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Sample records for fefe hydrogenase model

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

    PubMed

    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

    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 μ-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, Mössbauer, and susceptibility techniques indicates that for this compound initially [Fe(2){μ-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){μ-MeSi(CH(2)S)(3)}(2)(CO)(8)]. PMID:20873759

  2. The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

    PubMed Central

    Boxma, Brigitte; Ricard, Guenola; van Hoek, Angela HAM; Severing, Edouard; Moon-van der Staay, Seung-Yeo; van der Staay, Georg WM; van Alen, Theo A; de Graaf, Rob M; Cremers, Geert; Kwantes, Michiel; McEwan, Neil R; Newbold, C Jamie; Jouany, Jean-Pierre; Michalowski, Tadeusz; Pristas, Peter; Huynen, Martijn A; Hackstein, Johannes HP

    2007-01-01

    Background The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I. Results The [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome. Conclusion The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering. PMID:18021395

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

    DOEpatents

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

    2014-09-16

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

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

    PubMed Central

    Stapleton, James A.; Swartz, James R.

    2010-01-01

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

  5. Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase from Chlamydomonas reinhardtii.

    PubMed

    Rumpel, Sigrun; Siebel, Judith F; Diallo, Mamou; Farès, Christophe; Reijerse, Edward J; Lubitz, Wolfgang

    2015-07-27

    The transfer of photosynthetic electrons by the ferredoxin PetF to the [FeFe] hydrogenase HydA1 in the microalga Chlamydomonas reinhardtii is a key step in hydrogen production. Electron delivery requires a specific interaction between PetF and HydA1. However, because of the transient nature of the electron-transfer complex, a crystal structure remains elusive. Therefore, we performed protein-protein docking based on new experimental data from a solution NMR spectroscopy investigation of native and gallium-substituted PetF. This provides valuable information about residues crucial for complex formation and electron transfer. The derived complex model might help to pinpoint residue substitution targets for improved hydrogen production. PMID:26010059

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

    PubMed

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

    2015-06-01

    The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. PMID:25461840

  7. High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

    PubMed Central

    Kuchenreuther, Jon M.; Grady-Smith, Celestine S.; Bingham, Alyssa S.; George, Simon J.; Cramer, Stephen P.; Swartz, James R.

    2010-01-01

    Background The realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. Principal Findings In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H2 evolution with rates comparable to those of enzymes isolated from their respective native organisms. Significance The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H2-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments. PMID:21124800

  8. Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer.

    PubMed

    Baffert, Carole; Sybirna, Kateryna; Ezanno, Pierre; Lautier, Thomas; Hajj, Viviane; Meynial-Salles, Isabelle; Soucaille, Philippe; Bottin, Hervé; Léger, Christophe

    2012-09-18

    Direct electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling. In both cases, a surface patch of lysine residues makes it possible to favor an orientation that is efficient for fast, direct electron transfer. High hydrogen-oxidation current densities are maintained for up to one week, the only limitation being the intrinsic stability of the enzyme. We also show that covalent attachment has no effect on the catalytic properties of the enzyme, which means that this strategy can also used be for electrochemical studies of the catalytic mechanism. PMID:22891965

  9. Following [FeFe] Hydrogenase Active Site Intermediates by Time-Resolved Mid-IR Spectroscopy.

    PubMed

    Mirmohades, Mohammad; Adamska-Venkatesh, Agnieszka; Sommer, Constanze; Reijerse, Edward; Lomoth, Reiner; Lubitz, Wolfgang; Hammarström, Leif

    2016-08-18

    Time-resolved nanosecond mid-infrared spectroscopy is for the first time employed to study the [FeFe] hydrogenase from Chlamydomonas reinhardtii and to investigate relevant intermediates of the enzyme active site. An actinic 355 nm, 10 ns laser flash triggered photodissociation of a carbonyl group from the CO-inhibited state Hox-CO to form the state Hox, which is an intermediate of the catalytic proton reduction cycle. Time-resolved infrared spectroscopy allowed us to directly follow the subsequent rebinding of the carbonyl, re-forming Hox-CO, and determine the reaction half-life to be t1/2 ≈ 13 ± 5 ms at room temperature. This gives direct information on the dynamics of CO inhibition of the enzyme. PMID:27494400

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

    PubMed Central

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

    2015-01-01

    Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe–4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN–, and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-l-methionine family of enzymes that transforms Fe and l-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the “dangler” Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that l-cysteine (Cys) binds the auxiliary [4Fe–4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe–4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe2+ binding and synthon release. PMID:26324916

  11. Patterns of [FeFe] Hydrogenase Diversity in the Gut Microbial Communities of Lignocellulose-Feeding Higher Termites

    PubMed Central

    Ballor, Nicholas R.

    2012-01-01

    Hydrogen is the central free intermediate in the degradation of wood by termite gut microbes and can reach concentrations exceeding those measured for any other biological system. Degenerate primers targeting the largest family of [FeFe] hydrogenases observed in a termite gut metagenome have been used to explore the evolution and representation of these enzymes in termites. Sequences were cloned from the guts of the higher termites Amitermes sp. strain Cost010, Amitermes sp. strain JT2, Gnathamitermes sp. strain JT5, Microcerotermes sp. strain Cost008, Nasutitermes sp. strain Cost003, and Rhyncotermes sp. strain Cost004. Each gut sample harbored a more rich and evenly distributed population of hydrogenase sequences than observed previously in the guts of lower termites and Cryptocercus punctulatus. This accentuates the physiological importance of hydrogen for higher termite gut ecosystems and may reflect an increased metabolic burden, or metabolic opportunity, created by a lack of gut protozoa. The sequences were phylogenetically distinct from previously sequenced [FeFe] hydrogenases. Phylogenetic and UniFrac comparisons revealed congruence between host phylogeny and hydrogenase sequence library clustering patterns. This may reflect the combined influences of the stable intimate relationship of gut microbes with their host and environmental alterations in the gut that have occurred over the course of termite evolution. These results accentuate the physiological importance of hydrogen to termite gut ecosystems. PMID:22636002

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

    PubMed

    Ballor, Nicholas R; Leadbetter, Jared R

    2012-04-01

    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

  13. New redox states observed in [FeFe] hydrogenases reveal redox coupling within the H-cluster.

    PubMed

    Adamska-Venkatesh, Agnieszka; Krawietz, Danuta; Siebel, Judith; Weber, Katharina; Happe, Thomas; Reijerse, Edward; Lubitz, Wolfgang

    2014-08-13

    Active [FeFe] hydrogenases can be obtained by expressing the unmaturated enzyme in Escherichia coli followed by incubation with a synthetic precursor of the binuclear [2Fe] subcluster, namely: [NEt4]2[Fe2(adt)(CO)4(CN)2] (adt = [S-CH2-NH-CH2-S](2-)). The binuclear subsite Fe2(adt)(CO)3(CN)2 is attached through a bridging cysteine side chain to a [4Fe-4S] subcluster already present in the unmaturated enzyme thus yielding the intact native "H-cluster". We present FTIR electrochemical studies of the [FeFe] hydrogenase from Chlamydomonas reinhardtii, CrHydA1, maturated with the precursor of the native cofactor [Fe2(adt)(CO)4(CN)2](2-) as well as a non-natural variant [Fe2(pdt)(CO)4(CN)2](2-) in which the bridging amine functionality is replaced by CH2. The obtained active enzyme CrHydA1(adt) shows the same redox states in the respective potential range as observed for the native system (E(ox/red) = -400 mV, E(red/sred) = -470 mV). For the Hox → Hred transition the reducing equivalent is stored on the binuclear part, ([4Fe-4S](2+)Fe(II)Fe(I) → [4Fe-4S](2+)Fe(I)Fe(I)), while the Hred → Hsred transition is characterized by a reduction of the [4Fe-4S] part of the H-cluster ([4Fe-4S](2+)Fe(I)Fe(I) → [4Fe-4S](+)Fe(I)Fe(I)). A similar transition is reported here for the CO inhibited state of the H-cluster: ([4Fe-4S](2+)Fe(I)Fe(II)CO → [4Fe-4S](+)Fe(I)Fe(II)CO). An FTIR electrochemical study of the inactive variant with the pdt ligand, CrHydA1(pdt), identified two redox states H(pdt)-ox and H(pdt)-"red". Both EPR and FTIR spectra of H(pdt)-ox are virtually identical to those of the H(adt)-ox and the native Hox state. The H(pdt)-"red" state is also characterized by a reduced [4Fe-4S] subcluster. In contrast to CrHydA1(adt), the H(pdt)-ox state of CrHydA1(pdt) is stable up to rather high potentials (+200 mV). This study demonstrates the distinct redox coupling between the two parts of the H-cluster and confirms that the [4Fe-4S]H subsite is also redox active and as

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

    PubMed

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

    2016-08-10

    Hydrogenase enzymes efficiently process H2 and protons at organometallic FeFe, NiFe, or Fe active sites. Synthetic modeling of the many H2ase states has provided insight into H2ase structure and mechanism, as well as afforded catalysts for the H2 energy vector. Particularly important are hydride-bearing states, with synthetic hydride analogues now known for each hydrogenase class. These hydrides are typically prepared by protonation of low-valent cores. Examples of FeFe and NiFe hydrides derived from H2 have also been prepared. Such chemistry is more developed than mimicry of the redox-inactive monoFe enzyme, although functional models of the latter are now emerging. Advances in physical and theoretical characterization of H2ase enzymes and synthetic models have proven key to the study of hydrides in particular, and will guide modeling efforts toward more robust and active species optimized for practical applications. PMID:27353631

  15. N-Substituted Derivatives of the Azadithiolate Cofactor from the [FeFe] Hydrogenases: Stability and Complexation

    PubMed Central

    Angamuthu, Raja; Chen, Chi-Shian; Cochrane, Tyler R.; Gray, Danielle L.; Schilter, David; Ulloa, Olbelina A.; Rauchfuss, Thomas B.

    2015-01-01

    Experiments are described that probe the stability of N-substituted derivatives of the azadithiolate cofactor recently confirmed in the [FeFe] hydrogenases (Berggren, G., et al. Nature 2013, 499, 66). Acid-catalyzed hydrolysis of bis(thioester) BnN(CH2SAc)2 gives [BnNCH2SCH2]2 rather than azadithiol BnN(CH2SH)2. Treatment of BnN(CH2SAc)2 with NaOtBu generates BnN(CH2SNa)2, which was trapped with NiCl2(diphos) (diphos = 1,2-C2H4(PR2)2; R = Ph (dppe) and Cy (dcpe)) to give fully characterized complexes Ni[(SCH2)2NBn](diphos). The related N-aryl derivative Ni[(SCH2)2NC6H4Cl](diphos) was prepared analogously from 4-ClC6H4N(CH2SAc)2, NaOtBu, and NiCl2(dppe). Crystallographic analysis confirmed that these rare nonbridging [adtR]2− complexes feature distorted square planar Ni centers. The analogue Pd[(SCH2)2NBn](dppe) was also prepared. 31P NMR analysis indicates that Ni[(SCH2)2NBn](dppe) has basicity comparable to typical amines. As shown by cyclic voltammetry, the couple [M[(SCH2)2NBn](dppe)]+/0 is reversible near −2.0 V versus Fc+/0. The wave shifts to −1.78 V upon N-protonation. In the presence of CF3CO2H, Ni[(SCH2)2NBn](dppe) catalyzes hydrogen evolution at rate of 22 s−1 in the acid-independent regime, at room temperature in CH2Cl2 solution. In contrast to the instability of RN(CH2SH)2 (R = alkyl, aryl), the dithiol of tosylamide TsN(CH2SH)2 proved sufficiently stable to allow full characterization. This dithiol reacts with Fe3(CO)12 and, in the presence of base, NiCl2(dppe) to give Fe2[(SCH2)2NTs](CO)6 and Ni[(SCH2)2NTs](dppe), respectively. PMID:26000618

  16. N-Substituted Derivatives of the Azadithiolate Cofactor from the [FeFe] Hydrogenases: Stability and Complexation.

    PubMed

    Angamuthu, Raja; Chen, Chi-Shian; Cochrane, Tyler R; Gray, Danielle L; Schilter, David; Ulloa, Olbelina A; Rauchfuss, Thomas B

    2015-06-15

    Experiments are described that probe the stability of N-substituted derivatives of the azadithiolate cofactor recently confirmed in the [FeFe] hydrogenases (Berggren, G., et al. Nature 2013, 499, 66). Acid-catalyzed hydrolysis of bis(thioester) BnN(CH2SAc)2 gives [BnNCH2SCH2]2 rather than azadithiol BnN(CH2SH)2. Treatment of BnN(CH2SAc)2 with NaO(t)Bu generates BnN(CH2SNa)2, which was trapped with NiCl2(diphos) (diphos = 1,2-C2H4(PR2)2; R = Ph (dppe) and Cy (dcpe)) to give fully characterized complexes Ni[(SCH2)2NBn](diphos). The related N-aryl derivative Ni[(SCH2)2NC6H4Cl](diphos) was prepared analogously from 4-ClC6H4N(CH2SAc)2, NaO(t)Bu, and NiCl2(dppe). Crystallographic analysis confirmed that these rare nonbridging [adt(R)](2-) complexes feature distorted square planar Ni centers. The analogue Pd[(SCH2)2NBn](dppe) was also prepared. (31)P NMR analysis indicates that Ni[(SCH2)2NBn](dppe) has basicity comparable to typical amines. As shown by cyclic voltammetry, the couple [M[(SCH2)2NBn](dppe)](+/0) is reversible near -2.0 V versus Fc(+/0). The wave shifts to -1.78 V upon N-protonation. In the presence of CF3CO2H, Ni[(SCH2)2NBn](dppe) catalyzes hydrogen evolution at rate of 22 s(-1) in the acid-independent regime, at room temperature in CH2Cl2 solution. In contrast to the instability of RN(CH2SH)2 (R = alkyl, aryl), the dithiol of tosylamide TsN(CH2SH)2 proved sufficiently stable to allow full characterization. This dithiol reacts with Fe3(CO)12 and, in the presence of base, NiCl2(dppe) to give Fe2[(SCH2)2NTs](CO)6 and Ni[(SCH2)2NTs](dppe), respectively. PMID:26000618

  17. A Functional Model of [Fe]-Hydrogenase.

    PubMed

    Xu, Tao; Yin, Chih-Juo Madeline; Wodrich, Matthew D; Mazza, Simona; Schultz, Katherine M; Scopelliti, Rosario; Hu, Xile

    2016-03-16

    [Fe]-Hydrogenase catalyzes the hydrogenation of a biological substrate via the heterolytic splitting of molecular hydrogen. While many synthetic models of [Fe]-hydrogenase have been prepared, none yet are capable of activating H2 on their own. Here, we report the first Fe-based functional mimic of the active site of [Fe]-hydrogenase, which was developed based on a mechanistic understanding. The activity of this iron model complex is enabled by its unique ligand environment, consisting of biomimetic pyridinylacyl and carbonyl ligands, as well as a bioinspired diphosphine ligand with a pendant amine moiety. The model complex activates H2 and mediates hydrogenation of an aldehyde. PMID:26926708

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

    SciTech Connect

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

    2012-01-28

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

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

    PubMed Central

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

    2007-01-01

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

  20. Extended X-ray absorption fine structure of the [Fe]-hydrogenase Hmd active site

    NASA Astrophysics Data System (ADS)

    Salomone-Stagni, Marco; Vogt, Sonja; Shima, Seigo; Meyer-Klaucke, Wolfram

    2009-11-01

    Hydrogenases are enzymes that catalyze the reversible oxidation of molecular hydrogen. Although their structure and catalytic mechanism are of considerable applied interest as models for the development of efficient catalysts for hydrogen fueled processes, the understanding of how hydrogenases react with H2 is only in its infancy. Two of the three known types of hydrogenases are iron-sulfur proteins that contain a dinuclear metal center, either [NiFe] or [FeFe]. In contrast, [Fe]-hydrogenase is the only mononuclear hydrogenase and thus a perfect system for studying the structural and electronic determinants of these enzymes. Here we summarize recent improvements in modeling based on the EXAFS signal and the geometric structure of this metalloenzyme in its as isolated or reconstituted form. The individual contributions to the EXAFS resulting in two different structural models are presented and discussed. Inspired by the new crystal structure, we show an advanced EXAFS model for the enzyme from Methanothermobacter marburgensis.

