex was found to take place via a dissociative mechanism on a seconds time scale with activation parameters consistent with such a pathway. That is, the ?S(‡) values were positive and the ?H(‡) parameters closely agreed with bond dissociation enthalpies (BDEs) obtained from DFT calculations. For example, for cyclohexene replacement by pyridine, experimental ?H(‡) and ?S(‡) values were determined to be 19.7 ± 0.6 kcal/mol (versus a theoretical prediction of 19.8 kcal/mol) and 15 ± 2 eu, respectively. The ambidentate ligand 2,3-DHF was shown to initially bind to the iron center via its oxygen atom followed by an intramolecular rearrangement to the more stable ?(2)-olefin bound species. DFT calculations revealed a transition state structure with the iron atom almost equidistant from the oxygen and one edge of the olefinic bond. The computed ?H(‡) of 10.7 kcal/mol for this isomerization process was found to be in excellent agreement with the experimental value of 11.2 ± 0.3 kcal/mol. PMID:22680284

Muhammad, Sohail; Moncho, Salvador; Brothers, Edward N; Darensbourg, Marcetta Y; Darensbourg, Donald J; Bengali, Ashfaq A

2012-07-01

47

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

SciTech Connect

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) Ĺ between the protic N-H?+ and hydridic Fe-H?-. 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 as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

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

2014-05-19

48

Computational investigation of [FeFe]-hydrogenase models: characterization of singly and doubly protonated intermediates and mechanistic insights.  

PubMed

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 ligand, and intramolecular electron transfer between the Fe centers in the catalytic cycle. Such insights will assist in the design of more effective bioinspired catalysts for H2 production. PMID:25207842

Huynh, Mioy T; Wang, Wenguang; Rauchfuss, Thomas B; Hammes-Schiffer, Sharon

2014-10-01

49

Hydrogenase/ferredoxin charge-transfer complexes: effect of hydrogenase mutations on the complex association.  

PubMed

The [FeFe]-hydrogenases in the green alga Chlamydomonas reinhardtii utilize photogenerated electrons to reduce protons into hydrogen gas. The electrons are supplied from photosystem I and transferred to the [FeFe]-hydrogenase through specific hydrogenase-ferredoxin association. To understand how structural and kinetic factors control the association better, we used Brownian dynamics simulation methods to simulate the charge-transfer complex formation between both native and in silico mutants of the [FeFe]-hydrogenase HYDA2 and the [2Fe2S]-ferredoxin FDX1 from C. reinhardtii . The changes in binding free energy between different HYDA2 mutants and the native FDX1 were calculated by the free-energy perturbation method. Within the limits of our current models, we found that two HYDA2 mutations, T99K(H) and D102K(H), led to lower binding free energies and higher association rate with FDX1 and are thus promising targets for improving hydrogen production rates in engineered organisms. PMID:19317477

Long, Hai; King, Paul W; Ghirardi, Maria L; Kim, Kwiseon

2009-04-23

50

X-ray-absorption-spectroscopic evidence for a novel iron cluster in hydrogenase II from Clostridium pasteurianum.  

PubMed Central

Hydrogenase II from Clostridium pasteurianum contains three different iron-sulphur clusters. Two are [4Fe-4S](2+.1+) clusters, whereas the other, which is thought to be the site of interaction with H2 and is known as the 'H cluster', is of unknown structure and possesses unusual spectroscopic properties. Analysis of the iron e.x.a.f.s. spectra shows that the H cluster contains iron co-ordinated mostly to sulphur and possesses 2.8 A (1 A = 0.1 nm) Fe--Fe separations when oxidized and 3.3 A Fe--Fe separations when reduced with H2. The data suggest that the reduced H cluster represents a new structural type of iron-sulphur cluster. PMID:2655584

George, G N; Prince, R C; Stokley, K E; Adams, M W; Stockley, K E

1989-01-01

51

Nitrogenases and Hydrogenases in Cyanobacteria  

Microsoft Academic Search

\\u000a Cyanobacteria may contain different types of nitrogenases that catalyzes (di)nitrogen fixation: two types of Mo-nitrogenases\\u000a and the V-enzyme. The formation of ammonia by nitrogenases is accompanied with the production of hydrogen gas. This H2-formation is barely detectable in intact cyanobacteria, since the gas is immediately recycled by hydrogenase. Cyanobacteria\\u000a contain two types of Ni-containing hydrogenases that are defined by their

Hermann Bothe; Oliver Schmitz; M. Geoffrey Yates; William E. Newton

52

Fe@Fe2O3 core-shell nanowires enhanced Fenton oxidation by accelerating the Fe(III)/Fe(II) cycles.  

PubMed

In this study we demonstrate Fe@Fe2O3 core-shell nanowires can improve Fenton oxidation efficiency by two times with rhodamine B as a model pollutant at pH > 4. Active species trapping experiments revealed that the rhodamine B oxidation enhancement was attributed to molecular oxygen activation induced by Fe@Fe2O3 core-shell nanowires. The molecular oxygen activation process could generate superoxide radicals to assist iron core for the reduction of ferric ions to accelerate the Fe(III)/Fe(II) cycles, which favored the H2O2 decomposition to produce more hydroxyl radicals for the rhodamine B oxidation. The combination of Fe@Fe2O3 core-shell nanowires and ferrous ions (Fe@Fe2O3/Fe(2+)) offered a superior Fenton catalyst to decompose H2O2 for producing OH. We employed benzoic acid as a probe reagent to check the generation of OH and found the OH generation rate of Fe@Fe2O3/Fe(2+) was 2-4 orders of magnitude larger than those of commonly used iron based Fenton catalysts and 38 times that of Fe(2+). The reusability and the stability of Fe@Fe2O3 core-shell nanowires were studied. Total organic carbon and ion chromatography analyses revealed the mineralization of rhodamine B and the releasing of nitrate ions. Gas chromatograph-mass spectrometry was used to investigate the degradation intermediates to propose the possible rhodamine B Fenton oxidation pathway in the presence of Fe@Fe2O3 nanowires. This study not only provides a new Fenton oxidation system for pollutant control, but also widen the application of molecular oxygen activation induced by nanoscale zero valent iron. PMID:24793112

Shi, Jingu; Ai, Zhihui; Zhang, Lizhi

2014-08-01

53

Nitrogenase — hydrogenase relationships in Rhizobium japonicum  

Microsoft Academic Search

The conditions necessary for coordinate derepression of nitrogenase and O2-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate a-ketoglutarate was able to

Lennox A. Graham; Larry W. Stults; R. J. Maier

1984-01-01

54

Effect of Secondary Interactions on the Fundamental Properties of Small Molecule Models of the Diiron Hydrogenase Active Site  

E-print Network

of the model. The X-ray crystal structure of the inclusion complex, Na (?-SCH2N(C6H4SO3-)CH2S-)[Fe(CO)3]2?2 ?-cyclodextrin shows complete enclosure of the diiron model within two cyclodextrin units. Solution studies support the formation of an inclusion complex...

Singleton, Michael Lee

2012-02-14

55

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

PubMed

Parent complex (?-PDTe)Fe(2)(CO)(6) (1, PDTe = ?-TeCH(2)CH(2)CH(2)Te-?) is prepared via a new synthetic route involving the reaction of (?-Te(2))Fe(2)(CO)(6) with Et(3)BHLi, followed by treatment of (?-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 (?-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 (?-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 (?-PDTe)Fe(2)(CO)(4)(dppm) (6) and (?-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 [(?-PDTe)Fe(2)(CO)(5)](2)(dppp) (8), [(?-PDTe)Fe(2)(CO)(5)](2)(dppb) (9), and [(?-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. PMID:23143609

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

2013-02-01

56

The [4Fe-4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies.  

PubMed

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms. PMID:22985598

Berto, Paola; Di Valentin, Marilena; Cendron, Laura; Vallese, Francesca; Albertini, Marco; Salvadori, Enrico; Giacometti, Giorgio M; Carbonera, Donatella; Costantini, Paola

2012-12-01

57

[NiFe] hydrogenases: structural and spectroscopic studies of the reaction mechanism.  

PubMed

[NiFe] hydrogenases catalyze the reversible oxidation of dihydrogen. For this simple reaction the molecule has developed a complex catalytic mechanism, during which the enzyme passes through various redox states. The [NiFe] hydrogenase contains several metal centres, including the bimetallic Ni-Fe active site, iron-sulfur clusters and a Mg(2+) ion. The Ni-Fe active site is located in the inner part of the protein molecule, therefore a number of pathways are involved in the catalytic reaction route. These consist of an electron transfer pathway, a proton transfer pathway and a gas-access channel. Over the last 10-15 years we have been investigating the crystal structures of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F, which is a sulfate-reducing anaerobic bacterium. So far the crystal structures of the oxidized, H(2)-reduced and carbon monoxide inhibited states have been determined at high resolution and have revealed a rather unique structure of the hetero-bimetallic Ni-Fe active site. Furthermore, intensive spectroscopic studies have been performed on the enzyme. Based on the crystal structure, a water-soluble Ni-Ru complex has been synthesized as a functional model for the [NiFe] hydrogenases. The present review gives an overview of the catalytic reaction mechanism of the [NiFe] hydrogenases. PMID:19759926

Ogata, Hideaki; Lubitz, Wolfgang; Higuchi, Yoshiki

2009-10-01

58

The phylogeny of uptake hydrogenases in Frankia.  

PubMed

Uptake hydrogenase is an enzyme that is beneficial for nitrogen fixation in bacteria. Recent studies have shown that Frankia sp. has two sets of uptake hydrogenase genes, organized in synton 1 and synton 2. In the present study, phylogenetic analysis of the structural subunits of hydrogenase syntons 1 and 2 showed a distinct clustering pattern between the proteins of Frankia strains that were isolated from different host plants and non-Frankia organisms. The structural subunits of hydrogenase synton 1 of Frankia sp. CpI1, Frankia alni ACN14a, and F. alni AvCI1 were grouped together while those of Frankia spp. CcI3, KB5, UGL140104, and UGL011102 formed another group. The structural subunits of hydrogenase synton 2 of F. alni ACN14a and Frankia spp. CcI3 and BCU110501 grouped together, but those of Frankia spp. KB5 and CpI1, F. alni ArI3, and F. alniAvCI1 comprised a separate group. The structural subunits of hydrogenase syntons 1 and 2 of Frankia sp. EAN1pec were more closely related to those of non-Frankia bacteria, i.e., Streptomyces avermitilis and Anaeromyxobacter sp., respectively, than to those of other Frankia strains, suggesting the occurrence of lateral gene transfer between these organisms. In addition, the accessory Hyp proteins of hydrogenase syntons 1 and 2 of F. alni ACN14a and Frankia sp. CcI3 were shown to be phylogenetically more related to each other than to those of Frankia EAN1pec. PMID:19440980

Leul, Melakeselam; Normand, Philippe; Sellstedt, Anita

2009-03-01

59

[NiFe] hydrogenases: how close do structural and functional mimics approach the active site?  

PubMed

Hydrogen is being considered as a versatile alternative fuel with the ever increasing energy demand and oil prices. Hydrogenases (H2ases) found in bacteria, archaea and eukaryotes are very efficient catalysts for biological hydrogen production. An important and unique hydrogenase enzyme is the [NiFe] H2ase, with an unusual heterobimetallic site. Since the determination of its crystal structure, a variety of complexes have been synthesised and studied. Bioinspired and biomimetic complexes have been investigated as potential catalysts. So far, of all the reported complexes only a few of them have been found to be catalytically active. Moreover, most of the reports are on the reverse reaction, e.g. proton reduction rather than dihydrogen oxidation. This perspective article therefore reviews the structural and functional aspects of the very recently reported model complexes that mimic the [NiFe] hydrogenase active site either in structure or function or both. PMID:24846119

Kaur-Ghumaan, Sandeep; Stein, Matthias

2014-07-01

60

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

PubMed

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. PMID:24579799

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

2014-04-01

61

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

PubMed Central

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 Ĺ 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 PMID:24569482

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

2014-01-01

62

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

SciTech Connect

[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.

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

63

Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity  

E-print Network

1 Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased model of anaerobic metabolism in this and potentially other algae. Chlamydomonas reinhardtii

64

A model for the CO-inhibited form of [NiFe] hydrogenase: synthesis of (CO)3Fe(?-StBu)3Ni{SC6H3-2,6-(mesityl)2} and reversible CO addition at the Ni site  

PubMed Central

A [NiFe] hydrogenase model compound having a distorted trigonal-pyramidal nickel center, (CO)3Fe(?-StBu)3Ni(SDmp), 1 (Dmp = C6H3-2,6-(mesityl)2), was synthesized from the reaction of the tetranuclear Fe-Ni-Ni-Fe complex [(CO)3Fe(?-StBu)3Ni]2(?-Br)2, 2 with NaSDmp at -40 °C. The nickel site of complex 1 was found to add CO or CNtBu at -40 °C to give (CO)3Fe(StBu)(?-StBu)2Ni(CO)(SDmp), 3, or (CO)3Fe(StBu)(?-StBu)2Ni(CNtBu)(SDmp), 4, respectively. One of the CO bands of 3, appearing at 2055 cm-1 in the infrared spectrum, was assigned as the Ni-CO band, and this frequency is comparable to those observed for the CO-inhibited forms of [NiFe] hydrogenase. Like the CO-inhibited forms of [NiFe] hydrogenase, the coordination of CO at the nickel site of 1 is reversible, while the CNtBu adduct 4 is more robust. PMID:20147622

Ohki, Yasuhiro; Yasumura, Kazunari; Ando, Masaru; Shimokata, Satoko; Tatsumi, Kazuyuki

2010-01-01

65

Merging [FeFe]-Hydrogenases with Materials and Nanomaterials as Biohybrid Catalysts for Solar H2 Production  

SciTech Connect

The catalysts commonly used for the H{sub 2} producing reaction in artificial solar systems are typically platinum or particulate platinum composites. Biological catalysts, the hydrogenases, exist in a wide-variety of microbes and are biosynthesized from abundant, non-precious metals. By virtue of a unique catalytic metallo-cluster that is composed of iron and sulfur, [FeFe]-hydrogenases are capable of catalyzing H{sub 2} production at turnover rates of millimoles-per-second. In addition, these biological catalysts possess some of the characteristics that are desired for cost-effective solar H{sub 2} production systems, high solubilities in aqueous solutions and low activation energies, but are sensitive to CO and O{sub 2}. We are investigating ways to merge [FeFe]-hydrogenases with a variety of organic materials and nanomaterials for the fabrication of electrodes and biohybrids as catalysts for use in artificial solar H{sub 2} production systems. These efforts include designs that allow for the integration of [FeFe]-hydrogenase in dye-solar cells as models to measure solar conversion and H{sub 2} production efficiencies. In support of a more fundamental understanding of [FeFe]-hydrogenase for these and other applications the role of protein structure in catalysis is being investigated. Currently there is little known about the mechanism of how these and other enzymes couple multi-electron transfer to proton reduction. To further the mechanistic understanding of [FeFe]-hydrogenases, structural models for substrate transfer are being used to create enzyme variants for biochemical analysis. Here results are presented on investigations of proton-transfer pathways in [FeFe]-hydrogenase and their interaction with single-walled carbon nanotubes.

King, P. W.; Svedruzic, D.; Hambourger, M.; Gervaldo, M.; McDonald, T.; Blackburn, J.; Heben, M.; Gust, D.; Moore, A. L.; Moore, T. A.; Ghirardi, M. L.

2007-01-01

66

Merging [FeFe]-hydrogenases with materials and nanomaterials as biohybrid catalysts for solar H II production  

NASA Astrophysics Data System (ADS)

The catalysts commonly used for the H II producing reaction in artificial solar systems are typically platinum or particulate platinum composites. Biological catalysts, the hydrogenases, exist in a wide-variety of microbes and are biosynthesized from abundant, non-precious metals. By virtue of a unique catalytic metallo-cluster that is composed of iron and sulfur, [FeFe]-hydrogenases are capable of catalyzing H II production at turnover rates of millimoles-per-second. In addition, these biological catalysts possess some of the characteristics that are desired for cost-effective solar H II production systems, high solubilities in aqueous solutions and low activation energies, but are sensitive to CO and O II. We are investigating ways to merge [FeFe]-hydrogenases with a variety of organic materials and nanomaterials for the fabrication of electrodes and biohybrids as catalysts for use in artificial solar H II production systems. These efforts include designs that allow for the integration of [FeFe]-hydrogenase in dye-solar cells as models to measure solar conversion and H II production efficiencies. In support of a more fundamental understanding of [FeFe]-hydrogenase for these and other applications the role of protein structure in catalysis is being investigated. Currently there is little known about the mechanism of how these and other enzymes couple multi-electron transfer to proton reduction. To further the mechanistic understanding of [FeFe]-hydrogenases, structural models for substrate transfer are being used to create enzyme variants for biochemical analysis. Here results are presented on investigations of proton-transfer pathways in [FeFe]-hydrogenase and their interaction with single-walled carbon nanotubes.

King, Paul W.; Svedruzic, Drazenka; Hambourger, Michael; Gervaldo, Miguel; McDonald, Tim; Blackburn, Jeff; Heben, Michael; Gust, Devens; Moore, Ana L.; Moore, Thomas A.; Ghirardi, Maria L.

2007-09-01

67

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase  

PubMed Central

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

2014-01-01

68

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

PubMed

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 ?-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. PMID:23894098

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

2014-07-01

69

Hydrogenases and Hydrogen Metabolism of Cyanobacteria  

PubMed Central

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

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

2002-01-01

70

A bacterial electron-bifurcating hydrogenase.  

PubMed

The Wood-Ljungdahl pathway of anaerobic CO(2) 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

Schuchmann, Kai; Müller, Volker

2012-09-01

71

A Bacterial Electron-bifurcating Hydrogenase*  

PubMed Central

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

Schuchmann, Kai; Müller, Volker

2012-01-01

72

Improvements of the multichannel collimator set-up on ID27, ESRF: applications to the Fe-FeS system  

NASA Astrophysics Data System (ADS)

Sulfur is believed to be an alloying light element in iron-rich planetary cores such as those of the Earth, Mars or Ganymede 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 physical properties of the liquid Fe-FeS alloys in respect to pressure, temperature and sulphur content. A new multichannel collimator set-up 4 has been designed on the High Pressure Beamline ID27, ESRF. The signature from the slits on the diffraction signal could be removed using a transfer image, measured by the comparison of diffraction patterns of tin glass with and without the multichannel collimator set-up. Here we present an application of this set-up to the Fe-FeS system. Partial melting properties of Fe-16%at S samples were investigated at ~5.7 GPa, by in situ X-ray radiography to determine the absence or presence of solid Fe in coexistence with the S-rich liquid. Diffraction patterns of partially molten Fe-16%at S (composition of the liquid changed with increasing temperature) and pure liquid FeS have been acquired up to 5.9 GPa and 1870 K 5. After a careful data analysis, structural properties and densities have been extracted for different sulfur content. Structural properties changes from compact structure at 16%at S to a disordered structure at 50%at S. Density of Fe-S liquids could be fitted using a symmetric regular solution, with a non-ideality parameter constant over the pressure range studied. Such data set could be used to model small planetary cores, such as Ganymede. References 1 C.J. Allčgre, J.P. Poirier, E. Humler et al., Earth Planet. Sc. Lett. 134, 515 (1995). 2 F. Sohl and T. Spohn, J. Geophys. Res. 102 (E1), 1613 (1997). 3 C.F. Yoder, A.S. Konopliv, D.N. Yuan et al., Science 300, 299 (2003). 4 G. Morard, M. Mezouar, S. Bauchau et al., Rev. Sci. Inst., Submitted (2010). 5 G. Morard, M. Mezouar, M. Alvarez-Murga et al., High Press. Res., Submitted (2010).

Mezouar, M.; Morard, G.; Bauchau, S.; Álvarez-Murga, M.; Hodeau, J.; Garbarino, G.

2010-12-01

73

ASSESSING SHOOT-ROOT COMMUNICATION IN THE REGULATION OF IRON HOMEOSTASIS IN THE FEFE MELON MUTANT  

Technology Transfer Automated Retrieval System (TEKTRAN)

The fefe mutant of musk melon exhibits characteristics of iron deficiency such as interveinal chlorosis of leaves, retarded growth, and finally death unless supplemental Fe is provided. The seedlings have normal green cotyledons but the first true leaves are yellow with green veins. To determine the...

74

Hydrogenase activity in the thermophile mastigocladus laminosus  

SciTech Connect

Hydrogenase activity in the thermophilic cyanobacterium, Mastigocladus laminosus was studied both in vivo and in vitro. In vivo hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H/sub 2/-supported acetylene reduction in reductant-limited cultures was a light-dependent, but O/sub 2/-independent reaction. In vitro hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for Anabaena cylindrica.

Benemann, J.R.; Miyamoto, K.; Hallenbeck, P.C.; Murry, M.A.

1982-06-30

75

Activation and de novo synthesis of hydrogenase in Chlamydomonas  

SciTech Connect

Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogenase in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process. Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg/sup 2 +/, Ca/sup 2 +/, and iron does not lead to active hydrogenase formation. Furthermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place. The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.

Roessler, P.G.; Lien, S.

1984-12-01

76

Maturation of [NiFe]-hydrogenases in Escherichia coli.  

PubMed

Hydrogenases catalyze the reversible oxidation of dihydrogen. Catalysis occurs at bimetallic active sites that contain either nickel and iron or only iron and the nature of these active sites forms the basis of categorizing the enzymes into three classes, the [NiFe]-hydrogenases, the [FeFe]-hydrogenases and the iron sulfur cluster-free [Fe]-hydrogenases. The [NiFe]-hydrogenases and the [FeFe]-hydrogenases are unrelated at the amino acid sequence level but the active sites share the unusual feature of having diatomic ligands associated with the Fe atoms in the these enzymes. Combined structural and spectroscopic studies of [NiFe]-hydrogenases identified these diatomic ligands as CN- and CO groups. Major advances in our understanding of the biosynthesis of these ligands have been achieved primarily through the study of the membrane-associated [NiFe]-hydrogenases of Escherichia coli. A complex biosynthetic machinery is involved in synthesis and attachment of these ligands to the iron atom, insertion of the Fe(CN)2CO group into the apo-hydrogenase, introduction of the nickel atom into the pre-formed active site and ensuring that the holoenzyme is correctly folded prior to delivery to the membrane. Although much remains to be uncovered regarding each of the individual biochemical steps on the pathway to synthesis of a fully functional enzyme, our understanding of the initial steps in CN- synthesis have revealed that it is generated from carbamoyl phosphate. What is becoming increasingly clear is that the metabolic origins of the carbonyl group may be different. PMID:17216401

Forzi, Lucia; Sawers, R Gary

2007-06-01

77

Hydrogenase in actinorhizal root nodules and root nodule homogenates.  

PubMed

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

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

1980-04-01

78

Force Field Development and Molecular Dynamics of [NiFe] Hydrogenase  

SciTech Connect

Classical molecular force-field parameters describing the structure and motion of metal clusters in [NiFe] hydrogenase enzymes can be used to compare the dynamics and thermodynamics of [NiFe] under different oxidation, protonation, and ligation circumstances. Using density functional theory (DFT) calculations of small model clusters representative of the active site and the proximal, medial, and distal Fe/S metal centers and their attached protein side chains, we have calculated classical force-field parameters for [NiFe] in reduced and oxidized states, including internal coordinates, force constants, and atom-centered charges. Derived force constants revealed that cysteinate ligands bound to the metal ions are more flexible in the Ni-B active site, which has a bridging hydroxide ligand, than in the Ni-C active site, which has a bridging hydride. Ten nanosecond all-atom, explicit-solvent MD simulations of [NiFe] hydrogenase in oxidized and reduced catalytic states established the stability of the derived force-field parameters in terms of C{alpha} and metal cluster fluctuations. Average active site structures from the protein MD simulations are consistent with [NiFe] structures from the Protein Data Bank, suggesting that the derived force-field parameters are transferrable to other hydrogenases beyond the structure used for testing. A comparison of experimental H{sub 2}-production rates demonstrated a relationship between cysteinate side chain rotation and activity, justifying the use of a fully dynamic model of [NiFe] metal cluster motion.

Smith, Dayle MA; Xiong, Yijia; Straatsma, TP; Rosso, Kevin M.; Squier, Thomas C.

2012-05-09

79

Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene  

SciTech Connect

Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H{sub 2}S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen or PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H{sub 2}S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bioprocessing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology.

Woodward, C.A.; Kaufman, E.N. [Oak Ridge National Lab., TN (United States)] [Oak Ridge National Lab., TN (United States)

1996-11-05

80

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

PubMed Central

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

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

1987-01-01

81

Small molecule mimics of hydrogenases: hydrides and redox†  

PubMed Central

This tutorial review is aimed at chemical scientists interested in understanding and exploiting the remarkable catalytic behavior of the hydrogenases. The key structural features are analyzed for the active sites of the two most important hydrogenases. Reactivity is emphasized, focusing on mechanism and catalysis. Through this analysis, gaps are identified in the synthesis of functional replicas of these fascinating and potentially useful enzymes. PMID:19088969

Gloaguen, Frédéric; Rauchfuss, Thomas B.

2012-01-01

82

[FeFe]-hydrogenases and photobiological hydrogen production  

NASA Astrophysics Data System (ADS)

The promise of efficient, economic and renewable H II photoproduction from water can potentially be met by green algae. These organisms are able to functionally link photosynthetic water oxidation to the catalytic recombination of protons and electrons to generate H II gas through the activity of the hydrogenase enzyme. Green algal hydrogenases contain a unique metallo-catalytic H-cluster that performs the reversible H II oxidation /evolution reactions. The H-cluster, located in the interior of the protein structure is irreversibly inactivated by O II, the by-product of water oxidation. We developed an Escherichi coli expression system to produce [FeFe]-hydrogenases from different biological sources and demonstrated that clostridial [FeFe]-hydrogenases have higher tolerance to O II inactivation compared to their algal counterparts. We have been using computational simulations of gas diffusion within the Clostridium pasteurianum CpI hydrogenase to identify the pathways through which O II can reach its catalytic site. Subsequently, we modify the protein structure at specific sites along the O II pathways (identified by the computational simulations) by site-directed mutagenesis with the goal of generating recombinant enzymes with higher O II tolerance. In this paper, we review the computational simulation work and report on preliminary results obtained through this strategy.

Ghirardi, Maria L.; Cohen, Jordi; King, Paul; Schulten, Klaus; Kim, Kwiseon; Seibert, Michael

2006-08-01

83

Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production.  

PubMed

Escherichia coli can perform at least two modes of anaerobic hydrogen metabolism and expresses at least two types of hydrogenase activity. Respiratory hydrogen oxidation is catalysed by two 'uptake' hydrogenase isoenzymes, hydrogenase -1 and -2 (Hyd-1 and -2), and fermentative hydrogen production is catalysed by Hyd-3. Harnessing and enhancing the metabolic capability of E. coli to perform anaerobic mixed-acid fermentation is therefore an attractive approach for bio-hydrogen production from sugars. In this work, the effects of genetic modification of the genes encoding the uptake hydrogenases, as well as the importance of preculture conditions, on hydrogen production and fermentation balance were examined. In suspensions of resting cells pregrown aerobically with formate, deletions in Hyd-3 abolished hydrogen production, whereas the deletion of both uptake hydrogenases improved hydrogen production by 37% over the parent strain. Under fermentative conditions, respiratory H2 uptake activity was absent in strains lacking Hyd-2. The effect of a deletion in hycA on H2 production was found to be dependent upon environmental conditions, but H2 uptake was not significantly affected by this mutation. PMID:17995952

Redwood, Mark D; Mikheenko, Iryna P; Sargent, Frank; Macaskie, Lynne E

2008-01-01

84

Comparative characterization of two distinct hydrogenases from Anabaena sp. strain 7120.  

PubMed Central

Two distinct hydrogenases, hereafter referred to as "uptake" and "reversible" hydrogenase, were extracted from Anabaena sp. strain 7120 and partially purified. The properties of the two enzymes were compared in cell-free extracts. Uptake hydrogenase was largely particulate, and although membrane bound, it could catalyze an oxyhydrogen reaction. Particulate and solubilized uptake hydrogenase could catalyze H2 uptake with a variety of artificial electron acceptors which had midpoint potentials above 0 mV. Reversible hydrogenase was soluble, could donate electrons rapidly to electron acceptors of both positive and negative midpoint potential, and could evolve H2 rapidly when provided with reduced methyl viologen. Uptake hydrogenase was irreversibly inactivated by O2, whereas reversible hydrogenase was reversibly inactivated and could be reactivated by exposure to dithionite or H2. Reversible hydrogenase was stable to heating at 70 degrees C, but uptake hydrogenase was inactivated with a half-life of 12 min at this temperature. Uptake hydrogenase was eluted from Sephadex G-200 in a single peak of molecular weight 56,000, whereas reversible hydrogenase was eluted in two peaks with molecular weights of 165,000 and 113,000. CO was competitive with H2 for each enzyme; the Ki's for CO were 0.0095 atm for reversible hydrogenase and 0.039 atm for uptake hydrogenase. The pH optima for H2 evolution and H2 uptake by reversible hydrogenase were 6 and 9, respectively. Uptake hydrogenase existed in two forms with pH optima of 6 and 8.5. Both enzymes had very low Km's for H2, and neither was inhibited by C2H2. PMID:6783615

Houchins, J P; Burris, R H

1981-01-01

85

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

PubMed

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. PMID:24975482

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

2014-09-01

86

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum  

SciTech Connect

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 {sup 14}C-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.

Minamisawa, Kiwamu; Fukai, Kastuhiko; Asami, Teruo (Ibaraki Univ. (Japan))

1990-08-01

87

Nitrogen-doped Fe/Fe3C@graphitic layer/carbon nanotube hybrids derived from MOFs: efficient bifunctional electrocatalysts for ORR and OER.  

PubMed

A novel nitrogen-doped Fe/Fe3C@graphitic layer/carbon nanotube hybrid derived from MOFs has been first fabricated by a facile approach. The hybrid exhibited outstanding bifunctional electrocatalytic activity for ORR and OER, due to the merits of graphitic layer/carbon nanotube structures with highly active N and Fe/Fe3C sites. PMID:25575029

Li, Ji-Sen; Li, Shun-Li; Tang, Yu-Jia; Han, Min; Dai, Zhi-Hui; Bao, Jian-Chun; Lan, Ya-Qian

2015-02-14

88

Distribution and activity of hydrogenase enzymes in subsurface sediments  

NASA Astrophysics Data System (ADS)

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 turnover rate of a specific process like sulfate reduction. However, we can use the cell-specific hydrogenase activity to estimate the size of the metabolically active microbial population. The conversion factors vary according to the predominant electron-accepting process. In subsurface sediment standard methods for quantification of the metabolically active microbial population (e.g. CARD-FISH) are at their lower detection limit. The hydrogenase enzyme activity measurement provides an alternative and sensitive way of quantification.

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

2013-12-01

89

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus  

SciTech Connect

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O{sub 2}-dependent H{sub 2} uptake and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni{sup 2+}. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H{sub 2} uptake activity of the culture at that Ni{sup 2+} concentration. Between 0 and 30 nM added Ni{sup 2+}, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni{sup 2+}. Thus, all of the added Ni{sup 2+} could be accounted for in hydrogenase. Between 0 and 50 nM added Ni{sup 2+}, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni{sup 2+}, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni{sup 2+} in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.

Doyle, C.M.; Arp, D.J. (Univ. of California, Riverside (USA))

1988-09-01

90

Variation in Nitrogenase and Hydrogenase Activity of Alaska Pea Root Nodules 1  

PubMed Central

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H2 and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H2 and was not inhibited by the presence of 0.10 atmosphere C2H2, which prevented H2 evolution from nitrogenase. Total H2 production from nitogenase was estimated as net H2 evolution in air plus H2 exchange in 0.10 atmosphere C2H2. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H2 exchange and H2 uptake activity of hydrogenase, differences observed in H2 exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H2 production by nitrogenase. Total H2 production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H2 production declined, but the potential proportion of nitrogenase-produced H2 recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H2 had significantly higher rates of N2 reduction than those which did not exhibit hydrogenase activity. PMID:16660819

Bethlenfalvay, Gabor J.; Phillips, Donald A.

1979-01-01

91

Hydrogen fuel electrode based on bioelectrocatalysis by the enzyme hydrogenase  

Microsoft Academic Search

Our aim is to show, that the enzymes as electrocatalysts are able to improve the performance characteristics of the fuel cells. The hydrogen fuel electrode based on hydrogenase from Thiocapsa roseopersicina immobilized directly on carbon filament material has been made. The enzyme electrode has operated according to electron tunneling between the enzyme active site and the electrode support; this mechanism

A. A. Karyakin; S. V. Morozov; E. E. Karyakina; S. D. Varfolomeyev; N. A. Zorin; S. Cosnier

2002-01-01

92

Switchable transport strategy to deposit active Fe/Fe3C cores into hollow microporous carbons for efficient chromium removal.  

PubMed

Magnetic hollow structures with microporous shell and highly dispersed active cores (Fe/Fe3 C nanoparticles) are rationally designed and fabricated by solution-phase switchable transport of active iron species combined with a solid-state thermolysis technique, thus allowing selective encapsulation of functional Fe/Fe3 C nanoparticles in the interior cavity. These engineered functional materials show high loading (?54 wt%) of Fe, excellent chromium removal capability (100 mg g(-1)), fast adsorption rate (8766 mL mg(-1) h(-1)), and easy magnetic separation property (63.25 emu g(-1)). During the adsorption process, the internal highly dispersed Fe/Fe3 C nanoparticles supply a driving force for facilitating Cr(VI) diffusion inward, thus improving the adsorption rate and the adsorption capacity. At the same time, the external microporous carbon shell can also efficiently trap guest Cr(VI) ions and protect Fe/Fe3 C nanoparticles from corrosion and subsequent leaching problems. PMID:23749637

Liu, Dong-Hai; Guo, Yue; Zhang, Lu-Hua; Li, Wen-Cui; Sun, Tao; Lu, An-Hui

2013-11-25

93

Oxygen-resistant hydrogenases and methods for designing and making same  

DOEpatents

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.

King, Paul (Golden, CO); Ghirardi, Maria L (Lakewood, CO); Seibert, Michael (Lakewood, CO)

2009-03-10

94

Role of nickel in membrane-bound hydrogenase and nickel metabolism in Rhizobium japonicum  

SciTech Connect

The membrane-bound hydrogenase of Rhizobium japonicum requires nickel for activity. Radioactive /sup 63/Ni co-migrates with hydrogenase activity in native gel systems and co-elutes with purified hydrogenase form an affinity matrix column. A simplified scheme for the purification of hydrogenase has been developed and constitutes the first report of the aerobic purification of this enzyme from R. japonicum. The aerobic purification utilizes the general affinity matrix. Reactive Red 120-agarose and results in higher specific activity and yield of enzyme than previously reported. The stability of aerobically purified hydrogenase to oxygen is substantially greater than that reported for anaerobically isolated enzyme. Reduction of the aerobically purified enzyme in the presence of oxygen, however, results in the rapid loss of activity. R. japonicum cells accumulate nickel during heterotrophic growth and as non-growing cells. The hydrogenase constitutive mutant SR470 accumulates substantially greater amounts of nickel under both conditions. Kinetic studies indicate that the nickel uptake system in the hydrogenase constitutive mutant SR470 is upregulated relative to SRwt cells. The uptake system is specific for nickel, although a 10-fold excess (relative to nickel) of copper or zinc inhibits nickel uptake. The nickel uptake system appears to require energy. Under nickel-free conditions hydrogenase protein is not synthesized as determined by cross-reactivity with antibodies directed against hydrogenase, indicating that nickel regulates the formation of the enzyme as well as being a constituent of the active protein.

Stults, L.W.

1986-01-01

95

Oxygen-resistant hydrogenases and methods for designing and making same  

DOEpatents

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.

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

2014-03-04

96

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

PubMed Central

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

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

2013-01-01

97

Amidine Dications: Isolation and [Fe]-Hydrogenase-Related Hydrogenation  

PubMed Central

This commmunication demonstrates the preparation, isolation, and full characterization of superelectrophilic salts based on amidine dications in organic solvent, as their triflate salts. These dications are highly activated toward regiospecific reaction with hydrogen gas under mild conditions in the presence of a metal catalyst (Pd/C), mimicking the behavior of the natural substrate, N5,N10-methenyltetrahydromethanopterin, in the iron?sulfur cluster-free [Fe]-hydrogenase. PMID:19534467

2009-01-01

98

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

PubMed Central

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 hydrogenases might be useful as marker for bacteria living inside of marine snow particles. PMID:21079771

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

99

Experimental approaches to kinetics of gas diffusion in hydrogenase  

PubMed Central

Hydrogenases, which catalyze H2 to H+ conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these. PMID:18685111

Leroux, Fanny; Dementin, Sébastien; Burlat, Bénédicte; Cournac, Laurent; Volbeda, Anne; Champ, Stéphanie; Martin, Lydie; Guigliarelli, Bruno; Bertrand, Patrick; Fontecilla-Camps, Juan; Rousset, Marc; Léger, Christophe

2008-01-01

100

Competition between electron transfer, trapping, and recombination in CdS nanorod-hydrogenase complexes.  

PubMed

Electron transfer from photoexcited CdS nanorods to [FeFe]-hydrogenase is a critical step in photochemical H2 production by CdS-hydrogenase complexes. By accounting for the distributions in the numbers of electron traps and enzymes adsorbed, we determine rate constants and quantum efficiencies for electron transfer from transient absorption measurements. PMID:25623885

Utterback, James K; Wilker, Molly B; Brown, Katherine A; King, Paul W; Eaves, Joel D; Dukovic, Gordana

2015-02-10

101

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli  

PubMed Central

Background Hydrogenases catalyze reversible reaction between hydrogen (H2) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability. Results Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h·L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (~65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of ~12 nmol H2/(min·mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. Conclusions This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H2 production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions. PMID:20604966

2010-01-01

102

[FeFe]-Hydrogenase-Catalyzed H2 Production in a Photoelectrochemical Biofuel Cell  

SciTech Connect

The Clostridium acetobutylicum [FeFe]-hydrogenase HydA has been investigated as a hydrogen production catalyst in a photoelectrochemical biofuel cell. Hydrogenase was adsorbed to pyrolytic graphite edge and carbon felt electrodes. Cyclic voltammograms of the immobilized hydrogenase films reveal cathodic proton reduction and anodic hydrogen oxidation, with a catalytic bias toward hydrogen evolution. When corrected for the electrochemically active surface area, the cathodic current densities are similar for both carbon electrodes, and 40% of those obtained with a platinum electrode. The high surface area carbon felt/hydrogenase electrode was subsequently used as the cathode in a photoelectrochemical biofuel cell. Under illumination, this device is able to oxidize a biofuel substrate and reduce protons to hydrogen. Similar photocurrents and hydrogen production rates were observed in the photoelectrochemical biofuel cell using either hydrogenase or platinum cathodes.

Hambourger, M.; Gervaldo, M.; Svedruzic, D.; King, P. W.; Gust, D.; Ghirardi, M.; Moore, A. L.; Moore, T. A.

2008-01-01

103

Toward Functional Type?III [Fe]-Hydrogenase Biomimics for H2 Activation: Insights from Computation.  

PubMed

The chemistry of [Fe]-hydrogenase has attracted significant interest due to its ability to activate molecular hydrogen. The intriguing properties of this enzyme have prompted the synthesis of numerous small molecule mimics aimed at activating H2 . Despite considerable effort, a majority of these compounds remain nonfunctional for hydrogenation reactions. By using a recently synthesized model as an entry point, seven biomimetic complexes have been examined through DFT computations to probe the influence of ligand environment on the ability of a mimic to bind and split H2 . One mimic, featuring a bidentate diphosphine group incorporating an internal nitrogen base, was found to have particularly attractive energetics, prompting a study of the role played by the proton/hydride acceptor necessary to complete the catalytic cycle. Computations revealed an experimentally accessible energetic pathway involving a benzaldehyde proton/hydride acceptor and the most promising catalyst. PMID:25649221

Murray, Kevin A; Wodrich, Matthew D; Hu, Xile; Corminboeuf, Clémence

2015-03-01

104

H2 and O2 activation--a remarkable insight into hydrogenase.  

PubMed

This article summarizes the development of a range of organometallic, biomimetic analogues of [NiFe]hydrogenases and their employment in a new generation of H2 -O2 fuel cells. It begins with a summary of O2 -sensitive and O2 -tolerant enzyme chemistry before detailing the properties and functionality of our biomimetic complexes, including: the first ever fully functional model, selective H2 and O2 activation, and the first catalyst using only common metals. These systems are centered on Ni-Fe, Ni-Ru, Ir-Ir, and Rh-Rh cores and use a range of ligands that all follow a set of design principles described herein. PMID:24890792

Ogo, Seiji

2014-06-01

105

Structural and functional investigations of biological catalysts for optimization of solar-driven H II production systems  

NASA Astrophysics Data System (ADS)

Research efforts to develop efficient systems for H II production encompass a variety of biological and chemical approaches. For solar-driven H II production we are investigating an approach that integrates biological catalysts, the [FeFe] hydrogenases, with a photoelectrochemical cell as a novel bio-hybrid system. Structurally the [FeFe] hydrogenases consist of an iron-sulfur catalytic site that in some instances is electronically wired to accessory iron-sulfur clusters proposed to function in electron transfer. The inherent structural complexity of most examples of these enzymes is compensated by characteristics desired for bio-hybrid systems (i.e., low activation energy, high catalytic activity and solubility) with the benefit of utilizing abundant, less costly non-precious metals. Redesign and modification of [FeFe] hydrogenases is being undertaken to reduce complexity and to optimize structural properties for various integration strategies. The least complex examples of [FeFe] hydrogenase are found in the species of photosynthetic green algae and are being studied as design models for investigating the effects of structural minimization on substrate transfer, catalytic activity and oxygen sensitivity. Redesigning hydrogenases for effective use in bio-hybrid systems requires a detailed understanding of the relationship between structure and catalysis. To achieve better mechanistic understanding of [FeFe] hydrogenases both structural and dynamic models are being used to identify potential substrate transfer mechanisms which are tested in an experimental system. Here we report on recent progress of our investigations in the areas of [FeFe] hydrogenase overexpression, minimization and biochemical characterization.

King, Paul W.; Svedruzic, Drazenka; Cohen, Jordi; Schulten, Klaus; Seibert, Michael; Ghirardi, Maria L.

2006-08-01

106

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

NASA Astrophysics Data System (ADS)

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. 102, 1613-1635 (1997). 3. Yoder, C. F., Konopliv, A. S., Yuan, D. N., Standish, E. M. & Folkner, W. M. Fluid core size of Mars from detection of the solar tide. Science 300, 299-303 (2003). 4. Morard, G. et al. Optimization of Paris Edinburgh cell assemblies for in situ monochromatic X-ray diffraction and X-ray absorption. High Press. Res. 27, 1-11 (2007). 5. Morard, G. et al. Structure of eutectic Fe-FeS melts up to 17 GPa: Implications for planetary cores. Earth Planet. Sc. Lett. in press (2007). 6. Sanloup, C. & Fei, Y. Closure of the Fe-S-Si liquid miscibility gap at high pressure. Phys. Earth Plan. Int. 147, 57 (2004). 7. Mezouar, M. et al. Development of a new state-of-the-art beamline optimized for monochromatic single crystal and powder X-ray diffraction under extreme conditions at the ESRF. J. Synch. Rad. 12, 659-664 (2005). 8. Morard, G. et al. Experimental constraints on the Earth's core sulphur content. Nature (Submitted).

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

2007-12-01

107

Structure prediction and molecular simulation of gases diffusion pathways in hydrogenase.  

PubMed

Although hydrogen is considered to be one of the most promising future energy sources and the technical aspects involved in using it have advanced considerably, the future supply of hydrogen from renewable sources is still unsolved. The [Fe]- hydrogenase enzymes are highly efficient H(2) catalysts found in ecologically and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. While these enzymes can occur in several forms, H(2) catalysis takes place at a unique [FeS] prosthetic group or H-cluster, located at the active site. 3D structure of the protein hydA1 hydrogenase from Chlamydomonas reinhardtti was predicted using the MODELER 8v2 software. Conserved region was depicted from the NCBI CDD Search. Template selection was done on the basis NCBI BLAST results. For single template 1FEH was used and for multiple templates 1FEH and 1HFE were used. The result of the Homology modeling was verified by uploading the file to SAVS server. On the basis of the SAVS result 3D structure predicted using single template was chosen for performing molecular simulation. For performing molecular simulation three strategies were used. First the molecular simulation of the protein was performed in solvated box containing bulk water. Then 100 H(2) molecules were randomly inserted in the solvated box and two simulations of 50 and 100 ps were performed. Similarly 100 O(2) molecules were randomly placed in the solvated box and again 50 and 100 ps simulation were performed. Energy minimization was performed before each simulation was performed. Conformations were saved after each simulation. Analysis of the gas diffusion was done on the basis of RMSD, Radius of Gyration and no. of gas molecule/ps plot. PMID:21364783

Sundaram, Shanthy; Tripathi, Ashutosh; Gupta, Vipul

2010-01-01

108

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism  

Microsoft Academic Search

Background  FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases\\u000a are irreversibly inactivated by O2.\\u000a \\u000a \\u000a \\u000a \\u000a Results  We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the

Buz Barstow; Christina M Agapakis; Patrick M Boyle; Gerald Grandl; Pamela A Silver; Edwin H Wintermute

2011-01-01

109

Turning Cellulose Waste Into Electricity: Hydrogen Conversion by a Hydrogenase Electrode  

PubMed Central

Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L-1 h-1 and 1 mM L-1 h-1, respectively. An enzymatic fuel cell (EFC) with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h) to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value. PMID:24312437

Abramov, Sergey M.; Sadraddinova, Elmira R.; Shestakov, Andrey I.; Voronin, Oleg G.; Karyakin, Arkadiy A.; Zorin, Nikolay A.; Netrusov, Alexander I.

2013-01-01

110

Structure and magnetic properties of irradiated Fe/Fe oxide core-shell nanoclusters  

SciTech Connect

A cluster deposition method was used to produce a film of loosely aggregated particles of Fe-Fe3O4 core-shell nanoclusters with an 8 nm iron core size and 2 nm oxide shell thickness. The film of particles on a silicon substrate was irradiated with 5.5 MeV Si2+ ions to a fluence of 1016 cm-2 near room temperature, and computer simulations based on the SRIM (Stopping and Range of Ions in Matter) code show that the implanted Si species stops near the film-substrate interface. The ion irradiation creates a structural change in the film with corresponding chemical and magnetic changes. X-ray diffraction shows that the core size and chemistry stay the same but the shell becomes FeO that grows to a thickness of 17 nm. Helium ion microscopy shows that the previously separate particles have densified into a nearly continuous film. Major loop magnetic hysteresis measurements show a decrease in saturation magnetization that we attribute to the presence of the antiferromagnetic (AFM) FeO shell. First-order reversal curve measurements on the irradiated film performed with a vibrating sample magnetometer show that the AFM shell prevents the particles from interacting magnetically, leading to low coercivity from the iron core and little bias field from the core interactions. These results, and others reported previously on different compositions (Fe3O4 or FeO+Fe3N nanoclusters), show that the ion irradiation behavior of nanocluster films such as these depends strongly on the initial nanostructure and chemistry.

McCloy, John S.; Jiang, Weilin; Sundararajan, Jennifer A.; Qiang, You; Burks, Edward; Liu, Kai

2013-04-25

111

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

NASA Astrophysics Data System (ADS)

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.

Buono, Antonio S.; Walker, David

2015-04-01

112

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

USGS Publications Warehouse

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.

Brett, R.; Bell, P.M.

1969-01-01

113

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra.  

PubMed

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation. PMID:2766117

Murry, M A; Lopez, M F

1989-06-01

114

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

SciTech Connect

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.

Arp, D.J.

1997-10-01

115

Function of Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough? †  

PubMed Central

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

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

2007-01-01

116

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

SciTech Connect

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.

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

2007-09-24

117

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

SciTech Connect

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.

Arp, D.J.

1991-01-01

118

Gd[subscript 13]Fe[subscript 10]C[subscript 13]: Indications of Fe?Fe Multiple Bonding Emerging from Chemical Frustration  

SciTech Connect

We report the synthesis and crystal structure of the carbide Gd{sub 13}Fe{sub 10}C{sub 13}. This compound adopts a new structure type that is remarkable for its 'H'-shaped C{sub 2}FeFeC{sub 2} units, which have some of the shortest Fe-Fe contacts known. A bonding analysis using DFT-calibrated Hueckel calculations hints that Fe-Fe multiple bonding underlies these short distances. Gd{sub 13}Fe{sub 10}C{sub 13} undergoes ferromagnetic ordering at {approx}55 K.

Hadler, Amelia B.; Fredrickson, Daniel C. (UW)

2012-10-25

119

Synthesis and vibrational spectroscopy of 57Fe-labeled models of [NiFe] hydrogenase: first direct observation of a nickel–iron interaction† †Electronic supplementary information (ESI) available: Experimental procedures, spectral data, computational chemistry details, animated vibrational modes as GIFs. See DOI: 10.1039/c4cc04572f Click here for additional data file. Click here for additional data file.  

PubMed Central

A new route to iron carbonyls has enabled synthesis of 57Fe-labeled [NiFe] hydrogenase mimic (OC)3 57Fe(pdt)Ni(dppe). Its study by nuclear resonance vibrational spectroscopy revealed Ni–57Fe vibrations, as confirmed by calculations. The modes are absent for [(OC)3 57Fe(pdt)Ni(dppe)]+, which lacks Ni–57Fe bonding, underscoring the utility of the analyses in identifying metal–metal interactions. PMID:25237680

Pelmenschikov, Vladimir; Wang, Hongxin; Meier, Florian; Gee, Leland B.; Yoda, Yoshitaka; Kaupp, Martin; Rauchfuss, Thomas B.

2014-01-01

120

Reduction of Technetium(VII) by Desulfovibrio fructosovorans Is Mediated by the Nickel-Iron Hydrogenase  

PubMed Central

Resting cells of the sulfate-reducing bacterium Desulfovibrio fructosovorans grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase. The role of cytochrome c3 in the mechanism is also discussed. PMID:11571159

De Luca, Gilles; de Philip, Pascale; Dermoun, Zorah; Rousset, Marc; Verméglio, André

2001-01-01

121

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

SciTech Connect

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.

Changlin Fu; Maier, R.J. (John Hopkins Univ., Baltimore, MD (United States))

1991-12-01

122

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

SciTech Connect

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.

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

2014-02-01

123

Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis? †  

PubMed Central

In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs. PMID:20656864

Lim, Jae Kyu; Kang, Sung Gyun; Lebedinsky, Alexander V.; Lee, Jung-Hyun; Lee, Hyun Sook

2010-01-01

124

Hybrid molecular assemblies composed of hydrogenase enzymes and quantum dots helps to pave the way for the  

E-print Network

Hybrid molecular assemblies composed of hydrogenase enzymes and quantum dots helps to pave the way selectivity and fast turnover of hydrogenase enzymes to achieve light-driven hydrogen (H2) production was maximal at low enzyme coverages favoring one-to-one ratios. The efficiency of photocatalytic H2 production

125

Selenium increases hydrogenase expression is autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme  

SciTech Connect

The authors have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 {mu}M significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO{sub 3}{sup 2{minus}} enhanced the time course of hydrogenase derepression by 133%, whereas VO{sub 3}, AsO{sub 2}{sup 2{minus}}, SO{sub 2}{sup 2{minus}}, and TeO{sub 3}{sup 2{minus}} failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 {plus minus} 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H{sub 2} metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.

Boursier, P.; Hanus, F.J.; Papen, H.; Becker, M.M.; Russell, S.A.; Evans, H.J. (Oregon State Univ., Corvallis (USA))

1988-12-01

126

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

SciTech Connect

Hydrogenases are enzymes which catalyze reactions involving dihydrogen. They serve integral roles in a number of microbial metabolic pathways. Our research is focussed on investigations of the catalytic mechanism of the hydrogenases found in aerobic, N{sub 2}-fixing microorganisms such as Azotobacter vinelandii and the agronomically important Bradyrhizobium japonicum as well as microorganisms with similar hydrogenases. The hydrogenases isolated from these microorganisms are Ni- and Fe-containing heterodimers. Our work has focussed on three areas during the last grant period. In all cases, a central theme has been the role of inhibitors in the characteristics under investigation. In addition, a number of collaborative efforts have yielded interesting results. In metalloenzymes such as hydrogenase, inhibitors often influence the activity of the enzyme through ligand interactions with redox centers, often metals, within the enzyme. Therefore, investigations of the ability of various compounds to inhibit an enzyme's activity, as well as the mechanism of inhibition, can provide insight into the catalytic mechanism of the enzyme as well as the role of various redox centers in catalysis. We have investigated in detail four inhibitors of A. vinelandii and the results are summarized here. The influence of these inhibitors on the spectral properties of the enzyme are summarized. Electron paramagnetic resonance and ultraviolet spectra investigations are discussed. 9 figs.

Arp, D.J.

1990-01-01

127

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

PubMed Central

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 ?hupF 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

2012-01-01

128

Advances in the function and regulation of hydrogenase in the cyanobacterium Synechocystis PCC6803.  

PubMed

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

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

2014-01-01

129

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

PubMed Central

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

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

2014-01-01

130

Acetylene is an active-site-directed, slow-binding, reversible inhibitor of Azotobacter vinelandii hydrogenase  

SciTech Connect

The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H/sub 2/ and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 ..mu..M were derived for these gases, respectively. Both H/sub 2/-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 were also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria.

Hyman, M.R.; Arp, D.J.

1987-10-06

131

Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation.  

PubMed

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif. PMID:10727938

Theodoratou, E; Paschos, A; Magalon, A; Fritsche, E; Huber, R; Böck, A

2000-04-01

132

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

SciTech Connect

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.

Bolin, J.T.

1994-05-01

133

Flow-FISH analysis and isolation of clostridial strains in an anaerobic semi-solid bio-hydrogen producing system by hydrogenase gene target.  

PubMed

By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste. PMID:17277963

Jen, Chang Jui; Chou, Chia-Hung; Hsu, Ping-Chi; Yu, Sian-Jhong; Chen, Wei-En; Lay, Jiunn-Jyi; Huang, Chieh-Chen; Wen, Fu-Shyan

2007-04-01

134

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

PubMed Central

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

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

2012-01-01

135

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

PubMed

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. PMID:25747389

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

2015-04-01

136

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

SciTech Connect

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.

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

137

Electrochemical sensing the DNA damage in situ induced by a cathodic process based on Fe@Fe 2O 3 core–shell nanonecklace and Au nanoparticles mimicking metal toxicity pathways in vivo  

Microsoft Academic Search

Sensitive electrochemical sensing for the DNA damage in situ based on a cathodic process of Fe@Fe2O3 core–shell nanonecklace and Au nanoparticles was performed by a novel biosensor, which was constructed via a glassy carbon electrode (GCE) modified with a multilayer film comprising of separate layers of poly(dimethyldiallylammonium chloride) (PDDA), the mixture of Fe@Fe2O3 core–shell nanonecklace and Au nanoparticles, PDDA and

Xueliang Wang; Tao Yang; Kui Jiao

2009-01-01

138

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

PubMed Central

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 (?hupL), and we determined how this would affect the amount of H2 produced. The ?hupL 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

Zhang, Xiaohui; Sherman, Debra M.

2014-01-01

139

Proton electroreduction catalyzed by cobaloximes: functional models for hydrogenases.  

PubMed

Cobaloximes have been examined as electrocatalysts for proton reduction in nonaqueous solvent in the presence of triethylammonium chloride. [Co(III)(dmgH)2pyCl], working at moderate potentials (-0.90 V/(Ag/AgCl/3 mol x L(-1) NaCl) and in neutral conditions, is a promising catalyst as compared to other first-row transition metal complexes which generally function at more negative potentials and/or at lower pH. More than 100 turnovers can be achieved during controlled-potential electrolysis without detectable degradation of the catalyst. Cyclic voltammograms simulation is consistent with a heterolytic catalytic mechanism and allowed us to extract related kinetic parameters. Introduction of an electron-donating (electron-withdrawing) substituent in the axial pyridine ligand significantly increases (decreases) the rate constant of the catalytic cycle determining step. This effect linearly correlates with the Hammet coefficients of the introduced substituents. The influence of the equatorial glyoxime ligand was also investigated and the capability of the stabilized BF2-bridged species [Co(dmgBF2)2(OH2)2] for electrocatalyzed hydrogen evolution confirmed. PMID:15962987

Razavet, Mathieu; Artero, Vincent; Fontecave, Marc

2005-06-27

140

The [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough is a bacterial lipoprotein lacking a typical lipoprotein signal peptide.  

PubMed

Desulfovibrio vulgaris Hildenborough has a membrane-bound [NiFeSe] hydrogenase whose mode of membrane association was unknown since it is constituted by two hydrophilic subunits. This work shows that this hydrogenase is a bacterial lipoprotein bound to the membrane by lipidic groups found at the N-terminus of the large subunit, which is unusual since it is missing the typical lipoprotein signal peptide. Nevertheless, the large subunit has a conserved four residue lipobox and its synthesis is sensitive to the signal peptidase II inhibitor globomycin. The D. vulgaris [NiFeSe] hydrogenase is the first example of a bacterial lipoprotein translocated through the Tat pathway. PMID:17601576

Valente, Filipa M A; Pereira, Patrícia M; Venceslau, Sofia S; Regalla, Manuela; Coelho, Ana V; Pereira, Inęs A C

2007-07-24

141

A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage  

NASA Astrophysics Data System (ADS)

Hydrogenases are nature's efficient catalysts for both the generation of energy via oxidation of molecular hydrogen and the production of hydrogen via the reduction of protons. However, their O2 sensitivity and deactivation at high potential limit their applications in practical devices, such as fuel cells. Here, we show that the integration of an O2-sensitive hydrogenase into a specifically designed viologen-based redox polymer protects the enzyme from O2 damage and high-potential deactivation. Electron transfer between the polymer-bound viologen moieties controls the potential applied to the active site of the hydrogenase and thus insulates the enzyme from excessive oxidative stress. Under catalytic turnover, electrons provided from the hydrogen oxidation reaction induce viologen-catalysed O2 reduction at the polymer surface, thus providing self-activated protection from O2. The advantages of this tandem protection are demonstrated using a single-compartment biofuel cell based on an O2-sensitive hydrogenase and H2/O2 mixed feed under anode-limiting conditions.

Plumeré, Nicolas; Rüdiger, Olaf; Oughli, Alaa Alsheikh; Williams, Rhodri; Vivekananthan, Jeevanthi; Pöller, Sascha; Schuhmann, Wolfgang; Lubitz, Wolfgang

2014-09-01

142

Structural foundations for the O2 resistance of Desulfomicrobium baculatum [NiFeSe]-hydrogenase.  