  1. Artificial Hydrogenases

    PubMed Central

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

    2010-01-01

    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

  2. A [NiFe]hydrogenase model that catalyses the release of hydrogen from formic acid.

    PubMed

    Nguyen, Nga T; Mori, Yuki; Matsumoto, Takahiro; Yatabe, Takeshi; Kabe, Ryota; Nakai, Hidetaka; Yoon, Ki-Seok; Ogo, Seiji

    2014-11-11

    We report the decomposition of formic acid to hydrogen and carbon dioxide, catalysed by a NiRu complex originally developed as a [NiFe]hydrogenase model. This is the first example of H2 evolution, catalysed by a [NiFe]hydrogenase model, which does not require additional energy. PMID:25234420

  3. Modeling three-dimensional structure of two closely related Ni-Fe hydrogenases.

    PubMed

    Abdullatypov, A V; Tsygankov, A A

    2015-08-01

    The results of homology modeling of HydSL, a NiFe-hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina BBS, and deep-water bacterium Alteromonas macleodii deep ecotype are presented in this work. It is shown that the models have larger confidence level than earlier published ones; full-size models of these enzymes are presented for the first time. The C-end fragment of small subunit of T. roseopersicina hydrogenase is shown to have random orientation in relation to the main protein globule. The obtained models of this enzyme have a large number of ion pairs, as well as thermostable HydSL hydrogenase from Allochromatium vinosum, in contrast to thermostable HydSL hydrogenase from Alt. macleodii and thermolabile HydAB hydrogenase from Desulfovibrio vulgaris. The possible determinant of oxygen stability of studied hydrogenases could be the lack of several intramolecular tunnels. Hydrophobic and electrostatic surfaces were mapped in order to find out possible pathways of coupling hydrogenase to electron-transferring chains, as well as methods for construction of artificial photobiohydrogen-producing systems. PMID:25572109

  4. Model study of CO inhibition of [NiFe]hydrogenase.

    PubMed

    Matsumoto, Takahiro; Kabe, Ryota; Nonaka, Kyoshiro; Ando, Tatsuya; Yoon, Ki-Seok; Nakai, Hidetaka; Ogo, Seiji

    2011-09-19

    We propose a modified mechanism for the inhibition of [NiFe]hydrogenase ([NiFe]H(2)ase) by CO. We present a model study, using a NiRu H(2)ase mimic, that demonstrates that (i) CO completely inhibits the catalytic cycle of the model compound, (ii) CO prefers to coordinate to the Ru(II) center rather than taking an axial position on the Ni(II) center, and (iii) CO is unable to displace a hydrido ligand from the NiRu center. We combine these studies with a reevaluation of previous studies to propose that, under normal circumstances, CO inhibits [NiFe]H(2)ase by complexing to the Fe(II) center. PMID:21853978

  5. 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)

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

    2015-01-01

    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.

  6. Reconstitution of [Fe]-hydrogenase using model complexes

    NASA Astrophysics Data System (ADS)

    Shima, Seigo; Chen, Dafa; Xu, Tao; Wodrich, Matthew D.; Fujishiro, Takashi; Schultz, Katherine M.; Kahnt, Jörg; Ataka, Kenichi; Hu, Xile

    2015-12-01

    [Fe]-Hydrogenase catalyses the reversible hydrogenation of a methenyltetrahydromethanopterin substrate, which is an intermediate step during the methanogenesis from CO2 and H2. The active site contains an iron-guanylylpyridinol cofactor, in which Fe2+ is coordinated by two CO ligands, as well as an acyl carbon atom and a pyridinyl nitrogen atom from a 3,4,5,6-substituted 2-pyridinol ligand. However, the mechanism of H2 activation by [Fe]-hydrogenase is unclear. Here we report the reconstitution of [Fe]-hydrogenase from an apoenzyme using two FeGP cofactor mimics to create semisynthetic enzymes. The small-molecule mimics reproduce the ligand environment of the active site, but are inactive towards H2 binding and activation on their own. We show that reconstituting the enzyme using a mimic that contains a 2-hydroxypyridine group restores activity, whereas an analogous enzyme with a 2-methoxypyridine complex was essentially inactive. These findings, together with density functional theory computations, support a mechanism in which the 2-hydroxy group is deprotonated before it serves as an internal base for heterolytic H2 cleavage.

  7. The Model [NiFe]-Hydrogenases of Escherichia coli.

    PubMed

    Sargent, F

    2016-01-01

    In Escherichia coli, hydrogen metabolism plays a prominent role in anaerobic physiology. The genome contains the capability to produce and assemble up to four [NiFe]-hydrogenases, each of which are known, or predicted, to contribute to different aspects of cellular metabolism. In recent years, there have been major advances in the understanding of the structure, function, and roles of the E. coli [NiFe]-hydrogenases. The membrane-bound, periplasmically oriented, respiratory Hyd-1 isoenzyme has become one of the most important paradigm systems for understanding an important class of oxygen-tolerant enzymes, as well as providing key information on the mechanism of hydrogen activation per se. The membrane-bound, periplasmically oriented, Hyd-2 isoenzyme has emerged as an unusual, bidirectional redox valve able to link hydrogen oxidation to quinone reduction during anaerobic respiration, or to allow disposal of excess reducing equivalents as hydrogen gas. The membrane-bound, cytoplasmically oriented, Hyd-3 isoenzyme is part of the formate hydrogenlyase complex, which acts to detoxify excess formic acid under anaerobic fermentative conditions and is geared towards hydrogen production under those conditions. Sequence identity between some Hyd-3 subunits and those of the respiratory NADH dehydrogenases has led to hypotheses that the activity of this isoenzyme may be tightly coupled to the formation of transmembrane ion gradients. Finally, the E. coli genome encodes a homologue of Hyd-3, termed Hyd-4, however strong evidence for a physiological role for E. coli Hyd-4 remains elusive. In this review, the versatile hydrogen metabolism of E. coli will be discussed and the roles and potential applications of the spectrum of different types of [NiFe]-hydrogenases available will be explored. PMID:27134027

  8. Nickel-Substituted Rubredoxin as a Minimal Enzyme Model for Hydrogenase.

    PubMed

    Slater, Jeffrey W; Shafaat, Hannah S

    2015-09-17

    A simple, functional mimic of [NiFe] hydrogenases based on a nickel-substituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20-100 s(-1) at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation. PMID:26722748

  9. Electrochemistry of Simple Organometallic Models of Iron-Iron Hydrogenases in Organic Solvent and Water.

    PubMed

    Gloaguen, Frederic

    2016-01-19

    Synthetic models of the active site of iron-iron hydrogenases are currently the subjects of numerous studies aimed at developing H2-production catalysts based on cheap and abundant materials. In this context, the present report offers an electrochemist's view of the catalysis of proton reduction by simple binuclear iron(I) thiolate complexes. Although these complexes probably do not follow a biocatalytic pathway, we analyze and discuss the interplay between the reduction potential and basicity and how these antagonist properties impact the mechanisms of proton-coupled electron transfer to the metal centers. This question is central to any consideration of the activity at the molecular level of hydrogenases and related enzymes. In a second part, special attention is paid to iron thiolate complexes holding rigid and unsaturated bridging ligands. The complexes that enjoy mild reduction potentials and stabilized reduced forms are promising iron-based catalysts for the photodriven evolution of H2 in organic solvents and, more importantly, in water. PMID:26641526

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  11. Brownian dynamics and molecular dynamics study of the association between hydrogenase and ferredoxin from Chlamydomonas reinhardtii.

    PubMed

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

    2008-10-01

    The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer. PMID:18621810

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

    PubMed

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

    2014-11-26

    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

  13. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

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

  14. [NiFeSe]-hydrogenase chemistry.

    PubMed

    Wombwell, Claire; Caputo, Christine A; Reisner, Erwin

    2015-11-17

    The development of technology for the inexpensive generation of the renewable energy vector H2 through water splitting is of immediate economic, ecological, and humanitarian interest. Recent interest in hydrogenases has been fueled by their exceptionally high catalytic rates for H2 production at a marginal overpotential, which is presently only matched by the nonscalable noble metal platinum. The mechanistic understanding of hydrogenase function guides the design of synthetic catalysts, and selection of a suitable hydrogenase enables direct applications in electro- and photocatalysis. [FeFe]-hydrogenases display excellent H2 evolution activity, but they are irreversibly damaged upon exposure to O2, which currently prevents their use in full water splitting systems. O2-tolerant [NiFe]-hydrogenases are known, but they are typically strongly biased toward H2 oxidation, while H2 production by [NiFe]-hydrogenases is often product (H2) inhibited. [NiFeSe]-hydrogenases are a subclass of [NiFe]-hydrogenases with a selenocysteine residue coordinated to the active site nickel center in place of a cysteine. They exhibit a combination of unique properties that are highly advantageous for applications in water splitting compared with other hydrogenases. They display a high H2 evolution rate with marginal inhibition by H2 and tolerance to O2. [NiFeSe]-hydrogenases are therefore one of the most active molecular H2 evolution catalysts applicable in water splitting. Herein, we summarize our recent progress in exploring the unique chemistry of [NiFeSe]-hydrogenases through biomimetic model chemistry and the chemistry with [NiFeSe]-hydrogenases in semiartificial photosynthetic systems. We gain perspective from the structural, spectroscopic, and electrochemical properties of the [NiFeSe]-hydrogenases and compare them with the chemistry of synthetic models of this hydrogenase active site. Our synthetic models give insight into the effects on the electronic properties and reactivity of

  15. Photocatalytic hydrogen production from a simple water-soluble [FeFe]-hydrogenase model system.

    PubMed

    Cao, Wei-Ning; Wang, Feng; Wang, Hong-Yan; Chen, Bin; Feng, Ke; Tung, Chen-Ho; Wu, Li-Zhu

    2012-08-21

    Combined with a simple water soluble [FeFe]-hydrogenase mimic 1, Ru(bpy)(3)(2+) and ascorbic acid enable hydrogen production photocatalytically. More than 88 equivalents of H(2) were achieved in water, which is much better than that obtained in an organic solvent or a mixture of organic solvent and water. PMID:22772838

  16. Synthetic Active Site Model of the [NiFeSe] Hydrogenase

    PubMed Central

    Wombwell, Claire; Reisner, Erwin

    2015-01-01

    A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe(‘S2Se2’)(CO)3] (H2‘S2Se2’=1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni(‘S2Se2’)] with [Fe(CO)3bda] (bda=benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe(‘S2Se2’)(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe(‘S2Se2’)(CO)3] with the previously reported thiolate analogue [NiFe(‘S4’)(CO)3] (H2‘S4’=H2xbsms=1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe(‘S2Se2’)(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe(‘S2Se2’)(CO)3] and [NiFe(‘S4’)(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. PMID:25847470

  17. Interaction of [FeFe]-Hydrogenases with Single-walled Carbon Nanotubes

    SciTech Connect

    Chang, D. S.; McDonald, T. J.; Kim, Y.-H.; Blackburn, J. L.; Heben, M. J.; King, P. W.

    2007-01-01

    Single-walled carbon nanotubes (SWNT) are promising candidates for use in energy conversion devices as an active photo-collecting elements, for dissociation of bound excitons and charge-transfer from photo-excited chromophores, or as molecular wires to transport charge. Hydrogenases are enzymes that efficiently catalyze the reduction of protons from a variety of electron donors to produce molecular hydrogen. Hydrogenases together with SWNT suggest a novel biohybrid material for direct conversion of sunlight into H{sub 2}. Here, we report changes in SWNT optical properties upon addition of recombinant [FeFe] hydrogenases from Clostridium acetobutylicum and Chlamydomonas reinhardtii. We find evidence that novel and stable charge-transfer complexes are formed under conditions of the hydrogenase catalytic turnover, providing spectroscopic handles for further study and application of this hybrid system.

  18. Interaction of [FeFe]-hydrogenases with single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Svedruzic Chang, Drazenka; McDonald, Timothy J.; Kim, Yong-Hyun; Blackburn, Jeffrey L.; Heben, Michael J.; King, Paul W.

    2007-09-01

    Single-walled carbon nanotubes (SWNT) are promising candidates for use in energy conversion devices as an active photo-collecting elements, for dissociation of bound excitons and charge-transfer from photo-excited chromophores, or as molecular wires to transport charge. Hydrogenases are enzymes that efficiently catalyze the reduction of protons from a variety of electron donors to produce molecular hydrogen. Hydrogenases together with SWNT suggest a novel biohybrid material for direct conversion of sunlight into H II. Here, we report changes in SWNT optical properties upon addition of recombinant [FeFe] hydrogenases from Clostridium acetobutylicum and Chlamydomonas reinhardtii. We find evidence that novel and stable charge-transfer complexes are formed under conditions of the hydrogenase catalytic turnover, providing spectroscopic handles for further study and application of this hybrid system.

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

    PubMed Central

    Manor, Brian C.; Rauchfuss, Thomas B.

    2013-01-01

    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) Å. 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

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

    PubMed

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

    2016-04-28

    Metal hydrides are invoked as important intermediates in both chemical and biological H2 production. In the [NiFe] hydrogenase enzymes, pulsed EPR and high-resolution crystallography have argued that the hydride interacts primarily at the Ni site. In contrast, in [NiFe] hydrogenase model complexes, it is observed that the bridging hydride interacts primarily with the Fe. Herein, we utilize a combination of Ni and Fe X-ray absorption (XAS) and emission (XES) spectroscopies to examine the contribution of the bridging hydride to the observed spectral features in [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3](+). The corresponding data on (dppe)Ni(μ-pdt)Fe(CO)3 are used as a reference for the changes that occur in the absence of a hydride bridge. For further interpretation of the observed spectral features, all experimental spectra were calculated using a density functional theory (DFT) approach, with excellent agreement between theory and experiment. It is found that the iron valence-to-core (VtC) XES spectra reveal clear signatures for the presence of a Fe-H interaction in the hydride bridged model complex. In contrast, the Ni VtC XES spectrum largely reflects changes in the local Ni geometry and shows little contribution from a Ni-H interaction. A stepwise theoretical analysis of the hydride contribution and the Ni site symmetry provides insights into the factors, which govern the different metal-hydride interactions in both the model complexes and the enzyme. Furthermore, these results establish the utility of two-color XES to reveal important insights into the electronic structure of various metal-hydride species. PMID:26924248

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

    PubMed Central

    Kaiser, Manuel; Knör, Günther

    2015-01-01

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

  2. The iron-site structure of [Fe]-hydrogenase and model systems: an X-ray absorption near edge spectroscopy study†‡

    PubMed Central

    Salomone-Stagni, Marco; Stellato, Francesco; Whaley, C. Matthew; Vogt, Sonja; Morante, Silvia; Shima, Seigo; Rauchfuss, Thomas B.; Meyer-Klaucke, Wolfram

    2012-01-01

    The [Fe]-hydrogenase is an ideal system for studying the electronic properties of the low spin iron site that is common to the catalytic centres of all hydrogenases. Because they have no auxiliary iron-sulfur clusters and possess a cofactor containing a single iron centre, the [Fe]-hydrogenases are well suited for spectroscopic analysis of those factors required for the activation of molecular hydrogen. Specifically, in this study we shed light on the electronic and molecular structure of the iron centre by XAS analysis of [Fe]-hydrogenase from Methanocaldococcus jannashii and five model complexes (Fe(ethanedithiolate)-(CO)2(PMe3)2, [K(18-crown-6)]2[Fe(CN)2(CO)3], K[Fe(CN)(CO)4], K3[Fe(iii)(CN)6], K4[Fe(ii)(CN)6]). The different electron donors have a strong influence on the iron absorption K-edge energy position, which is frequently used to determine the metal oxidation state. Our results demonstrate that the K-edges of Fe(ii) complexes, achieved with low-spin ferrous thiolates, are consistent with a ferrous centre in the [Fe]-hydrogenase from Methanocaldococcus jannashii. The metal geometry also strongly influences the XANES and thus the electronic structure. Using in silico simulation, we were able to reproduce the main features of the XANES spectra and describe the effects of individual donor contributions on the spectra. Thereby, we reveal the essential role of an unusual carbon donor coming from an acyl group of the cofactor in the determination of the electronic structure required for the activity of the enzyme. PMID:20221540