PubMed

This study shows how the NiFeSe site of an anaerobically purified O2-resistant hydrogenase reacts with air to give a seleninate as the first product. Less oxidized states of the active site are readily reduced in the presence of X-rays. Reductive enzyme activation requires an efficient pathway for water escape. PMID:23811828

Volbeda, Anne; Amara, Patricia; Iannello, Marina; De Lacey, Antonio L; Cavazza, Christine; Fontecilla-Camps, Juan Carlos

2013-08-14

143

Designed Surface Residue Substitutions in [NiFe] Hydrogenase that Improve Electron Transfer Characteristics  

PubMed Central

Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii “Deep Ecotype” [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme. PMID:25603181

Yonemoto, Isaac T.; Smith, Hamilton O.; Weyman, Philip D.

2015-01-01

144

Hydrogenases in green algae: do they save the algae's life and solve our energy problems?  

Microsoft Academic Search

Green algae are the only known eukaryotes with both oxygenic photosynthesis and a hydrogen metabolism. Recent physiological and genetic discoveries indicate a close connection between these metabolic pathways. The anaerobically inducible hydA genes of algae encode a special type of highly active [Fe]-hydrogenase. Electrons from reducing equivalents generated during fermentation enter the photosynthetic electron transport chain via the plastoquinone pool.

Thomas Happe; Anja Hemschemeier; Martin Winkler; Annette Kaminski

2002-01-01

145

A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage.  

PubMed

Hydrogenases are nature's efficient catalysts for both the generation of energy via oxidation of molecular hydrogen and the production of hydrogen via the reduction of protons. However, their O2 sensitivity and deactivation at high potential limit their applications in practical devices, such as fuel cells. Here, we show that the integration of an O2-sensitive hydrogenase into a specifically designed viologen-based redox polymer protects the enzyme from O2 damage and high-potential deactivation. Electron transfer between the polymer-bound viologen moieties controls the potential applied to the active site of the hydrogenase and thus insulates the enzyme from excessive oxidative stress. Under catalytic turnover, electrons provided from the hydrogen oxidation reaction induce viologen-catalysed O2 reduction at the polymer surface, thus providing self-activated protection from O2. The advantages of this tandem protection are demonstrated using a single-compartment biofuel cell based on an O2-sensitive hydrogenase and H2/O2 mixed feed under anode-limiting conditions. PMID:25143219

Plumeré, Nicolas; Rüdiger, Olaf; Oughli, Alaa Alsheikh; Williams, Rhodri; Vivekananthan, Jeevanthi; Pöller, Sascha; Schuhmann, Wolfgang; Lubitz, Wolfgang

2014-09-01

146

Hydrogenases, their cloning, preparation, characterization and modification for use in photobioreactors. Final report  

SciTech Connect

The screen for hydrogen evolving activity involved some 40 organisms and strains including photosynthetic bacteria, sulfate reducers, several thermophilic blue green algae, as well as soil bacteria such as Alcaligenes eutrophus and various clostridia. Desulfovibrio vulgaris was found to produce abundant readily extracted hydrogenease of very high specific activity. Attempts at molecular cloning of this hydrogenase gene are described.

Not Available

1984-01-01

147

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...

148

Polymyxin-Coated Au and Carbon Nanotube Electrodes for Stable [NiFe]-Hydrogenase Film Voltammetry  

E-print Network

Polymyxin-Coated Au and Carbon Nanotube Electrodes for Stable [NiFe]-Hydrogenase Film Voltammetry report on the use of polymyxin (PM), a cyclic cationic lipodecapeptide, as an electrode modifier-planegraphite(EPG)withthecycliccationic lipodecapeptide polymyxin7 (PM, Chart 1) has been particularly successful in obtaining an active enzyme coverage.2

Dekker, Cees

149

Importance of the Protein Framework for Catalytic Activity of [FeFe]-Hydrogenases  

PubMed Central

The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met415 and CrHydA1Lys228 resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet353 (CrHydA1Met223) and CpICys299 (CrHydA1Cys169) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the Htrans state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM353 (CrHydA1M223) to leucine led to a strong decrease in turnover without affecting the Km value of the electron donor. We suggest that this methionine constitutes a “fine-tuning” element of hydrogenase activity. PMID:22110126

Knörzer, Philipp; Silakov, Alexey; Foster, Carina E.; Armstrong, Fraser A.; Lubitz, Wolfgang; Happe, Thomas

2012-01-01

150

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

NASA Astrophysics Data System (ADS)

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 ?1 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 ?4 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 ?2 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.

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

2013-11-01

151

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

E-print Network

Photosynthetic water splitting, coupled to hydrogenase-catalyzed hydrogen production, is considered a promising clean, renewable source of energy. It is widely accepted that the oxygen sensitivity of hydrogen production, ...

Yacoby, Iftach

152

Reversible Active Site Sulfoxygenation Can Explain the Oxygen Tolerance of a NAD(+)-Reducing [NiFe] Hydrogenase and Its Unusual Infrared Spectroscopic Properties.  

PubMed

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD(+)-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD(+) and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2. PMID:25647259

Horch, Marius; Lauterbach, Lars; Mroginski, Maria Andrea; Hildebrandt, Peter; Lenz, Oliver; Zebger, Ingo

2015-02-25

153

Electrochemical kinetic investigations of the reactions of [FeFe]-hydrogenases with carbon monoxide and oxygen: comparing the importance of gas tunnels and active-site electronic/redox effects.  

PubMed

A major obstacle for future biohydrogen production is the oxygen sensitivity of [FeFe]-hydrogenases, the highly active catalysts produced by bacteria and green algae. The reactions of three representative [FeFe]-hydrogenases with O(2) have been studied by protein film electrochemistry under conditions of both H(2) oxidation and H(2) production, using CO as a complementary probe. The hydrogenases are DdHydAB and CaHydA from the bacteria Desulfovibrio desulfuricans and Clostridium acetobutylicum , and CrHydA1 from the green alga Chlamydomonas reinhardtii . Rates of inactivation depend on the redox state of the active site 'H-cluster' and on transport through the protein to reach the pocket in which the H-cluster is housed. In all cases CO reacts much faster than O(2). In the model proposed, CaHydA shows the most sluggish gas transport and hence little dependence of inactivation rate on H-cluster state, whereas DdHydAB shows a large dependence on H-cluster state and the least effective barrier to gas transport. All three enzymes show a similar rate of reactivation from CO inhibition, which increases upon illumination: the rate-determining step is thus assigned to cleavage of the labile Fe-CO bond, a reaction likely to be intrinsic to the atomic and electronic state of the H-cluster and less sensitive to the surrounding protein. PMID:19824734

Goldet, Gabrielle; Brandmayr, Caterina; Stripp, Sven T; Happe, Thomas; Cavazza, Christine; Fontecilla-Camps, Juan C; Armstrong, Fraser A

2009-10-21

154

[FeFe]-Hydrogenase Maturation: Insights into the Role HydE Plays in Dithiomethylamine Biosynthesis.  

PubMed

HydE and HydG are radical S-adenosyl-l-methionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the nonprotein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxides, three cyanides, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatic, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-l-methionine. Incorporation of deuterium from D2O into 5'-deoxyadenosine, the cleavage product of S-adenosyl-l-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, has allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster is likely derived from HydE's thiol containing substrate. PMID:25654171

Betz, Jeremiah N; Boswell, Nicholas W; Fugate, Corey J; Holliday, Gemma L; Akiva, Eyal; Scott, Anna G; Babbitt, Patricia C; Peters, John W; Shepard, Eric M; Broderick, Joan B

2015-03-10

155

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

PubMed

Hydrogen (H?) 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 H? in microbial communities. In this study, we examined the [FeFe]-hydrogenase gene (hydA) as a proxy for fermentative bacterial H? 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 H?. 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. PMID:20535223

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

2010-12-01

156

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5  

Microsoft Academic Search

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H2), 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 O2 ,C O 2 ,N 2, and H2 was followed simultaneously with a mass

Pia Lindberg; Peter Lindblad; Laurent Cournac

2004-01-01

157

The [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough is a bacterial lipoprotein lacking a typical lipoprotein signal peptide  

Microsoft Academic Search

Desulfovibrio vulgaris Hildenborough has a membrane-bound [NiFeSe] hydrogenase whose mode of membrane association was unknown since it is constituted by two hydrophilic subunits. This work shows that this hydrogenase is a bacterial lipoprotein bound to the membrane by lipidic groups found at the N-terminus of the large subunit, which is unusual since it is missing the typical lipoprotein signal peptide.

Filipa M. A. Valente; Patrícia M. Pereira; Sofia S. Venceslau; Manuela Regalla; Ana V. Coelho; Inęs A. C. Pereira

2007-01-01

158

A simplified method for assay of hydrogenase activities of H 2 evolution and uptake in Enterobacter aerogenes  

Microsoft Academic Search

Assay of hydrogenase activity pertaining to H2 production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobateraerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H2 uptake and evolution activities of the

YunLi Ren; Xin Hui Xing; Chong Zhang; ZhongXuan Gou

2005-01-01

159

Biocatalysts for fuel cells: efficient hydrogenase orientation for H 2 oxidation at electrodes modified with carbon nanotubes  

Microsoft Academic Search

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization\\u000a of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs),\\u000a and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed:\\u000a covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at

É. Lojou; X. Luo; M. Brugna; N. Candoni; S. Dementin; M. T. Giudici-Orticoni

2008-01-01

160

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

PubMed Central

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

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

2010-01-01

161

Effect of H2 Binding on the Nonadiabatic Transition Probability between Singlet and Triplet States of the [NiFe]-Hydrogenase Active Site.  

PubMed

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. PMID:25603170

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

2015-02-12

162

Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: Evidence for a sulfur-reducing hydrogenase ancestor  

SciTech Connect

Microorganisms growing near and above 100[degrees]C have recently been discovered near shallow and deep sea hydrothermal vents. Most are obligately dependent upon the reduction of elemental sulfur (S[sup 0]) to hydrogen sulfide (H[sub 2]S) for optimal growth, even though S[sup 0] reduction readily occurs abiotically at their growth temperatures. The sulfur reductase activity of the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100[degrees]C by a metabolism that produces H[sub 2]S if S[sup 0] is present, was found in the cytoplasm. It was purified anaerobically and was shown to be identical to the hydrogenase that had been previously purified from this organism. Both S[sup 0] and polysulfide served as substrates for H[sub 2]S production, and the S[sub 0] reduction activity but not the H[sub 2]-oxidation activity was enhanced by the redox protein rubredoxin. The H[sub 2]-oxidizing and S[sup 0]-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional [open quotes]sulfhydrogenase[close quotes] enzyme can, therefore, dispose of the excess reductant generated during fermentation using either protons or polysulfides as the electron acceptor. In addition, purified hydrogenases from both hyperthermophilic and mesophilic representatives of the archaeal and bacterial domains were shown to reduce S[sup 0] to H[sub 2]S. It is suggested that the function of some form of ancestral hydrogenase was S[sup 0] reduction rather than, or in addition, to the reduction of protons. 33 refs., 4 figs., 2 tabs.

Ma, K.; Adams, M.W.W. (Univ. of Georgia, Athens (United States)); Schicho, R.N. (Johns Hopkins Univ., Baltimore, MD (United States)); Kelly, R.M. (North Carolina State Univ., Raleigh (United States))

1993-06-01

163

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

NASA Astrophysics Data System (ADS)

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.

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

1996-03-01

164

Two membrane anchors of Wolinella succinogenes hydrogenase and their function in fumarate and polysulfide respiration.  

PubMed

Wolinella succinogenes can grow by anaerobic respiration with fumarate or polysulfide as the terminal electron acceptor, and H2 or formate as the electron donor. A DeltahydABC mutant lacking the hydrogenase structural genes did not grow with H2 and either fumarate or polysulfide. In contrast to the wild-type strain, the mutant grown with fumarate and with formate instead of H2 did not catalyze the reduction of fumarate, polysulfide, dimethylnaphthoquinone, or benzyl viologen by H2. Growth and enzymic activities were restored upon integration of a plasmid carrying hydABC into the genome of the DeltahydABC mutant. The DeltahydABC mutant was complemented with hydABC operons modified by artificial stop codons in hydA (StopA) or at the 5'-end of hydC (StopC). The StopC mutant lacked HydC, and the hydrophobic C-terminus of HydA was missing in the hydrogenase of the StopA mutant. The two mutants catalyzed benzyl viologen reduction by H2. The enzyme activity was located in the membrane of the mutants. A mutant with both modifications (StopAC) contained the activity in the periplasm. The three mutants did not grow with H2 and either fumarate or polysulfide, and did not catalyze dimethylnaphthoquinone reduction by H2. We conclude that the same hydrogenase serves in the anaerobic respiration with fumarate and with polysulfide. HydC and the C-terminus of HydA appear to be required for both routes of electron transport and for dimethylnaphthoquinone reduction by H2. The hydrogenase is anchored in the membrane by HydC and by the C-terminus of HydA. The catalytic subunit HydB is oriented towards the periplasmic side of the membrane. PMID:9639603

Gross, R; Simon, J; Theis, F; Kröger, A

1998-07-01

165

Bacterial Genes Involved in Incorporation of Nickel Into a Hydrogenase Enzyme  

Microsoft Academic Search

Nickel is an essential component of all H_2-uptake hydrogenases. A fragment of DNA that complements a H_2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The

Changlin Fu; Sam Javedan; Farhad Moshiri; Robert J. Maier

1994-01-01

166

Rhizobium leguminosarum hupE Encodes a Nickel Transporter Required for Hydrogenase Activity?  

PubMed Central

Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster (hupSLCDEFGHIJK-hypABFCDEX). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of hupE2, a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX5DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A (hupE hupE2), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (Lens culinaris L.) root nodules but not in pea (Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE. These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions. PMID:20023036

Brito, Belén; Prieto, Rosa-Isabel; Cabrera, Ezequiel; Mandrand-Berthelot, Marie-Andrée; Imperial, Juan; Ruiz-Argüeso, Tomás; Palacios, José-Manuel

2010-01-01

167

Reversible Electrocatalytic Production and Oxidation of Hydrogen at Low Overpotentials by a Functional Hydrogenase Mimic  

SciTech Connect

A new bis(diphosphine) nickel(II) complex, [Ni(PPh2NR2)2](BF4)2, 1, (R = CH2CH2OCH3) is described. A {Delta}G{sup o} of 0.84 kcal/mol{sup -1} for hydrogen addition for this complex was calculated from the experimentally determined equilibrium constant. This complex displays reversible electrocatalytic activity for hydrogen production and oxidation at low overpotentials, a characteristic most commonly associated with hydrogenase enzymes.

Smith, Stuart E.; Yang, Jenny Y.; DuBois, Daniel L.; Bullock, Morris

2012-03-26

168

Studies on the Iron-Sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismane protein  

Microsoft Academic Search

Iron-sulfur clusters are present in a large number of proteins. Sofar structures of four types of protein-bound iron-sulfur clusters have been determined by X-ray diffraction: rubredoxin-like, [2Fe-2S], [3Fe-4S] and [4Fe-4S] centers. The presence of any of these clusters in a protein can be predicted by comparison of spectroscopic properties. However a number of multiple-electron transferring enzymes, like the Fe-only hydrogenase,

A. J. Pierik

1993-01-01

169

Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium Anabaena variabilis ATCC 29413  

Microsoft Academic Search

A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase (hupSL) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation

THOMAS HAPPE; KATHRIN SCHUTZ; HERBERT BOHME

2000-01-01

170

Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase*  

PubMed Central

The O2-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H2 conversion in the presence of ambient O2. Oxygen represents not only a challenge for catalysis but also for the complex assembling process of the [NiFe] active site. Apart from nickel and iron, the catalytic center contains unusual diatomic ligands, namely two cyanides (CN?) and one carbon monoxide (CO), which are coordinated to the iron. One of the open questions of the maturation process concerns the origin and biosynthesis of the CO group. Isotope labeling in combination with infrared spectroscopy revealed that externally supplied gaseous 13CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding 13CO titration experiments showed that a concentration 130-fold higher than ambient CO (0.1 ppmv) caused a 50% labeling of the carbonyl ligand in the [NiFe] hydrogenase, leading to the conclusion that the carbonyl ligand originates from an intracellular metabolite. A novel setup allowed us to the study effects of CO depletion on maturation in vivo. Upon induction of CO depletion by addition of the CO scavenger PdCl2, cells cultivated on H2, CO2, and O2 showed severe growth retardation at low cell concentrations, which was on the basis of partially arrested hydrogenase maturation, leading to reduced hydrogenase activity. This suggests gaseous CO as a metabolic precursor under these conditions. The addition of PdCl2 to cells cultivated heterotrophically on organic substrates had no effect on hydrogenase maturation. These results indicate at least two different pathways for biosynthesis of the CO ligand of [NiFe] hydrogenase. PMID:22049085

Bürstel, Ingmar; Hummel, Philipp; Siebert, Elisabeth; Wisitruangsakul, Nattawadee; Zebger, Ingo; Friedrich, Bärbel; Lenz, Oliver

2011-01-01

171

A soil actinobacterium scavenges atmospheric H2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases.  

PubMed

In the Earth's lower atmosphere, H2 is maintained at trace concentrations (0.53 ppmv/0.40 nM) and rapidly turned over (lifetime ? 2.1 y(-1)). It is thought that soil microbes, likely actinomycetes, serve as the main global sink for tropospheric H2. However, no study has ever unambiguously proven that a hydrogenase can oxidize this trace gas. In this work, we demonstrate, by using genetic dissection and sensitive GC measurements, that the soil actinomycete Mycobacterium smegmatis mc(2)155 constitutively oxidizes subtropospheric concentrations of H2. We show that two membrane-associated, oxygen-dependent [NiFe] hydrogenases mediate this process. Hydrogenase-1 (Hyd1) (MSMEG_2262-2263) is well-adapted to rapidly oxidize H2 at a range of concentrations [Vmax(app) = 12 nmol?g?dw(-1)?min(-1); Km(app) = 180 nM; threshold = 130 pM in the ?hyd23 (Hyd1 only) strain], whereas Hyd2 (MSMEG_2719-2720) catalyzes a slower-acting, higher-affinity process [Vmax(app) = 2.5 nmol?g?dw(-1)?min(-1); Km(app) = 50 nM; threshold = 50 pM in the ?hyd13 (Hyd2 only) strain]. These observations strongly support previous studies that have linked group 5 [NiFe] hydrogenases (e.g., Hyd2) to the oxidation of tropospheric H2 in soil ecosystems. We further reveal that group 2a [NiFe] hydrogenases (e.g., Hyd1) can contribute to this process. Hydrogenase expression and activity increases in carbon-limited cells, suggesting that scavenging of trace H2 helps to sustain dormancy. Distinct physiological roles for Hyd1 and Hyd2 during the adaptation to this condition are proposed. Soil organisms harboring high-affinity hydrogenases may be especially competitive, given that they harness a highly dependable fuel source in otherwise unstable environments. PMID:24591586

Greening, Chris; Berney, Michael; Hards, Kiel; Cook, Gregory M; Conrad, Ralf

2014-03-18

172

[NiFe] hydrogenase from Alteromonas macleodii with unusual stability in the presence of oxygen and high temperature.  

PubMed

Hydrogenases are enzymes involved in the bioproduction of hydrogen, a clean alternative energy source whose combustion generates water as the only end product. In this article we identified and characterized a [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "deep ecotype" with unusual stability toward oxygen and high temperature. The A. macleodii hydrogenase (HynSL) can catalyze both H(2) evolution and H(2) uptake reactions. HynSL was expressed in A. macleodii under aerobic conditions and reached the maximum activity when the cells entered the late exponential phase. The higher level of hydrogenase activity was accompanied by a greater abundance of the HynSL protein in the late-log or stationary phase. The addition of nickel to the growth medium significantly enhanced the hydrogenase activity. Ni treatment affected the level of the protein, but not the mRNA, indicating that the effect of Ni was exerted at the posttranscriptional level. Hydrogenase activity was distributed ?30% in the membrane fraction and ?70% in the cytoplasmic fraction. Thus, HynSL appears to be loosely membrane-bound. Partially purified A. macleodii hydrogenase demonstrated extraordinary stability. It retained 84% of its activity after exposure to 80°C for 2 h. After exposure to air for 45 days at 4°C, it retained nearly 100% of its activity when assayed under anaerobic conditions. Its catalytic activity in the presence of O(2) was evaluated by the hydrogen-deuterium (H-D) exchange assay. In 1% O(2), 20.4% of its H-D exchange activity was retained. The great stability of HynSL makes it a potential candidate for biotechnological applications. PMID:21257809

Vargas, Walter A; Weyman, Philip D; Tong, Yingkai; Smith, Hamilton O; Xu, Qing

2011-03-01

173

Photosynthetic hydrogen production by a hybrid complex of photosystem I and [NiFe]-hydrogenase.  

PubMed

Nature provides key components for generating fuels from renewable resources in the form of enzymatic nanomachines which catalyze crucial steps in biological energy conversion, for example, the photosynthetic apparatus, which transforms solar power into chemical energy, and hydrogenases, capable of generating molecular hydrogen. As sunlight is usually used to synthesize carbohydrates, direct generation of hydrogen from light represents an exception in nature. On the molecular level, the crucial step for conversion of solar energy into H(2) lies in the efficient electronic coupling of photosystem I and hydrogenase. Here we show the stepwise assembly of a hybrid complex consisting of photosystem I and hydrogenase on a solid gold surface. This device gave rise to light-induced H(2) evolution. Hydrogen production is possible at far higher potential and thus lower energy compared to those of previously described (bio)nanoelectronic devices that did not employ the photosynthesis apparatus. The successful demonstration of efficient solar-to-hydrogen conversion may serve as a blueprint for the establishment of this system in a living organism with the paramount advantage of self-replication. PMID:19947646

Krassen, Henning; Schwarze, Alexander; Friedrich, Bärbel; Ataka, Kenichi; Lenz, Oliver; Heberle, Joachim

2009-12-22

174

Proton-Coupled Electron Transfer Dynamics in the Catalytic Mechanism of a [NiFe]-Hydrogenase.  

PubMed

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. PMID:25790178

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

2015-04-01

175

[FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster.  

PubMed

The [FeFe]-hydrogenase catalytic site H cluster is a complex iron sulfur cofactor that is sensitive to oxygen (O2). The O2 sensitivity is a significant barrier for production of hydrogen as an energy source in water-splitting, oxygenic systems. Oxygen reacts directly with the H cluster, which results in rapid enzyme inactivation and eventual degradation. To investigate the progression of O2-dependent [FeFe]-hydrogenase inactivation and the process of H cluster degradation, the highly O2-sensitive [FeFe]-hydrogenase HydA1 from the green algae Chlamydomonas reinhardtii was exposed to defined concentrations of O2 while monitoring the loss of activity and accompanying changes in H cluster spectroscopic properties. The results indicate that H cluster degradation proceeds through a series of reactions, the extent of which depend on the initial enzyme reduction/oxidation state. The degradation process begins with O2 interacting and reacting with the 2Fe subcluster, leading to degradation of the 2Fe subcluster and leaving an inactive [4Fe-4S] subcluster state. This final inactive degradation product could be reactivated in vitro by incubation with 2Fe subcluster maturation machinery, specifically HydF(EG), which was observed by recovery of enzyme activity. PMID:25579778

Swanson, Kevin D; Ratzloff, Michael W; Mulder, David W; Artz, Jacob H; Ghose, Shourjo; Hoffman, Andrew; White, Spencer; Zadvornyy, Oleg A; Broderick, Joan B; Bothner, Brian; King, Paul W; Peters, John W

2015-02-11

176

Theoretical study of dioxygen induced inhibition of [FeFe]-hydrogenase.  

PubMed

Hydrogenases comprise a variety of enzymes that catalyze the reversible oxidation of molecular hydrogen. Out of this group, [FeFe]-hydrogenase shows the highest activity for hydrogen production which is, therefore, of great interest in the field of renewable energies. Unfortunately, this comes with the flaw of a generally very high sensitivity against molecular oxygen that irreversibly inhibits this enzyme. While many studies have already addressed the mechanism of hydrogen formation by [FeFe]-hydrogenase, little is known about the molecular and mechanistic details leading to enzyme inactivation by O(2). In order to elucidate this process, we performed density functional theory calculations on several possible O(2) adducts of the catalytic center--the so-called H-cluster--and show that the direct interaction of the [2Fe](H) subsite with dioxygen is an exothermic and specific reaction in which O(2) most favorably binds in an end-on manner to the distal Fe(d). Based on the results, we propose a protonation mechanism that can explain the irreversibility of dioxygen-induced enzyme inactivation by water release and degradation of the ligand environment of the H-cluster. PMID:19580299

Stiebritz, Martin T; Reiher, Markus

2009-08-01

177

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

NASA Technical Reports Server (NTRS)

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 high pH that make it the unique subject for different biochemical research and detects the possibility for biotechnological application.

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

2004-01-01

178

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

PubMed

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. PMID:25678135

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

2015-04-10

179

The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities  

PubMed Central

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H2-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H2 production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays. PMID:21993396

Marshall, Ian PG; Berggren, Dusty RV; Azizian, Mohammad F; Burow, Luke C; Semprini, Lewis; Spormann, Alfred M

2012-01-01

180

Novel [NiFe]- and [FeFe]-Hydrogenase Gene Transcripts Indicative of Active Facultative Aerobes and Obligate Anaerobes in Earthworm Gut Contents?†  

PubMed Central

The concomitant occurrence of molecular hydrogen (H2) and organic acids along the alimentary canal of the earthworm is indicative of ongoing fermentation during gut passage. Fermentative H2 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 H2 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. PMID:21784904

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

2011-01-01

181

Theoretical studies of [FeFe]-hydrogenase: infrared fingerprints of the dithiol-bridging ligand in the active site.  

PubMed

An unresolved structural issue for [FeFe]-hydrogenases is the nature of the dithiol-bridging ligand in the diiron subcluster of the active site. The two most probable candidates are 1,3-dithiopropane (propane dithiol, PDT) and di-(thiomethyl)-amine (DTN). In the latter case, the dithiol-bridging ligand is assumed to play a major role in the reaction cycle. We report density-functional theory studies of the differing roles of these dithiol-bridging ligands in the infrared spectra of synthetic models and of computational representations of the diiron cluster of the active site. Our analysis shows distinct spectral features associated with the dithiol-bridging NH mode for compounds having a DTN bridge, which, however, would have been obscured by the H2O vibrations in existing measurements. However, if indeed nitrogen is present in the dithiol-bridging ligand, a combination of selective deuteration and chemical inactivation with CO would create a unique signature in an accessible region of the infrared spectrum, whose position and intensity are predicted. PMID:17256840

Zilberman, Silviu; Stiefel, Edward I; Cohen, Morrel H; Car, Roberto

2007-02-19

182

Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules 1  

PubMed Central

A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O2). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H2. The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H2 uptake efficiency of intact nodules. PMID:16660568

Lim, Soo T.

1978-01-01

183

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate.  

PubMed

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H(2)) are also produced. The effect of hydrogenase inhibitors (H(2), carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N(2) to H(2), acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H(2) or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD(+) from NADH by H(2), lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H(2) production for the conversion of synthetic gases to chemicals. PMID:22218768

Li, Hsin-Fen; Knutson, Barbara L; Nokes, Sue E; Lynn, Bert C; Flythe, Michael D

2012-02-01

184

Naphthalene adsorptions on graphene using Cr/Cr2/Fe/Fe2 linkages: Stability and spin perspectives from first-principles calculations  

NASA Astrophysics Data System (ADS)

We present a first-principles study of naphthalene adsorption on graphene via coordination bonds with Cr/Cr2/Fe/Fe2. The obtained structures possess great binding stability, and the geometry alignment of C10H8 is distorted. Especially, the use of Cr/Fe dimer further enhances the binding stability of C10H8 on graphene. From binding energy analysis, the adsorption of C10H8 on metal-graphene is observed to be more favorable than the adsorption of metal-C10H8 on graphene. When empirical dispersion corrections are introduced, the binding energy is improved by 0.78-1.40 eV. Interestingly, various degrees of magnetism are observed with respect to the metal identity, atom/dimer utilization, and bonding interactions.

Bui, Viet Q.; Le, Hung M.

2014-10-01

185

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase  

PubMed Central

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Ĺ and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. PMID:24428762

Bowman, Lisa; Flanagan, Lindsey; Fyfe, Paul K.; Parkin, Alison; Hunter, William N.; Sargent, Frank

2014-01-01

186

Involvement of hyp Gene Products in Maturation of the H2-Sensing [NiFe] Hydrogenase of Ralstonia eutropha  

PubMed Central

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H2-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H2-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of 63Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH. PMID:11717266

Buhrke, Thorsten; Bleijlevens, Boris; Albracht, Simon P. J.; Friedrich, Bärbel

2001-01-01

187

Strategies for reliable and improved large-scale production of Pyrococcus furiosus with integrated purification of hydrogenase I.  