  3. Hydrogen Production Catalyzed by Bidirectional, Biomimetic Models of the [FeFe]-Hydrogenase Active Site.

    PubMed

    Lansing, James C; Camara, James M; Gray, Danielle E; Rauchfuss, Thomas B

    2014-10-27

    Active site mimics of [FeFe]-hydrogenase are shown to be bidirectional catalysts, producing H2 upon treatment with protons and reducing equivalents. This reactivity complements the previously reported oxidation of H2 by these same catalysts in the presence of oxidants. The complex Fe2(adt(Bn))(CO)3(dppv)(PFc*(Et2) ) ([1](0); adt(Bn) = (SCH2)2NBn, dppv = cis-1,2-bis(diphenylphosphino)ethylene, PFc*(Et2) = Et2PCH2C5Me4FeCp*) reacts with excess [H(OEt2)2]BAr(F) 4 (BAr(F) 4 (-) = B(C6H3-3,5-(CF3)2)4 (-)) to give ∼0.5 equiv of H2 and [Fe2(adt(Bn)H)(CO)3(dppv)(PFc*(Et2) )](2+) ([1H](2+)). The species [1H](2+) consists of a ferrocenium ligand, an N-protonated amine, and an Fe(I)Fe(I) core. In the presence of additional reducing equivalents in the form of decamethylferrocene (Fc*), hydrogen evolution is catalytic, albeit slow. The related catalyst Fe2(adt(Bn))(CO)3(dppv)(PMe3) (3) behaves similarly in the presence of Fc*, except that in the absence of excess reducing agent it converts to the catalytically inactive μ-hydride derivative [μ-H3](+). Replacement of the adt in [1](0) with propanedithiolate (pdt) results in a catalytically inactive complex. In the course of synthesizing [FeFe]-hydrogenase mimics, new routes to ferrocenylphosphine ligands and nonamethylferrocene were developed. PMID:25364093

  4. Hydrogen Production Catalyzed by Bidirectional, Biomimetic Models of the [FeFe]-Hydrogenase Active Site

    PubMed Central

    2015-01-01

    Active site mimics of [FeFe]-hydrogenase are shown to be bidirectional catalysts, producing H2 upon treatment with protons and reducing equivalents. This reactivity complements the previously reported oxidation of H2 by these same catalysts in the presence of oxidants. The complex Fe2(adtBn)(CO)3(dppv)(PFc*Et2) ([1]0; adtBn = (SCH2)2NBn, dppv = cis-1,2-bis(diphenylphosphino)ethylene, PFc*Et2 = Et2PCH2C5Me4FeCp*) reacts with excess [H(OEt2)2]BArF4 (BArF4– = B(C6H3-3,5-(CF3)2)4–) to give ∼0.5 equiv of H2 and [Fe2(adtBnH)(CO)3(dppv)(PFc*Et2)]2+ ([1H]2+). The species [1H]2+ consists of a ferrocenium ligand, an N-protonated amine, and an FeIFeI core. In the presence of additional reducing equivalents in the form of decamethylferrocene (Fc*), hydrogen evolution is catalytic, albeit slow. The related catalyst Fe2(adtBn)(CO)3(dppv)(PMe3) (3) behaves similarly in the presence of Fc*, except that in the absence of excess reducing agent it converts to the catalytically inactive μ-hydride derivative [μ-H3]+. Replacement of the adt in [1]0 with propanedithiolate (pdt) results in a catalytically inactive complex. In the course of synthesizing [FeFe]-hydrogenase mimics, new routes to ferrocenylphosphine ligands and nonamethylferrocene were developed. PMID:25364093

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

    SciTech Connect

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

    2008-01-01

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

  6. Studies on hydrogenase

    PubMed Central

    YAGI, Tatsuhiko; HIGUCHI, Yoshiki

    2013-01-01

    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

  7. Hydride binding to the active site of [FeFe]-hydrogenase.

    PubMed

    Chernev, Petko; Lambertz, Camilla; Brünje, Annika; Leidel, Nils; Sigfridsson, Kajsa G V; Kositzki, Ramona; Hsieh, Chung-Hung; Yao, Shenglai; Schiwon, Rafael; Driess, Matthias; Limberg, Christian; Happe, Thomas; Haumann, Michael

    2014-11-17

    [FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases. PMID:25369169

  8. H2 binding and splitting on a new-generation [FeFe]-hydrogenase model featuring a redox-active decamethylferrocenyl phosphine ligand: a theoretical investigation.

    PubMed

    Greco, Claudio

    2013-02-18

    [FeFe]-hydrogenases are dihydrogen-evolving metalloenzymes that are able to combine substrate binding and redox functionalities, a feature that has important bearing on their efficiency. New-generation bioinspired systems such as Fe(2)[(SCH(2))(2)NBn](CO)(3)(Cp*Fe(C(5)Me(4)CH(2)PEt(2)))(dppv) were shown to mimic H(2) oxidation and splitting processes performed by the [FeFe]-hydrogenase/ferredoxin system, and key mechanistic aspects of such reaction are theoretically investigated in the present contribution. We found that H(2) binding and heterolytic cleavage take place concomitantly on DFT models of the synthetic catalyst, due to a substrate-dependent intramolecular redox process that promotes dihydrogen activation. Therefore, formation of an iron-dihydrogen complex as a reaction intermediate is excluded in the biomimetic system, at variance with the case of the enzyme. H(2) uptake at the synthetic system also requires an energetically disfavored isomerization of the amine group acting as a base during splitting. A possible strategy to stabilize the conformation competent for H(2) binding is proposed, along with an analysis of the reactivity of a triiron complex in which di(thiomethyl)amine--the chelating group naturally occurring in [FeFe]-hydrogenases--substitutes the benzyl-containing dithiolate ligand. PMID:23374093

  9. Ligand Displacement Reaction Paths in a Diiron Hydrogenase Active Site Model Complex.

    PubMed

    Blank, Jan H; Moncho, Salvador; Lunsford, Allen M; Brothers, Edward N; Darensbourg, Marcetta Y; Bengali, Ashfaq A

    2016-08-26

    The mechanism and energetics of CO, 1-hexene, and 1-hexyne substitution from the complexes (SBenz)2 [Fe2 (CO)6 ] (SBenz=SCH2 Ph) (1-CO), (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexene)] (1-(η(2) -1-hexene)), and (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexyne)] (1-(η(2) -1-hexyne)) were studied by using time-resolved infrared spectroscopy. Exchange of both CO and 1-hexyne by P(OEt)3 and pyridine, respectively, proceeds by a bimolecular mechanism. As similar activation enthalpies are obtained for both reactions, the rate-determining step in both cases is assumed to be the rotation of the Fe(CO)2 L (L=CO or 1-hexyne) unit to accommodate the incoming ligand. The kinetic profile for the displacement of 1-hexene is quite different than that for the alkyne and, in this case, both reaction channels, that is, dissociative (SN 1) and associative (SN 2), were found to be competitive. Because DFT calculations predict similar binding enthalpies of alkene and alkyne to the iron center, the results indicate that the bimolecular pathway in the case of the alkyne is lower in free energy than that of the alkene. In complexes of this type, subtle changes in the departing ligand characteristics and the nature of the mercapto bridge can influence the exchange mechanism, such that more than one reaction pathway is available for ligand substitution. The difference between this and the analogous study of (μ-pdt)[Fe(CO)3 ]2 (pdt=S(CH2 )3 S) underscores the unique characteristics of a three-atom S-S linker in the active site of diiron hydrogenases. PMID:27482938

  10. Iron Hydride Detection and Intramolecular Hydride Transfer in a Synthetic Model of Mono-Iron Hydrogenase with a CNS Chelate.

    PubMed

    Durgaprasad, Gummadi; Xie, Zhu-Lin; Rose, Michael J

    2016-01-19

    We report the identification and reactivity of an iron hydride species in a synthetic model complex of monoiron hydrogenase. The hydride complex is derived from a phosphine-free CNS chelate that includes a Fe-C(NH)(═O) bond (carbamoyl) as a mimic of the active site iron acyl. The reaction of [((O═)C(HN)N(py)S(Me))Fe(CO)2(Br)] (1) with NaHBEt3 generates the iron hydride intermediate [((O═)C(HN)N(py)S(Me))Fe(H)(CO)2] (2; δFe-H = -5.08 ppm). Above -40 °C, the hydride species extrudes CH3S(-) via intramolecular hydride transfer, which is stoichiometrically trapped in the structurally characterized dimer μ2-(CH3S)2-[((O═)C(HN)N(Ph))Fe(CO)2]2 (3). Alternately, when activated by base ((t)BuOK), 1 undergoes desulfurization to form a cyclometalated species, [((O═)C(NH)NC(Ph))Fe(CO)2] (5); derivatization of 5 with PPh3 affords the structurally characterized species [((O═)C(NH)NC)Fe(CO)(PPh3)2] (6), indicating complex 6 as the common intermediate along each pathway of desulfurization. PMID:26405810

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

    PubMed Central

    Camara, James M.; Rauchfuss, Thomas B.

    2012-01-01

    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

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

    NASA Astrophysics Data System (ADS)

    Chang, Christopher H.; Kim, Kwiseon

    2007-03-01

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

  13. Reactivity of Fe/FeS nanoparticles: electrolyte composition effects on corrosion electrochemistry.

    PubMed

    Turcio-Ortega, David; Fan, Dimin; Tratnyek, Paul G; Kim, Eun-Ju; Chang, Yoon-Seok

    2012-11-20

    Zerovalent iron nanoparticles (Fe(0) NPs or nZVI) synthesized by reductive precipitation in aqueous solution (Fe/FeO) differ in composition and reactivity from the NPs obtained by reductive precipitation in the presence of a S-source such as dithionite (Fe/FeS). To compare the redox properties of these types of NPs under a range of environmentally relevant solution conditions, stationary powder disk electrodes (PDEs) made from Fe/FeO and Fe/FeS were characterized using a series of complementary electrochemical techniques: open-circuit chronopotentiometry (CP), linear polarization resistance (LPR), electrochemical impedance spectroscopy (EIS), and linear sweep voltammetry (LSV). The passive films on these materials equilibrate within minutes of first immersion and do not show further breakdown until >1 day of exposure. During this period, the potentials and currents measured by LPR and LSV suggest that Fe/FeS undergoes more rapid corrosion and is more strongly influence by solution chemical conditions than Fe/FeO. Chloride containing media were strongly activating and natural organic matter (NOM) was mildly passivating for both materials. These effects were also seen in the impedance data obtained by EIS, and equivalent circuit modeling of the electrodes composed of these powders suggested that the higher reactivity of Fe/FeS is due to greater abundance of defects in its passive film. PMID:23078203

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

    SciTech Connect

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

    2014-05-19

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

  15. Hydrogenase from Acetobacterium woodii.

    PubMed

    Ragsdale, S W; Ljungdahl, L G

    1984-11-01

    Hydrogenase from fructose-grown cells of Acetobacterium woodii has been purified 70-fold to a specific activity of 3,500 mumol hydrogen oxidized per min per mg of protein measured at 35 degrees C and pH 7.6 with methyl viologen as electron acceptor. At the same conditions with reduced methyl viologen as electron donor the enzyme catalyzes the evolvement of 440 mumol of H2 per min per mg of protein. The enzyme was found in the soluble portion of the cell, indicating that it is either not membrane-bound or is loosely associated with the membrane. The purified enzyme, which does not contain nickel, exhibits spectroscopic properties similar to the iron-sulfur hydrogenase of Clostridium pasteurianum. The enzyme is strongly inhibited by carbon monoxide, with 50% inhibition occurring at approximately 7 nM CO. Ferredoxin, flavodoxin, and carbon monoxide dehydrogenase are reduced in hydrogen-dependent reaction by the A. woodi hydrogenase. PMID:6393895

  16. Production and Engineering of Hydrogenase as a Biocatalyst for Hydrogen Fuel

    SciTech Connect

    Wang, Guangyi

    2010-04-09

    Hydrogenases are fascinating redox proteins, showing tremendous promise in the utilization of hydrogen fuel as a bioelectrocatalyst. They play critical roles in both biohydrogen production and hydrogen oxidation. Specifically, the recently-established comparability of the oxidative activity of the [NiFe]-hydrogenase active site to that of the fuel cell catalyst platinum marks a significant milestone for the potential application of hydrogenase in hydrogen fuel cells to replace platinum. However, the ability of producing hydrogenase in heterologous expression hosts and the sensitivity of hydrogenases to oxygen and carbon monoxide, etc. have seriously limited the viable macroscale utilization and production of hydrogen from the renewable source. A new technology for the production of up-take hydrogenase is being developed for the utilization of hydrogenase as a hydrogen catalyst. The development of this new technology integrates knowledge of structural biology, molecular biology, and principles of metabolic engineering to produce and engineer a stable hydrogenase as a hydrogen bioelectrocatalyst. It contributes to the critical issues of “expensive noble metal catalysts (i.e., platinum) and their limited reserves threatening the long-term sustainability of a hydrogen economy”. It also provides a model to “design natural materials and enzyme catalyst” for “efficient and cost-effective technologies” for a clean and sustainable energy in 21st century. This new technology includes 3 major components. The first component is the synthetic operons, which carry hydrogenase maturation pathways of Ralstonia eutropha. These synthetic operons are engineered to produce RH hydrogenase in the Escherichia coli strains based on our current molecular and genetic information of hydrogenase maturation mechanisms and pathways of R. eutropha. It presents the first example of producing hydrogenase in the conventional expression host using synthetic biology principles and tool

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

    PubMed

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

    2009-04-23

    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

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

    PubMed

    Shi, Jingu; Ai, Zhihui; Zhang, Lizhi

    2014-08-01

    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

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

    PubMed

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

    2010-01-01

    In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow. PMID:19966788

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

    PubMed Central

    Khanna, Namita; Lindblad, Peter

    2015-01-01

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

  1. Cyanobacterial hydrogenases and hydrogen metabolism revisited: recent progress and future prospects.

    PubMed

    Khanna, Namita; Lindblad, Peter

    2015-01-01

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

  2. pH-Dependent isotope exchange and hydrogenation catalysed by water-soluble NiRu complexes as functional models for [NiFe]hydrogenases.