PubMed

The hyperthermophilic archaeon Pyrococcus furiosus is an interesting organism for research and application, especially owing to its unique NADPH-dependent hydrogenase I. However, mass production of P. furiosus through fermentation is susceptible to fault because of its sensitivity to oxygen, a short exponential and stationary phase and a rapid cell lysis in typical cultivation process. In this study, significant improvement for pilot plant scale production processes for P. furiosus biomass was made by investigations of the fermentation process with subsequent hydrogenase I enzyme purification. Scale-up in a 300-L stirred tank bioreactor was successfully achieved. A repeated-batch cultivation process with high reproducibility and productivity was realized. Furthermore, the enzyme hydrogenase I was purified, and its activity tested and verified. The improvements in this production process for the production of large amount of P. furiosus biomass and hydrogenase I have been achieved, especially by successfully implementing the following key measures and steps: unsterile cultivation setup, skipping typical intermediate preculture and inoculation steps, accelerating the cultivation process by defining an optimal state of the inoculation, optimal time point of biomass harvesting and finally by choosing a one-step purification procedure for enzyme recovery. PMID:24894374

Rieckenberg, Fabian; Götz, Katharina; Hilterhaus, Lutz; Liese, Andreas; Zeng, An-Ping

2014-12-01

188

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

PubMed Central

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

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

2012-01-01

189

Applications of bacterial hydrogenases in waste decontamination, manufacture of novel bionanocatalysts and in sustainable energy.  

PubMed

Bacterial hydrogenases have been harnessed to the removal of heavy metals from solution by reduction to less soluble metal species. For Pd(II), its bioreduction results in the deposition of cell-bound Pd(0)-nanoparticles that are ferromagnetic and have a high catalytic activity. Hydrogenases can also be used synthetically in the production of hydrogen from sugary wastes through breakdown of formate produced by fermentation. The Bio-H(2) produced can be used to power an electrical device using a fuel cell to provide clean electricity. Production of hydrogen from confectionery wastes by one organism (Escherichia coli) can be used as the electron donor for the production of Bio-Pd(0) from soluble Pd(II) by a second organism. The resulting Bio-Pd(0) can then be used as a bioinorganic catalyst in the remediation of Cr(VI)-contaminated solutions or polychlorinated biphenyls at the expense of Bio-H(2), as a hydrogenation catalyst for industry or as a component of a fuel cell electrode. PMID:15667270

Macaskie, L E; Baxter-Plant, V S; Creamer, N J; Humphries, A C; Mikheenko, I P; Mikheenko, P M; Penfold, D W; Yong, P

2005-02-01

190

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

NASA Astrophysics Data System (ADS)

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 A2-, 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 A2- by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.

Sode, Olaseni; Voth, Gregory A.

2014-12-01

191

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

PubMed

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(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(2-) by closing a cavity that could otherwise fill with water near the proximal Fe of the active site. PMID:25494798

Sode, Olaseni; Voth, Gregory A

2014-12-14

192

Fast and efficient molecular electrocatalysts for H{sub 2} production: Using hydrogenase enzymes as guides  

SciTech Connect

Hydrogen generation using solar energy will require the development of efficient electrocatalysts for proton reduction. This article discusses the important role that proton movement plays in hydrogenase enzymes and potential devices for solar generation. Studies of hydrogenase enzymes provide many important design principles for the development of simpler molecular catalysts. These principles are illustrated with examples from the literature and from the authors’ laboratories. In particular, pendant bases incorporated in the second coordination sphere of catalytic molecules play a number of important roles that are crucial to efficient catalysis. These roles include acting as relays to move protons between the metal center and solution, promoting intra- and intermolecular proton transfer reactions, coupling proton and electron transfer reactions, assisting heterolytic cleavage of hydrogen, and stabilizing critical reaction intermediates. The importance of controlling proton movement on the molecular scale underscores the importance of a similar degree of control in devices designed for the solar production of hydrogen or any fuel generation process involving multiple electrons and protons.

Yang, Jenny Y.; Bullock, R. Morris; DuBois, M. Rakowski; DuBois, Daniel L.

2011-01-01

193

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases  

PubMed Central

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H2 + O2 = 2 H2O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H2O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H2 and 10% O2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H2 oxidation. The oxidase activity is too slow to be useful in removing O2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation. PMID:24715724

Wulff, Philip; Day, Christopher C.; Sargent, Frank; Armstrong, Fraser A.

2014-01-01

194

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases.  

PubMed

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H2 + O2 = 2 H2O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H2O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H2 and 10% O2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H2 oxidation. The oxidase activity is too slow to be useful in removing O2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation. PMID:24715724

Wulff, Philip; Day, Christopher C; Sargent, Frank; Armstrong, Fraser A

2014-05-01

195

hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system.  

PubMed

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus. PMID:8631353

Dernedde, J; Eitinger, T; Patenge, N; Friedrich, B

1996-01-15

196

Octa­carbon­yl(5-meth­oxy-2,3-dihydro-1H-benzimidazol-2-yl)di-?3-sulfido-diiron(I)iron(II)(2 Fe—Fe)  

PubMed Central

The title compound, [Fe3(C8H8N2O)S2(CO)8], was prepared by the direct reaction of Fe3(CO)12 and 5-meth­oxy-1H-benzoimidazole-2-thiol in tetra­hydro­furan. Desulfurization took place readily to form a sulfide carbonyl cluster. The mol­ecule contains a triangle consisting of three Fe atoms capped by two S atoms above and below. There are two Fe—Fe bonds [2.6322?(5) and 2.5582?(5)?Ĺ] in the triangle; the length of the third edge [3.3987?(5)?Ĺ] is too long to represent an Fe—Fe bond. PMID:21200564

Zhang, Tingting; Wang, Mei; Li, Ping; Sun, Licheng

2008-01-01

197

Density comparison between liquid and solid phases in the Fe-FeS system by the sink/float method: Implications for the structural evolution of the cores  

NASA Astrophysics Data System (ADS)

At the early stage of the differentiated planetary bodies, a metallic component separated from a silicate component and formed a metallic core at the center of bodies. Cooling the early molten cores would lead to partial solidification of the core. For a sulfur-bearing iron core, the partially solidified core would consist of Fe-S liquid coexisting with either solid Fe or FeS depending on the S concentration. The stratification of the solid and liquid core is largely controlled by the density contrast between the solid and liquid phases. Based on the previous high-pressure and high-temperature density data of Fe-S liquid, solid Fe, and solid FeS [Nishida et al., 2008, 2011; Balog et al., 2003; Sakamaki et al., 2009; Urakawa et al., 2004], the Fe-S liquid at the Fe-rich side is predictably less dense than the coexisting solid Fe along the liquidus temperatures, whereas the Fe-S liquid at the FeS-rich side is denser than the coexisting solid FeS except only much near FeS. Such density relation implies that a S-rich core would consist of an "outer solid" core and an "inner liquid" core, in contrast to the Earth's core. The predicted density contrast between liquid and solid is relatively small and it is derived from the previous density measurements of the individual phases with large uncertainties, particularly for the liquid phase. In order to directly compare the densities between the liquid and solid phases in the Fe-FeS system, we have performed sink/float experiments to precisely determine the density relationship at high pressure and temperature. High-pressure and high-temperature melting experiments in the Fe-FeS system were carried out using the multi-anvil apparatus at the Geophysical Laboratory. An Fe or FeS pellet was placed at the center in a MgO capsule and surrounded by an Fe-FeS powder mixture (20 wt% S or 28 wt% S, respectively). From the sink-float behavior of the centered pellet at high temperature, we determine whether the solid phase (Fe or FeS) is denser than the coexisting liquid phase or less dense. The experiment at 4 GPa and 1100 °C, using the Fe pellet and Fe-20 wt% S mixture (Fe-rich side) as the starting materials, showed that the solid Fe sank in the liquid with a composition of Fe-22.8 wt% S. Similar experiments in the FeS-rich side were performed and the results will be discussed in conjunction with the stratification and evolution of the cores.

Shibazaki, Y.; Fei, Y.

2012-12-01

198

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase  

PubMed Central

Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine ?-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 ?mole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 ?mole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin. PMID:22217184

2012-01-01

199

Enhanced photocatalytic hydrogen production from an MCM-41-immobilized photosensitizer-[Fe-Fe] hydrogenase mimic dyad.  

PubMed

A covalently linked photosensitizer-catalytic center dyad Ps-Hy, consisting of two bis(2-phenylpyridine)(2,2'-bipyridine)iridium(iii) chromophores (Ps) and a diiron hydrogenase mimic (Hy) was constructed by using click reaction. Ps-Hy was incorporated into K(+)-exchanged molecular sieve MCM-41 to form a composite (Ps-Hy@MCM-41), which has been successfully applied to the photochemical production of hydrogen. The catalytic activity of Ps-Hy@MCM-41 is ?3-fold higher as compared with that of Ps-Hy in the absence of MCM-41. The incorporation of Ps-Hy into MCM-41 stabilizes the catalyst, and consequently, advances the photocatalysis. The present study provides a potential strategy for improving catalytic efficiency of artificial photosynthesis systems using mesoporous molecular sieves. PMID:25238441

Wang, Wen; Yu, Tianjun; Zeng, Yi; Chen, Jinping; Yang, Guoqiang; Li, Yi

2014-11-01

200

Photocatalytic hydrogen production using polymeric carbon nitride with a hydrogenase and a bioinspired synthetic Ni catalyst.  

PubMed

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 (CN(x)), 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 (?>420?nm). PMID:25205168

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

2014-10-20

201

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme*  

PubMed Central

In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ?340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ˝) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ˝) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS. PMID:23649626

Raleiras, Patrícia; Kellers, Petra; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2013-01-01

202

The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase.  

PubMed

The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression. In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JH delta Eg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JH delta 23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JH delta 23H was low in comparison to the wild type in 10-50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 microM nickel during the derepression incubation. Studies on strains harbouring the hup promoter-lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 microM NiCl2. Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O2, 10% partial pressure H2) HypB was detected, and its expression preceded hydrogenase synthesis by 3-6 h. 63Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation. Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JH delta 23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps. PMID:9140970

Olson, J W; Fu, C; Maier, R J

1997-04-01

203

Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha  

PubMed Central

Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen–oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane. PMID:24866391

2014-01-01

204

Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha.  

PubMed

Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen-oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane. PMID:24866391

Radu, Valentin; Frielingsdorf, Stefan; Evans, Stephen D; Lenz, Oliver; Jeuken, Lars J C

2014-06-18

205

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

PubMed Central

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

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

2014-01-01

206

Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli.  

PubMed

In a previous work (Trchounian et al., Biol. Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5. The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity. In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5. The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress. The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5. Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4. K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain. If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress. The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E. coli grown at pH 7.5. In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production. PMID:11817571

Bagramyan, K; Vassilian, A; Mnatsakanyan, N; Trchounian, A

2001-01-01

207

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

SciTech Connect

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.

Osuka, Hisao [Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan) [Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma-shi, Nara 630-0192 (Japan); Shomura, Yasuhito; Komori, Hirofumi; Shibata, Naoki [Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan)] [Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); Nagao, Satoshi [Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma-shi, Nara 630-0192 (Japan)] [Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma-shi, Nara 630-0192 (Japan); Higuchi, Yoshiki, E-mail: hig@sci.u-hyogo.ac.jp [Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan) [Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); CREST, JST, Gobancho, Chiyoda-ku, Tokyo 102-0076 (Japan); Hirota, Shun, E-mail: hirota@ms.naist.jp [Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma-shi, Nara 630-0192 (Japan) [Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma-shi, Nara 630-0192 (Japan); CREST, JST, Gobancho, Chiyoda-ku, Tokyo 102-0076 (Japan)

2013-01-04

208

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.  

PubMed Central

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome. PMID:3884595

Sankar, P; Lee, J H; Shanmugam, K T

1985-01-01

209

Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis ? †  

PubMed Central

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S0) or H2 generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H2S- and H2-evolving conditions. H2S generation in PHY1 was equivalent to that of the host strain, and H2 generation was higher in PHY1, suggesting that Hyh functions in the direction of H2 uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H2-evolving conditions. This fact, as well as the impaired H2 generation activity in MHD1, indicated that Mbh is mainly responsible for H2 evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H2 transfer (i.e., H2 evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H2S-evolving conditions and exhibited a lower H2S generation activity, indicating the involvement of Mbx in the S0 reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales. PMID:21515783

Kanai, Tamotsu; Matsuoka, Ryoji; Beppu, Haruki; Nakajima, Akihito; Okada, Yoshihiro; Atomi, Haruyuki; Imanaka, Tadayuki

2011-01-01

210

Using Gas Chromatography/Isotope Ratio Mass Spectrometry to Determine the Fractionation Factor for H2 Production by Hydrogenases  

SciTech Connect

Hydrogenases catalyze the reversible formation of H2, and they are key enzymes in the biological cycling of H2. H isotopes should be a very useful tool in quantifying proton trafficking in biological H2 production processes, but there are several obstacles that have thus far limited the use of this tool. In this manuscript, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H2 evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. A custom-designed high-throughput gas chromatography-isotope ratio mass spectrometer is employed to measure the isotope ratio of the H2. Using this method, we determined that the fractionation factor of H2 production by the [NiFe]-hydrogenase from Desulfivibrio fructosovran is 0.27. This result indicates that, as expected, protons are highly favored over deuterons during H2 evolution. Potential applications of this new method are discussed.

Yang, Hui; Ghandi, H.; Shi, Liang; Kreuzer, Helen W.; Ostrom, Nathaniel; Hegg, Eric L.

2012-01-15

211

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

PubMed Central

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 demonstrated ntrA dependence for the labile hydrogenase activity. Images PMID:2666400

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

1989-01-01

212

A density functional theory study on the active center of Fe-only hydrogenase: characterization and electronic structure of the redox states.  

PubMed

We have carried out extensive density functional theory (DFT) calculations for possible redox states of the active center in Fe-only hydrogenases. The active center is modeled by [(H(CH(3))S)(CO)(CN(-))Fe(p)(mu-DTN)(mu-CO)Fe(d)(CO)(CN(-))(L)](z)() (z is the net charge in the complex; Fe(p)= the proximal Fe, Fe(d) = the distal Fe, DTN = (-SCH(2)NHCH(2)S-), L is the ligand that bonds with the Fe(d) at the trans position to the bridging CO). Structures of possible redox states are optimized, and CO stretching frequencies are calculated. By a detailed comparison of all the calculated structures and the vibrational frequencies with the available experimental data, we find that (i) the fully oxidized, inactive state is an Fe(II)-Fe(II) state with a hydroxyl (OH(-)) group bonded at the Fe(d), (ii) the oxidized, active state is an Fe(II)-Fe(I) complex which is consistent with the assignment of Cao and Hall (J. Am. Chem. Soc. 2001, 123, 3734), and (iii) the fully reduced state is a mixture with the major component being a protonated Fe(I)-Fe(I) complex and the other component being its self-arranged form, Fe(II)-Fe(II) hydride. Our calculations also show that the exogenous CO can strongly bond with the Fe(II)-Fe(I) species, but cannot bond with the Fe(I)-Fe(I) complex. This result is consistent with experiments that CO tends to inhibit the oxidized, active state, but not the fully reduced state. The electronic structures of all the redox states have been analyzed. It is found that a frontier orbital which is a mixing state between the e(g) of Fe and the 2 pi of the bridging CO plays a key role concerning the reactivity of Fe-only hydrogenases: (i) it is unoccupied in the fully oxidized, inactive state, half-occupied in the oxidized, active state, and fully occupied in the fully reduced state; (ii) the e(g)-2 pi orbital is a bonding state, and this is the key reason for stability of the low oxidation states, such as Fe(I)-Fe(I) complexes; and (iii) in the e(g)-2 pi orbital more charge accumulates between the bridging CO and the Fe(d) than between the bridging CO and the Fe(p), and the occupation increase in this orbital will enhance the bonding between the bridging CO and the Fe(d), leading to the bridging-CO shift toward the Fe(d). PMID:11982382

Liu, Zhi-Pan; Hu, P

2002-05-01

213

Molecular Dynamics Study of the Proposed Proton Transport Pathways in [FeFe]-Hydrogenase  

SciTech Connect

Possible proton channels in Clostridium pasteurianum [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to discern proposed channels, compare their properties, evaluate the functional channel, and to provide insight into the features of an active proton channel. Our simulations suggest that protons are not transported through water wires. Instead, a five-residue motif (E282, S319, E279, HOH, C299) was found to be the likely channel, consistent with experimental observations. This channel connects the surface of the enzyme and the di-thiomethylamine bridge of the catalytic H-cluster, permitting the transport of protons. The channel was found to have a persistent hydrogen bonded core (residues E279 to S319), with less persistent hydrogen bonds at the ends of the channel. The hydrogen bond occupancy in this network was found to be sensitive to the protonation state of the residues in the channel, with different protonation states enhancing or stabilizing hydrogen bonding in different regions of the network. Single site mutations to non-hydrogen bonding residues break the hydrogen bonding network at that residue, consistent with experimental observations showing catalyst inactivation. In many cases, these mutations alter the hydrogen bonding in other regions of the channel which may be equally important in catalytic failure. A correlation between the protein dynamics near the proton channel and the redox partner binding regions was also found as a function of protonation state. The likely mechanism of proton movement in [FeFe]-hydrogenases is discussed based on the structural analysis presented here. This work was funded by the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences. 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 Biological and Environmental Research located at Pacific Northwest National Laboratory, and a portion of the research was performed using PNNL Institutional Computing at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

Ginovska-Pangovska, Bojana; Ho, Ming-Hsun; Linehan, John C.; Cheng, Yuhui; Dupuis, Michel; Raugei, Simone; Shaw, Wendy J.

2014-01-15

214

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

SciTech Connect

Nickel-deficient Nic{sup {minus}} mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterizied. The Nic{sup {minus}} 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{sup +} 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{sup {minus}} mutants was enhanced by increasing the concentrations of magnesium. This suggests that the Nic{sup {minus}} mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.

Eberz, G.; Eitinger, T.; Friedrich, B. (Freien Universitaet Berlin (Germany, F.R.))

1989-03-01

215

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

PubMed Central

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

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

2002-01-01

216

Crystal Structure of HydG from Carboxydothermus hydrogenoformans: A Trifunctional [FeFe]-Hydrogenase Maturase.  

PubMed

The structure of the radical S-adenosyl-L-methionine (SAM) [FeFe]-hydrogenase maturase HydG involved in CN(-) /CO synthesis is characterized by two internal tunnels connecting its tyrosine-binding pocket with the external medium and the C-terminal Fe4 S4 cluster-containing region. A comparison with a tryptophan-bound NosL structure suggests that substrate binding causes the closing of the first tunnel and, along with mutagenesis studies, that tyrosine binds to HydG with its amino group well positioned for H-abstraction by SAM. In this orientation the dehydroglycine (DHG) fragment caused by tyrosine C??C? bond scission can readily migrate through the second tunnel towards the C-terminal domain where both CN(-) and CO are synthesized. Our HydG structure appears to be in a relaxed state with its C-terminal cluster CysX2 CysX22 Cys motif exposed to solvent. A rotation of this domain coupled to Fe4 S4 cluster assembly would bury its putatively reactive unique Fe ion thereby allowing it to interact with DHG. PMID:25504963

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

2015-02-01

217

Synthesis of nickel-iron hydrogenase in Cupriavidus metallidurans is controlled by metal-dependent silencing and un-silencing of genomic islands.  

PubMed

Cupriavidus metallidurans CH34 is able to grow autotrophically as a hydrogen-oxidizing bacterium and produces nickel-dependent hydrogenases, even under heterotrophic conditions. Loss of its two native plasmids resulted in inability of the resulting strain AE104 to synthesize the hydrogenases and to grow autotrophically in phosphate-poor, Tris-buffered mineral salts medium (TMM). Three of eleven previously identified catabolic genomic islands (CMGIs; Van Houdt et al., 2009), two of which harbor the genes for the membrane-bound (CMGI-2) and the soluble hydrogenase (CMGI-3), were silenced in strain AE104 when cultivated in phosphate-poor TMM, explaining its inability to produce hydrogenases. Production of the soluble hydrogenase from the aut region 1 of CMGI-3, and concomitant autotrophic growth, was recovered when the gene for the zinc importer ZupT was deleted in strain AE104. The transcriptome of the ?zupT mutant exhibited two up-regulated gene regions compared to its parent strain AE104. Expression of the genes in the aut region 1 increased independently of the presence of added zinc. A second gene region was expressed only under metal starvation conditions. This region encoded a TonB-dependent outer membrane protein, a putative metal chaperone plus paralogs of essential zinc-dependent proteins, indicating the presence of a zinc allocation pathway in C. metallidurans. Thus, expression of the genes for the soluble hydrogenase and the Calvin cycle enzymes on aut region 1 of CMGI-3 of C. metallidurans is under global control and needs efficient ZupT-dependent zinc allocation for a regulatory role, which might be discrimination of nickel. PMID:25720835

Herzberg, Martin; Schüttau, Marcel; Reimers, Matthias; Große, Cornelia; Hans-Günther-Schlegel; Nies, Dietrich H

2015-04-01

218

Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*  

PubMed Central

The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

2014-01-01

219

Rubredoxin-related maturation factor guarantees metal cofactor integrity during aerobic biosynthesis of membrane-bound [NiFe] hydrogenase.  

PubMed

The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

2014-03-14

220

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

PubMed

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 (?cycA) 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. PMID:23805134

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

2013-01-01

221

Fractionation of sulfur isotopes by Desulfovibrio vulgaris mutants lacking hydrogenases or type I tetraheme cytochrome c3  

PubMed Central

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-c3). 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-c3 mutant (?cycA) 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. PMID:23805134

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

2013-01-01

222

Crystallization and preliminary X-ray diffraction analysis of membrane-bound respiratory [NiFe] hydrogenase from Hydrogenovibrio marinus  

PubMed Central

Membrane-bound respiratory [NiFe] hydrogenase is an H2-uptake enzyme found in the periplasmic space of bacteria that plays a crucial role in energy-conservation processes. The heterodimeric unit of the enzyme from Hydrogeno­vibrio marinus was purified to homogeneity using chromatographic procedures. Crystals were grown using the sitting-drop vapour-diffusion method at room temperature. Preliminary crystallographic analysis revealed that the crystals belonged to space group P21, with unit-cell parameters a = 75.72, b = 116.59, c = 113.40?Ĺ, ? = 91.3°, indicating that two heterodimers were present in the asymmetric unit. PMID:21795805

Shomura, Yasuhito; Hagiya, Keisuke; Yoon, Ki-Seok; Nishihara, Hirofumi; Higuchi, Yoshiki

2011-01-01

223

Hybrid [FeFe]-Hydrogenases with Modified Active Sites Show Remarkable Residual Enzymatic Activity.  

PubMed

[FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ?50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brřnsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

2015-02-24

224

Proton management as a design principle for hydrogenase-inspired catalysts  

SciTech Connect

The properties of the hydrogenase-inspired [Ni(PNP)2]2+ (PNP Ľ Et2PCH2NMeCH2PEt2) 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 H2 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 pKa values of these potential intermediates, as they control both the species in solution at a given pH and the freeenergy profile of reaction pathways. Optimal relationships among these pKa values can be identified, and it is demonstrated that ‘‘proton management’’, i.e., the manipulation of these pKa values (e.g., through choice of metal or substituents on ligands), can serve as a design principle for improved catalytic behavior. 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.

Small, Yolanda A.; DuBois, Daniel L.; Fujita, Etsuko; Muckerman, J. T.

2011-06-01

225

Photochemical dihydrogen production using an analogue of the active site of [NiFe] hydrogenase.  

PubMed

Photoproduction of dihydrogen (H2) by a low molecular weight analogue of the active site of [NiFe] hydrogenase has been investigated by reduction of the [NiFe2] cluster, 1, by a photosensitier PS (PS = [ReCl(CO)3(bpy)] or [Ru(bpy)3][PF6]2). Reductive quenching of the (3)MLCT excited state of the photosensitizer by NEt3 or N(CH2CH2OH)3 (TEOA) generates PS(•-), and subsequent intermolecular electron transfer to 1 produces the reduced anionic form of 1. Time-resolved infrared spectroscopy (TRIR) has been used to probe the intermediates throughout the reduction of 1 and subsequent photocatalytic H2 production from [HTEOA][BF4], which was monitored by gas chromatography. Two structural isomers of the reduced form of 1 (1a(•-) and 1b(•-)) were detected by Fourier transform infrared spectroscopy (FTIR) in both CH3CN and DMF (dimethylformamide), while only 1a(•-) was detected in CH2Cl2. Structures for these intermediates are proposed from the results of density functional theory calculations and FTIR spectroscopy. 1a(•-) is assigned to a similar structure to 1 with six terminal carbonyl ligands, while calculations suggest that in 1b(•-) two of the carbonyl groups bridge the Fe centers, consistent with the peak observed at 1714 cm(-1) in the FTIR spectrum for 1b(•-) in CH3CN, assigned to a ?(CO) stretching vibration. Formation of 1a(•-) and 1b(•-) and production of H2 was studied in CH3CN, DMF, and CH2Cl2. Although the more catalytically active species (1a(•-) or 1b(•-)) could not be determined, photocatalysis was observed only in CH3CN and DMF. PMID:24749646

Summers, Peter A; Dawson, Joe; Ghiotto, Fabio; Hanson-Heine, Magnus W D; Vuong, Khuong Q; Davies, E Stephen; Sun, Xue-Z; Besley, Nicholas A; McMaster, Jonathan; George, Michael W; Schröder, Martin

2014-05-01

226

Light-induced reactivation of O2-tolerant membrane-bound [Ni-Fe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus  

E-print Network

Light-induced reactivation of O2-tolerant membrane-bound [Ni-Fe] hydrogenase from, 35042 Rennes, France Electronic Supplementary Information (ESI) available. See DOI: 10.1039/b000000x/10 Received 21 Jun 2013, Accepted 12 Aug 2013 DOI: 10.1039/c3cp52596a We report the effect of UV-Vis light

Paris-Sud XI, Université de

227

Structure of the Ni sites in hydrogenases by x-ray absorption spectroscopy. Species variation and the effects of redox poise  

SciTech Connect

Structural information obtained from the analysis of nickel K-edge X-ray absorption spectroscopic data of [NiFe]hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alcaligenes eutrophus H16 (NAD{sup +}-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme is compared with the structural features obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutrophus hydrogenase are quite similar. Analysis of the XANES features assigned to 1s {yields} 3d electronic transitions indicates that the shift in energy that occurs for reduction of the enzymes to the SI level may be attributed at least in part to an increase in the coordination number from five to six. The results of EXAFS analysis are shown to be in general agreement with the published crystal structure of the D. gigas enzyme. 42 refs., 6 figs., 2 tabs.

Gu, Z.; Maroney, M.J.; Allan, C.B.; Choudhury, S.B. [Univ. of Massachusetts, Amherst, MA (United States)] [and others] [Univ. of Massachusetts, Amherst, MA (United States); and others

1996-11-13

228

A Universal Scaffold for Synthesis of the Fe(CN)2(CO) Moiety of [NiFe] Hydrogenase*  

PubMed Central

Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN?) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN? ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)2(CO) entity of [NiFe] hydrogenase. PMID:23019332

Bürstel, Ingmar; Siebert, Elisabeth; Winter, Gordon; Hummel, Philipp; Zebger, Ingo; Friedrich, Bärbel; Lenz, Oliver

2012-01-01

229

The Influence of Oxygen on [NiFe]–Hydrogenase Cofactor Biosynthesis and How Ligation of Carbon Monoxide Precedes Cyanation  

PubMed Central

The class of [NiFe]–hydrogenases is characterized by a bimetallic cofactor comprising low–spin nickel and iron ions, the latter of which is modified with a single carbon monoxide (CO) and two cyanide (CN?) molecules. Generation of these ligands in vivo requires a complex maturation apparatus in which the HypC–HypD complex acts as a ‘construction site’ for the Fe–(CN)2CO portion of the cofactor. The order of addition of the CO and CN– ligands determines the ultimate structure and catalytic efficiency of the cofactor; however much debate surrounds the succession of events. Here, we present an FT–IR spectroscopic analysis of HypC–HypD isolated from a hydrogenase–competent wild–type strain of Escherichia coli. In contrast to previously reported samples, HypC–HypD showed spectral contributions indicative of an electron–rich Fe–CO cofactor, at the same time lacking any Fe–CN– signatures. This immature iron site binds external CO and undergoes oxidative damage when in contact with O2. Binding of CO protects the site against loss of spectral features associated with O2 damage. Our findings strongly suggest that CO ligation precedes cyanation in vivo. Furthermore, the results provide a rationale for the deleterious effects of O2 on in vivo cofactor biosynthesis. PMID:25211029

Stripp, Sven T.; Lindenstrauss, Ute; Granich, Claudia; Sawers, R. Gary; Soboh, Basem

2014-01-01

230

Molecular basis of [FeFe]-hydrogenase function: an insight into the complex interplay between protein and catalytic cofactor.  