    PubMed

    Kure, Bunsho; Matsumoto, Takahiro; Ichikawa, Koji; Fukuzumi, Shunichi; Higuchi, Yoshiki; Yagi, Tatsuhiko; Ogo, Seiji

    2008-09-21

    The pH-dependent hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes and hydrogenation of the carbonyl compounds have been investigated with water-soluble bis(mu-thiolate)(mu-hydride)NiRu complexes, Ni(II)(mu-SR)(2)(mu-H)Ru(II) {(mu-SR)(2) = N,N'-dimethyl-N,N'-bis(2-mercaptoethyl)-1,3-propanediamine}, as functional models for [NiFe]hydrogenases. In acidic media (at pH 4-6), the mu-H ligand of the Ni(II)(mu-SR)(2)(mu-H)Ru(II) complexes has H(+) properties, and the complexes catalyse the hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes. A mechanism of the hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes through a low-valent Ni(I)(mu-SR)(2)Ru(I) complex is proposed. In contrast, in neutral-basic media (at pH 7-10), the mu-H ligand of the Ni(II)(mu-SR)(2)(mu-H)Ru(II) complexes acts as H(-), and the complexes catalyse the hydrogenation of carbonyl compounds. PMID:18728883

  3. Characterization of the Fe Site in Iron-Sulfur-Cluster-Free Hydrogenase (Hmd) and of a Model Compound via Nuclear Resonance Vibrational Spectroscopy (NRVS)

    PubMed Central

    Guo, Yisong; Wang, Hongxin; Xiao, Yuming; vogt, Sonja; Shima, Seigo; Volkers, Phillip I.; Pelmentschikov, Vladimir; Alp, Ercan E.; Sturhahn, Wolfgang; Yada, Yoshitaka

    2009-01-01

    We have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron site in the iron-sulfur-cluster-free hydrogenase Hmd from the methanogenic archaeon Methanothermobacter marburgensis. The spectra have been interpreted by comparison with a cis-(CO)2-ligated Fe model compound, Fe(S2C2H4)(CO)2(PMe3)2, as well as by normal mode simulations of plausible active site structures. For this model complex, normal mode analyses both from an optimized Urey-Bradley force field and from complementary density functional theory (DFT) calculations produced consistent results. Previous IR spectroscopic studies found strong CO stretching modes at 1944 and 2011 cm−1, interpreted as evidence for cis-Fe(CO)2 ligation. The NRVS data provide further insight into the dynamics of the Fe site, revealing Fe-CO stretch and Fe-CO bend modes at 494, 562, 590, and 648 cm−1, consistent with the proposed cis-Fe(CO)2 ligation. The NRVS also reveals a band assigned to Fe-S stretching motion at ~311 cm−1, and another reproducible feature at ~380 cm−1. The 57Fe partial vibrational densities of states (PVDOS) for Hmd can be reasonably well simulated by a normal mode analysis based on a Urey-Bradley force field for a 5-coordinate cis-(CO)2-ligated Fe site with additional cysteine, water, and pyridone cofactor ligands. A final interpretation of the Hmd NRVS data, including DFT analysis, awaits a 3-dimensional structure for the active site. PMID:18407624

  4. Effects of impurity states on exchange coupling in Fe/Fe3O4 junctions

    NASA Astrophysics Data System (ADS)

    Inoue, J.; Honda, S.; Itoh, H.; Mibu, K.; Yanagihara, H.; Kita, E.

    2012-05-01

    Exchange coupling (EC) in Fe/Fe3O4 junctions containing magnetic impurities and in-gap states at the interface is calculated using a formula obtained by a cleaved layer method. The model for EC is constructed by performing first-principles calculations of the electronic and magnetic states of Co, Mn, and Cr impurities on the Fe surface and those of in-gap states in a bulk γ-Fe2O3, which has the same lattice structure as Fe3O4 but contains Fe defects. We show that the effect of Co impurities on EC is opposite to that of Cr and Mn impurities and that in-gap states tend to cause parallel magnetization alignment of two ferromagnets. These results are attributed to the change in electronic states caused by the presence of impurities. Further, we compare calculated results with experimental ones obtained in Fe/Fe3O4 junctions and suggest that doping magnetic impurities at the interface could be a useful way to control the magnitude and sign of the EC.

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

    SciTech Connect

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

    2015-02-01

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

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

    DOE PAGESBeta

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

    2015-02-01

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

  7. Cyanide inactivation of hydrogenase from Azotobacter vinelandii

    SciTech Connect

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

    1989-06-01

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

  8. Synthetic and structural studies on L-cysteinyl group-containing diiron/triiron azadithiolates as active site models of [FeFe]-hydrogenases.

    PubMed

    Song, Li-Cheng; Yan, Jing; Li, Yu-Long; Wang, De-Fu; Hu, Qing-Mei

    2009-12-01

    Five new L-cysteinyl group-containing diiron/triiron azadithiolate complexes (3-6, 10), which could be regarded as the active site models of [FeFe]-hydrogenases, have been successfully synthesized. Treatment of L-cysteinyl sodium mercaptide CytSNa (1, Cyt = CH(2)CH(CO(2)Et)NH(CO(2)Bu-t) with complex [(mu-SCH(2))(2)NCH(2)CH(2)Br]Fe(2)(CO)(6) (2) in THF at room temperature resulted in formation of model complex [(mu-SCH(2))(2)NCH(2)CH(2)SCyt]Fe(2)(CO)(6) (3). Further treatment of 3 with decarbonylating agent Me(3)NO in MeCN at room temperature afforded model complex [(mu-SCH(2))(2)NCH(2)CH(2)SCyt]Fe(2)(CO)(5) (4). Similarly, treatment of 3 with an equimolar mixture of Me(3)NO and Ph(3)P gave model complex [(mu-SCH(2))(2)NCH(2)CH(2)SCyt]Fe(2)(CO)(5)(Ph(3)P) (5) and further treatment of 5 with Me(3)NO produced model complex [(mu-SCH(2))(2)NCH(2)CH(2)SCyt]Fe(2)(CO)(4)(Ph(3)P) (6). More interestingly, model complex [(mu-SCH(2))(2)NCH(CO(2)Et)CH(2)SFe(CO)(2)Cp]Fe(2)(CO)(5) (10) could be synthesized by a "one pot" reaction of the in situ prepared (mu-HS)(2)Fe(2)(CO)(6) (9) with 37% aqueous formaldehyde followed by treatment with the N-deprotected L-cysteinyl iron mercaptide Cp(CO)(2)FeSCH(2)CH(CO(2)Et)NH(2) (8). Complex 8 is new, which was prepared by treatment of complex Cp(CO)(2)FeSCyt (7) with CF(3)CO(2)H followed by 25% aqueous NH(3). All the new complexes 3-6, 8, and 10 were characterized by elemental analysis and various spectroscopic techniques, whereas complexes 5 and 10 were further characterized by X-ray crystallography. PMID:19860376

  9. Modeling the Active Sites in Metalloenzymes 5. The Heterolytic Bond Cleavage of H2 in the [NiFe] Hydrogenase of DesulfoWibrio gigas by a Nucleophilic Addition Mechanism

    SciTech Connect

    Niu, Shuqiang; Hall, Michael B.

    2001-11-19

    The H2 activation catalyzed by an Fe(II)-Ni(III) model of the [NiFe] hydrogenase of DesulfoVibrio gigas has been investigated by density functional theory (DFT/B3LYP) calculations on the neutral and anionic active site complexes, [(CO)(CN)2Fe(Mu-SH)2Ni(SH)(SH2)]0 and [(CO)(CN)2Fe(Mu-SH)2Ni(SH)2]-. The results suggest that the reaction proceeds by a nucleophilic addition mechanism that cleaves the H-H bond heterolytically. The terminal cysteine residue Cys530 in the [NiFe] hydrogenase active site of the D. gigas enzyme plays a crucial role in the catalytic process by accepting the proton. The active site is constructed to provide access by this cysteine residue, and this role explains the change in activity observed when this cysteine is replaced by a selenocysteine. Furthermore, the optimized geometry of the transition state in the model bears a striking resemblance to the geometry of the active site as determined by X-ray crystallography.

  10. Hydrogenase polypeptide and methods of use

    DOEpatents

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

    2016-02-02

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

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

    PubMed

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

    2014-04-01

    Nano-zerovalent iron (nZVI) formed under sulfidic conditions results in a biphasic material (Fe/FeS) that reduces trichloroethene (TCE) more rapidly than nZVI associated only with iron oxides (Fe/FeO). Exposing Fe/FeS to dissolved metals (Pd(2+), Cu(2+), Ni(2+), Co(2+), and Mn(2+)) results in their sequestration by coprecipitation as dopants into FeS and FeO and/or by electroless precipitation as zerovalent metals that are hydrogenation catalysts. Using TCE reduction rates to probe the effect of metal amendments on the reactivity of Fe/FeS, it was found that Mn(2+) and Cu(2+) decreased TCE reduction rates, while Pd(2+), Co(2+), and Ni(2+) increased them. Electrochemical characterization of metal-amended Fe/FeS showed that aging caused passivation by growth of FeO and FeS phases and poisoning of catalytic metal deposits by sulfide. Correlation of rate constants for TCE reduction (kobs) with electrochemical parameters (corrosion potentials and currents, Tafel slopes, and polarization resistance) and descriptors of hydrogen activation by metals (exchange current density for hydrogen reduction and enthalpy of solution into metals) showed the controlling process changed with aging. For fresh Fe/FeS, kobs was best described by the exchange current density for activation of hydrogen, whereas kobs for aged Fe/FeS correlated with electrochemical descriptors of electron transfer. PMID:24579799

  12. Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina.

    PubMed

    Maróti, Gergely; Tong, Yingkai; Yooseph, Shibu; Baden-Tillson, Holly; Smith, Hamilton O; Kovács, Kornél L; Frazier, Marvin; Venter, J Craig; Xu, Qing

    2009-09-01

    Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner. PMID:19633107

  13. The phylogeny of uptake hydrogenases in Frankia.

    PubMed

    Leul, Melakeselam; Normand, Philippe; Sellstedt, Anita

    2009-03-01

    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

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

    PubMed

    Ogata, Hideaki; Lubitz, Wolfgang; Higuchi, Yoshiki

    2009-10-01

    [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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

    PubMed Central

    Hexter, Suzannah V.; Grey, Felix; Happe, Thomas; Climent, Victor; Armstrong, Fraser A.

    2012-01-01

    The extraordinary ability of Fe- and Ni-containing enzymes to catalyze rapid and efficient H+/H2 interconversion—a property otherwise exclusive to platinum metals—has been investigated in a series of experiments combining variable-temperature protein film voltammetry with mathematical modeling. The results highlight important differences between the catalytic performance of [FeFe]-hydrogenases and [NiFe]-hydrogenases and justify a simple model for reversible catalytic electron flow in enzymes and electrocatalysts that should be widely applicable in fields as diverse as electrochemistry, catalysis, and bioenergetics. The active site of [FeFe]-hydrogenases, an intricate Fe-carbonyl complex known as the “H cluster,” emerges as a supreme catalyst. PMID:22802675

  18. Cobaloxime-based artificial hydrogenases.

    PubMed

    Bacchi, Marine; Berggren, Gustav; Niklas, Jens; Veinberg, Elias; Mara, Michael W; Shelby, Megan L; Poluektov, Oleg G; Chen, Lin X; Tiede, David M; Cavazza, Christine; Field, Martin J; Fontecave, Marc; Artero, Vincent

    2014-08-01

    Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions. PMID:25029381

  19. Hydride transfer and dihydrogen elimination from osmium and ruthenium metalloporphyrin hydrides: Model processes for hydrogenase enzymes and the hydrogen electrode reaction

    SciTech Connect

    Collman, J.P.; Wagenknecht, P.S.; Lewis, N.S.

    1992-07-01

    A series of metalloporphyrin hydride complexes of the type K[M(Por)(L)(H)] (M - Ru, Os; Por - OEP, TMP; L = THF, *Im, PPh{sub 3}, pyridine) has been synthesized by stoichiometric protonation of the corresponding K{sub 2}[M(Por)], followed by addition of L. The addition of excess acids to these hydrides resulted in the elimination of dihydrogen. The kinetics showed no evidence for a bimolecular mechanism for this process and suggest simple protonation of the metal-hydride bond followed by dihydrogen loss. One-electron oxidation of the metal hydrides also resulted in dihydrogen formation. The kinetics of the oxidatively induced hydrogen evolution step from K[Ru(OEP)(THF)(H)] were examined and indicate a biomolecular mechanism in which two metal hydrides reductively eliminate one dihydrogen molecule. The rate constant was determined to be 88 {+-} 14 M{sup -1} s{sup -1}. These reaction mechanisms are discussed in the context of designing bimetallic proton reduction catalysts. The metal hydride K[Ru(OEP)(THF)(H)], was also synthesized by heterolytic activation of H{sub 2}. This hydride is a good one-electron reductant (-1.15 V vs FeCp{sub 2}) and is capable of reducing, by hydride transfer, the NAD{sup +} analogue, 1-benzyl-N,N-diethyl-nicotinamide. This nicotinamide reduction by a hydride formed from heterolytic dihydrogen activation is suggested as the mechanism for hydrogenase enzymes. 38 refs., 4 figs., 3 tabs.

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

    PubMed Central

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

    2010-01-01

    A [NiFe] hydrogenase model compound having a distorted trigonal-pyramidal nickel center, (CO)3Fe(μ-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(μ-StBu)3Ni]2(μ-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)(μ-StBu)2Ni(CO)(SDmp), 3, or (CO)3Fe(StBu)(μ-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

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

    SciTech Connect

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

    2011-10-01

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

  2. 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...

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

    SciTech Connect

    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

    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.

  4. Mechanism of H2 production by the [FeFe]H subcluster of di-iron hydrogenases: implications for abiotic catalysts.

    PubMed

    Sbraccia, Carlo; Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Dismukes, G Charles; Selloni, Annabella

    2008-10-23

    To explore the possibility that the active center of the di-iron hydrogenases, the [FeFe] H subcluster, can serve by itself as an efficient hydrogen-producing catalyst, we perform comprehensive calculations of the catalytic properties of the subcluster in vacuo using first principles density functional theory. For completeness, we examine all nine possible geometrical isomers of the Fe(II)Fe(I) active-ready state and report in detail on the relevant ones that lead to the production of H 2. These calculations, carried out at the generalized gradient approximation level, indicate that the most efficient catalytic site in the isolated [FeFe] H subcluster is the Fe d center distal (d) to the [4Fe-4S] H cluster; the other iron center site, the proximal Fe p, also considered in this study, has much higher energy barriers. The pathways with the most favorable kinetics (lowest energy barrier to reaction) proceed along configurations with a CO ligand in a bridging position. The most favorable of these CO-bridging pathways start from isomers where the distal CN (-) ligand is in up position, the vacancy V in down position, and the remaining distal CO is either cis or trans with respect to the proximal CO. These isomers, not observed in the available enzyme X-ray structures, are only marginally less stable than the most stable nonbridging Fe d-CO-terminal isomer. Our calculations indicate that this CO-bridging CN-up isomer has a small barrier to production of H 2 that is compatible with the observed rate for the enzyme. These results suggest that catalysis of H 2 production could proceed on this stereochemically modified [FeFe] H subcluster alone, thus offering a promising target for functional bioinspired catalyst design. PMID:18826265

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

    PubMed Central

    2014-01-01

    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

  6. In situ determination of Fe-Fe 3S phase diagram and liquid structural properties up to 65 GPa

    NASA Astrophysics Data System (ADS)

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

    2008-08-01

    Lighter elements than iron such as sulphur are required in the Earth's core to account of the core density deficit. Accurate determination of the evolution of the Fe-FeS phase diagram at high pressure is essential to determine sulphur amount in the Earth's core. Ab initio calculations predict extensive solubility of S in solid Fe at core pressures of 330 GPa, whereas multi anvil quench analysis exhibits deep eutectic system at moderate pressure of 21 GPa. In this study, we investigated the Fe-rich part of Fe-FeS phase diagram up to 65 GPa and 2200 K using in situ angle dispersive X-ray diffraction. We report a uniform increase with pressure of the eutectic temperatures ( TEut), of about 15 K/GPa. Above 50 GPa, we evidence a decrease of S content in eutectic liquid with increasing pressure. Extrapolating this trend to inner core boundary pressures suggests that S cannot account for the 10 wt.% outer core density deficit and that other light elements, such as Si and O, are needed. Diffraction pattern recorded at 42 GPa and 2150 K was selected for structural investigations of the Fe-S liquid. By applying liquid structure simulation based on Gaussian distribution of atoms around crystalline positions, a good agreement has been found with hcp Fe model-structure, rather than with Fe 3S structure. It suggests that S acts as an interstitial impurity in the liquid state. Therefore, S could have a relatively minor effect on sound velocities in liquid outer core.

  7. Hydrogenases and Hydrogen Metabolism of Cyanobacteria

    PubMed Central

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

    2002-01-01

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

  8. A bacterial electron-bifurcating hydrogenase.

    PubMed

    Schuchmann, Kai; Müller, Volker

    2012-09-01

    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

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

    PubMed

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

    2012-12-01

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

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

    PubMed

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

    2016-01-19

    A series of pendant amine-containing [FeFe]-hydrogenase models, [X(CH2S-μ)2{Fe(CO)3}{Fe(CO)(P2(Ph)N2(Bn))}] (1H, X = CH2; 2Me, C(CH3)2; 3Et, C(CH2CH3)2; and P2(Ph)N2(Bn) = 1,5-dibenzyl-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane) with different groups at the bridgehead carbon of the S-to-S linker were synthesized. The oxidations of these complexes as well as the reverse reduction reaction were studied by cyclic voltammetry and in situ IR spectroscopy. Regardless of the bridgehead steric bulk, all three complexes demonstrate intramolecular iron-mediated C(sp(3))-H bond heterolytic cleavage with the assistance of the pendant amine base within the chelating diphosphine ligand in the two-electron oxidation process. X-ray crystallographic analysis shows that the doubly oxidized products, [1'H](+), [2'Me](+), and [3'Et](+), all have a rigid FeSC three-membered ring at the open apical site of the rotated iron center. The most noticeable difference in structures of the oxidized complexes is that the single CO ligand of the rotated Fe(P2(Ph)N2(Bn))(CO) unit in [1'H](+) and [2'Me](+) is found below the Fe···Fe vector, while in [3'Et](+) an unusually rotated Fe(P2(Ph)N2(Bn))(CO) moiety positions one of the P donors within the bidentate ligand under the Fe···Fe vector. The starting Fe(I)Fe(I) complexes can be recovered from their corresponding doubly oxidized complexes by reduction in the presence of Brönsted acid. PMID:26230977

  11. Mechanism of electrocatalytic hydrogen production by a di-iron model of iron-iron hydrogenase: a density functional theory study of proton dissociation constants and electrode reduction potentials.

    PubMed

    Surawatanawong, Panida; Tye, Jesse W; Darensbourg, Marcetta Y; Hall, Michael B

    2010-03-28

    Simple dinuclear iron dithiolates such as (mu-SCH2CH2CH2S)[Fe(CO)3]2, (1) and (mu-SCH2CH2S)[Fe(CO)3]2 (2) are functional models for diiron-hydrogenases, [FeFe]-H2ases, that catalyze the reduction of protons to H2. The mechanism of H2 production with 2 as the catalyst and with both toluenesulfonic (HOTs) and acetic (HOAc) acids as the H+ source in CH3CN solvent has been examined by density functional theory (DFT). Proton dissociation constants (pKa) and electrode reduction potentials (E(o)) are directly computed and compared to the measured pKa of HOTs and HOAc acids and the experimental reduction potentials. Computations show that when the strong acid, HOTs, is used as a proton source the one-electron reduced species 2- can be protonated to form a bridging hydride complex as the most stable structure. Then, this species can be reduced and protonated to form dihydrogen and regenerate 2. This cycle produces H2 via an ECEC process at an applied potential of -1.8 V vs. Fc/Fc+. A second faster process opens for this system when the species produced at the ECEC step above is further reduced and H2 release returns the system to 2- rather than 2, an E[CECE] process. On the other hand, when the weak acid, HOAc, is the proton source a more negative applied reduction potential (-2.2 V vs. Fc/Fc+) is necessary. At this potential two one-electron reductions yield the dianion 2(2-) before the first protonation, which in this case occurs on the thiolate. Subsequent reduction and protonation form dihydrogen and regenerate 2- through an E[ECEC] process. PMID:20221544

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

    PubMed Central

    Bailleul, Benjamin; Berne, Nicolas

    2015-01-01

    The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP+ oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments. PMID:25931521

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

    PubMed

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

    2015-06-01

    The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments. PMID:25931521

  14. Simple ligand effects switch a hydrogenase mimic between H2 and O2 activation.

    PubMed

    Kim, Kyoungmok; Matsumoto, Takahiro; Robertson, Andrew; Nakai, Hidetaka; Ogo, Seiji

    2012-06-01

    Herein, we report a [NiRu] biomimetic system for O(2)-tolerant [NiFe]hydrogenases and demonstrate that electron donation to the [NiRu] center can switch the system between the activation of H(2) and O(2) through simple ligand effects by using hexamethylbenzene and pentamethylcyclopentadienyl ligands, respectively. Furthermore, we present the synthesis and direct observations of a [NiRu]-peroxo species, which was formed by the oxygenation of a Ni-SIa model [NiRu] complex, that we propose as a biomimetic analogue of O(2)-bound species (OBS) of O(2)-tolerant [NiFe]hydrogenases. The [NiRu]-peroxo complex was fully characterized by X-ray analysis, X-ray photoelectron spectroscopy (XPS), mass spectrometry, and (1)H NMR spectroscopy. The OBS analogue was capable of oxidizing p-hydroquinone and sodium borohydride to turn back into the Ni-SIa model complex. PMID:22383335

  15. Activation and de novo synthesis of hydrogenase in Chlamydomonas

    SciTech Connect

    Roessler, P.G.; Lien, S.

    1984-12-01

    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.

  16. Biomimetic assembly and activation of [FeFe]-hydrogenases.

    PubMed

    Berggren, G; Adamska, A; Lambertz, C; Simmons, T R; Esselborn, J; Atta, M; Gambarelli, S; Mouesca, J-M; Reijerse, E; Lubitz, W; Happe, T; Artero, V; Fontecave, M

    2013-07-01

    Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery. PMID:23803769

  17. Maturation of [NiFe]-hydrogenases in Escherichia coli.

    PubMed

    Forzi, Lucia; Sawers, R Gary

    2007-06-01

    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

  18. Force Field Development and Molecular Dynamics of [NiFe] Hydrogenase

    SciTech Connect

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

    2012-05-09

    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.

  19. Photosensitized production of hydrogen by hydrogenase in reversed micelles

    PubMed Central

    Hilhorst, Riet; Laane, Colja; Veeger, Cees

    1982-01-01

    Hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from Desulfovibrio vulgaris was encapsulated in reversed micelles with cetyltrimethylammonium bromide as surfactant and a chloroform/octane mixture as solvent. Reducing equivalents for hydrogenase-catalyzed hydrogen production were provided by vectorial photosensitized electron transfer from a donor (thiophenol) in the organic phase through a surfactant-Ru2+ sensitizer located in the interphase to methyl viologen concentrated in the aqueous core of the reversed micelle. The results show that reversed micelles provide a microenvironment that (i) stabilizes hydrogenase against inactivation and (ii) allows an efficient vectorial photosensitized electron and proton flow from the organic phase to hydrogenase in the aqueous phase. Images PMID:16593204

  20. Maturation of Rhizobium leguminosarum Hydrogenase in the Presence of Oxygen Requires the Interaction of the Chaperone HypC and the Scaffolding Protein HupK*

    PubMed Central

    Albareda, Marta; Pacios, Luis F.; Manyani, Hamid; Rey, Luis; Brito, Belén; Imperial, Juan; Ruiz-Argüeso, Tomás; Palacios, Jose M.

    2014-01-01

    [NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O2) and bacteroids from legume nodules (∼10–100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen. PMID:24942742

  1. Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase.

    PubMed

    Ogata, Hideaki; Nishikawa, Koji; Lubitz, Wolfgang

    2015-04-23

    The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H(-)) bridging the Ni and Fe and a proton (H(+)) attached to the sulphur of a cysteine ligand. The Ni-H(-) and Fe-H(-) bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN(-) ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis. PMID:25624102

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

    SciTech Connect

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

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  3. Activation and de novo synthesis of hydrogenase in chlamydomonas.

    PubMed

    Roessler, P G; Lien, S

    1984-12-01

    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 a 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 hydrogense 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(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, 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. PMID:16663954

  4. Fabrication and physical properties of [Fe/Fe4N]N multilayers with high saturation magnetization

    NASA Astrophysics Data System (ADS)

    Yu, B.; Lin, L.; Ma, B.; Zhang, Z. Z.; Jin, Q. Y.; Wang, J. P.

    2016-05-01

    [Fe/Fe4N]N multilayers with high saturation magnetization were prepared on MgO(200) substrate, by the DC reactive magnetron sputtering and then annealed at higher temperature. Their structural and magnetic properties were investigated. Epitaxial growth of α-Fe and γ'-Fe4N were demonstrated on MgO, and then excellent [Fe/Fe4N]N was obtained. Though the saturation magnetizations of the as-deposited [Fe/Fe4N]N are slightly below the average value of those of α-Fe and γ'-Fe4N, the saturation magnetization of the annealed [Fe(3.04 nm)/Fe4N(3.04 nm)]5 increases up to 1850 emu/cc, 32 % larger than that of α-Fe film. N atom diffusion from the γ'-Fe4N to the α-Fe layer at high temperature greatly improves the saturation magnetization.

  5. Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia.

    PubMed

    Richau, K H; Kudahettige, R L; Pujic, P; Kudahettige, N P; Sellstedt, A

    2013-11-01

    The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this 'waste' product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under freeliving conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  7. [FeFe]-hydrogenases and photobiological hydrogen production

    NASA Astrophysics Data System (ADS)

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

    2006-08-01

    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.

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

    SciTech Connect

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

    2014-09-04

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

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

    DOE PAGESBeta

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

    2014-09-04

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

  10. Hydrogen activation by [NiFe]-hydrogenases.

    PubMed

    Carr, Stephen B; Evans, Rhiannon M; Brooke, Emily J; Wehlin, Sara A M; Nomerotskaia, Elena; Sargent, Frank; Armstrong, Fraser A; Phillips, Simon E V

    2016-06-15

    Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2 The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg(509)), which interacts with two conserved aspartate residues (Asp(118) and Asp(574)) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu(28)) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg(509) (the base). PMID:27284053

  11. Distribution and activity of hydrogenase enzymes in subsurface sediments

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

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

    PubMed

    Petrenko, Alexander; Stein, Matthias

    2015-10-29

    Hydrogenase enzymes are being used in enzymatic fuel cells immobilized on a graphite or carbon electrode surface, for example. The enzyme is used for the anodic oxidation of molecular hydrogen (H2) to produce protons and electrons. The association and orientation of the enzyme at the anode electrode for a direct electron transfer is not completely resolved. The distal FeS-cluster in [NiFe]-hydrogenases contains a histidine residue which is known to play a critical role in the intermolecular electron transfer between the enzyme and the electrode surface. The [NiFe]-hydrogenase graphite electrode association was investigated using Brownian Dynamics simulations. Residues that were shown to be in proximity to the electrode surface were identified (His184, Ser196, Glu461, Glu464), and electron transfer routes connecting the distal FeS-cluster with the surface residues were investigated. Several possible pathways for electron transfer between the distal FeS-cluster and the terminal amino acid residues were probed in terms of their rates of electron transfer using DFT methods. The reorganization energies λ of the distal iron-sulfur cluster and coronene as a molecular model for graphite were calculated. The reorganization energy of the distal (His)(Cys)3 cluster was found to be not very different from that of a standard cubane clusters with a (Cys)4 coordination. Electronic coupling matrix elements and rates of electron transfer for the different pathways were calculated according to the Marcus equation. The rates for glutamate-mediated electrode binding were found to be incompatible with experimental data. A direct electron transfer from the histidine ligand of the distal FeS-cluster to the electrode yielded rates of electron transfer in excellent agreement with experiment. A second pathway, however, from the distal FeS-cluster to the Ser196 residue was found to be equally efficient and feasible. PMID:26218232

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

    PubMed

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

    2015-08-18

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

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

    PubMed

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

    2016-05-17

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

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

    PubMed

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

    2015-12-21

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

  17. Energy harvesting based on FE-FE transition in ferroelectric single crystals.

    PubMed

    Guyomar, Daniel; Pruvost, Sebastien; Sebald, Gael

    2008-02-01

    The pyroelectric properties of Pb(Zn(1/3)Nb(2/3))(0955)Ti(0.045)O(3) single crystals versus an electric field have been studied for energy harvesting in this paper. Two thermodynamic cycles (Stirling and Ericsson) were used for this purpose. By applying an electric field, a FE-FE transition was induced, abruptly increasing the polarization. This transition minimized the supplied energy and improved the harvested energy. By discharging the single crystal at a higher temperature, a gain of 1100% was obtained with the Stirling cycle at 1 kV/mm (gain is defined as harvested energy divided by supplied energy). The study revealed that Stirling cycles are more interesting for low electric fields. Based on experimental results, simulations were carried out to estimate energy harvesting in high electric fields to evaluate the performances of thin samples (single crystals or oriented thin films). At high electric fields, both cycles gave almost the same energy harvesting, but Ericsson cycles were more appropriate to control the voltage on the sample. The simulation led to a harvested energy of 500 mJ/g for an applied electric field equal to 50 kV/mm. The efficiency with respect to Carnot was raised 20%. PMID:18334334

  18. Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states.

    PubMed

    Volbeda, Anne; Martin, Lydie; Barbier, Elodie; Gutiérrez-Sanz, Oscar; De Lacey, Antonio L; Liebgott, Pierre-Pol; Dementin, Sébastien; Rousset, Marc; Fontecilla-Camps, Juan C

    2015-01-01

    Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species. PMID:25315838

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

    SciTech Connect

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

    2007-08-01

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

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

    SciTech Connect

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

    2007-08-01

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

  1. Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion

    SciTech Connect

    Chatelus, C.; Carrier, P.; Saignes, P.; Libert, M.F.; Berlier, Y.; Lespinat, P.A.; Fauque, G.; Legall, J.

    1987-07-01

    Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena. (Refs. 16).

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

    SciTech Connect

    Stults, L.W.

    1986-01-01

    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.

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

    DOEpatents

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

    2009-03-10

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

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

    DOEpatents

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

    2014-03-04

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

  5. Multiscale simulations give insight into the hydrogen in and out pathways of [NiFe]-hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans.

    PubMed

    Oteri, Francesco; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

    2014-12-01

    [NiFe]-hydrogenases catalyze the cleavage of molecular hydrogen into protons and electrons and represent promising tools for H2-based technologies such as biofuel cells. However, many aspects of these enzymes remain to be understood, in particular how the catalytic center can be protected from irreversible inactivation by O2. In this work, we combined homology modeling, all-atom molecular dynamics, and coarse-grain Brownian dynamics simulations to investigate and compare the dynamic and mechanical properties of two [NiFe]-hydrogenases: the soluble O2-sensitive enzyme from Desulfovibrio fructosovorans, and the O2-tolerant membrane-bound hydrogenase from Aquifex aeolicus. We investigated the diffusion pathways of H2 from the enzyme surface to the central [NiFe] active site, and the possible proton pathways that are used to evacuate hydrogen after the oxidation reaction. Our results highlight common features of the two enzymes, such as a Val/Leu/Arg triad of key residues that controls ligand migration and substrate access in the vicinity of the active site, or the key role played by a Glu residue for proton transfer after hydrogen oxidation. We show specificities of each hydrogenase regarding the enzymes internal tunnel network or the proton transport pathways. PMID:25399809

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. Experimental approaches to kinetics of gas diffusion in hydrogenase.

    PubMed

    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

    2008-08-12

    Hydrogenases, which catalyze H(2) 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 H(2) 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

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

    PubMed

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

    2015-07-01

    In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions. PMID:26046591

  10. Experimental approaches to kinetics of gas diffusion in hydrogenase

    PubMed Central

    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

    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

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

    PubMed Central

    Suess, Daniel L. M.

    2015-01-01

    Proton reduction and H2 oxidation are key elementary reactions for solar fuel production. Hydrogenases interconvert H+ and H2 with remarkable efficiency and have therefore received much attention in this context. For [FeFe]-hydrogenases, catalysis occurs at a unique cofactor called the H-cluster. In this article, we discuss ways in which EPR spectroscopy has elucidated aspects of the bioassembly of the H-cluster, with a focus on four case studies: EPR spectroscopic identification of a radical en route to the CO and CN− ligands of the H-cluster, tracing 57Fe from the maturase HydG into the H-cluster, characterization of the auxiliary Fe–S cluster in HydG, and isotopic labeling of the CN− ligands of HydA for electronic structure studies of its Hox state. Advances in cell-free maturation protocols have enabled several of these mechanistic studies, and understanding H-cluster maturation may in turn provide insights leading to improvements in hydrogenase production for biotechnological applications. PMID:26508821

  12. Mechanism of hydrogen activation by [NiFe] hydrogenases.

    PubMed

    Evans, Rhiannon M; Brooke, Emily J; Wehlin, Sara A M; Nomerotskaia, Elena; Sargent, Frank; Carr, Stephen B; Phillips, Simon E V; Armstrong, Fraser A

    2016-01-01

    The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niμ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases. PMID:26619250

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

    PubMed Central

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

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Buono, Antonio S.; Walker, David

    2015-04-01

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

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

    USGS Publications Warehouse

    Brett, R.; Bell, P.M.