PubMed

The precise electrochemical features of metal cofactors that convey the functions of redox enzymes are essentially determined by the specific interaction pattern between cofactor and enclosing protein environment. However, while biophysical techniques allow a detailed understanding of the features characterizing the cofactor itself, knowledge about the contribution of the protein part is much harder to obtain. [FeFe]-hydrogenases are an interesting class of enzymes that catalyze both, H2 oxidation and the reduction of protons to molecular hydrogen with significant efficiency. The active site of these proteins consists of an unusual prosthetic group (H-cluster) with six iron and six sulfur atoms. While H-cluster architecture and catalytic states during the different steps of H2 turnover have been thoroughly investigated during the last 20 years, possible functional contributions from the polypeptide framework were only assumed according to the level of conservancy and X-ray structure analyses. Due to the recent development of simpler and more efficient expression systems the role of single amino acids can now be experimentally investigated. This article summarizes, compares and categorizes the results of recent investigations based on site directed and random mutagenesis according to their informative value about structure function relationships in [FeFe]-hydrogenases. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. PMID:23507618

Winkler, Martin; Esselborn, Julian; Happe, Thomas

2013-01-01

231

Purification and Characterization of [NiFe]-Hydrogenase of Shewanella oneidensis MR-1  

SciTech Connect

The ?-proteobacterium Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that was implicated in both H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned into a protein expression vector. The resulting plasmid was transformed into a MR-1 mutant deficient in H2 formation. Expression of MR-1 [NiFe]-H2ase in trans restored the mutant’s ability to produce H2 at 37% of that for wild type. Following expression, MR-1 [NiFe]-H2ase was purified to near homogeneity. The purified MR-1 [NiFe]-H2ase could couple H2 oxidation to reduction of Tc(VII) and methyl viologen directly. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated Tc(VII) but not methyl viologen reductions. Under the conditions tested, Tc(VII) reduction was complete in Tris buffer but not in HEPES buffer. The reduced Tc(IV) was soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc(IV) precipitates formed in HEPES buffer were packed with crystallites. Although X-ray absorption near-edge spectroscopy measurements confirmed that the reduction products found in both buffers were Tc(IV), extended X-ray adsorption fine-structure measurements revealed that these products were very different. While the product in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2•nH2O. These results shows for the first time that MR-1 [NiFe]-H2ase is a bidirectional enzyme that catalyzes both H2 formation and oxidation as well as Tc(VII) reduction directly by coupling H2 oxidation.

Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.; Heald, Steve M.; Dohnalkova, Alice; Sybirna, Kateryna; Bottin, Herve; Squier, Thomas C.; Zachara, John M.; Fredrickson, Jim K.

2011-08-02

232

The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound [NiFe] Hydrogenase in Ralstonia eutropha H16 ? †  

PubMed Central

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

Fritsch, Johannes; Lenz, Oliver; Friedrich, Bärbel

2011-01-01

233

Protonation of nickel-iron hydrogenase models proceeds after isomerization at nickel.  

PubMed

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; pdt(2-) = (-)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

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

2014-09-01

234

Occurrence of H2-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus1  

PubMed Central

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 conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains. Images Figure 1 Figure 2 PMID:16666550

Murillo, Jesús; Villa, Ana; Chamber, Manuel; Ruiz-Argüeso, Tomás

1989-01-01

235

[NiFe]Hydrogenase from Citrobacter sp. S-77 surpasses platinum as an electrode for H2 oxidation reaction.  

PubMed

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. PMID:24895095

Matsumoto, Takahiro; Eguchi, Shigenobu; Nakai, Hidetaka; Hibino, Takashi; Yoon, Ki-Seok; Ogo, Seiji

2014-08-18

236

Evidence for selenocysteine coordination to the active site nickel in the [NiFeSe]hydrogenases from Desulfovibrio baculatus.  

PubMed Central

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

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

237

Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity  

SciTech Connect

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.

Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

2014-09-30

238

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold  

PubMed Central

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydFEG), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydFEG. The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP. PMID:20498089

Shepard, Eric M.; McGlynn, Shawn E.; Bueling, Alexandra L.; Grady-Smith, Celestine S.; George, Simon J.; Winslow, Mark A.; Cramer, Stephen P.; Peters, John W.; Broderick, Joan B.

2010-01-01

239

The Alcaligenes eutrophus membrane-bound hydrogenase gene locus encodes functions involved in maturation and electron transport coupling.  

PubMed

Alcaligenes eutrophus H16 produces two [NiFe] hydrogenases which catalyze the oxidation of hydrogen and enable the organism to utilize H2 as the sole energy source. The genes (hoxK and hoxG) for the heterodimeric, membrane-bound hydrogenase (MBH) are located adjacent to a series of eight accessory genes (hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, and hoxV). In the present study, we generated a set of isogenic mutants with in-frame deletions in the two structural genes and in each of the eight accessory genes. The resulting mutants can be grouped into two classes on the basis of the H2-oxidizing activity of the MBH. Class I mutants (hoxKdelta, hoxGdelta, hoxMdelta, hoxOdelta, and hoxQdelta) were totally devoid of MBH-mediated, H2-oxidizing activity. The hoxM deletion strain was the only mutant in our collection which was completely blocked in carboxy-terminal processing of large subunit HoxG, indicating that hoxM encodes a specific protease. Class II mutants (hoxZdelta, hoxLdelta, hoxRdelta, hoxTdelta, and hoxVdelta) contained residual amounts of MBH activity in the membrane fraction of the extracts. Immunochemical analysis and 63Ni incorporation experiments revealed that the mutations affect various steps in MBH maturation. A lesion in hoxZ led to the production of a soluble MBH which was highly active with redox dye. PMID:8755880

Bernhard, M; Schwartz, E; Rietdorf, J; Friedrich, B

1996-08-01

240

[FeFe]-Hydrogenase Abundance and Diversity along a Vertical Redox Gradient in Great Salt Lake, USA  

PubMed Central

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

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

241

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation.  

PubMed

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-Ĺ 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

Tominaga, Taiga; Watanabe, Satoshi; Matsumi, Rie; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

2013-12-17

242

Investigations on the role of proton-coupled electron transfer in hydrogen activation by [FeFe]-hydrogenase.  

PubMed

Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion. The [FeFe]-hydrogenases catalyze the reversible activation of molecular H2 through a unique metallocofactor, the H-cluster, which is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The correlation of electronic and structural transitions at the H-cluster with proton-transfer (PT) steps has not been well-resolved experimentally. Here, we explore how modification of the conserved PT network via a Cys ? Ser substitution at position 169 proximal to the H-cluster of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1) affects the H-cluster using electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. Despite a substantial decrease in catalytic activity, the EPR and FTIR spectra reveal different H-cluster catalytic states under reducing and oxidizing conditions. Under H2 or sodium dithionite reductive treatments, the EPR spectra show signals that are consistent with a reduced [4Fe-4S]H(+) subcluster. The FTIR spectra showed upshifts of ?CO modes to energies that are consistent with an increase in oxidation state of the [2Fe]H subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA1, spectra associated with Hred and Hsred states are less populated in the Cys ? Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions. PMID:25286239

Mulder, David W; Ratzloff, Michael W; Bruschi, Maurizio; Greco, Claudio; Koonce, Evangeline; Peters, John W; King, Paul W

2014-10-29

243

[FeFe]-hydrogenase abundance and diversity along a vertical redox gradient in Great Salt Lake, USA.  

PubMed

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

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

244

Identification of an Uptake Hydrogenase Required for Hydrogen-Dependent Reduction of Fe(III) and Other Electron Acceptors by Geobacter sulfurreducens  

PubMed Central

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens. PMID:15126463

Coppi, Maddalena V.; O'Neil, Regina A.; Lovley, Derek R.

2004-01-01

245

Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study  

Microsoft Academic Search

The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium Vinosum can exist in three different redox states. In the most oxidized state (Nia-S) the nickel is divalent. The most reduced state (Nia-SR) likewise has Ni2+, while the intermediate state (Nia-C*) has Ni3+. The transitions between these states have been studied by stopped-flow Fourier transform infrared spectroscopy. It is inferred

S. J. George; S. Kurkin; R. N. Thorneley; S. P. J. Albracht

2004-01-01

246

Influence of the fusion of two subunits of the F 420 -non-reducing hydrogenase of Methanococcus voltae on its biochemical properties  

Microsoft Academic Search

In Methanococcus voltae, one of the two [NiFeSe] hydrogenases is unusual in that the large subunit is split into two subunits, each contributing two ligands to the [NiFe] center that catalyzes the heterolytic cleavage of the dihydrogen molecule. We have engineered a fusion of these two subunits. The resulting new enzyme showed no significant difference in hydrogen uptake activity or

Ruth Bingemann; Antonio J. Pierik; Albrecht Klein

2000-01-01

247

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.  

PubMed

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system. PMID:22403188

Howlett, Robert M; Hughes, Bethan M; Hitchcock, Andrew; Kelly, David J

2012-06-01

248

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus  

PubMed Central

Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygen-tolerant Hydrogenase I from Aquifex aeolicus such electron “wires” form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O2 to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a [3Fe4S] and two [4Fe4S], but also a unique high-potential (HP) state was found. On the basis of 57Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the [4Fe4S]3+ state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3+, 2+, and 1+ oxidation states within a very small potential range. We suggest that the (1 + /2+) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress. PMID:21444783

Pandelia, Maria-Eirini; Nitschke, Wolfgang; Infossi, Pascale; Giudici-Orticoni, Marie-Thérčse; Bill, Eckhard; Lubitz, Wolfgang

2011-01-01

249

Direct probing of photoinduced electron transfer in a self-assembled biomimetic [2Fe2S]-hydrogenase complex using ultrafast vibrational spectroscopy.  

PubMed

A pyridyl-functionalized diiron dithiolate complex, [?-(4-pyCH2-NMI-S2)Fe2(CO)6] (3, py = pyridine (ligand), NMI = naphthalene monoimide) was synthesized and fully characterized. In the presence of zinc tetraphenylporphyrin (ZnTPP), a self-assembled 3·ZnTPP complex was readily formed in CH2Cl2 by the coordination of the pyridyl nitrogen to the porphyrin zinc center. Ultrafast photoinduced electron transfer from excited ZnTPP to complex 3 in the supramolecular assembly was observed in real time by monitoring the ?(C?O) and ?(C?O)NMI spectral changes with femtosecond time-resolved infrared (TRIR) spectroscopy. We have confirmed that photoinduced charge separation produced the monoreduced species by comparing the time-resolved IR spectra with the conventional IR spectra of 3(•-) generated by reversible electrochemical reduction. The lifetimes for the charge separation and charge recombination processes were found to be ?CS = 40 ± 3 ps and ?CR = 205 ± 14 ps, respectively. The charge recombination is much slower than that in an analogous covalent complex, demonstrating the potential of a supramolecular approach to extend the lifetime of the charge-separated state in photocatalytic complexes. The observed vibrational frequency shifts provide a very sensitive probe of the delocalization of the electron-spin density over the different parts of the Fe2S2 complex. The TR and spectro-electrochemical IR spectra, electron paramagnetic resonance spectra, and density functional theory calculations all show that the spin density in 3(•-) is delocalized over the diiron core and the NMI bridge. This delocalization explains why the complex exhibits low catalytic dihydrogen production even though it features a very efficient photoinduced electron transfer. The ultrafast porphyrin-to-NMI-S2-Fe2(CO)6 photoinduced electron transfer is the first reported example of a supramolecular Fe2S2-hydrogenase model studied by femtosecond TRIR spectroscopy. Our results show that TRIR spectroscopy is a powerful tool to investigate photoinduced electron transfer in potential dihydrogen-producing catalytic complexes, and that way to optimize their performance by rational approaches. PMID:24766080

Li, Ping; Amirjalayer, Saeed; Hartl, František; Lutz, Martin; de Bruin, Bas; Becker, René; Woutersen, Sander; Reek, Joost N H

2014-05-19

250

Protein-pyridinol thioester precursor for biosynthesis of the organometallic acyl-iron ligand in [Fe]-hydrogenase cofactor.  

PubMed

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation. PMID:25882909

Fujishiro, Takashi; Kahnt, Jörg; Ermler, Ulrich; Shima, Seigo

2015-01-01

251

X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima.  

PubMed

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes. PMID:18400755

Nicolet, Yvain; Rubach, Jon K; Posewitz, Matthew C; Amara, Patricia; Mathevon, Carole; Atta, Mohamed; Fontecave, Marc; Fontecilla-Camps, Juan C

2008-07-01

252

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase.  

PubMed

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

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

253

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

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. PMID:24462971

Trujillo-Reyes, J; Majumdar, S; Botez, C E; Peralta-Videa, J R; Gardea-Torresdey, J L

2014-02-28

254

Enhanced nematic and antiferromagnetic phases in the spin-fermion model for strained iron pnictides  

NASA Astrophysics Data System (ADS)

The effects of anisotropic superexchange and Fe-Fe hoppings on phase transitions in the undoped three-orbital spin-fermion model are investigated to understand the experimentally reported strain effect in BaFe2As2. Monte Carlo simulated phase diagrams show that both the collinear antiferromagnetic and nematic transitions shift toward high temperature with the increasing magnitude of anisotropies, qualitatively consistent with experimental observation. Thus, both the anisotropic superexchange and Fe-Fe hoppings are suggested to be responsible for the variation of the transition temperatures of BaFe2As2 with uniaxial stress. In addition, we observed a 90 degree rotation of the collinear antiferromagnetic order, accompanied with a reversal of the orbital occupancy at the Fermi surface when the sign of the superexchange anisotropy changes, further supporting previous predictions by first principles calculation.

Qin, Minghui; Dong, Shuai; Liu, Junming; Ren, Zhifeng

2015-01-01

255

Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen.  

PubMed

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography. PMID:12296727

Ogata, Hideaki; Mizoguchi, Yasutaka; Mizuno, Nobuhiro; Miki, Kunio; Adachi, Shin-ichi; Yasuoka, Noritake; Yagi, Tatsuhiko; Yamauchi, Osamu; Hirota, Shun; Higuchi, Yoshiki

2002-10-01

256

Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins.  

PubMed

Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (P(nikA)), but not to the urease or the hydrogenase promoter; NikR-P(nikA) binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen. PMID:23139401

Benoit, Stéphane L; Seshadri, Susmitha; Lamichhane-Khadka, Reena; Maier, Robert J

2013-01-01

257

Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins  

PubMed Central

Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (PnikA), but not to the urease or the hydrogenase promoter; NikR-PnikA binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen. PMID:23139401

Benoit, Stéphane L.; Seshadri, Susmitha; Lamichhane-Khadka, Reena

2013-01-01

258

Influence of the fusion of two subunits of the F420-non-reducing hydrogenase of Methanococcus voltae on its biochemical properties.  

PubMed

In Methanococcus voltae, one of the two [NiFeSe] hydrogenases is unusual in that the large subunit is split into two subunits, each contributing two ligands to the [NiFe] center that catalyzes the heterolytic cleavage of the dihydrogen molecule. We have engineered a fusion of these two subunits. The resulting new enzyme showed no significant difference in hydrogen uptake activity or in the Ni-C or Ni-L EPR spectra compared to the the wild-type enzyme, but exhibited a tenfold increase in both the Km for hydrogen and the Ki for the competitive inhibitor carbon monoxide. PMID:11131028

Bingemann, R; Pierik, A J; Klein, A

2000-11-01

259

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

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.

Kreuzer, Helen W.; Hill, Eric A.; Moran, James J.; Bartholomew, Rachel A.; Hui, Yang; Hegg, Eric L.

2014-03-01

260

Branched Polyethylenimine Improves Hydrogen Photoproduction from a CdSe Quantum Dot/[FeFe]-Hydrogenase Mimic System in Neutral Aqueous Solutions.  

PubMed

Nature uses hydrogenase enzyme to catalyze proton reduction at pH?7 with overpotentials and catalytic efficiencies that rival platinum electrodes. Over the past several years, [FeFe]-hydrogenase ([FeFe]-H2 ase) mimics have been demonstrated to be effective catalysts for light-driven H2 evolution. However, it remains a significant challenge to realize H2 production by such an artificial photosynthetic system in neutral aqueous solution. Herein, we report a new system for photocatalytic H2 evolution working in a broad pH range, especially under neutral conditions. This unique system is consisted of branched polyethylenimine (PEI)-grafted [FeFe]-H2 ase mimic (PEI-g-Fe2 S2 ), MPA-CdSe quantum dots (MPA=mercaptopropionic acid), and ascorbic acid (H2 A) in water. Due to the secondary coordination sphere of PEI, which has high buffering capacity and stabilizing ability, the system is able to produce H2 under visible-light irradiation with turnover number of 10?600 based on the Fe2 S2 active site in PEI-g-Fe2 S2 . The stability and activity are much better than that of the same system under acidic or basic conditions and they are, to the best of our knowledge, the highest known to date for photocatalytic H2 evolution from a [FeFe]-H2 ase mimic in neutral aqueous solution. PMID:25572459

Liang, Wen-Jing; Wang, Feng; Wen, Min; Jian, Jing-Xin; Wang, Xu-Zhe; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

2015-02-16

261

Novel Arrangement of Enhancer Sequences for NifA-Dependent Activation of the Hydrogenase Gene Promoter in Rhizobium leguminosarum bv. viciae? †  

PubMed Central

The transcriptional activation of the NifA-dependent ?54 promoter of the Rhizobium leguminosarum hydrogenase structural genes hupSL (P1) has been studied through gel retardation analysis and detailed mutagenesis. Gel retardation analysis indicated the existence of a physical interaction between NifA and the promoter. Extensive mutagenesis followed by in vivo expression analysis showed that three sequences of 4 bases each (?170 ACAA ?167, ?161 ACAA ?158, and ?145 TTGT ?142) are required for maximal stimulation of in vivo transcription of the P1 promoter. The arrangement of these upstream activating sequences (ACAA N5 ACAA N12 TTGT) differs from the canonical 5?ACA N10 TGT 3? UAS structure involved in NifA-dependent activation of nif/fix genes. Mutant promoter analysis indicated that the relative contribution of each of these sequences to P1 promoter activity increases with its proximity to the transcription start site. Analysis of double mutants altered in two out of the three enhancer sequences suggests that each of these sequences functions in NifA-dependent activation of the P1 promoter in an independent but cooperative mode. The similarities and differences between cis elements of hup and nif/fix promoters suggest that the structure of the P1 promoter has adapted to activation by NifA in order to coexpress hydrogenase and nitrogenase activities in legume nodules. PMID:18310336

Martínez, Marta; Colombo, Maria-Victoria; Palacios, Jose-Manuel; Imperial, Juan; Ruiz-Argüeso, Tomás

2008-01-01

262

Thiolate-bridged dinuclear iron(tris-carbonyl)–nickel complexes relevant to the active site of [NiFe] hydrogenase  

PubMed Central

The reaction of NiBr2(EtOH)4 with a 1:2–3 mixture of FeBr2(CO)4 and Na(SPh) generated a linear trinuclear Fe–Ni–Fe cluster (CO)3Fe(?-SPh)3Ni(?-SPh)3Fe(CO)3, 1, whereas the analogous reaction system FeBr2(CO)4/Na(StBu)/NiBr2(EtOH)4 (1:2–3:1) gave rise to a linear tetranuclear Fe–Ni–Ni–Fe cluster [(CO)3Fe(?-StBu)3Ni(?-Br)]2, 2. By using this tetranuclear cluster 2 as the precursor, we have developed a new synthetic route to a series of thiolate-bridged dinuclear Fe(CO)3–Ni complexes, the structures of which mimic [NiFe] hydrogenase active sites. The reactions of 2 with SC(NMe2)2 (tmtu), Na{S(CH2)2SMe} and ortho-NaS(C6H4)SR (R = Me, tBu) led to isolation of (CO)3Fe(?-StBu)3NiBr(tmtu), 3, (CO)3Fe(StBu)(?-StBu)2Ni{S(CH2)2SMe}, 4, and (CO)3Fe(StBu)(?-StBu)2Ni{S(C6H4)SR}, 5a (R = Me) and 5b (R = tBu), respectively. On the other hand, treatment of 2 with 2-methylthio-phenolate (ortho-O(C6H4)SMe) in methanol resulted in (CO)3Fe(?-StBu)3Ni(MeOH){O(C6H4)SMe}, 6a. The methanol molecule bound to Ni is labile and is readily released under reduced pressure to afford (CO)3Fe(StBu)(?-StBu)2Ni{O(C6H4)SMe}, 6b, and the coordination geometry of nickel changes from octahedral to square planar. Likewise, the reaction of 2 with NaOAc in methanol followed by crystallization from THF gave (CO)3Fe(?-StBu)3Ni(THF)(OAc), 7. The dinuclear complexes, 3-7, are thermally unstable, and a key to their successful isolation is to carry out the reactions and manipulations at ?40°C. PMID:18511566

Ohki, Yasuhiro; Yasumura, Kazunari; Kuge, Katsuaki; Tanino, Soichiro; Ando, Masaru; Li, Zilong; Tatsumi, Kazuyuki

2008-01-01

263

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

PubMed Central

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

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

2013-01-01

264

EPR characterization of the new Qrc complex from sulfate reducing bacteria and its ability to form a supercomplex with hydrogenase and TpIc3.  

PubMed

The Quinone-reductase complex (Qrc) is a respiratory complex with Type I cytochrome c(3):menaquinone reductase activity, recently described in sulfate-reducing bacteria. Qrc is related to the complex iron-sulfur molybdoenzyme family and to the alternative complex III. In this work we report a detailed characterization of the redox properties of the metal cofactors of Qrc using EPR spectroscopy, which allowed the determination of the reduction potentials of five out of six hemes c, one [3Fe-4S](1+/0) center and the three [4Fe-4S](2+/1+) centers. In addition, we show that Qrc forms a supercomplex with [NiFe] hydrogenase and TpIc(3), its physiological electron donors. PMID:21651911

Venceslau, Sofia S; Matos, Daniela; Pereira, Inęs A C

2011-07-21

265

Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation  

PubMed Central

Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H2 and is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). To elucidate the physiological role(s) of Ech a mutant lacking this enzyme was constructed. The mutant was unable to grow on methanol/H2/CO2, H2/CO2, or acetate as carbon and energy sources but showed wild-type growth rates with methanol as sole substrate. Addition of pyruvate to the growth medium restored growth on methanol/H2/CO2 but not on H2/CO2 or acetate. Results obtained from growth experiments, cell suspension experiments, and enzyme activity measurements in cell extracts provide compelling evidence for essential functions of Ech and a 2[4Fe-4S] ferredoxin in the metabolism of M. barkeri. The following conclusions were made. (i) In acetoclastic methanogenesis, Ech catalyzes H2 formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO2. (ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H2, most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway. (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H2/CO2, the reduction of CO2 to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis. PMID:11929975

Meuer, Jörn; Kuettner, H. Craig; Zhang, Jun Kai; Hedderich, Reiner; Metcalf, William W.

2002-01-01

266

Differential expression of the Chlamydomonas [FeFe]-hydrogenase-encoding HYDA1 gene is regulated by the copper response regulator1.  

PubMed

The unicellular green alga Chlamydomonas reinhardtii adapts to anaerobic or hypoxic conditions by developing a complex fermentative metabolism including the production of molecular hydrogen by [FeFe]-hydrogenase isoform1 (HYDA1). HYDA1 transcript and hydrogenase protein accumulate in the absence of oxygen or copper (Cu). Factors regulating this differential gene expression have been unknown so far. In this study, we report on the isolation of a Chlamydomonas mutant strain impaired in HYDA1 gene expression by screening an insertional mutagenesis library for HYDA1 promoter activity using the arylsulfatase-encoding ARYLSULFATASE2 gene as a selection marker. The mutant strain has a deletion of the COPPER RESPONSE REGULATOR1 (CRR1) gene encoding for CRR1, indicating that this SQUAMOSA-PROMOTER BINDING PROTEIN (SBP) domain transcription factor is involved in the regulation of HYDA1 transcription. Treating the C. reinhardtii wild type with mercuric ions, which were shown to inhibit the binding of the SBP domain to DNA, prevented or deactivated HYDA1 gene expression. Reporter gene analyses of the HYDA1 promoter revealed that two GTAC motifs, which are known to be the cores of CRR1 binding sites, are necessary for full promoter activity in hypoxic conditions or upon Cu starvation. However, mutations of the GTAC sites had a much stronger impact on reporter gene expression in Cu-deficient cells. Electrophoretic mobility shift assays showed that the CRR1 SBP domain binds to one of the GTAC cores in vitro. These combined results prove that CRR1 is involved in HYDA1 promoter activation. PMID:22669892

Pape, Miriam; Lambertz, Camilla; Happe, Thomas; Hemschemeier, Anja

2012-08-01

267

Designing interfaces of hydrogenase-nanomaterial hybrids for efficient solar conversion.  

PubMed

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. PMID:23541891

King, Paul W

2013-01-01

268

Borane-Protected Cyanides as Surrogates of H-Bonded Cyanides in [FeFe]-Hydrogenase Active Site Models  

PubMed Central

Triarylborane Lewis acids bind [Fe2(pdt)-(CO)4(CN)2]2? (pdt2? = 1,3-propanedithiolate) and [Fe2(adt)(CO)4(CN)2]2? [3]2? (adt2? = 1,3-azadithiolate, HN(CH2S?)2) to give the 2:1 adducts [Fe2(xdt)-(CO)4(CNBAr3)2]2?. Attempts to prepare the 1:1 adducts [1(BAr3)]2? (Ar = Ph, C6F5) were unsuccessful, but related 1:1 adducts were obtained using the bulky borane B(C6F4-o-C6F5)3 (BArF*3). By virtue of the N-protection by the borane, salts of [Fe2(pdt)(CO)4(CNBAr3)2]2? sustain protonation to give hydrides that are stable (in contrast to [H1]?). The hydrides [H1(BAr3)2]? are 2.5–5 pKa units more acidic than the parent [H1]?. The adducts [1(BAr3)2]2? oxidize quasi-reversibly around ?0.3 V versus Fc0/+ in contrast to ca. ?0.8 V observed for the [1]2?/? couple. A simplified synthesis of [1]2?, [3]2?, and [Fe2(pdt)(CO)5(CN)]? ([2]?) was developed, entailing reaction of the diiron hexacarbonyl complexes with KCN in MeCN. PMID:24992155

Manor, Brian C.; Ringenberg, Mark R.; Rauchfuss, Thomas B.

2015-01-01

269

Terminal Hydride in [FeFe]-Hydrogenase Model Has Lower Potential for H2 Production Than the Isomeric Bridging Hydride  

PubMed Central

Protonation of the symmetrical tetraphosphine complexes Fe2(S2CnH2n)(CO)2(dppv)2 afforded the corresponding terminal hydrides, establishing that even symmetrical diiron(I) dithiolates undergo protonation at terminal sites. The terminal hydride [HFe2(S2C3H6)(CO)2(dppv)2]+ was found to catalyze proton reduction at potentials 200 mV milder than the isomeric bridging hydride, thereby establishing a thermodynamic advantage for catalysis operating via terminal hydride. The azadithiolate protonates to afford, [Fe2[(SCH2)2NH2](CO)2(dppv)2]+, [HFe2[(SCH2)2NH](CO)2?(dppv)2]+, and [HFe2[(SCH2)2NH2](CO)2(dppv)2]2+, depending on conditions. PMID:18333613

Barton, Bryan E.; Rauchfuss, Thomas B.

2008-01-01

270

Steady-state catalytic wave-shapes for 2-electron reversible electrocatalysts and enzymes.  

PubMed

Using direct electrochemistry to learn about the mechanism of electrocatalysts and redox enzymes requires that kinetic models be developed. Here we thoroughly discuss the interpretation of electrochemical signals obtained with adsorbed enzymes and molecular catalysts that can reversibly convert their substrate and product. We derive analytical relations between electrochemical observables (overpotentials for catalysis in each direction, positions, and magnitudes of the features of the catalytic wave) and the characteristics of the catalytic cycle (redox properties of the catalytic intermediates, kinetics of intramolecular and interfacial electron transfer, etc.). We discuss whether or not the position of the wave is determined by the redox potential of a redox relay when intramolecular electron transfer is slow. We demonstrate that there is no simple relation between the reduction potential of the active site and the catalytic bias of the enzyme, defined as the ratio of the oxidative and reductive limiting currents; this explains the recent experimental observation that the catalytic bias of NiFe hydrogenase depends on steps of the catalytic cycle that occur far from the active site [Abou Hamdan et al., J. Am. Chem. Soc. 2012, 134, 8368]. On the experimental side, we examine which models can best describe original data obtained with various NiFe and FeFe hydrogenases, and we illustrate how the presence of an intramolecular electron transfer chain affects the voltammetry by comparing the data obtained with the FeFe hydrogenases from Chlamydomonas reinhardtii and Clostridium acetobutylicum, only one of which has a chain of redox relays. The considerations herein will help the interpretation of electrochemical data previously obtained with various other bidirectional oxidoreductases, and, possibly, synthetic inorganic catalysts. PMID:23362993

Fourmond, Vincent; Baffert, Carole; Sybirna, Kateryna; Lautier, Thomas; Abou Hamdan, Abbas; Dementin, Sébastien; Soucaille, Philippe; Meynial-Salles, Isabelle; Bottin, Hervé; Léger, Christophe

2013-03-13

271

The electronic structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: a Q-band 57Fe-ENDOR and HYSCORE study.  

PubMed

The active site of the (57)Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H(ox)) and the CO inhibited form (H(ox)-CO) all six (57)Fe hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six (57)Fe nuclei in the H-cluster of the H(ox)-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S](H) (cubane) subcluster. Two weak (57)Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H(ox) state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An (57)Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six (57)Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe](H) assumes a [Fe(I)Fe(II)] redox configuration where the paramagnetic Fe(I) atom is attached to the [4Fe-4S](H) subcluster. The (57)Fe hyperfine interactions of the formally diamagnetic [4Fe-4S](H) are due to an exchange interaction between the two subclusters as has been discussed earlier by Popescu and Münck [Popescu, C.V.; Münck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase. PMID:17722921

Silakov, Alexey; Reijerse, Eduard J; Albracht, Simon P J; Hatchikian, E Claude; Lubitz, Wolfgang

2007-09-19

272

An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli  

PubMed Central

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 presented cloning platform, the system might represent an important step towards new routes in synthetic biology. PMID:23861944

Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A. A.; Baumann, Marcus E. M.; Selmer, Thorsten

2013-01-01

273

X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution.  

PubMed

A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems. PMID:9836629

Peters, J W; Lanzilotta, W N; Lemon, B J; Seefeldt, L C

1998-12-01

274

Structural insights into the active-ready form of [FeFe]-hydrogenase and mechanistic details of its inhibition by carbon monoxide.  