    1969-01-01

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

  16. Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria.

    PubMed Central

    Kärst, U; Suetin, S; Friedrich, C G

    1987-01-01

    In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system. Of these, the B peptide was purified to homogeneity. This protein (Mr, 37,500) was composed of two identical subunits (Mr, 18,800). Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis. About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1. The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200. The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens. Immunochemical analysis of other species and genera than A. eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens. The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown. Images PMID:3553156

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

    PubMed Central

    2010-01-01

    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

  18. Hydrogenase-based nanomaterials as anode electrode catalyst in polymer electrolyte fuel cells

    NASA Astrophysics Data System (ADS)

    Tsuda, Muneyuki; Diño, Wilson Agerico; Kasai, Hideaki

    2005-03-01

    We consider hydrogenase-based nanomaterials for possible use as anode electrode catalysts in polymer electrolyte fuel cells (PEFCs). We choose Fe-only hydrogenase component of Desulfovibrio desulfuricans (DdHase) as a hydrogenase complex, and investigate its catalytic activity for H 2 dissociation using ab initio calculations based on density functional theory (DFT). We found two possible H-H bond cleavage pathways, which are heterolytic and possess low activation barriers. Moreover, the H 2 dissociation can be promoted by inducing spin polarization of the H 2 adduct. We report that hydrogenase or hydrogenase-based nanomaterials can manipulate to exhibit the catalytic activity equivalent to the well-known platinum catalyst.

  19. Understanding the High Activity of Fe-N-C Electrocatalysts in Oxygen Reduction: Fe/Fe3C Nanoparticles Boost the Activity of Fe-N(x).

    PubMed

    Jiang, Wen-Jie; Gu, Lin; Li, Li; Zhang, Yun; Zhang, Xing; Zhang, Lin-Juan; Wang, Jian-Qiang; Hu, Jin-Song; Wei, Zidong; Wan, Li-Jun

    2016-03-16

    Understanding the origin of high activity of Fe-N-C electrocatalysts in oxygen reduction reaction (ORR) is critical but still challenging for developing efficient sustainable nonprecious metal catalysts in fuel cells and metal-air batteries. Herein, we developed a new highly active Fe-N-C ORR catalyst containing Fe-N(x) coordination sites and Fe/Fe3C nanocrystals (Fe@C-FeNC), and revealed the origin of its activity by intensively investigating the composition and the structure of the catalyst and their correlations with the electrochemical performance. The detailed analyses unambiguously confirmed the coexistence of Fe/Fe3C nanocrystals and Fe-N(x) in the best catalyst. A series of designed experiments disclosed that (1) N-doped carbon substrate, Fe/Fe3C nanocrystals or Fe-N(x) themselves did not deliver the high activity; (2) the catalysts with both Fe/Fe3C nanocrystals and Fe-N(x) exhibited the high activity; (3) the higher content of Fe-N(x) gave the higher activity; (4) the removal of Fe/Fe3C nanocrystals severely degraded the activity; (5) the blocking of Fe-N(x) downgraded the activity and the recovery of the blocked Fe-N(x) recovered the activity. These facts supported that the high ORR activity of the Fe@C-FeNC electrocatalysts should be ascribed to that Fe/Fe3C nanocrystals boost the activity of Fe-N(x). The coexistence of high content of Fe-N(x) and sufficient metallic iron nanoparticles is essential for the high ORR activity. DFT calculation corroborated this conclusion by indicating that the interaction between metallic iron and Fe-N4 coordination structure favored the adsorption of oxygen molecule. These new findings open an avenue for the rational design and bottom-up synthesis of low-cost highly active ORR electrocatalysts. PMID:26906342

  20. Insight into core-shell dependent anoxic Cr(VI) removal with Fe@Fe2O3 nanowires: indispensable role of surface bound Fe(II).

    PubMed

    Mu, Yi; Ai, Zhihui; Zhang, Lizhi; Song, Fahui

    2015-01-28

    In this study, we investigated the anoxic Cr(VI) removal with core-shell Fe@Fe2O3 nanowires. It was found the surface area normalized Cr(VI) removal rate constants of Fe@Fe2O3 nanowires first increased with increasing the iron oxide shell thickness and then decreased, suggesting that Fe@Fe2O3 nanowires possessed an interesting core-shell structure dependent Cr(VI) removal property. Meanwhile, the Cr(VI) removal efficiency was positively correlated to the amount of surface bound Fe(II). This result revealed that the core-shell structure dependent Cr(VI) removal property of Fe@Fe2O3 nanowires was mainly attributed to the reduction of Cr(VI) by the surface bound Fe(II) besides the reduction of Cr(VI) adsorbed on the iron oxide shell via the electrons transferred from the iron core. The indispensable role of surface bound Fe(II) was confirmed by Tafel polarization and high-resolution X-ray photoelectron spectroscopic depth profiles analyses. X-ray diffraction patterns and scanning electron microscope images of the fresh and used Fe@Fe2O3 nanowires revealed the formation of Fe(III)/Cr(III)/Cr(VI) composite oxides during the anoxic Cr(VI) removal process. This study sheds a deep insight into the anoxic Cr(VI) removal mechanism of core-shell Fe@Fe2O3 nanowires and also provides an efficient Cr(VI) removal method. PMID:25543716

  1. MR/SPECT Imaging Guided Photothermal Therapy of Tumor-Targeting Fe@Fe3O4 Nanoparticles in Vivo with Low Mononuclear Phagocyte Uptake.

    PubMed

    Wang, Jing; Zhao, Heng; Zhou, Zhiguo; Zhou, Ping; Yan, Yuping; Wang, Mingwei; Yang, Hong; Zhang, Yingjian; Yang, Shiping

    2016-08-10

    The (125)I-c(RGDyK) peptide PEGylated Fe@Fe3O4 nanoparticles ((125)I-RGD-PEG-MNPs) with the average hydrodynamic diameter of ∼40 nm as a novel multifunctional platform were developed for tumor-targeting MR/SPECT imaging guided photothermal therapy in vivo. On the αvβ3-positive U87MG glioblastoma xenograft model, the signals of tumor from T2-weighted MR and SPECT imaging were much higher than those in the blocking group at 6 h post injection (p.i.) of RGD-PEG-MNPs and (125)I-RGD-PEG-MNPs intravenously, respectively. The pharmacokinetics and biodistribution were analyzed quantitatively by gamma counter ex vivo. The fact suggested that RGD-PEG-MNPs exhibited excellent targeting property and low mononuclear phagocyte uptake. At 6 h p.i. for (125)I-RGD-PEG-MNPs, the maximum uptake of 6.75 ± 1.24% of the percentage injected dose per gram (ID/g) was accumulated in the tumor. At 48 h p.i., only 1.11 ± 0.21% and 0.16 ± 0.09% ID/g were accumulated in the liver and spleen, respectively. With the guidance of MR/SPECT imaging, the multifunctional nanoparticles achieved a good photothermal therapeutic efficacy in vivo. PMID:27428929

  2. Hydrogenase synthesis in Bradyrhizobium japonicum Hupc mutants is altered in sensitivity to DNA gyrase inhibitors.

    PubMed Central

    Novak, P D; Maier, R J

    1989-01-01

    In the Hupc mutants of Bradyrhizobium japonicum SR, regulation of expression of hydrogenase is altered; the mutants synthesize hydrogenase constitutively in the presence of atmospheric levels of oxygen. The DNA gyrase inhibitors nalidixic acid, novobiocin, and coumermycin were used to inhibit growth of wild-type and mutant cells. For each inhibitor tested, growth of mutant and wild-type strains was equally sensitive. However, in contrast to the wild type, the Hupc mutants synthesized hydrogenase in the presence of high levels of any inhibitor. Cells were incubated with the drugs and simultaneously labeled with 14C-labeled amino acids, and hydrogenase was immunoprecipitated with antibody to the large subunit of the enzyme. Fluorograms of antibody blots then were scanned to determine the relative amount of hydrogenase (large subunit) synthesized in the presence or absence of the gyrase inhibitors. The amount of hydrogenase synthesized by the Hupc mutants in the presence of 300 micrograms of nalidixic acid per ml was near the level of enzyme synthesized in the absence of the inhibitor. No hydrogenase was detected in antibody blots of wild-type cultures which were derepressed for hydrogenase in the presence of 100 micrograms of coumermycin or novobiocin per ml. In contrast, hydrogenase was synthesized by the Hupc mutants in the presence of 100 micrograms of either drug per ml. The amount synthesized ranged from 5 to 32% and 20 to 49%, respectively, of that in the absence of those inhibitors, but nevertheless, hydrogenase synthesis was detected in all of the mutants examined.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2547335

  3. Detection and localization of two hydrogenases in Methylococcus capsulatus (Bath) and their potential role in methane metabolism.

    PubMed

    Hanczár, Tímea; Csáki, Robert; Bodrossy, Levente; Murrell, J Colin; Kovács, Kornél L

    2002-02-01

    Methylococcus capsulatus (Bath) was shown to contain two distinct hydrogenases, a soluble hydrogenase and a membrane-bound hydrogenase. This is the first report of a membrane-bound hydrogenase in methanotrophs. Both enzymes were expressed apparently constitutively under normal growth conditions. The soluble hydrogenase was capable of reducing NAD(+) with molecular hydrogen. The activities of both soluble and particulate methane monooxygenases could be driven by molecular hydrogen. This confirmed that molecular hydrogen could be used as a source of reducing power for methane oxidation. Hydrogen-driven methane monooxygenase activities tolerated elevated temperatures and moderate oxygen concentrations. The significance of these findings for biotechnological applications of methanotrophs is discussed. PMID:11807566

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

    PubMed Central

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

    2011-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-08-01

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  9. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.

    1996-10-01

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

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

    PubMed

    Sundaram, Shanthy; Tripathi, Ashutosh; Gupta, Vipul

    2010-01-01

    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

  11. Ferrous Carbonyl Dithiolates as Precursors to FeFe, FeCo, and FeMn Carbonyl Dithiolates

    PubMed Central

    2015-01-01

    Reported are complexes of the formula Fe(dithiolate)(CO)2(diphos) and their use to prepare homo- and heterobimetallic dithiolato derivatives. The starting iron dithiolates were prepared by a one-pot reaction of FeCl2 and CO with chelating diphosphines and dithiolates, where dithiolate = S2(CH2)22– (edt2–), S2(CH2)32– (pdt2–), S2(CH2)2(C(CH3)2)2– (Me2pdt2–) and diphos = cis-C2H2(PPh2)2 (dppv), C2H4(PPh2)2 (dppe), C6H4(PPh2)2 (dppbz), C2H4[P(C6H11)2]2 (dcpe). The incorporation of 57Fe into such building block complexes commenced with the conversion of 57Fe into 57Fe2I4(iPrOH)4, which then was treated with K2pdt, CO, and dppe to give 57Fe(pdt)(CO)2(dppe). NMR and IR analyses show that these complexes exist as mixtures of all-cis and trans-CO isomers, edt2– favoring the former and pdt2– the latter. Treatment of Fe(dithiolate)(CO)2(diphos) with the Fe(0) reagent (benzylideneacetone)Fe(CO)3 gave Fe2(dithiolate)(CO)4(diphos), thereby defining a route from simple ferrous salts to models for hydrogenase active sites. Extending the building block route to heterobimetallic complexes, treatment of Fe(pdt)(CO)2(dppe) with [(acenaphthene)Mn(CO)3]+ gave [(CO)3Mn(pdt)Fe(CO)2(dppe)]+ ([3d(CO)]+). Reduction of [3d(CO)]+ with BH4– gave the Cs-symmetric μ-hydride (CO)3Mn(pdt)(H)Fe(CO)(dppe) (H3d). Complex H3d is reversibly protonated by strong acids, the proposed site of protonation being sulfur. Treatment of Fe(dithiolate)(CO)2(diphos) with CpCoI2(CO) followed by reduction by Cp2Co affords CpCo(dithiolate)Fe(CO)(diphos) (4), which can also be prepared from Fe(dithiolate)(CO)2(diphos) and CpCo(CO)2. Like the electronically related (CO)3Fe(pdt)Fe(CO)(diphos), these complexes undergo protonation to afford the μ-hydrido complexes [CpCo(dithiolate)HFe(CO)(diphos)]+. Low-temperature NMR studies indicate that Co is the kinetic site of protonation. PMID:24803716

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

    PubMed Central

    2011-01-01

    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 are irreversibly inactivated by O2. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts. PMID:21615937

  13. Purification and characterization of membrane-associated hydrogenase from the deep-sea epsilonproteobacterium Hydrogenimonas thermophila.

    PubMed

    Nishimura, Hiroshi; Kitano, Yuki; Inoue, Takahiro; Nomura, Keigo; Sako, Yoshihiko

    2010-01-01

    Membrane-associated hydrogenase was purified from the chemolithoautotrophic epsilonproteobacterium Hydrogenimonas thermophila at 152-fold purity. The hydrogenase was found to be localized in the periplasmic space, and was easily solubilized with 0.1% Triton X-100 treatment. Hydrogen oxidation activity was 1,365 micromol H(2)/min/mg of protein at 80 degrees C at pH 9.0, with phenazine methosulphate as the electron acceptor. Hydrogen production activity was 900 micromol H(2)/min/mg of protein at 80 degrees C and pH 6.0, with reduced methyl viologen as the electron donor. The hydrogenase from this organism showed higher oxygen tolerance than those from other microorganisms showing hydrogen oxidation activity. The structural genes of this hydrogenase, which contains N-terminal amino acid sequences from both small and large subunits of purified hydrogenase, were successfully elucidated. The hydrogenase from H. thermophila was found to be phylogenetically related with H(2) uptake hydrogenases from pathogenic Epsilonproteobacteria. PMID:20699572

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

    DOE PAGESBeta

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

    2015-01-01

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

  15. Molecular characterization of structural genes coding for a membrane bound hydrogenase in Methylococcus capsulatus (Bath).

    PubMed

    Csáki, R; Hanczár, T; Bodrossy, L; Murrell, J C; Kovács, K L

    2001-12-18

    The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A DeltahupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo. PMID:11750803

  16. Radical S-Adenosyl-l-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors*

    PubMed Central

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; Broderick, Joan B.

    2015-01-01

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. The FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-l-methionine enzymes are vital for the assembly of all three of these diverse cofactors. This minireview presents and discusses the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors. PMID:25477518

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

    SciTech Connect

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

    2007-09-24

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

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

    SciTech Connect

    Arp, D.J.

    1997-10-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120.

    PubMed Central

    Houchins, J P; Burris, R H

    1981-01-01

    Two distinct types of hydrogenase occur in Anabaena 7120 and are distinguishable in whole filaments by the application of selective assay methods. A reversible hydrogenase occurs both in heterocysts and vegetative cells and can be selectively assayed by measuring H2 evolution from reduced methyl viologen. Activities in aerobically grown filaments were low but could be increased by 2 to 3 orders of magnitude by growing cells microaerobically. The presence of the reversible hydrogenase was independent of the N2-fixing properties of the organism, and activity did not respond to added H2 in the culture. Illumination was necessary during derepression of the reversible hydrogenase, and addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea increased the amount of enzyme that was synthesized. An uptake hydrogenase occurred only in heterocysts of aerobically grown filaments, but a small amount of activity also was present in the vegetative cells of filaments grown microaerobically with 20% H2. It was assayed selectively by measuring an oxyhydrogen reaction at atmospheric levels of O2. Additional uptake hydrogenase could be elicited by including H2 or by removing O2 from the sparging gas of a culture. PMID:6783614

  3. Effects of alcohols on the reactivity and stability of Azotobacter vinelandii hydrogenase.

    PubMed

    Arp, D J

    1988-02-15

    The effects of alcohols on the reactivity of Azotobacter vinelandii hydrogenase were investigated. Hydrogenase catalyzed H2 oxidation coupled to methylene blue, benzyl viologen, or phenazine methosulfate when in the presence of solvents containing 15 or 40% ethanol or 40% methanol or 2-propanol. In general, the Km's for the electron acceptors were increased substantially by the presence of the alcohols, while the Km for H2 was not altered in a solvent containing 40% ethanol. Calculation of the apparent maximum velocities for H2 oxidation in the presence of alcohols indicated that the maximum velocity was not decreased in most cases. In contrast, the rates of both H2 evolution and isotope exchange by hydrogenase were substantially decreased when solvent containing alcohol. Hydrogenase was inactivated by 100% ethanol with a half-life of 17 s. Hydrogenase from A. vinelandii was stable when stored in alcohol/buffer solvents at 20 degrees C or below. However, the thermal stability of hydrogenase was greatly decreased by inclusion of an alcohol in the solvent. When incubated at 55 degrees C in a solvent containing 40% ethanol, activity decreased in a first-order process with a half-life of 7 min. When incubated at the same temperature in aqueous buffer, no loss of activity was observed over 30 min. PMID:3277540

  4. A TiO₂ nanoparticle system for sacrificial solar H₂ production prepared by rational combination of a hydrogenase with a ruthenium photosensitizer.

    PubMed

    Reisner, Erwin; Armstrong, Fraser A

    2011-01-01

    A hybrid system comprising a hydrogenase and a photosensitizer co-attached to a nanoparticle serves as a rational model for fast dihydrogen (H(2)) production using visible light. This chapter describes a stepwise procedure for preparing TiO(2) nanoparticles functionalized with a hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) and a tris(bipyridyl)ruthenium photosensitizer (RuP). Upon irradiation with visible light, these particles produce H(2) from neutral water at room temperature in the presence of a sacrificial electron donor - a test-system for the cathodic half reaction of water splitting. In particular, we describe how a hydrogenase and a photosensitizer with desired properties, including strong adsorption on TiO(2), can be selected by electrochemical methods. The catalyst Db [NiFeSe]-H is selected for its high H(2) production activity even when H(2) and traces of O(2) are present. Adsorption of Db [NiFeSe]-H and RuP on TiO(2) electrodes results in high electrochemical and photocatalytic activities that translate into nanoparticles exhibiting efficient light harvesting, charge separation, and sacrificial H(2) generation. PMID:21553186

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

    PubMed

    Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady; Marquis, Christopher P

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2014-02-01

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

  8. Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis

    PubMed Central

    Lie, Thomas J.; Costa, Kyle C.; Lupa, Boguslaw; Korpole, Suresh; Whitman, William B.; Leigh, John A.

    2012-01-01

    Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H2 metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H2 suggests that its role is anaplerotic. Indeed, H2 via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for. PMID:22872868

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

    PubMed

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

    2016-10-01

    Efficient sludge dewatering methods are highly desired by municipal wastewater treatment plants. In this study, Fe@Fe2O3 nanomaterial, combined with polydiallyldimethylammonium chloride (PDMDAAC) and H2SO4, was used for sludge dewatering. This composite conditioner exhibited an excellent dewatering capability. By using uniform design, the optimized dosages of Fe@Fe2O3, H2SO4 and PDMDAAC were determined to be 40, 136 and 4.8mg/gDS (dry solids), respectively. The moisture content of sludge cake decreased from 78.1% to 64.8%, and the capillary suction time from 56 to 21s. The sludge extracellular polymeric substances (EPS) were decomposed, resulting in greater conversion of the bound water into free water and the release of free water. The electron spin resonance results show that the molecular oxygen activation process induced by Fe@Fe2O3 produced hydroxyl radicals, which were mainly responsible for the EPS decomposition. In this way, an efficient composite conditioner for enhancing sludge dewatering was developed. PMID:27395000

  10. Broad Negative Thermal Expansion Operation-Temperature Window Achieved by Adjusting Fe-Fe Magnetic Exchange Coupling in La(Fe,Si)13 Compounds.

    PubMed

    Li, Shaopeng; Huang, Rongjin; Zhao, Yuqiang; Li, Wen; Wang, Wei; Huang, Chuanjun; Gong, Pifu; Lin, Zheshuai; Li, Laifeng

    2015-08-17

    Cubic La(Fe,Si)13-based compounds have been recently developed as promising negative thermal expansion(NTE) materials, but the narrow NTE operation-temperature window(∼110 K) restricts their actual applications. In this work, we demonstrate that the NTE operation-temperature window of LaFe(13-x)Si(x) can be significantly broadened by adjusting Fe-Fe magnetic exchange coupling as x ranges from 2.8 to 3.1. In particular, the NTE operation-temperature window of LaFe10.1Si2.9 is extended to 220 K. More attractively, the coefficients of thermal expansion of LaFe10.0Si3.0 and LaFe9.9Si3.1 are homogeneous in the NTE operation-temperature range of about 200 K, which is much valuable for the stability of fabricating devices. The further experimental characterizations combined with first-principles studies reveal that the tetragonal phase is gradually introduced into the cubic phase as the Si content increases, hence modifies the Fe-Fe interatomic distance. The reduction of the overall Fe-Fe magnetic exchange interactions contributes to the broadness of NTE operation-temperature window for LaFe(13-x)Si(x). PMID:26196377

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

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

    SciTech Connect

    Arp, D.J.

    1990-01-01

    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.

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

    PubMed Central

    Hansen, Moritz; Perner, Mirjam

    2016-01-01

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

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

    PubMed

    Hansen, Moritz; Perner, Mirjam

    2016-01-01

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

  15. A modular system for regeneration of NAD cofactors using graphite particles modified with hydrogenase and diaphorase moieties.

    PubMed

    Reeve, Holly A; Lauterbach, Lars; Ash, Philip A; Lenz, Oliver; Vincent, Kylie A

    2012-02-01

    Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase. PMID:21986817

  16. [NiFe]-hydrogenase is essential for cyanobacterium Synechocystis sp. PCC 6803 aerobic growth in the dark

    PubMed Central

    De Rosa, Edith; Checchetto, Vanessa; Franchin, Cinzia; Bergantino, Elisabetta; Berto, Paola; Szabò, Ildikò; Giacometti, Giorgio M.; Arrigoni, Giorgio; Costantini, Paola

    2015-01-01

    The cyanobacterium Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged darkness of three Synechocystis knock-out strains, lacking either Hox hydrogenase (ΔHoxE-H) or one of the proteins responsible for the assembly of its NiFe active site (ΔHypA1 and ΔHypB1). We found that Hox hydrogenase is required for Synechocystis growth under this condition, regardless of the functional status of its catalytic site, suggesting an additional role beside hydrogen metabolism. Moreover, quantitative proteomic analyses revealed that the expression levels of several subunits of the respiratory NADPH/plastoquinone oxidoreductase (NDH-1) are reduced when Synechocystis is grown in the dark. Our findings suggest that the Hox hydrogenase could contribute to electron transport regulation when both photosynthetic and respiratory pathways are down-regulated, and provide a possible explanation for the close evolutionary relationship between mitochondrial respiratory Complex I and cyanobacterial [NiFe]-hydrogenases. PMID:26215212

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

    SciTech Connect

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

    1989-06-01

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

  18. Efficiency of hydrogen photoproduction by chloroplast-bacterial hydrogenase systems.

    PubMed

    Krasnovsky, A A; Van Ni, C; Nikandrov, V V; Brin, G P

    1980-11-01

    A comparative study of H(2) photoproduction by chloroplasts and solubilized chlorophyll was performed in the presence of hydrogenase preparations of Clostridium butyricum. The photoproduction of H(2) by chloroplasts in the absence of exogenous electron donors, and with irreversibly oxidized dithiothreitol and cysteine, is thought to be limited by a cyclic transport of electrons wherein methylviologen short-circuits the electron transport in photosystem I. The efficiency of H(2) photoproduction by chloroplasts with ascorbate and NADPH is limited by a back reaction between light-reduced methylviologen and the oxidized electron donors. The use of a combination of electron donors (dithiothreitol and ascorbate), providing anaerobiosis without damage to chloroplasts, makes it possible to avoid consumption of reduced methylviologen for the reduction of oxidized electron donors and to exclude the short-circuiting of electron transfer. Under these conditions, photoproduction of H(2) was observed to occur with a rate of 350 to 400 micromoles H(2) per milligram chlorophyll per hour. In this case, the full electron-transferring capability of photosystem I (measured by irreversible photoreduction of methyl red or O(2)) is used to produce H(2). PMID:16661554

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

    PubMed Central

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  1. Mechanism of inhibition of NiFe hydrogenase by nitric oxide.

    PubMed

    Ceccaldi, Pierre; Etienne, Emilien; Dementin, Sébastien; Guigliarelli, Bruno; Léger, Christophe; Burlat, Bénédicte

    2016-04-01

    Hydrogenases reversibly catalyze the oxidation of molecular hydrogen and are inhibited by several small molecules including O2, CO and NO. In the present work, we investigate the mechanism of inhibition by NO of the oxygen-sensitive NiFe hydrogenase from Desulfovibrio fructosovorans by coupling site-directed mutagenesis, protein film voltammetry (PFV) and EPR spectroscopy. We show that micromolar NO strongly inhibits NiFe hydrogenase and that the mechanism of inhibition is complex, with NO targeting several metallic sites in the protein. NO reacts readily at the NiFe active site according to a two-step mechanism. The first and faster step is the reversible binding of NO to the active site followed by a slower and irreversible transformation at the active site. NO also induces irreversible damage of the iron-sulfur centers chain. We give direct evidence of preferential nitrosylation of the medial [3Fe-4S] to form dinitrosyl-iron complexes. PMID:26827939

  2. Immobilization of isolated and cellular hydrogenase of D. desulfuricans in radiation-polymerized polyacrylamides

    SciTech Connect

    Ziomek, E.; Martin, W.G.; Williams, R.E.

    1984-02-01

    Purified hydrogenase from Desulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6-7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65 degrees C) and long term storage (4 degrees C) of the hydrogenase activity of both the purified enzyme and whole cells.

  3. Immobilization of isolated and cellular hydrogenase of D. desulfuricans in radiation-polymerized polyacrylamides.

    PubMed

    Ziomek, E; Martin, W G; Williams, R E

    1984-02-01

    Purified hydrogenase from Desulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6-7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65 degrees C) and long term storage (4 degrees C) of the hydrogenase activity of both the purified enzyme and whole cells. PMID:6383215

  4. [Stability of the hydrogenase from Tetraselmis subcordiformis and its preliminary purification].

    PubMed

    Yan, Fei; Chen, Zhao'an; Cao, Xupeng; Lu, Hongbin; Xue, Song; Zhang, Wei

    2010-07-01

    Tetraselmis subcordiformis, a marine green alga, can produce hydrogen by photobiologically hydrolyzing seawater with hydrogenase. In this study, the preliminary purification of the enzyme was explored by ammonium sulfate precipitation, and the impact of sodium dithionite, beta-mercaptoethanol and glycerol on the enzyme stability during the process was investigated. The experimental results illustrated that sodium dithionite provided significant protection on the hydrogenase by depleting oxygen, while glycerol, a protectant against the structure instability of the enzyme, also presented protection. Crude enzyme with specific activity of 0.557 U/mg protein was extracted using 60%-70% saturated ammonium sulfate solution supplemented with 200 mmol/L sodium dithionite and 5% glycerol, and the hydrogenase recovery yield was about 30%. PMID:20954403

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

    PubMed

    Happe, Thomas; Hemschemeier, Anja; Winkler, Martin; Kaminski, Annette

    2002-06-01

    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. They are transferred to the hydrogenase by photosystem I and ferredoxin. Thus, the [Fe]-hydrogenase is an electron 'valve' that enables the algae to survive under anaerobic conditions. During sulfur deprivation, illuminated algal cultures evolve large quantities of hydrogen gas, and this promises to be an alternative future energy source. PMID:12049920

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

    SciTech Connect

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

    1987-10-06

    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.

  7. Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

    PubMed Central

    Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

    2012-01-01

    The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

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

    PubMed Central

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

    2014-01-01

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

  9. Evolutionary and biotechnological implications of robust hydrogenase activity in halophilic strains of Tetraselmis.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Proton electroreduction catalyzed by cobaloximes: functional models for hydrogenases.

    PubMed

    Razavet, Mathieu; Artero, Vincent; Fontecave, Marc

    2005-06-27

    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

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

    PubMed

    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

    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

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

    SciTech Connect

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

    2008-10-22

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

  14. Function of the chloroplast hydrogenase in the microalga Chlamydomonas: the role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis.

    PubMed

    Ghysels, Bart; Godaux, Damien; Matagne, René F; Cardol, Pierre; Franck, Fabrice

    2013-01-01

    Like a majority of photosynthetic microorganisms, the green unicellular alga Chlamydomonas reinhardtii may encounter O2 deprived conditions on a regular basis. In response to anaerobiosis or in a respiration defective context, the photosynthetic electron transport chain of Chlamydomonas is remodeled by a state transition process to a conformation that favours the photoproduction of ATP at the expense of reductant synthesis. In some unicellular green algae including Chlamydomonas, anoxia also triggers the induction of a chloroplast-located, oxygen sensitive hydrogenase, which accepts electrons from reduced ferredoxin to convert protons into molecular hydrogen. Although microalgal hydrogen evolution has received much interest for its biotechnological potential, its physiological role remains unclear. By using specific Chlamydomonas mutants, we demonstrate that the state transition ability and the hydrogenase function are both critical for induction of photosynthesis in anoxia. These two processes are thus important for survival of the cells when they are transiently placed in an anaerobic environment. PMID:23717558

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

    PubMed

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

    2016-01-01

    In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3. PMID:26672442

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

    SciTech Connect

    Krasna, A.I.

    1988-10-01

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

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

    PubMed Central

    Zhang, Xiaohui; Sherman, Debra M.

    2014-01-01

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

  18. Powerful fermentative hydrogen evolution of photosynthate in the cyanobacterium Lyngbya aestuarii BL J mediated by a bidirectional hydrogenase

    PubMed Central

    Kothari, Ankita; Parameswaran, Prathap; Garcia-Pichel, Ferran

    2014-01-01

    Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H2 under certain physiological conditions. However, most produce only little H2, revert readily to H2 consumption, and suffer from hydrogenase sensitivity to O2. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H2 production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H2 production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H2 concentrations during fermentation of photosynthate, which proceeded via a mixed acid fermentation pathway to yield acetate, ethanol, lactate, H2, CO2, and pyruvate. Contrary to expectations, the H2 yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(P)H in Lyngbya hydrogenases as the basis for its strong H2 production ability. In any event, the high specific rates and H2 concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H2 production. PMID:25540642

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

    PubMed

    Ma, K; Schicho, R N; Kelly, R M; Adams, M W

    1993-06-01

    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 (S0) to hydrogen sulfide (H2S) for optimal growth, even though S0 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 H2S if S0 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 S0 and polysulfide served as substrates for H2S production, and the S0 reduction activity but not the H2-oxidation activity was enhanced by the redox protein rubredoxin. The H2-oxidizing and S0-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional "sulfhydrogenase" 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 S0 to H2S. It is suggested that the function of some form of ancestral hydrogenase was S0 reduction rather than, or in addition to, the reduction of protons. PMID:8389482

  20. Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

    PubMed Central

    Eismann, K; Mlejnek, K; Zipprich, D; Hoppert, M; Gerberding, H; Mayer, F

    1995-01-01

    Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm. PMID:7592402

  1. Designed surface residue substitutions in [NiFe] hydrogenase that improve electron transfer characteristics.

    PubMed

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

    2015-01-01

    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

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

    PubMed Central

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

    2015-01-01

    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

  3. 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...