PubMed

[FeFe]-hydrogenases harbor a {2Fe3S} assembly bearing two CO and two CN- groups, a mu-CO ligand, and a vacant coordination site trans to the mu-CO group. Recent theoretical results obtained studying the isolated {2Fe3S} subsite indicated that one of the CN- ligands can easily move from the crystallographic position to the coordination site trans to the mu-CO group; such an isomerization would have a major impact on substrates and inhibitors binding regiochemistry and, consequently, on the catalytic mechanism. To shed light on this crucial issue, we have carried out hybrid QM/MM and free energy perturbation calculations on the whole enzyme, which demonstrate that the protein environment plays a crucial role and maintains the CN- group fixed in the position observed in the crystal structure; these results strongly support the hypothesis that the vacant coordination site trans to the mu-CO group has a crucial functional relevance both in the context of CO-mediated inhibition of the enzyme and in dihydrogen oxidation/evolution catalysis. PMID:17676838

Greco, Claudio; Bruschi, Maurizio; Heimdal, Jimmy; Fantucci, Piercarlo; De Gioia, Luca; Ryde, Ulf

2007-09-01

275

Investigating the Role of the Outer-Coordination Sphere in [Ni(PPh2NPh-R2)2]2+ Hydrogenase Mimics  

SciTech Connect

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.

Jain, Avijita; Reback, Matthew L.; Lindstrom, Mary L.; Thogerson, Colleen E.; Helm, Monte L.; Appel, Aaron M.; Shaw, Wendy J.

2012-06-18

276

Relationship of proton motive force and the F(0)F (1)-ATPase with bio-hydrogen production activity of Rhodobacter sphaeroides: effects of diphenylene iodonium, hydrogenase inhibitor, and its solvent dimethylsulphoxide.  

PubMed

Rhodobacter sphaeroides MDC 6521 was able to produce bio-hydrogen (H(2)) in anaerobic conditions under illumination. In this study the effects of the hydrogenase inhibitor-diphenylene iodonium (Ph(2)I) and its solvent dimethylsulphoxide (DMSO) on growth characteristics and H(2) production by R. sphaeroides were investigated. The results point out the concentration dependent DMSO effect: in the presence of 10 mM DMSO H(2) yield was ~6 fold lower than that of the control. The bacterium was unable to produce H(2) in the presence of Ph(2)I. In order to examine the mediatory role of proton motive force (?p) or the F(0)F(1)-ATPase in H(2) production by R. sphaeroides, the effects of Ph(2)I and DMSO on ?p and its components (membrane potential (??) and transmembrane pH gradient), and ATPase activity were determined. In these conditions ?? was of -98 mV and the reversed ?pH was +30 mV, resulting in ?p of -68 mV. Ph(2)I decreased ?? in concentrations of 20 ?M and higher; lower concentrations of Ph(2)I as DMSO had no valuable effect on ??. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide. The 10-20 ?M Ph(2)I did not affect the ATPase activity, whereas 40 ?M Ph(2)I caused a marked inhibition (~2 fold) in ATPase activity. The obtained results provide novel evidence on the involvement of hydrogenase and the F(0)F(1)-ATPase in H(2) production by R. sphaeroides. Moreover, these data indicate the role of hydrogenase and the F(0)F(1)-ATPase in ?p generation. In addition, DMSO might increase an interaction of nitrogenase with CO(2), decreasing nitrogenase activity and affecting H(2) production. PMID:22689145

Hakobyan, Lilit; Gabrielyan, Lilit; Trchounian, Armen

2012-08-01

277

Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study.  

PubMed

The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium vinosum can exist in three different redox states. In the most oxidized state (Ni(a)-S) the nickel is divalent. The most reduced state (Ni(a)-SR) likewise has Ni(2+), while the intermediate state (Ni(a)-C) has Ni(3+). The transitions between these states have been studied by stopped-flow Fourier transform infrared spectroscopy. It is inferred from the data that the Ni(a)-S --> Ni(a)-C* and Ni(a)-C* --> Ni(a)-SR transitions induced by dihydrogen require one of the [4Fe-4S] clusters to be oxidized. Enzyme in the Ni(a)-S* state with all of the iron-sulfur clusters reduced reacts with dihydrogen to form the Ni(a)-SR state in milliseconds. By contrast, when one of the cubane clusters is oxidized, the Ni(a)-S state reacts with dihydrogen to form the Ni(a)-C state with all of the iron-sulfur clusters reduced. The competition between dihydrogen and carbon monoxide for binding to the active site was dependent on the redox state of the nickel ion. Formation of the Ni(a)-S.CO state (Ni(2+)) by reacting CO with enzyme in the Ni(a)-SR and Ni(a)-S states (Ni(2+)) is considerably faster than its formation from enzyme in the Ni(a)-C* (Ni(3+)) state. Excess oxygen converted hydrogen-reduced enzyme to the inactive Ni(r)* state within 158 ms, suggesting a direct reaction at the Ni-Fe site. With lower O(2) concentrations the formation of intermediate states was observed. The results are discussed in the light of the present knowledge of the structure and mechanism of action of the A. vinosum enzyme. PMID:15157115

George, Simon J; Kurkin, Sergei; Thorneley, Roger N F; Albracht, Simon P J

2004-06-01

278

Temperature tolerance of hydrogenase expression in Alcaligenes eutrophus is conferred by a single amino acid exchange in the transcriptional activator HoxA.  

PubMed Central

Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor sigma 54. Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragments containing the SH upstream region and purified beta-galactosidase-HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A. eutrophus failed to detect specific binding. In contrast, A. eutrophus extracts enriched for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA. This effect was not observed with comparable extracts prepared from hoxA mutants. A similar experiment using antibodies against HoxA confirmed that HoxA was responsible for the observed mobility shift. Extracts prepared from a temperature-tolerant mutant of A. eutrophus gave a stronger retardation than did those from the wild type. Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at temperatures above 33 degrees C because of a mutation in the regulatory gene hoxA. In this paper, we show that a single amino acid substitution (Gly-468-->Val) in the C-terminal part of HoxA is responsible for temperature tolerance. The SH upstream region also contains sequence motifs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator fragment. PMID:7730267

Zimmer, D; Schwartz, E; Tran-Betcke, A; Gewinner, P; Friedrich, B

1995-01-01

279

Hydrogen photoproduction by immobilized n2-fixing cyanobacteria: understanding the role of the uptake hydrogenase in the long-term process.  

PubMed

We have investigated two approaches to enhance and extend H2 photoproduction yields in heterocystous, N2-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H2 production over time. Our results demonstrated that alginate-entrapped ?hupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ?hupL mutant, and Calothrix strain 336/3 films were supplemented with N2 by periodic treatments of air, or air plus CO2. These treatments restored the photosynthetic activity of the cells and led to a high level of H2 production in Calothrix 336/3 and ?hupL cells (except for the treatment air plus CO2) but not in the Anabaena PCC 7120 strain (for which H2 yields did not change after air treatments). The highest H2 yield was obtained by the air treatment of ?hupL cells. Notably, the supplementation of CO2 under an air atmosphere led to prominent symptoms of N deficiency in the ?hupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells. PMID:25015894

Kosourov, Sergey; Leino, Hannu; Murukesan, Gayathri; Lynch, Fiona; Sivonen, Kaarina; Tsygankov, Anatoly A; Aro, Eva-Mari; Allahverdiyeva, Yagut

2014-09-01

280

Hydrogen Photoproduction by Immobilized N2-Fixing Cyanobacteria: Understanding the Role of the Uptake Hydrogenase in the Long-Term Process  

PubMed Central

We have investigated two approaches to enhance and extend H2 photoproduction yields in heterocystous, N2-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H2 production over time. Our results demonstrated that alginate-entrapped ?hupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ?hupL mutant, and Calothrix strain 336/3 films were supplemented with N2 by periodic treatments of air, or air plus CO2. These treatments restored the photosynthetic activity of the cells and led to a high level of H2 production in Calothrix 336/3 and ?hupL cells (except for the treatment air plus CO2) but not in the Anabaena PCC 7120 strain (for which H2 yields did not change after air treatments). The highest H2 yield was obtained by the air treatment of ?hupL cells. Notably, the supplementation of CO2 under an air atmosphere led to prominent symptoms of N deficiency in the ?hupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells. PMID:25015894

Kosourov, Sergey; Leino, Hannu; Murukesan, Gayathri; Lynch, Fiona; Sivonen, Kaarina; Tsygankov, Anatoly A.; Aro, Eva-Mari

2014-01-01

281

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2009, p. 46204623 Vol. 75, No. 13 0099-2240/09/$08.00 0 doi:10.1128/AEM.00582-09  

E-print Network

) is an important intermediate in the decom- position of organic matter in anaerobic environments and is the basis for many syntrophic interactions that occur in the food chain such as those between acetogens cycling in the GN saline mat environment. The [FeFe] hydrogenase has a central role in primary

282

A computational library for multiscale modeling of material failure  

NASA Astrophysics Data System (ADS)

We present an open-source software framework called PERMIX for multiscale modeling and simulation of fracture in solids. The framework is an object oriented open-source effort written primarily in Fortran 2003 standard with Fortran/C++ interfaces to a number of other libraries such as LAMMPS, ABAQUS, LS-DYNA and GMSH. Fracture on the continuum level is modeled by the extended finite element method (XFEM). Using several novel or state of the art methods, the piece software handles semi-concurrent multiscale methods as well as concurrent multiscale methods for fracture, coupling two continuum domains or atomistic domains to continuum domains, respectively. The efficiency of our open-source software is shown through several simulations including a 3D crack modeling in clay nanocomposites, a semi-concurrent FE-FE coupling, a 3D Arlequin multiscale example and an MD-XFEM coupling for dynamic crack propagation.

Talebi, Hossein; Silani, Mohammad; Bordas, Stéphane P. A.; Kerfriden, Pierre; Rabczuk, Timon

2014-05-01

283

Connecting [NiFe]- and [FeFe]-Hydrogenases: Mixed-Valence Nickel-Iron Dithiolates With Rotated Structures  

PubMed Central

A series of mixed-valence iron-nickel dithiolates is described that exhibits structures similar to those of mixed-valence diiron dithiolates. Interaction of tricarbonyl salt [(dppe)Ni(pdt)Fe(CO)3]BF4 ([1]BF4, dppe = Ph2PCH2CH2PPh2, pdtH2 = HSCH2CH2CH2SH) with P-donor ligands (L) afforded the substituted derivatives [(dppe)Ni(pdt)Fe(CO)2L]BF4 incorporating L = PHCy2 ([1a]BF4), PPh(NEt2)2 ([1b]BF4), P(NMe2)3 ([1c]BF4), P(i-Pr)3 ([1d]BF4) and PCy3 ([1e]BF4). The related precursor [(dcpe)Ni(pdt)Fe(CO)3]BF4 ([2]BF4, dcpe = Cy2PCH2CH2PCy2) gave the more electron-rich family of compounds [(dcpe)Ni(pdt)Fe(CO)2L]BF4 for L = PPh2(2-pyridyl) ([2a]BF4), PPh3 ([2b]BF4) and PCy3 ([2c]BF4). For bulky and strongly basic monophosphorus ligands, the salts feature distorted Fe coordination geometries: crystallographic analyses of [1e]BF4 and [2c]BF4 showed they adopt ‘rotated’ Fe(I) centers, in which PCy3 occupies a basal site and one CO ligand partially bridges the Ni and Fe centers. Like the undistorted mixed-valence derivatives, the new class of complexes are described as Ni(II)Fe(I) (S = ˝) systems according to EPR spectroscopy, although with attenuated 31P hyperfine interactions. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the spin for [1e]+ is localized in a Fe(I)-centered d(z2) orbital, orthogonal to the Fe-P bond. The PCy3 complexes, rare examples of species featuring ‘rotated’ Fe centers, both structurally and spectroscopically resemble mixed-valence diiron dithiolates. Also reproducing the NiS2Fe core of the [NiFe]-H2ase active site, the hybrid models incorporate key features of the two major classes of H2ase. Furthermore, cyclic voltammetry experiments suggest that the highly basic phosphine ligands enable a second oxidation corresponding to the couple [(dxpe)Ni(pdt)Fe(CO)2L]+/2+. The resulting unsaturated 32e? dications represent the closest approach to modeling the highly electrophilic Ni-SIa state. In the case of L = PPh2(2-pyridyl) chelation of this ligand accompanies the second oxidation. PMID:22838645

Schilter, David; Rauchfuss, Thomas B.; Stein, Matthias

2012-01-01

284

Precursors to [FeFe]-Hydrogenase Models: Syntheses of Fe2(SR)2(CO)6 from CO-Free Iron Sources  

PubMed Central

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

Chen, Jinzhu; Boyke, Christine; Rauchfuss, Thomas B.; Volkers, Phillip I.; Whaley, C. Matthew; Wilson, Scott R.; Yao, Haijun

2008-01-01

285

Coordination Chemistry of [HFe(CN)2(CO)3? and Its Derivatives: Toward a Model for the Iron Subsite of the [NiFe]-Hydrogenases  

PubMed Central

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 pKa ? 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)2L]? (L = P(OPh)3 and PPh3). 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 °C) with ?H‡ = 51.6 kJ mol?1 and ?S‡ = ?83.0 J K?1 mol?1. These ligands are displaced under an atmosphere of CO. With cis-Ph2PCH=CHPPh2 (dppv), we obtained the monocarbonyl, [HFe(CN)2(CO)(dppv)]?, a highly basic hydride (pKa > 23.3) that rearranges in solution to a single isomer. Treatment of [K(18-crown-6)][HFe(CN)2(CO)3] with Et4NCN resulted in rapid proton transfer 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(PPh3)]? 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 H2 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 °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, CH3). 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. PMID:19374433

Whaley, C. Matthew; Wilson, Scott R.

2009-01-01

286

Hydrogen as an energy source for the human pathogen Bilophila wadsworthia.  

PubMed

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H(2), formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia. PMID:18066702

da Silva, Sofia M; Venceslau, Sofia S; Fernandes, Cláudia L V; Valente, Filipa M A; Pereira, Inęs A C

2008-05-01

287

Nitrosyl Derivatives of Diiron(I) Dithiolates Mimic the Structure and Lewis Acidity of the FeFe-Hydrogenase Active Site  

PubMed Central

This study probes the impact of electronic asymmetry of diiron(I) dithiolato carbonyls. Treatment of Fe2(S2CnH2n)(CO)6-x(PMe3)x compounds (n = 2, 3) with NOBF4 gave the derivatives [Fe2(S2CnH2n)(CO)5-x(PMe3)x(NO)]BF4 (x = 1, 2, 3) which are electronically unsymmetrical due to the presence of a single NO+ ligand. Whereas the mono phosphine derivative is largely undistorted, the bis PMe3 derivatives are distorted such that the CO ligand on the Fe(CO)(PMe3)(NO)+ subunit is semibridging. Two isomers of [Fe2(S2C3H6)(CO)3(PMe3)2(NO)]BF4 were characterized spectroscopically and crystallographically. Each isomer features electron-rich [Fe(CO)2PMe3] and electrophilic [Fe(CO)(PMe3)(NO)]+ subunits. These species are in equilibrium with an unobserved isomer that reversibly binds CO (?H = ?35 kJ/mol, ?S = ?139 J/mol•K) to give the symmetrical adduct [Fe2(S2C3H6)(?-NO)(CO)4(PMe3)2]BF4. In contrast to Fe2(S2C3H6)(CO)4(PMe3)2, the bis(PMe3) nitrosyls readily undergo CO-substitution to give the (PMe3)3 derivatives. The nitrosyl complexes reduce at potentials that are ~1 V milder than their carbonyl counterparts. DFT calculations, specifically NBO values, reinforce the electronic resemblance of the nitrosyl complexes with the corresponding mixed-valence diiron complexes. Unlike other diiron dithiolato carbonyls, these species undergo reversible reductions at mild conditions. The results show that the novel structural and chemical features associated with mixed valence diiron dithiolates – the so-called Hox models - can be replicated in the absence of mixed-valency by introducing electronic asymmetry. PMID:18700771

Olsen, Matthew T.; Bruschi, Maurizio; Wilson, Scott R.

2008-01-01

288

Modelling the Thermal History of Asteroid 4 Vesta  

NASA Technical Reports Server (NTRS)

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.

Solano, James M.; Kiefer, W. S.; Mittlefehldt, D. W.

2012-01-01

289

Using Stable Isotopes to Trace Microbial Hydrogen Production Pathways  

NASA Astrophysics Data System (ADS)

Biological H2 production by hydrogenase enzymes (H2ases) plays an important role in anaerobic microbial metabolism and community structure. Despite considerable progress in elucidating H2 metabolism, the regulation of and flux through key H2 production pathways remain largely undefined. Our goal is to improve understanding of biological H2 production by using H isotope ratios to dissect proton fluxes through different H2ase enzymes and from different substrates. We hypothesized that the isotope ratio of H2 produced by various hydrogenases (H2ase) would differ, and that the H isotope ratios would allow us to define the contribution of different enzymes when more than one is present in vivo. We chose Shewanella oneidensis (S.o.) MR-1, a facultative anaerobe capable of transferring electrons to a variety of terminal acceptors, including protons, as a model system for in vivo studies. S. o. encodes one [FeFe]- and one [NiFe]-H2ase. We purified three [FeFe]-H2ases (S.o., Clostridium pasteurianum, and Chlamydomonas reinhardtii) and two [NiFe]-H2ases (S. o. and Desulfovibrio fructosovorans) to test the isotope fractionation associated with activity by each enzyme in vitro. For in vivo analysis we used wild-type S.o. as well as electron transfer-deficient and H2ase-deficient strains. We employed batch cultures using lactate as an electron donor and O2 as an initial electron acceptor (with H2 production after O2 consumption). The five H2ases we tested all had a unique isotope fractionation. Measurements of H2 produced in vivo showed distinct periods of H2 production having isotope signatures consistent with in vitro results. Isotope data as well as studies of H2 production by mutants in the genes encoding either the [NiFe]-H2ase or the [FeFe]-H2ase, respectively, show that the [NiFe]- and [FeFe]- H2ases became active at different times. The [NiFe]-H2ase both produces and consumes H2 before the [FeFe]-H2ase becomes active. RNA analysis is consistent with up regulation of different hydrogenases at different points in the culture’s growth, but presents a mystery. Transcription of the [NiFe]-H2ase is more coincident with detection of H2 production and uptake by the protein. The [FeFe]-H2ase gene, however, undergoes a burst of transcription long before H2 production by the protein is detected. A second burst of transcription of the gene coincides with H2 production. We are working towards identifying key conditions that direct hydrogenase activity (including redox conditions and availability of auxiliary electron acceptors). Taken together we show that different H2ases express different fractionation factors in vitro, and H isotope ratios can be exploited to dissect pathways of H2 production in vivo.

Moran, J.; Hill, E.; Bartholomew, R.; Yang, H.; Shi, L.; Ostrom, N. E.; Gandhi, H.; Hegg, E.; Kreuzer, H.

2010-12-01

290

Mechanistic modeling of sulfur-deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii  

PubMed Central

The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron-hydrogenase is well known. However, the oxygen-sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo-production. Under illumination, sulfur-deprivation has been shown to accommodate the production of hydrogen gas by partially-deactivating O2 evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H2 production during sulfur-deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. Biotechnol. Bioeng. 2014;111: 320–335. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:24026984

Williams, C R; Bees, MA

2014-01-01

291

MetREx: A protein design approach for the exploration of sequence-reactivity relationships in metalloenzymes.  

PubMed

Metalloenzymes represent a particular challenge for any rational (re)design approach because the modeling of reaction events at their metallic cofactors requires time-consuming quantum mechanical calculations, which cannot easily be reconciled with the fast, knowledge-based approaches commonly applied in protein design studies. Here, an approach for the exploration of sequence-reactivity relationships in metalloenzymes is presented (MetREx) that consists of force field-based screening of mutants that lie energetically between a wild-type sequence and the global minimum energy conformation and which should, therefore, be compatible with a given protein fold. Mutant candidates are subsequently evaluated with a fast and approximate quantum mechanical/molecular mechanical-like procedure that models the influence of the protein environment on the active site by taking partial charges and van der Waals repulsions into account. The feasibility of the procedure is demonstrated for the active site of [FeFe] hydrogenase from Desulfovibrio desulfuricans. The method described allows for the identification of mutants with altered properties, such as inhibitor-coordination energies, and the understanding of the robustness of enzymatic reaction steps with respect to variations in sequence space. © 2015 Wiley Periodicals, Inc. PMID:25649465

Stiebritz, Martin T

2015-03-30

292

Integrated analysis of transcriptomic and proteomic data of Desulfovibrio vulgaris: Zero-Inflated Poisson regression models to predict abundance of undetected proteins  

SciTech Connect

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.

Nie, Lei; Wu, Gang; Brockman, Fred J.; Zhang, Weiwen

2006-05-04

293

Theoretical Studies of Structures and Mechanisms in Organometallic and Bioinorganic Chemistry: Heck Reaction with Palladium Phosphines, Active Sites of Superoxide Reductase and Cytochrome P450 Monooxygenase, and Tetrairon Hexathiolate Hydrogenase Model  

E-print Network

-orbital can introduce a polarization to the s-orbital for a bound hydrogen atom. Many properties depend on the wave function tail, far from the nucleus; to describe molecules with loosely bound electrons, such as anions, the basis functions with small...

Surawatanawong, Panida

2010-07-14

294

Combined DFT and BS study on the exchange coupling of dinuclear sandwich-type POM: comparison of different functionals and reliability of structure modeling.  

PubMed

The exchange coupling of a group of three dinuclear sandwich-type polyoxomolybdates [MM'(AsMo7O27)2](12-) with MM' = CrCr, FeFe, FeCr are theoretically predicted from combined DFT and broken-symmetry (BS) approach. Eight different XC functionals are utilized to calculate the exchange-coupling constant J from both the full crystalline structures and model structures of smaller size. The comparison between theoretical values and accurate experimental results supports the applicability of DFT-BS method in this new type of sandwich-type dinuclear polyoxomolybdates. However, a careful choice of functionals is necessary to achieve the desired accuracy. The encouraging results obtained from calculations on model structures highlight the great potential of application of structure modeling in theoretical study of POM. Structural modeling may not only reduce the computational cost of large POM species but also be able to take into account the external field effect arising from solvent molecules in solution or counterions in crystal. PMID:21975539

Yin, Bing; Xue, GangLin; Li, JianLi; Bai, Lu; Huang, YuanHe; Wen, ZhenYi; Jiang, ZhenYi

2012-05-01

295

Spin relaxation in antiferromagnetic Fe–Fe dimers slowed down by anisotropic DyIII ions  

PubMed Central

Summary By using Mössbauer spectroscopy in combination with susceptibility measurements it was possible to identify the supertransferred hyperfine field through the oxygen bridges between DyIII and FeIII in a {Fe4Dy2} coordination cluster. The presence of the dysprosium ions provides enough magnetic anisotropy to “block” the hyperfine field that is experienced by the iron nuclei. This has resulted in magnetic spectra with internal hyperfine fields of the iron nuclei of about 23 T. The set of data permitted us to conclude that the direction of the anisotropy in lanthanide nanosize molecular clusters is associated with the single ion and crystal field contributions and 57Fe Mössbauer spectroscopy may be informative with regard to the the anisotropy not only of the studied isotope, but also of elements interacting with this isotope. PMID:24367750

Klöwer, Frederik; Lan, Yanhua; Clérac, Rodolphe; Wolny, Juliusz A; Schünemann, Volker; Anson, Christopher E

2013-01-01

296

Microstructure, mechanical property, biodegradation behavior, and biocompatibility of biodegradable FeFe2O3 composites  

E-print Network

properties.1,2 Preliminary in vivo animal tests3­5 on pure iron stents show good biocompatibility of ironO instead of Fe2O3. Both eletro- chemical measurements and immersion test showed a faster degradation rate is a promising alternative for biodegradable stent material with elevated corrosion rate, enhanced mechanical

Zheng, Yufeng

297

Cluster molecular orbital description of the electronic structures of mixed-valence iron oxides and silicates  

USGS Publications Warehouse

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.

Sherman, D.M.

1986-01-01

298

Modeling and computations of the intramolecular electron transfer process in the two-heme protein cytochrome c(4).  

PubMed

The di-heme protein Pseudomonas stutzeri cytochrome c(4) (cyt c(4)) has emerged as a useful model for studying long-range protein electron transfer (ET). Recent experimental observations have shown a dramatically different pattern of intramolecular ET between the two heme groups in different local environments. Intramolecular ET in homogeneous solution is too slow (>10 s) to be detected but fast (ms-?s) intramolecular ET in an electrochemical environment has recently been achieved by controlling the molecular orientation of the protein assembled on a gold electrode surface. In this work we have performed computational modeling of the intramolecular ET process by a combination of density functional theory (DFT) and quantum mechanical charge transfer theory to disclose reasons for this difference. We first address the electronic structures of the model heme core with histidine and methionine axial ligands in both low- and high-spin states by structure-optimized DFT. The computations enable estimating the intramolecular reorganization energy of the ET process for different combinations of low- and high-spin heme couples. Environmental reorganization free energies, work terms ("gating") and driving force were determined using dielectric continuum models. We then calculated the electronic transmission coefficient of the intramolecular ET rate using perturbation theory combined with the electronic wave functions determined by the DFT calculations for different heme group orientations and Fe-Fe separations. The reactivity of low- and high-spin heme groups was notably different. The ET rate is exceedingly low for the crystallographic equilibrium orientation but increases by several orders of magnitude for thermally accessible non-equilibrium configurations. Deprotonation of the propionate carboxyl group was also found to enhance the ET rate significantly. The results are discussed in relation to the observed surface immobilization effect and support the notion of conformationally gated ET. PMID:22430606

Nazmutdinov, Renat R; Bronshtein, Michael D; Zinkicheva, Tamara T; Chi, Qijin; Zhang, Jingdong; Ulstrup, Jens

2012-05-01

299

Survey of Hydrogenase Activity in Algae: Final Report  

SciTech Connect

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.

Brand, J. J.

1982-04-01

300

Hydrogen production using hydrogenase-containing oxygenic photosynthetic organisms  

DOEpatents

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.

Melis, Anastasios; Zhang, Liping; Benemann, John R.; Forestier, Marc; Ghirardi, Maria; Seibert, Michael

2006-01-24

301

Hydrogen Production Using Hydrogenase-Containing Oxygenic Photosynthetic Organisms  

DOEpatents

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.

Melis, A.; Zhang, L.; Benemann, J. R.; Forestier, M.; Ghirardi, M.; Seibert, M.

2006-01-24

302

An atomistic model for the graphite-alumina/liquid iron system: Monte-Carlo simulations on carbon dissolution  

SciTech Connect

A Monte-Carlo (MC) simulation study has been carried out at 1600 deg C on graphite-alumina/liquid iron system with an aim to develop an atomistic model with optimum interaction parameters. The atomistic model of the graphite/liquid iron system was extended to include alumina molecules in the solid graphitic lattice. A molecule of alumina was represented as a rigid unified group of five atoms and occupied five neighbouring lattice sites. Using a canonical ensemble, simulations were carried out for a range of compositions and alumina interaction parameters. As non-wetting of alumina by liquid iron was expected to significantly affect carbon dissolution from graphite-alumina mixtures, interaction between alumina and iron was assumed to be strongly repulsive. Dissolution of carbon from alumina-graphite into liquid iron was monitored and compared with the experimental results. Even for a wide choice of alumina interaction parameters, it was not possible to get a good fit between simulation and experimental results. Another attempt was made to incorporate non-wetting at an atomic level by modifying the nature of interactions on the solid/liquid interface. While these simulations produced a much better agreement with experiment, some gaps still persisted at high alumina concentrations. A third attempt was then made to simulate non-wetting behaviour in terms of mutual exclusion of alumina and iron from their immediate neighbourhood. An excellent fit was obtained with experimental results in the entire composition range. In addition, alumina interaction parameters were found to have no effect on carbon dissolution and were therefore redundant. The optimum interaction parameters for the graphite-alumina/liquid iron system are: J{sub 1}(C-C) = -J,J{sub 1}(C-Fe) = 0.6J,J{sub 1}(Fe-Fe) = J and no interaction parameters for alumina.

Khanna, R. [School of Materials Science and Engineering, University of New South Wales, UNSW, Sydney NSW 2052 (Australia)]. E-mail: ritakhanna@unsw.edu.au; Sahajwalla, V. [School of Materials Science and Engineering, University of New South Wales, UNSW, Sydney NSW 2052 (Australia)

2005-02-01

303

Toward a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough  

SciTech Connect

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.

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

304

Modeling modeling.  

PubMed Central

Models are tools; they need to fit both the hand and the task. Presence or absence of a feature such as a pacemaker or a cascade is not in itself good. Or bad. Criteria for model evaluation involve benefit-cost ratios, with the numerator a function of the range of phenomena explained, goodness of fit, consistency with other nearby models, and intangibles such as beauty. The denominator is a function of complexity, the number of phenomena that must be ignored, and the effort necessary to incorporate the model into one's parlance. Neither part of the ratio can yet be evaluated for MTS, whose authors provide some cogent challenges to SET. PMID:10220934

Killeen, P R

1999-01-01

305

Genome Annotation Provides Insight into Carbon Monoxide and Hydrogen Metabolism in Rubrivivax gelatinosus  

PubMed Central

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. 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. PMID:25479613

Wawrousek, Karen; Noble, Scott; Korlach, Jonas; Chen, Jin; Eckert, Carrie; Yu, Jianping; Maness, Pin-Ching

2014-01-01

306

Single-Neuron Activity and Tissue Oxygenation in the Cerebral Cortex Jeffrey K.Thompson, Matthew R.Peterson, Ralph D.Freeman  

E-print Network

Axinyssa n. sp. has been proposed to involve sulfuration of cyanide to give ­ SCN followed by reaction cycle (23). 15. For evidence supporting the relevancy of modeling the active site of [NiFe] hydrogenase

Freeman, Ralph D.

307

Ion irradiation of Fe-Fe oxide core-shell nanocluster films: Effect of interface on stability of magnetic properties  

SciTech Connect

A cluster deposition method was used to produce films of loosely aggregated nanoclusters (NC) 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 nanocluster films depends strongly on the initial nanostructure and chemistry, but in general saturation magnetization decreases slightly.

McCloy, John S.; Jiang, Weilin; Droubay, Timothy C.; Varga, Tamas; Kovarik, Libor; Sundararajan, Jennifer A.; Kaur, Maninder; Qiang, You; Burks, Edward; Liu, Kai

2013-08-23

308

Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus*  

PubMed Central

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

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

309

Metal-Metal Bonds in Biology  

PubMed Central

Nickel-containing carbon monoxide dehydrogenases, acetyl-CoA synthases, nickel-iron hydrogenases, and diron hydrogenases are distinct metalloenzymes yet they share a number of important characteristics. All are O2-sensitive, with active-sites composed of iron and/or nickel ions coordinated primarily by sulfur ligands. In each case, two metals are juxtaposed at the “heart” of the active site, within range of forming metal-metal bonds. These active-site clusters exhibit multielectron redox abilities and must be reductively activated for catalysis. Reduction potentials are milder than expected based on formal oxidation state changes. When reductively activated, each cluster attacks an electrophilic substrate via an oxidative addition reaction. This affords a two-electron-reduced substrate bound to one or both metals of an oxidized cluster. M-M bonds have been established in hydrogenases where they serve to initiate the oxidative addition of protons and perhaps stabilize active sites in multiple redox states. The same may be true of the CODH and ACS active sites – Ni-Fe and Ni-Ni bonds in these sites may play critical roles in catalysis, stabilizing low-valence states and initiating oxidative addition of CO2 and methyl group cations, respectively. In this article, the structural and functional commonalities of these metalloenzyme active sites are described, and the case is made for the formation and use of metal-metal bonds in each enzyme mentioned. As a post-script, the importance of Fe-Fe bonds in the nitrogenase FeMoco active site is discussed. PMID:22119810

Lindahl, Paul A.

2011-01-01

310

Insights Into the P-To-Q Conversion in the Catalytic Cycle of Methane Monooxygenase From a Synthetic Model System  

SciTech Connect

For the catalytic cycle of soluble methane monooxygenase (sMMO), it has been proposed that cleavage of the O-O bond in the ({mu}-peroxo)diiron(III) intermediate P gives rise to the diiron(IV) intermediate Q with an Fe{sub 2}({mu}-O){sub 2} diamond core, which oxidizes methane to methanol. As a model for this conversion, ({mu}-oxo) diiron(III) complex 1 ([Fe{sup III}{sub 2}({mu}-O)({mu}-O{sub 2}H{sub 3})(L){sub 2}]{sup 3+}, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) has been treated consecutively with one eq of H{sub 2}O{sub 2} and one eq of HClO{sub 4} to form 3 ([Fe{sup IV}{sub 2}({mu}-O){sub 2}(L){sub 2}]{sup 4+}). In the course of this reaction a new species, 2, can be observed before the protonation step; 2 gives rise to a cationic peak cluster by ESI-MS at m/z 1,399, corresponding to the [Fe{sub 2}O{sub 3}L{sub 2}H](OTf){sub 2}{sup +} ion in which 1 oxygen atom derives from 1 and the other two originate from H{sub 2}O{sub 2}. Moessbauer studies of 2 reveal the presence of two distinct, exchange coupled iron(IV) centers, and EXAFS fits indicate a short Fe-O bond at 1.66 {angstrom} and an Fe-Fe distance of 3.32 {angstrom}. Taken together, the spectroscopic data point to an HO-Fe{sup IV}-O-Fe{sup IV} = O core for 2. Protonation of 2 results in the loss of H{sub 2}O and the formation of 3. Isotope labeling experiments show that the [Fe{sup IV}{sub 2}({mu}-O){sub 2}] core of 3 can incorporate both oxygen atoms from H{sub 2}O{sub 2}. The reactions described here serve as the only biomimetic precedent for the conversion of intermediates P to Q in the sMMO reaction cycle and shed light on how a peroxodiiron(III) unit can transform into an [Fe{sup IV}{sub 2}({mu}-O){sub 2}] core.

Xue, G.; Fiedler, A.T.; Martinho, M.; Munck, E.; Que, L.; Jr.

2009-05-28

311

Change in the magnetic properties of polycrystalline thin-film magnetite upon introduction of an iron sublayer  

NASA Astrophysics Data System (ADS)

The field dependences of the magnetic moment of polycrystalline magnetite films formed by pulsed laser deposition on a silicon substrate with the addition of an iron sublayer have been investigated. The influence of the sequence of layers Fe/Fe3O4 and Fe3O4/Fe on the magnetic characteristics of these structures has been analyzed. It has turned out that an increase in the saturation magnetization and the formation of a rectangular hysteresis loop with the coercive force acceptable for applications of thin-film magnetite as a hard magnetic electrode of the magnetic tunnel junction are observed only for the sequence of layers Fe/Fe3O4. The effect of the vacuum annealing temperature on the magnetic properties of polycrystalline samples of the Fe/Fe3O4 structure has been studied. It has been found that the best result is achieved at an annealing temperature of 500°C. The phenomenological model describing the magnetic properties of the polycrystalline two-layer magnetic structure Fe/Fe3O4 has been formulated. The results of numerical calculations have demonstrated that the introduction of only two phenomenological anisotropic interactions into the expression for the energy of the film provides a qualitative description of the observed experimental data in the form of hysteresis loops.

Anisimov, A. V.; Goikhman, A. Yu.; Kupriyanova, G. S.; Nevolin, V. N.; Popov, A. P.; Rodionova, V. V.

2012-06-01

312

Fermentation of biomass-generated synthesis gas: effects of nitric oxide.  

PubMed

The production of renewable fuels, such as ethanol, has been steadily increasing owing to the need for a reduced dependency on fossil fuels. It was demonstrated previously that biomass-generated synthesis gas (biomass-syngas) can be converted to ethanol and acetic acid using a microbial catalyst. The biomass-syngas (primarily CO, CO(2), H(2), and N(2)) was generated in a fluidized-bed gasifier and used as a substrate for Clostridium carboxidivorans P7(T). Results showed that the cells stopped consuming H(2) when exposed to biomass-syngas, thus indicating that there was an inhibition of the hydrogenase enzyme due to some biomass-syngas contaminant. It was hypothesized that nitric oxide (NO) detected in the biomass-syngas could be the possible cause of this inhibition. The specific activity of hydrogenase was monitored with time under varying concentrations of H(2) and NO. Results indicated that NO (at gas concentrations above 40 ppm) was a non-competitive inhibitor of hydrogenase activity, although the loss of hydrogenase activity was reversible. In addition, NO also affected the cell growth and increased the amount of ethanol produced. A kinetic model of hydrogenase activity with inhibition by NO was demonstrated with results suggesting there are multiple binding sites of NO on the hydrogenase enzyme. Since other syngas-fermenting organisms utilize the same metabolic pathways, this study estimates that NO < 40 ppm can be tolerated by cells in a syngas-fermentation system without compromising the hydrogenase activity, cell growth, and product distribution. PMID:17171719

Ahmed, Asma; Lewis, Randy S

2007-08-01

313

Experimental partitioning studies near the Fe-FeS eutectic, with an emphasis on elements important to iron meteorite chronologies (Pb, Ag, Pd, and Tl)  

NASA Technical Reports Server (NTRS)

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.

Jones, J. H.; Hart, S. R.; Benjamin, T. M.

1993-01-01

314

Das Verhalten von Nitratreductase, Nitritreductase, Hydrogenase und anderen Enzymen von Ankistrodesmus braunii bei Stickstoffmangel  

Microsoft Academic Search

An oxidation of organic nitrogen compounds leading to an intracellular formation of nitrite and nitrate (heterotrophic nitrification) was found in nitrogen-deficient Ankistrodesmus braunii. This explains the rather high levels of nitrate and nitrite reductases observed in algae after the supply of nitrogen has been exhausted.

Heinz Oesterheld

1971-01-01

315

The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans*  

PubMed Central

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H2 has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H2 with a limiting rate constant of 5.3 s?1 and a dissociation constant Kd of 525 ?m; both kred and kred/Kd, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H2, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site 63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H2 oxidation is proposed which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center. PMID:24165123

Wilcoxen, Jarett; Hille, Russ

2013-01-01

316

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

E-print Network

, SEM and zeta-potential measurement) in an aqueous electron donor solution (0.1 M, 3 mL). The catalyst (H2ase or NiP, see below) was added to the suspension and the light-protected reactor was sealed and purged with 2 % CH4 (as an internal gas... site for hydrogen bonding interactions between –NH–, terminal –NH2 or Lewis basic heptazine edge nitrogens in CNx.[10a, 17] The isoelectric point of CNx was determined by zeta potential measurements as 3.3[18] (Figure S4) and, at pH 6...

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

2014-09-09

317

Cobalt complexes as artificial hydrogenases for the reductive side of water splitting.  

PubMed

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. PMID:23689026

Eckenhoff, William T; McNamara, William R; Du, Pingwu; Eisenberg, Richard

2013-01-01

318

Chelate Control of Diiron(I) Dithiolates Relevant to the [Fe–Fe]-Hydrogenase Active Site  

PubMed Central

The reaction of Fe2(S2C2H4)(CO)6 with cis-Ph2PCH=CHPPh2 (dppv) yields Fe2(S2C2H4)(CO)4(dppv), 1(CO)4, wherein the dppv ligand is chelated to a single iron center. NMR analysis indicates that in 1(CO)4, the dppv ligand spans axial and basal coordination sites. In addition to the axial–basal isomer, the 1,3-propanedithiolate and azadithiolate derivatives exist as dibasal isomers. Density functional theory (DFT) calculations indicate that the axial–basal isomer is destabilized by nonbonding interactions between the dppv and the central NH or CH2 of the larger dithiolates. The Fe(CO)3 subunit in 1(CO)4 undergoes substitution with PMe3 and cyanide to afford 1(CO)3(PMe3) and (Et4N)[1(CN)(CO)3], respectively. Kinetic studies show that 1(CO)4 reacts faster with donor ligands than does its parent Fe2(S2C2H4)(CO)6. The rate of reaction of 1(CO)4 with PMe3 was first order in each reactant, k = 3.1 × 10? 4 M?1 s?1. The activation parameters for this substitution reaction, ?H‡ = 5.8(5) kcal/mol and ?S‡ = ?48(2) cal/deg·mol, indicate an associative pathway. DFT calculations suggest that, relative to Fe2(S2C2H4)(CO)6, the enhanced electrophilicity of 1(CO)4 arises from the stabilization of a “rotated” transition state, which is favored by the unsymmetrically disposed donor ligands. Oxidation of MeCN solutions of 1(CO)3(PMe3) with Cp2FePF6 yielded [Fe2(S2C2H4)(?-CO)(CO)2(dppv)(PMe3)(NCMe)](PF6)2. Reaction of this compound with PMe3 yielded [Fe2(S2C2H4)(?-CO)(CO)(dppv)(PMe3)2(NCMe)](PF6)2. PMID:17279743

Justice, Aaron K.; Zampella, Giuseppe; De Gioia, Luca; Rauchfuss, Thomas B.; van der Vlugt, Jarl Ivar; Wilson, Scott R.

2008-01-01

319

From Hydrogenases to Noble Metal-Free Catalytic Nanomaterials for H2 Production and Uptake  

Microsoft Academic Search

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

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

2009-01-01

320

Models and Modeling.  

ERIC Educational Resources Information Center

In this workshop, we will continue to reflect on a models and modeling perspective to understand how students and teachers learn and reason about real life situations encountered in a mathematics and science classroom. We will discuss the idea of a model as a conceptual system that is expressed by using external representational media, and that is…

Lesh, Richard; Carmona, Guadalupe; Post, Thomas

321

Thermodynamics and phase relationships of transition metal-sulfur systems: Part III. Thermodynamic properties of the Fe-S liquid phase and the calculation of the Fe-S phase diagram  

Microsoft Academic Search

An associated solution model is applied to describe the thermodynamic behavior of Fe-S liquid. This model assumes the existence\\u000a of ‘FeS’ species in addition to Fe and S in the liquid. With two solution parameters for each of the binaries Fe-‘FeS’ and\\u000a ‘FeS’-S, this model accounts for the compositional dependence of the thermodynamic properties of Fe-S liquid from pure Fe

R. C. Sharma; Y. A. Chang

1979-01-01

322

Models, Fiction, and Fictional Models  

NASA Astrophysics Data System (ADS)

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

Liu, Chuang

2014-03-01

323

Flexibility of Syntrophic Enzyme Systems in Desulfovibrio Species Ensures Their Adaptation Capability to Environmental Changes  

PubMed Central

The mineralization of organic matter in anoxic environments relies on the cooperative activities of hydrogen producers and consumers obligately linked by interspecies metabolite exchange in syntrophic consortia that may include sulfate reducing species such as Desulfovibrio. To evaluate the metabolic flexibility of syntrophic Desulfovibrio to adapt to naturally fluctuating methanogenic environments, we studied Desulfovibrio alaskensis strain G20 grown in chemostats under respiratory and syntrophic conditions with alternative methanogenic partners, Methanococcus maripaludis and Methanospirillum hungatei, at different growth rates. Comparative whole-genome transcriptional analyses, complemented by G20 mutant strain growth experiments and physiological data, revealed a significant influence of both energy source availability (as controlled by dilution rate) and methanogen on the electron transfer systems, ratios of interspecies electron carriers, energy generating systems, and interspecies physical associations. A total of 68 genes were commonly differentially expressed under syntrophic versus respiratory lifestyle. Under low-energy (low-growth-rate) conditions, strain G20 further had the capacity to adapt to the metabolism of its methanogenic partners, as shown by its differing gene expression of enzymes involved in the direct metabolic interactions (e.g., periplasmic hydrogenases) and the ratio shift in electron carriers used for interspecies metabolite exchange (hydrogen/formate). A putative monomeric [Fe-Fe] hydrogenase and Hmc (high-molecular-weight-cytochrome c3) complex-linked reverse menaquinone (MQ) redox loop become increasingly important for the reoxidation of the lactate-/pyruvate oxidation-derived redox pair, DsrCred and Fdred, relative to the Qmo-MQ-Qrc (quinone-interacting membrane-bound oxidoreductase; quinone-reducing complex) loop. Together, these data underscore the high enzymatic and metabolic adaptive flexibility that likely sustains Desulfovibrio in naturally fluctuating methanogenic environments. PMID:23974031

Meyer, Birte; Kuehl, Jennifer V.; Deutschbauer, Adam M.; Arkin, Adam P.

2013-01-01

324

A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea.  

PubMed

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H(+)-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H(2), formate, pyruvate, NAD(P)H, ?-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation. PMID:21747791

Pereira, Inęs A Cardoso; Ramos, Ana Raquel; Grein, Fabian; Marques, Marta Coimbra; da Silva, Sofia Marques; Venceslau, Sofia Santos

2011-01-01

325

A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea  

PubMed Central

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, ?-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation. PMID:21747791

Pereira, Inęs A. Cardoso; Ramos, Ana Raquel; Grein, Fabian; Marques, Marta Coimbra; da Silva, Sofia Marques; Venceslau, Sofia Santos

2011-01-01

326

Niche Modeling: Model Evaluation  

E-print Network

of carelessness. Among these aspects is that of model evaluation or validation. This unit provides a simple introduction to the basic concepts that are important in model validation, using some very simple examples. The focus is on two solutions to the challenge...

Peterson, A. Townsend

2012-08-29

327

Quantum Circuit Model Topological Model  

E-print Network

Quantum Circuit Model Topological Model Comparison of Models Topological Quantum Computation Eric Rowell Texas A&M University October 2010 Eric Rowell Topological Quantum Computation #12;Quantum Circuit Model Topological Model Comparison of Models Outline 1 Quantum Circuit Model Gates, Circuits

Rowell, Eric C.

328

Synthesis, Purification, and Characterization of a [mu]-(1,3-Propanedithiolato)-Hexacarbonyldiiron  

ERIC Educational Resources Information Center

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,…

Works, Carmen F.

2007-01-01

329

Genome-Guided Analysis of Physiological Capacities of Tepidanaerobacter acetatoxydans Provides Insights into Environmental Adaptations and Syntrophic Acetate Oxidation  

PubMed Central

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

Niazi, Adnan; Bongcam-Rudloff, Erik; Schnürer, Anna

2015-01-01

330

Genome-Guided Analysis of Physiological Capacities of Tepidanaerobacter acetatoxydans Provides Insights into Environmental Adaptations and Syntrophic Acetate Oxidation.  

PubMed

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

Müller, Bettina; Manzoor, Shahid; Niazi, Adnan; Bongcam-Rudloff, Erik; Schnürer, Anna

2015-01-01

331

MODEL DEVELOPMENT - DOSE MODELS  

EPA Science Inventory

Model Development Humans are exposed to mixtures of chemicals from multiple pathways and routes. These exposures may result from a single event or may accumulate over time if multiple exposure events occur. The traditional approach of assessing risk from a single chemica...

332

Modeling Malaria  

NSDL National Science Digital Library

In this module, we develop models of the effects of malaria on various populations of humans and mosquitoes. After considering differential equations to model a system, we create a model using the systems modeling tool STELLA. Projects involve various refinements of the model.

Angela B. Shiflet

333

Fair Model  

NSDL National Science Digital Library

The Fair model web site includes a freely available United States macroeconomic econometric model and a multicounty econometric model. The models run on the Windows OS. Instructors can use the models to teach forecasting, run policy experiments, and evaluate historical episodes of macroeconomic behavior. The web site includes extensive documentation for both models. The simulation is for upper-division economics courses in macroeconomics or econometrics. The principle developer is Ray Fair at Yale University.

Betty Blecha

334

Toilet Model  

NSDL National Science Digital Library

In this activity, PVC pipe, plastic water bottles and vinyl tubing are used to make a simple working toilet model. The model shows the role of a siphon in the flushing of a toilet. Educators can pre-assemble this model and use it for demonstration purposes or engage learners in the model building process.

Don Rathjen

2005-01-01

335

The UCSD HIRES/Keck I Damped Ly? Abundance Database. III. An Empirical Study of Photoionization in the Damped Ly? System toward GB 1759+7539  

NASA Astrophysics Data System (ADS)

We investigate the ionization state of the damped Ly? system at z=2.62 toward GB 1759+7539 through an analysis of ionic ratios sensitive to photoionization: Ar0/S+, Fe++/Fe+, N+/N0, and Al++/Al+. Approximately half of the metals arise in a mostly neutral velocity component with H I/H>0.9, based on Fe++/Fe+<0.013. In contrast, the remaining half exhibit Fe++/Fe+~0.3, indicative of a partially ionized medium with H I/H~0.5. These conclusions are supported by the observed N+/N0, Al++/Al+, and Ar0/Si+ ratios. We assess ionization corrections for the observed column densities through photoionization models derived from the CLOUDY software package. In the neutral gas, the ionization corrections are negligible, except for Ar0. However, for the partially ionized gas, element abundance ratios differ from the ionic ratios by 0.1-0.3 dex for (Si+, S+, Ni+, Al+)/Fe+ ratios and more for (N0, Ar0)/Fe+. Independent of the shape of the photoionizing spectrum and assumptions of the number of ionization phases, these ionization corrections have minimal impact (<~0.1 dex) on the total metallicity inferred for this damped Ly? system. Measurements of the relative elemental abundances of the partially ionized gas, however, have a greater than ~0.15 dex uncertainty, which hides the effects of nucleosynthesis and differential dust depletion. We caution the reader that this damped system is unusual for a number of reasons (e.g., a very low Ar0/S+ ratio), and we believe its ionization properties are special but not unique. Nevertheless, it clearly shows the value of examining photoionization diagnostics such as Fe++/Fe+ in a larger sample of damped systems. Visiting Astronomer, W. M. Keck Telescope. The Keck Observatory is a joint facility of the University of California and the California Institute of Technology.

Prochaska, Jason X.; Howk, J. Christopher; O'Meara, John M.; Tytler, David; Wolfe, Arthur M.; Kirkman, David; Lubin, Dan; Suzuki, Nao

2002-06-01

336

Understanding Models  

NSDL National Science Digital Library

Chapter 1 defines and discusses models in a broad, and perhaps unusual, way. In particular, the chapter stresses the framework of personal models that underlie science and learning across fields. Subsequent chapters will deal more with particular kinds of expressed models that are important in science and science teaching: physical models, analog models and plans, mathematical models, and computer simulations. Throughout, the book examines how all models are important to science, how they are used, and how to use them effectively. They can and should be used not only to teach science, but also to teach students something about the process of learning and about the nature of knowledge itself.

Shirley Watt Ireton

2003-01-01

337

MODEL ABSTRACTION IN HYDROLOGIC MODELING  

Technology Transfer Automated Retrieval System (TEKTRAN)

Model abstraction (MA) is a methodology for reducing the complexity of a simulation model while maintaining the validity of the simulation results with respect to the question that the simulation is being used to address. The MA explicitly deals with uncertainties in model structure and in model par...

338

Supermatrix models  

SciTech Connect

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.

Yost, S.A.

1991-05-01

339

Modeling Transformation  

E-print Network

pGLO plasmids Bacterial chromosomal DNA Cell membrane #12;Heat-shock @ 42°C IncreasesModeling Transformation What does each step do? #12;Transformation Procedure #12;Transformation Procedure #12;Building Your Model Yarn = chromosomal DNA Beads

Rose, Michael R.

340

Modeling Convection  

NSDL National Science Digital Library

Typically, teachers use simple models that employ differences in temperature and density to help students visualize convection. However, most of these models are incomplete or merely hint at (instead of model) convective circulation. In order to make the use of models more effective, the authors developed an alternative system that uses a simple, low-cost apparatus that not only maintains dynamic convective circulation, but also illustrates two adjacent cells that teaches students about Earth's processes.

Amanda Schulz

2004-09-01

341

Landscape Models  

NSDL National Science Digital Library

In this assignment students model different scenarios of landscape evolution using an on-line landscape evolution model. The assignment takes them through several situations involving changes in commonly modeled landscape variables like overland flow, faulting and uplift, erosivity, and drainage incision. At the end I have students devise a situation (of variables) that tests a hypothesis or the sensitivity of the model to changes in a variable. Designed for a geomorphology course Uses online and/or real-time data

David Marchetti

342

Why Model?  

Microsoft Academic Search

This address treats some enduring misconceptions about modeling. One of these is that the goal is always prediction. The lecture distinguishes between explanation and prediction as modeling goals, and offers sixteen reasons other than prediction to build a model. It also challenges the common assumption that scientific theories arise from and 'summarize' data, when often, theories precede and guide data

Joshua M. Epstein

2008-01-01

343

Phoenix model  

EPA Science Inventory

Phoenix (formerly referred to as the Second Generation Model or SGM) is a global general equilibrium model designed to analyze energy-economy-climate related questions and policy implications in the medium- to long-term. This model disaggregates the global economy into 26 industr...

344

Modeling Arcs  

Microsoft Academic Search

Although vacuum arcs were first identified over 110 years ago, they are not yet well understood. We have since developed a model of breakdown and gradient limits that tries to explain, in a self-consistent way: arc triggering, plasma initiation, plasma evolution, surface damage and gra- dient limits. We use simple PIC codes for modeling plasmas, molecular dynamics for modeling surface

Zeke Insepov; Jim Norem; Seth Veitzer; Sudhakar Mahalingam

2011-01-01

345

SIR Model  

NSDL National Science Digital Library

This worksheet implements an SIR (Susceptible/ Infected/ Resistant) model of epidemiology for vector-borne diseases. Up to three microbial strains with different virulence and transmission parameters can be modeled and the results graphed. Originally designed to explore coevolution of myxoma and rabbits, the model is easily generalized to other systems.

Tony Weisstein (Truman State University; Biology)

2007-06-20

346

Animal models.  

PubMed

Epilepsy accounts for a significant portion of the dis-ease burden worldwide. Research in this field is fundamental and mandatory. Animal models have played, and still play, a substantial role in understanding the patho-physiology and treatment of human epilepsies. A large number and variety of approaches are available, and they have been applied to many animals. In this chapter the in vitro and in vivo animal models are discussed,with major emphasis on the in vivo studies. Models have used phylogenetically different animals - from worms to monkeys. Our attention has been dedicated mainly to rodents.In clinical practice, developmental aspects of epilepsy often differ from those in adults. Animal models have often helped to clarify these differences. In this chapter, developmental aspects have been emphasized.Electrical stimulation and chemical-induced models of seizures have been described first, as they represent the oldest and most common models. Among these models, kindling raised great interest, especially for the study of the epileptogenesis. Acquired focal models mimic seizures and occasionally epilepsies secondary to abnormal cortical development, hypoxia, trauma, and hemorrhage.Better knowledge of epileptic syndromes will help to create new animal models. To date, absence epilepsy is one of the most common and (often) benign forms of epilepsy. There are several models, including acute pharmacological models (PTZ, penicillin, THIP, GBL) and chronic models (GAERS, WAG/Rij). Although atypical absence seizures are less benign, thus needing more investigation, only two models are so far available (AY-9944,MAM-AY). Infantile spasms are an early childhood encephalopathy that is usually associated with a poor out-come. The investigation of this syndrome in animal models is recent and fascinating. Different approaches have been used including genetic (Down syndrome,ARX mutation) and acquired (multiple hit, TTX, CRH,betamethasone-NMDA) models.An entire section has been dedicated to genetic models, from the older models obtained with spontaneous mutations (GEPRs) to the new engineered knockout, knocking, and transgenic models. Some of these models have been created based on recently recognized patho-genesis such as benign familial neonatal epilepsy, early infantile encephalopathy with suppression bursts, severe myoclonic epilepsy of infancy, the tuberous sclerosis model, and the progressive myoclonic epilepsy. The contribution of animal models to epilepsy re-search is unquestionable. The development of further strategies is necessary to find novel strategies to cure epileptic patients, and optimistically to allow scientists first and clinicians subsequently to prevent epilepsy and its consequences. PMID:22938964

Coppola, Antonietta; Moshé, Solomon L

2012-01-01

347

Hydrological models are mediating models  

NASA Astrophysics Data System (ADS)

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 more importance to identifying and communicating on the many factors involved in model development might increase transparency of model building.

Babel, L. V.; Karssenberg, D.

2013-08-01

348

Station Models  

NSDL National Science Digital Library

This project will allow users to become acquainted with station models that are found on weather maps. Students will study the various atmospheric variables that are depicted on a station model and then practice on an interactive station model program. Part 1 - Being able to read and interpret weather maps is a very important skill in meteorology. One of the most basic skills of predicting the weather is being able to interpret a station model of a given location. A station model is a bundle of information that ...

Mr. Ertl

2007-11-03

349

Computer Models  

NSDL National Science Digital Library

This undergraduate meteorology tutorial from Texas A&M University focuses on computer models that are run by the National Weather Service (NWS) National Centers for Environmental Prediction (NCEP) and are used for forecasting day-to-day weather in the United States. NCEP has four basic models: the Eta Model, the Nested Grid model (NGM), the Rapid Update Cycle (RUC), and the Global Forecast System (GFS). Each model is a self-contained set of computer programs, which include means of analyzing data and computing the evolution of the atmosphere's winds, temperature, pressure, and moisture based on the analyses. Students are given some basic terminology and learn to identify the models and to read model output.

John Nielsen-Gammon

1996-01-01

350

ICRF modelling  

SciTech Connect

This lecture provides a survey of the methods used to model fast magnetosonic wave coupling, propagation, and absorption in tokamaks. The validity and limitations of three distinct types of modelling codes, which will be contrasted, include discrete models which utilize ray tracing techniques, approximate continuous field models based on a parabolic approximation of the wave equation, and full field models derived using finite difference techniques. Inclusion of mode conversion effects in these models and modification of the minority distribution function will also be discussed. The lecture will conclude with a presentation of time-dependent global transport simulations of ICRF-heated tokamak discharges obtained in conjunction with the ICRF modelling codes. 52 refs., 15 figs.

Phillips, C.K.

1985-12-01

351

Functions and Models: Mathematical Models  

NSDL National Science Digital Library

Describe the process of mathematical modeling;Name and describe some methods of modeling;Classify a symbolically represented function as one of the elementary algebraic or transcendental functions;Appraise the suitability of different models for interpreting a given set of data.

Michael Freeze

352

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

E-print Network

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 ...

Sim, Min Sub

353

Climate Models  

NASA Technical Reports Server (NTRS)

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.

Druyan, Leonard M.