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

    PubMed

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

    2015-01-01

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

  5. How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure.

    PubMed

    Wulff, Philip; Thomas, Claudia; Sargent, Frank; Armstrong, Fraser A

    2016-03-01

    'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins. PMID:26861789

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

    PubMed Central

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

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H2) are also produced. The effect of hydrogenase inhibitors (H2, carbon monoxide (CO) and methyl viologen) on product selectivity was investigated....

  8. A Fe/Fe3O4/N-carbon composite with hierarchical porous structure and in situ formed N-doped graphene-like layers for high-performance lithium ion batteries.

    PubMed

    Li, Yao; Meng, Qing; Zhu, Shen-min; Sun, Zeng-hui; Yang, Hao; Chen, Zhi-xin; Zhu, Cheng-ling; Guo, Zai-ping; Zhang, Di

    2015-03-14

    A Fe/Fe3O4/N-carbon composite consisting of a porous carbon matrix containing a highly conductive N-doped graphene-like network and Fe/Fe3O4 nanoparticles was prepared. The porous carbon has a hierarchical structure which is inherited from rice husk and the N-doped graphene-like network formed in situ. When used as an anode material for lithium batteries, the composite delivered a reversible capacity of approximately 610 mA h g(-1) at a current density of 200 mA g(-1) even after 100 cycles, due to the synergism between the unique hierarchical porous structures, highly electrically conductive N-doped graphene-like networks and nanosized particles of Fe/Fe3O4. This work provides a simple approach to prepare N-doped porous carbon activated nanoparticle composites which could be used to improve the electrochemical performance of lithium ion batteries. PMID:25655996

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

    PubMed Central

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

    2012-01-01

    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

  10. Solid particle erosion of an Fe-Fe3C metal matrix composite

    NASA Astrophysics Data System (ADS)

    Lindsley, B. A.; Marder, A. R.

    1998-03-01

    The erosion resistance and morphology of spheroidized Fe-C alloys containing 0.2 to 1.4 wt pct carbon was investigated. The Fe-C alloy system was chosen as a model metal-matrix composite for the study of the effect on erosion of a hard second phase in a ductile matrix. Alloys were austenitized and water quenched to form martensite, then tempered at 690 °C for different times to produce carbide sizes of 0.4, 0.8, 1.6, and 2.4 μm. Utilizing these materials, it was found that the erosion resistance increased as the microstructural features decreased in size, with the important microstructural variables being carbide spacing and ferrite grain size. These variables control dislocation motion in the ferrite and, in turn, affect the plastic deformation and the erosion resistance of the spheroidized alloys. For the 0.4 to 1.4 pct C alloys, the carbide spacing was sufficient to determine erosion rate, whereas, for the 0.2 pct C alloys, ferrite grain size became the controlling structure. Microstructural spacing, which is a measure of the mean free path between both the grain boundaries and the carbides, was found to describe all of the erosion data. A Hall-Petch-type relationship was found between microstructural spacing and both erosion rate and hardness.

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

    PubMed

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

    2016-08-16

    The preparation of novel one-dimensional core-shell Fe/Fe2 O3 nanowires as anodes for high-performance lithium-ion batteries (LIBs) is reported. The nanowires are prepared in a facile synthetic process in aqueous solution under ambient conditions with subsequent annealing treatment that could tune the capacity for lithium storage. When this hybrid is used as an anode material for LIBs, the outer Fe2 O3 shell can act as an electrochemically active material to store and release lithium ions, whereas the highly conductive and inactive Fe core functions as nothing more than an efficient electrical conducting pathway and a remarkable buffer to tolerate volume changes of the electrode materials during the insertion and extraction of lithium ions. The core-shell Fe/Fe2 O3 nanowire maintains an excellent reversible capacity of over 767 mA h g(-1) at 500 mA g(-1) after 200 cycles with a high average Coulombic efficiency of 98.6 %. Even at 2000 mA g(-1) , a stable capacity as high as 538 mA h g(-1) could be obtained. The unique composition and nanostructure of this electrode material contribute to this enhanced electrochemical performance. Due to the ease of large-scale fabrication and superior electrochemical performance, these hybrid nanowires are promising anode materials for the next generation of high-performance LIBs. PMID:27406922

  12. Negative impact of oxygen molecular activation on Cr(VI) removal with core-shell Fe@Fe2O3 nanowires.

    PubMed

    Mu, Yi; Wu, Hao; Ai, Zhihui

    2015-11-15

    In this study, we demonstrate that the presence of oxygen molecule can inhibit Cr(VI) removal with core-shell Fe@Fe2O3 nanowires at neutral pH of 6.1. 100% of Cr(VI) removal was achieved by the Fe@Fe2O3 nanowires within 60 min in the anoxic condition, in contrast, only 81.2% of Cr(VI) was sequestrated in the oxic condition. Removal kinetics analysis indicated that the presence of oxygen could inhibit the Cr(VI) removal efficiency by near 3 times. XRD, SEM, and XPS analysis revealed that either the anoxic or oxic Cr(VI) removal was involved with adsorption, reduction, co-precipitation, and re-adsorption processes. More Cr(VI) was bound in a reduced state of Cr(III) in the anoxic process, while a thicker Cr(III)/Fe(III)/Cr(VI) oxyhydroxides shell, leading to inhibiting the electron transfer, was found under the oxic process. The negative impact of oxygen molecule was attributed to the oxygen molecular activation which competed with Cr(VI) adsorbed for the consumption of donor electrons from Fe(0) core and ferrous ions bound on the iron oxides surface under the oxic condition. This study sheds light on the understanding of the fate and transport of Cr(VI) in oxic and anoxic environment, as well provides helpful guide for optimizing Cr(VI) removal conditions in real applications. PMID:25988715

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

    PubMed

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

    2015-10-12

    The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2 . [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2 . Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2 , demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2 /O2 mixed feed. PMID:26073322

  14. The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different.

    PubMed

    Roseboom, Winfried; Blokesch, Melanie; Böck, August; Albracht, Simon P J

    2005-01-17

    The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in [NiFe]-hydrogenases are different. PMID:15642360

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

    PubMed

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

    2016-06-01

    The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.1M pH7 Tris-HCl medium with or without pure hydrogenase. The results showed that hydrogenase and the additional molecules were in competition, and the electrochemical response could not be attributed solely to hydrogenase. Then, solutions with additional chemicals of different compositions were studied electrochemically. DTT polluted the electrochemical signal by increasing the Eoc by 35mV 24h after the injection of 300μL of control solutions with DTT, whereas it drastically decreased the corrosion rate by increasing the charge transfer resistance (Rct 10 times the initial value). Thus, DTT was shown to have a strong antagonistic effect on corrosion and was removed from the purification process. An optimal composition of the medium was selected (0.5mM dithionite, 7.5mM desthiobiotin) that simultaneously allowed a high activity of hydrogenase and a lower impact on the electrochemical response for corrosion tests. PMID:26774688

  16. 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

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

    2014-01-01

    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

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

    PubMed

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

    2015-02-12

    We investigate the effect of H2 binding on the spin-forbidden nonadiabatic transition probability between the lowest energy singlet and triplet electronic states of [NiFe]-hydrogenase active site model, using a velocity averaged Landau-Zener theory. Density functional and multireference perturbation theories were used to provide parameters for the Landau-Zener calculations. It was found that variation of the torsion angle between the terminal thiolate ligands around the Ni center induces an intersystem crossing between the lowest energy singlet and triplet electronic states in the bare active site and in the active site with bound H2. Potential energy curves between the singlet and triplet minima along the torsion angle and H2 binding energies to the two spin states were calculated. Upon H2 binding to the active site, there is a decrease in the torsion angle at the minimum energy crossing point between the singlet and triplet states. The probability of nonadiabatic transitions at temperatures between 270 and 370 K ranges from 35% to 32% for the active site with bound H2 and from 42% to 38% for the bare active site, thus indicating the importance of spin-forbidden nonadiabatic pathways for H2 binding on the [NiFe]-hydrogenase active site. PMID:25603170

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

    SciTech Connect

    Ma, K.; Adams, M.W.W. ); Schicho, R.N. ); Kelly, R.M. )

    1993-06-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    1996-03-01

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

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

    PubMed

    Flanagan, Lindsey A; Parkin, Alison

    2016-02-15

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

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

    PubMed Central

    Flanagan, Lindsey A.; Parkin, Alison

    2016-01-01

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

  2. Converting the NiFeS Carbon Monoxide Dehydrogenase to a Hydrogenase and a Hydroxylamine Reductase

    PubMed Central

    Heo, Jongyun; Wolfe, Marcus T.; Staples, Christopher R.; Ludden, Paul W.

    2002-01-01

    Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys531 exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His265 exhibits hydroxylamine reductase activity. Both Cys531 and His265 are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity. PMID:12374822

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

    PubMed Central

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

    2014-01-01

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

  4. Reversible and irreversible effects of nitric oxide on the soluble hydrogenase from Alcaligenes eutrophus H16.

    PubMed Central

    Hyman, M R; Arp, D J

    1988-01-01

    The effects of NO on the H2-oxidizing and diaphorase activities of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. With fully activated enzyme, NO (8-150 nM in solution) inhibited H2 oxidation in a time- and NO-concentration-dependent process. Neither H2 nor NAD+ appeared to protect the enzyme against the inhibition. Loss of activity in the absence of an electron acceptor was about 10 times slower than under turnover conditions. The inhibition was partially reversible; approx. 50% of full activity was recoverable after removal of the NO. Recovery was slower in the absence of an electron acceptor than in the presence of H2 plus an electron acceptor. The diaphorase activity of the unactivated hydrogenase was not affected by NO concentrations of up to 200 microM in solution. Exposure of the unactivated hydrogenase to NO irreversibly inhibited the ability of the enzyme to be fully activated for H2-oxidizing activity. The enzyme also lost its ability to respond to H2 during activation in the presence of NADH. The results are interpreted in terms of a complex inhibition that displays elements of (1) a reversible slow-binding inhibition of H2-oxidizing activity, (2) an irreversible effect on H2-oxidizing activity and (30 an irreversible inhibition of a regulatory component of the enzyme. Possible sites of action for NO are discussed. PMID:3052436

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

    PubMed

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

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes. PMID:27343449

  6. Metal transfer within the E. coli HypB-HypA complex of hydrogenase accessory proteins†

    PubMed Central

    Douglas, Colin D.; Ngu, Thanh T.; Kaluarachchi, Harini; Zamble, Deborah B.

    2015-01-01

    The maturation of [NiFe]-hydrogenase in E. coli is a complex process involving many steps and multiple accessory proteins. The two accessory proteins, HypA and HypB, interact with each other and are thought to cooperate to insert nickel into the active site of the hydrogenase-3 precursor protein. Both of these accessory proteins bind metal individually, but little is known about the metal-binding activities of the proteins once they assemble together into a functional complex. In this study, we investigate how complex formation modulates metal binding to the E. coli proteins HypA and HypB. This work lead to a re-evaluation of the HypA nickel affinity, revealing a KD on the order of 10−8 M. HypA can efficiently remove nickel, but not zinc, from the metal-binding site in the GTPase domain of HypB, a process that is less efficient when complex formation between HypA and HypB is disrupted. Furthermore, nickel release from HypB to HypA is specifically accelerated when HypB is loaded with GDP, but not GTP. These results are consistent with the HypA-HypB complex serving as a transfer step in the relay of nickel from membrane transporter to its final destination in the hydrogenase active site, and suggest that this complex contributes to the metal fidelity of this pathway. PMID:23899293

  7. [FeFe]-hydrogenase oxygen inactivation is initiated at the H cluster 2Fe subcluster.

    PubMed

    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

    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

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

    PubMed

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

    2016-06-15

    Metalloenzymes featuring earth-abundant metal-based cores exhibit rates for catalytic processes such as hydrogen evolution comparable to those of noble metals. Realizing these superb catalytic properties in artificial systems is challenging owing to the difficulty of effectively interfacing metalloenzymes with an electrode surface in a manner that supports efficient charge-transfer. Here, we demonstrate that a nanoporous "black" silicon (b-Si) photocathode provides a unique interface for binding an adsorbed [FeFe]-hydrogenase enzyme ([FeFe]-H2ase). The resulting [FeFe]-H2ase/b-Si photoelectrode displays a 280 mV more positive onset potential for hydrogen generation than bare b-Si without hydrogenase, similar to that observed for a b-Si/Pt photoelectrode at the same light intensity. Additionally, we show that this H2ase/b-Si electrode exhibits a turnover frequency of ≥1300 s(-1) and a turnover number above 10(7) and sustains current densities of at least 1 mA/cm(2) based on the actual surface area of the electrode (not the smaller projected geometric area), orders of magnitude greater than that observed for previous enzyme-catalyzed electrodes. While the long-term stability of hydrogenase on the b-Si surface remains too low for practical applications, this work extends the proof-of-concept that biologically derived metalloenzymes can be interfaced with inorganic substrates to support technologically relevant current densities. PMID:27219350

  9. Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen 1

    PubMed Central

    Erbes, David L.; King, Dan; Gibbs, Martin

    1979-01-01

    O2 irreversibly inactivates hydrogenase from Chlamydomonas reinhardi. The mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of O2 (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres−1 minute−1 (1.4 × 105 molar−1 minute−1). At levels above 0.01 atmosphere O2, the increased numbers of O2 molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one O2 molecule and resulting in lowered rates of inactivation. CO is a reversible inhibitor of hydrogenase acting competitively against H2. The Ki for CO is 0.0010 atmosphere. CO antagonizes O2 inactivation. In a period when complete inactivation by O2 would usually occur, the presence of CO greatly reduces the inactivation rate. After 3 hours of adaptation in whole cells, the presence of H2 lowers the rate of deadaptation of hydrogenase. Inasmuch as H2 promotes increased O2 uptake the cellular concentration of O2 is likely to be lower. After 48 hours of adaptation O2 uptake is reduced even when H2 is present and the pattern of deadaptation under O2 with and without H2 and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of cell-free hydrogenase by O2 may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells. PMID:16660871

  10. Probing the origin of the metabolic precursor of the CO ligand in the catalytic center of [NiFe] hydrogenase.

    PubMed

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

    2011-12-30

    The O(2)-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H(2) conversion in the presence of ambient O(2). 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 (13)CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding (13)CO 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 PdCl(2), cells cultivated on H(2), CO(2), and O(2) 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 PdCl(2) 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

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

    PubMed Central

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

    2011-01-01

    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

  12. [NiFe]-hydrogenases revisited: nickel-carboxamido bond formation in a variant with accrued O2-tolerance and a tentative re-interpretation of Ni-SI states.

    PubMed

    Volbeda, Anne; Martin, Lydie; Liebgott, Pierre-Pol; De Lacey, Antonio L; Fontecilla-Camps, Juan C

    2015-04-01

    [NiFe]-hydrogenases are well-studied enzymes capable of oxidizing molecular hydrogen and reducing protons. EPR and FTIR spectroscopic studies have shown that these enzymes can be isolated in several redox states that include paramagnetic oxidized inactive Ni-A and Ni-B species and a reduced Ni-C form. The latter and the diamagnetic respectively more oxidized Ni-SI and more reduced Ni-R forms are generally thought to be involved in the catalytic cycle of [NiFe]-hydrogenases. With the exception of Ni-SI, these different stable states have been well characterized. Here, based on the crystal structure of a partially reduced Desulfovibrio fructosovorans (Df) enzyme and data from the literature we propose that at least one of the Ni-SI sub-states contains an unexpected combination of hydride and sulfenic acid moieties. We have also determined the structure of the less oxygen-sensitive Df [NiFe]-hydrogenase V74C mutant and found that more than half of the active site nickel occupies a novel position, called Ni'. In this new position, the metal ion is coordinated by two cysteine thiolates, a bridging species modeled as SH(-) and a main chain carboxamido N atom. The Ni' coordination is similar to the one found in Ni superoxide dismutase, an enzyme that operates at significantly more positive potentials than [NiFe]-hydrogenases. We propose that the oxygen-tolerance of the V74C variant results from a high potential stabilization of a