2012-01-01

354

Atmospheric Modeling  

Microsoft Academic Search

\\u000a Air quality models simulate the atmospheric concentrations and deposition fluxes to the Earth’s surface of air pollutants\\u000a by solving the mass conservation equations that represent the emissions, transport, dispersion, transformations and removal\\u000a of those air pollutants and associated chemical species. Contemporary air quality models can be grouped into two major categories:\\u000a (1) models that calculate the concentrations of air pollutants

Christian Seigneur; Robin Dennis

355

SCARP Model  

NSDL National Science Digital Library

SCARP is the first in a sequence of spreadsheet modeling exercises (SCARP2, LONGPRO, and GLACPRO). In this exercise, students use a simple arithmetic model (a running mean) to simulate the evolution of a scarp (escarpment) across time. Although the output closely resembles an evolving scarp, no real variables are included in the model. The purpose of the exercise, in addition to the simulation, is to develop basic skills in spreadsheeting and especially in graphical display.

Bill Locke

356

Model Volcanoes  

NSDL National Science Digital Library

In this lesson, students will explore volcanoes by constructing models and reflect upon their learning through drawing sketches of their models. Once they have finished making their models, they will experiment with making their volcanoes erupt. They will observe how eruption changes the original form of their volcano models. In this way, students see first hand how this type of phenomena creates physical change. While students at this level may struggle to understand larger and more abstract geographical concepts, they will work directly with material that will help them build a foundation for understanding concepts of phenomena that sculpt the earth.

357

Cloud Modeling  

NASA Technical Reports Server (NTRS)

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.

Tao, Wei-Kuo; Moncrieff, Mitchell; Einaud, Franco (Technical Monitor)

2001-01-01

358

Ventilation Model  

SciTech Connect

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-closure thermal models (Section 6.6). (3) To satisfy the remainder of KTI agreement TEF 2.07 (Reamer and Williams 2001b). Specifically to provide the results of post-test ANSYS modeling of the Atlas Facility forced convection tests (Section 7.1.2). This portion of the model report also serves as a validation exercise per AP-SIII.10Q, Models, for the ANSYS ventilation model. (4) To further satisfy KTI agreements RDTME 3.01 and 3.14 (Reamer and Williams 2001a) by providing the source documentation referred to in the KTI Letter Report, ''Effect of Forced Ventilation on Thermal-Hydrologic Conditions in the Engineered Barrier System and Near Field Environment'' (Williams 2002). Specifically to provide the results of the MULTIFLUX model which simulates the coupled processes of heat and mass transfer in and around waste emplacement drifts during periods of forced ventilation. This portion of the model report is presented as an Alternative Conceptual Model with a numerical application, and also provides corroborative results used for model validation purposes (Section 6.3 and 6.4).

V. Chipman

2002-10-05

359

Model Selection for Geostatistical Models  

SciTech Connect

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.

Hoeting, Jennifer A.; Davis, Richard A.; Merton, Andrew A.; Thompson, Sandra E.

2006-02-01

360

Turbulence modeling  

NASA Technical Reports Server (NTRS)

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.

Bardina, Jorge E.

1995-01-01

361

Budget Model.  

ERIC Educational Resources Information Center

Computerized formula-driven budget models are used by the Washington community college system to define resource needs for legislative budget requests and to distribute legislative appropriations among 22 community college districts. This manual outlines the sources of information needed to operate the model and illustrates the principles on which…

Washington State Board for Community Coll. Education, Olympia.

362

Modeling Sunspots  

ERIC Educational Resources Information Center

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…

Oh, Phil Seok; Oh, Sung Jin

2013-01-01

363

Minibeast Models  

NSDL National Science Digital Library

In this activity, learners create models of bugs. Learners use household materials like plastic cups and straws to create models of bugs like centipedes and spiders. The activity is covered in the first 5 pages of the document. There are also a number of related activities that introduce learners to the world of invertebrates.

2012-06-26

364

Daisyworld Model  

NSDL National Science Digital Library

The simulation exercise uses a STELLA-based model called Daisyworld to explore concepts associated with Earth's energy balance and climate change. Students examine the evolution of a simplified model of an imaginary planet with only two species of life on its surface -- white and black daisies -- with different albedos. The daisies can alter the temperature of the surface where they are growing.

James Lovelock

365

Modeling Daisyworld  

NSDL National Science Digital Library

Daisyworld is a classic model of complex feedbacks in a simple climate system; this activity guides students through the construction of a STELLA model that can be used to experiment with the system, exploring the somewhat surprising dynamics that arise from the interplay of positive and negative feedbacks between daisies and the temperature of their environment.

David Bice

366

Scale Models  

NSDL National Science Digital Library

In this activity, learners explore the relative sizes and distances of objects in the solar system. Without being informed of the expected product, learners will make a Play-doh model of the Earth-Moon system, scaled to size and distance. The facilitator reveals the true identity of the system at the conclusion of the activity. During the construction phase, learners try to guess what members of the solar system their model represents. Each group receives different amounts of Play-doh, with each group assigned a color (red, blue, yellow, white). At the end, groups set up their models and inspect the models of other groups. They report patterns of scale that they notice; as the amount of Play-doh increases, for example, so do the size and distance of the model. This resource guide includes background information about the Earth to Moon ratio and solar eclipses.

2012-06-26

367

Protein structure modeling with MODELLER.  

PubMed

Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized at atomic resolution using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. In this chapter, we present an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of a similar protocol has resulted in models of useful accuracy for domains in more than half of all known protein sequences. PMID:24573470

Webb, Benjamin; Sali, Andrej

2014-01-01

368

OSPREY Model  

SciTech Connect

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 OSPREY to used and evaluate the model.

Veronica J. Rutledge

2013-01-01

369

Model Center  

NSDL National Science Digital Library

A human is a complicated organism, and it is considered unethical to do many kinds of experiments on human subjects. For these reasons, biologists often use simpler 'model' organisms that are easy to keep and manipulate in the laboratory. Despite obvious differences, model organisms share with humans many key biochemical and physiological functions that have been conserved (maintained) by evolution. Each of the following model organisms has its advantages and disadvantages in different research applications. This tool allows you to examine the similarities between different systems by comparing the proteins they share and the proportion of DNA they have in common. Choose a gene from the drop-down menu and select the species you want to compare. Rolling over the images will give you a more detailed description of each model. Clicking on a gene�s name will take you to the National Center for Biological Information, where you can explore the latest relevant scientific literature.

2009-04-14

370

Programming models  

SciTech Connect

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.

Daniel, David J [Los Alamos National Laboratory; Mc Pherson, Allen [Los Alamos National Laboratory; Thorp, John R [Los Alamos National Laboratory; Barrett, Richard [SNL; Clay, Robert [SNL; De Supinski, Bronis [LLNL; Dube, Evi [LLNL; Heroux, Mike [SNL; Janssen, Curtis [SNL; Langer, Steve [LLNL; Laros, Jim [SNL

2011-01-14

371

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 fuels and renewables; power plants, refineries, and other technologies to process and convert these r...

372

Modeling Arcs  

E-print Network

Although vacuum arcs were first identified over 110 years ago, they are not yet well understood. We have since developed a model of breakdown and gradient limits that tries to explain, in a self-consistent way: arc triggering, plasma initiation, plasma evolution, surface damage and gra- dient limits. We use simple PIC codes for modeling plasmas, molecular dynamics for modeling surface breakdown, and surface damage, and mesoscale surface thermodynamics and finite element electrostatic codes for to evaluate surface properties. Since any given experiment seems to have more variables than data points, we have tried to consider a wide variety of arcing (rf structures, e beam welding, laser ablation, etc.) to help constrain the problem, and concentrate on common mechanisms. While the mechanisms can be comparatively simple, modeling can be challenging.

Insepov, Zeke; Veitzer, Seth; Mahalingam, Sudhakar

2011-01-01

373

Modeling Arcs  

SciTech Connect

Although vacuum arcs were first identified over 110 years ago, they are not yet well understood. We have since developed a model of breakdown and gradient limits that tries to explain, in a self-consistent way: arc triggering, plasma initiation, plasma evolution, surface damage and gradient limits. We use simple PIC codes for modeling plasmas, molecular dynamics for modeling surface breakdown, and surface damage, and mesoscale surface thermodynamics and finite element electrostatic codes for to evaluate surface properties. Since any given experiment seems to have more variables than data points, we have tried to consider a wide variety of arcing (rf structures, e beam welding, laser ablation, etc.) to help constrain the problem, and concentrate on common mechanisms. While the mechanisms can be comparatively simple, modeling can be challenging.

Insepov, Z.; Norem, J. [Argonne National Lab, Argonne, IL 60439 (United States); Vetizer, S.; Mahalingam, S. [Tech-X Corp., Boulder, CO (United States)

2011-12-23

374

Micromolecular modeling  

NASA Technical Reports Server (NTRS)

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.

Guillet, J. E.

1984-01-01

375

Device modeling  

NASA Technical Reports Server (NTRS)

A summary report is given of the activities of the device modeling workshop which was held as a part of the Space Photovoltaic Research and Technology Conference at the Lewis Research Center, October 7 to 9, 1986. The purpose of this workshop was to access the status of solar cell device modeling to see if it is meeting present and future needs of the photovoltaic community.

Schwartz, Richard

1987-01-01

376

Molecular Models  

NSDL National Science Digital Library

Created and maintained by Dr. Dave Woodcock of the Chemistry Department at Okanagan University College in British Columbia, Canada, this site features models of over 1,100 molecules in .pdb, or Chemscape Chime, format (link to free download provided). Users may search the molecular database using an internal search engine or browse by category or alphabetically. Index page entries include the molecule's name, formula, molar mass, and comments. The site also features more detailed models of selected molecular fragments.

Woodcock, Dave.

377

Modeling depression in animal models.  

PubMed

Animal models and preclinical tests have played large roles in the development of antidepressant drugs and are likely to continue to play important roles. In the present communication, the main animal models of depression have been described and reviewed. These models include the Flinders sensitive line (FSL) rat, the Wistar Kyoto (WKY) rat, the fawn-hooded (FH) rat, and the learned helpless (LH) rat. In addition, the materials used to assess the behavior of these rats, including swim tanks, drinking tubes, and an open field apparatus, have been discussed. Finally, the methods used in collecting the relevant behaviors in the animal models are described. These include the procedures used in the forced swim test and chronic mild stress protocols, including the sucrose preference test. It is concluded that the behavioral tests used to infer depressed-like behavior in rats will continue to provide useful data if the appropriate animals and proper methods are used. PMID:22231810

Overstreet, David H

2012-01-01

378

Mechanistic models  

SciTech Connect

Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) ``interaction`` of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

Curtis, S.B.

1990-09-01

379

Mechanistic models  

SciTech Connect

Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) interaction'' of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

Curtis, S.B.

1990-09-01

380

Measurement and Calculation of Electrochemical Potentials in Hydrogenated High Temperature Water, including an Evaluation of the Yttria-Stabilized Zirconia/Iron-Iron Oxide (Fe/Fe3O4) Probe as Reference Electrode  

SciTech Connect

The importance of knowing the electrochemical corrosion potential (ECP, also referred to as E{sub con}) of nickel-base alloys in hydrogenated water is related to the need to understand the effects of dissolved (i.e., aqueous) hydrogen concentration ([H{sub 2}]) on primary water stress corrosion cracking (PWSCC). Also, the use of a reference electrode (RE) can improve test quality by heightening the ability to detect instances of out-of-specification or unexpected chemistry. Three methods are used to measure and calculate the ECP of nickel-based alloys in hydrogenated water containing {approx} 1 to 150 scc/kg H{sub 2} (0.1 to 13.6 ppm H{sub 2}) at 260 to 360 C. The three methods are referred to as the specimen/component method, the platinum (Pt) method, and the yttria-stabilized zirconia/iron-iron oxide (YSZ/Fe-Fe{sub 3}O{sub 4}) RE method. The specimen/component method relies upon the assumption that the specimen or component behaves as a hydrogen electrode, and its E{sub corr} is calculated using the Nernst equation. The present work shows that this method is valid for aqueous H{sub 2} levels {ge} {approx} 5 to 10 scc/kg H{sub 2}. The Pt method uses a voltage measurement between the specimen or component and a Pt electrode, with the Pt assumed to behave as a hydrogen electrode; this method is valid as long as the aqueous H{sub 2}level is known. The YSZ/Fe-Fe{sub 3}O{sub 4}, which represents a relatively new approach for measuring E{sub corr} in this environment, can be used even if the aqueous H{sub 2} level is unknown. The electrochemical performance of the YSZ/Fe-Fe{sub 3}O{sub 4} probe supports its viability as a RE for use in high temperature hydrogenated water. Recent design modifications incorporating a teflon sealant have improved the durability of this RE (however, some of the REs do still fail prematurely due to water in-leakage). The Pt method is judged to represent the best overall approach, though there are cases where the other methods are superior. For example, the specimen/component method provides the simplest approach for calculating the E{sub corr} of plant components, and the YSZ/Fe-Fe{sub 3}O{sub 4} RE method provides the best approach if the H{sub 2} level is unknown, or in off-nominal chemistry conditions. The present paper describes the use of these methods to determine the ECP of a specimen or component versus the ECP of the nickel/nickel oxide (Ni/NiO) phase transition, which is important since prior work has shown that this parameter (ECP-ECP{sub Ni/NiO}) can be used to assess aqueous H{sub 2} effects on PWSCC.

Steven A. Attanasio; David S. Morton; Mark A. Ando

2001-10-22

381

Effects of an intensive hog farming operation on groundwater in east Mediterranean (II): a study on K?, Na?, Cl ?, PO?ł?-P, Ca˛?, Mg˛?, Feł?/Fe˛?, Mn˛?, Cu˛?, Zn˛? and Ni˛?.  

PubMed

The application of treated animal wastewater generated in concentrated animal feeding operations on surface soil (within farm borders) leads to degradation of groundwater. Effects of an intensive hog farming operation, located at a Mediterranean limestone soil coastal area, on groundwater were investigated. Treated animal wastewater was discharged on a small plot (~10.8 ha) with a geologic fault. Samples were taken from seven groundwater monitoring wells close to the farm. A significant increase of K(+), Na(+), Cl(-), PO4 (3-)-P, Ca(2+) and Mg(2+) concentrations was found in monitoring wells which are affected by the subsurface flow of groundwater. Concentrations of Fe(3+)/Fe(2+), Mn(2+), Cu(2+), Zn(2+) and Ni(2+) in all groundwater monitoring wells were extremely low. During the winter, significant increases in concentrations of K(+) and PO4 (3-)-P were noted and attributed to high precipitation, which assisted in the leaching of K and P to groundwater. PMID:25370904

Michalopoulos, Charalampos; Tzamtzis, Nikolaos; Liodakis, Stylianos

2014-12-01

382

Modeling reality  

NASA Technical Reports Server (NTRS)

Although powerful computers have allowed complex physical and manmade hardware systems to be modeled successfully, we have encountered persistent problems with the reliability of computer models for systems involving human learning, human action, and human organizations. This is not a misfortune; unlike physical and manmade systems, human systems do not operate under a fixed set of laws. The rules governing the actions allowable in the system can be changed without warning at any moment, and can evolve over time. That the governing laws are inherently unpredictable raises serious questions about the reliability of models when applied to human situations. In these domains, computers are better used, not for prediction and planning, but for aiding humans. Examples are systems that help humans speculate about possible futures, offer advice about possible actions in a domain, systems that gather information from the networks, and systems that track and support work flows in organizations.

Denning, Peter J.

1990-01-01

383

Supernova models  

SciTech Connect

Recent progress in understanding the observed properties of Type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the /sup 56/Ni produced therein is reviewed. Within the context of this model for Type I explosions and the 1978 model for Type II explosions, the expected nucleosynthesis and gamma-line spectra from both kinds of supernovae are presented. Finally, a qualitatively new approach to the problem of massive star death and Type II supernovae based upon a combination of rotation and thermonuclear burning is discussed.

Woosley, S.E.; Weaver, T.A.

1980-01-01

384

Model Behavior  

ERIC Educational Resources Information Center

There are dozens of books and hundreds of resources that address the issue of character development in students: how to raise them to be good people, how to teach them to be good citizens, how to help them to make good decisions. Little is written, however, about the character development of principals and school leaders, whose behavior is a model

Holloway, John

2006-01-01

385

Atmospheric Modeling  

EPA Science Inventory

Although air quality models have been applied historically to address issues specific to ambient air quality standards (i.e., one criteria pollutant at a time) or welfare (e.g.. acid deposition or visibility impairment). they are inherently multipollutant based. Therefore. in pri...

386

Modeling Convection  

ERIC Educational Resources Information Center

Students must understand the fundamental process of convection before they can grasp a wide variety of Earth processes, many of which may seem abstract because of the scales on which they operate. Presentation of a very visual, concrete model prior to instruction on these topics may facilitate students' understanding of processes that are largely…

Ebert, James R.; Elliott, Nancy A.; Hurteau, Laura; Schulz, Amanda

2004-01-01

387

Model thinking.  

PubMed

Nancy Roper, Win Logan and Alison Tierney published their ground-breaking model of nursing in 1980, sparking the evolution of nursing theory from staid, biomedical thinking to an individualised, independent approach. Jane Salvage looks back at the lasting impact their research had on her and the profession as whole. PMID:16425763

Salvage, Jane

388

Model Organisms  

NSDL National Science Digital Library

A human is a complicated organism, and it is considered unethical to do many kinds of experiments on human subjects. For these reasons, biologists often use simpler �model� organisms that are easy to keep and manipulate in the laboratory. Despite ob

2009-04-14

389

Marshmallow Models  

NSDL National Science Digital Library

No glue is needed for learners of any age to become marshmallow architects or engineers. Using marshmallows and water (and maybe edible decorations like peanut butter, pretzels, gumdrops, etc.), learners wet a few marshamallows at a time and stick them together bit by bit to construct whatever models they want.

Lawrence Hall of Science

2010-01-01

390

Daisyworld Model  

NSDL National Science Digital Library

The Daisyworld model created by Andrew Watson and James Lovelock (1983, Tellus, v. 35B, p. 284-289) is a wonderful example of a self-regulating system incorporating positive and negative feedbacks. The model consists of a planet on which black and white daisies are growing. The growth of these daisies is governed by a parabolic shaped growth function regulated by planetary temperature and is set to zero for temperatures less than 5 şC or greater than 40 şC and optimized at 22.5 şC. The model explores the effect of a steadily increasing solar luminosity on the growth of daisies and the resulting planetary temperature. The growth function for the daisies allows them to modulate the planet's temperature for many years, warming it early on as black daisies grow, and cooling it later as white daisies grow. Eventually, the solar luminosity increases beyond the daisies' capability to modulate the temperature and they die out, leading to a rapid rise in the planetary temperature. Students read Watson and Lovelock's original paper, and then use STELLA to create their own Daisyworld model with which they can experiment. Experiments include changing the albedos of the daisies, changing their death rates, and changing the rate at which energy is conducted from one part of the planet to another. In all cases, students keep track of daisy populations and of planetary temperature over time.

Kirsten Menking

391

Groundwater Model  

NSDL National Science Digital Library

In this activity, students build a model to demonstrate how aquifers are formed and ground water becomes polluted. For younger students, the teacher can perform this activity as a demonstration, or older students can perform it themselves. A materials list, instructions, and extension activities are provided.

392

Modeling Muscles  

ERIC Educational Resources Information Center

Teaching the anatomy of the muscle system to high school students can be challenging. Students often learn about muscle anatomy by memorizing information from textbooks or by observing plastic, inflexible models. Although these mediums help students learn about muscle placement, the mediums do not facilitate understanding regarding integration of…

Goodwyn, Lauren; Salm, Sarah

2007-01-01

393

Urban Modelling  

Microsoft Academic Search

Urban models are computer-based simulations used for testing theories about spatial location and interaction between land uses and related activities. They also provide digital environments for testing the consequences of physical planning policies on the future form of cities. As computers, software and data have become richer, and as our conception of the way complex systems such as cities grow

Michael Batty; Rob Kitchin; Nigel Thrift

394

Diffusion Models  

NSDL National Science Digital Library

Web-based intructional material describing the use of diffusion models in population ecology. This page is part of a set of on-line lectures on Quantitative Population Ecology produced by Alexei Sharov in the Department of Entomology at Virginia Tech.

Alexei Sharov

395

Criticality Model  

SciTech Connect

The ''Disposal Criticality Analysis Methodology Topical Report'' (YMP 2003) presents the methodology for evaluating potential criticality situations in the monitored geologic repository. As stated in the referenced Topical Report, the detailed methodology for performing the disposal criticality analyses will be documented in model reports. Many of the models developed in support of the Topical Report differ from the definition of models as given in the Office of Civilian Radioactive Waste Management procedure AP-SIII.10Q, ''Models'', in that they are procedural, rather than mathematical. These model reports document the detailed methodology necessary to implement the approach presented in the Disposal Criticality Analysis Methodology Topical Report and provide calculations utilizing the methodology. Thus, the governing procedure for this type of report is AP-3.12Q, ''Design Calculations and Analyses''. The ''Criticality Model'' is of this latter type, providing a process evaluating the criticality potential of in-package and external configurations. The purpose of this analysis is to layout the process for calculating the criticality potential for various in-package and external configurations and to calculate lower-bound tolerance limit (LBTL) values and determine range of applicability (ROA) parameters. The LBTL calculations and the ROA determinations are performed using selected benchmark experiments that are applicable to various waste forms and various in-package and external configurations. The waste forms considered in this calculation are pressurized water reactor (PWR), boiling water reactor (BWR), Fast Flux Test Facility (FFTF), Training Research Isotope General Atomic (TRIGA), Enrico Fermi, Shippingport pressurized water reactor, Shippingport light water breeder reactor (LWBR), N-Reactor, Melt and Dilute, and Fort Saint Vrain Reactor spent nuclear fuel (SNF). The scope of this analysis is to document the criticality computational method. The criticality computational method will be used for evaluating the criticality potential of configurations of fissionable materials (in-package and external to the waste package) within the repository at Yucca Mountain, Nevada for all waste packages/waste forms. The criticality computational method is also applicable to preclosure configurations. The criticality computational method is a component of the methodology presented in ''Disposal Criticality Analysis Methodology Topical Report'' (YMP 2003). How the criticality computational method fits in the overall disposal criticality analysis methodology is illustrated in Figure 1 (YMP 2003, Figure 3). This calculation will not provide direct input to the total system performance assessment for license application. It is to be used as necessary to determine the criticality potential of configuration classes as determined by the configuration probability analysis of the configuration generator model (BSC 2003a).

A. Alsaed

2004-09-14

396

Micrometer Model  

NSDL National Science Digital Library

This interactive simulation gives students practice in the operation and the physical parts of a real micrometer, a measuring device that employs a screw to amplify distances that are too small to measure easily. The accuracy of a micrometer derives from the accuracy of the thread that is at its heart. The basic operating principle of a micrometer is that the rotation of an accurately made screw can be directly and precisely correlated to a certain amount of axial movement (and vice-versa), through the constant known as the screw's lead. The Micrometer model was created using the Easy Java Simulations (EJS) modeling tool. It is distributed as a ready-to-run (compiled) Java archive. Double click the ejs_ntnu_Micrometer.jar file to run the program (Java must be installed).

2009-09-10

397

Modelling Coalitions: . . .  

E-print Network

In the last few years, argumentation frameworks have been successfully applied to multi agent systems. Recently, argumentation has been used to provide a framework for reasoning about coalition formation. At the same time alternatingtime temporal logic has been used to reason about the behavior and abilities of coalitions of agents. However, ATL operators account only for the existence of successful strategies of coalitions. They do not consider whether coalitions can be actually formed. This paper is an attempt to combine both frameworks and to develop a logic through which we can reason at the same time (1) about abilities of coalitions of agents and (2) about the formation of coalitions. We provide a formal extension of ATL, ATL c, in which the actual computation of the coalition is modelled in terms of argumentation semantics. We show that ATL c ’s proof theory can be understood as a natural extension of the model checking procedure used in ATL.

Nils Bulling; Jürgen Dix; Carlos I. Chesńevar

2008-01-01

398

Simple Models  

NSDL National Science Digital Library

As stated in "About This Book," the author isn't going to take the usual approach to the subject of chemistry. Because virtually all explanations of chemical reactions are based on our current model of atoms and molecules, the first thing to do here is to help you understand why we believe that atoms and molecules look and act the way they do. That's not a trivial issue, because despite the impression you might have gotten from textbooks, no one has ever seen an atom in the sense that you can see this page in front of you. What we have are observations and experiments that lead us to formulate models of atoms. This free selection includes the Table of Contents, Preface, About This Book section, a Safety Note, and the Glossary.

William C. Robertson, Ph.D.

2007-01-01

399

Fibre Models  

NASA Astrophysics Data System (ADS)

Fibre models have been introduced as simple models to describe failure. They are based on the probability distribution of broken fibres. The load redistribution after a fibre yields can be global or local and the first case can often be solved analytically. We will present an interpolation between these the local and the global case and apply it to experimental situations like the compression of granular packings. Introducing viscoelastic fibres allows to describe the creep of wood. It is even possible to deal analytically with a gradual degradation of fibres and consider damage as well as healing. In this way Basquin's law of fatigue can be reproduced and new universalities concerning the histograms of bursts and waiting times can be uncovered.

Herrmann, H. J.; Kun, F.

2007-12-01

400

Linear Models  

NSDL National Science Digital Library

This site uses linear models to demonstrate the change in bird populations on a barren island over time, supply and demand, and the natural cleaning of a polluted lake by fresh water over time. The problems are laid out and turned into both graphic and equation form in order to understand the rate of change happening in each scenario. There are also links to previously covered materials that can help student review material from past math lessons.

Frank Wattenberg

1997-01-01

401

Gas Model  

NSDL National Science Digital Library

This highly visual model demonstrates the atomic theory of matter which states that a gas is made up of tiny particles of atoms that are in constant motion, smashing into each other. Balls, representing molecules, move within a cage container to simulate this phenomenon. A hair dryer provides the heat to simulate the heating and cooling of gas: the faster the balls are moving, the hotter the gas. Learners observe how the balls move at a slower rate at lower "temperatures."

The Exploratorium

2013-01-30

402

Model Well  

NSDL National Science Digital Library

In this quick activity about pollutants and groundwater (page 2 of PDF), learners build a model well with a toilet paper tube. Learners use food coloring to simulate pollutants and observe how they can be carried by groundwater and eventually enter water sources such as wells, rivers, and streams. This activity is associated with nanotechnology and relates to linked video, DragonflyTV Nano: Water Clean-up.

Twin Cities Public Television, Inc.

2008-01-01

403

Temperature optima of enzyme-catalysed reactions in microemulsion systems  

Microsoft Academic Search

Ternary phase systems (water\\/surfactant\\/organic solvent) were utilised to increase and broaden the temperature optima of enzyme-catalysed reactions. Alcohol dehydrogenases from yeast and Thermoanaerobium brockii (EC 1.1.1.1 and EC 1.1.1.2), lactate dehydrogenase from Lactobacillus delbrueckii (EC 1.1.1.28) and the particulate hydrogenase from Ralstonia eutropha (EC 1.18.99.1) were used as model enzymes in microemulsions, consisting of the surfactant Aerosol OT, and various

K. Mlejnek; B. Seiffert; T. Demberg; M. Kämper; M. Hoppert

2004-01-01

404

Model checking  

NASA Technical Reports Server (NTRS)

Automatic formal verification methods for finite-state systems, also known as model-checking, successfully reduce labor costs since they are mostly automatic. Model checkers explicitly or implicitly enumerate the reachable state space of a system, whose behavior is described implicitly, perhaps by a program or a collection of finite automata. Simple properties, such as mutual exclusion or absence of deadlock, can be checked by inspecting individual states. More complex properties, such as lack of starvation, require search for cycles in the state graph with particular properties. Specifications to be checked may consist of built-in properties, such as deadlock or 'unspecified receptions' of messages, another program or implicit description, to be compared with a simulation, bisimulation, or language inclusion relation, or an assertion in one of several temporal logics. Finite-state verification tools are beginning to have a significant impact in commercial designs. There are many success stories of verification tools finding bugs in protocols or hardware controllers. In some cases, these tools have been incorporated into design methodology. Research in finite-state verification has been advancing rapidly, and is showing no signs of slowing down. Recent results include probabilistic algorithms for verification, exploitation of symmetry and independent events, and the use symbolic representations for Boolean functions and systems of linear inequalities. One of the most exciting areas for further research is the combination of model-checking with theorem-proving methods.

Dill, David L.

1995-01-01

405

Modeling biomembranes.  

SciTech Connect

Understanding the properties and behavior of biomembranes is fundamental to many biological processes and technologies. Microdomains in biomembranes or ''lipid rafts'' are now known to be an integral part of cell signaling, vesicle formation, fusion processes, protein trafficking, and viral and toxin infection processes. Understanding how microdomains form, how they depend on membrane constituents, and how they act not only has biological implications, but also will impact Sandia's effort in development of membranes that structurally adapt to their environment in a controlled manner. To provide such understanding, we created physically-based models of biomembranes. Molecular dynamics (MD) simulations and classical density functional theory (DFT) calculations using these models were applied to phenomena such as microdomain formation, membrane fusion, pattern formation, and protein insertion. Because lipid dynamics and self-organization in membranes occur on length and time scales beyond atomistic MD, we used coarse-grained models of double tail lipid molecules that spontaneously self-assemble into bilayers. DFT provided equilibrium information on membrane structure. Experimental work was performed to further help elucidate the fundamental membrane organization principles.

Plimpton, Steven James; Heffernan, Julieanne; Sasaki, Darryl Yoshio; Frischknecht, Amalie Lucile; Stevens, Mark Jackson; Frink, Laura J. Douglas

2005-11-01

406

10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: ...  

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: 1' = 400' HORIZONTAL, 1' = 100' VERTICAL), AND GREENVILLE BRIDGE MODEL (MODEL SCALE: 1' = 360' HORIZONTAL, 1' = 100' VERTICAL). - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS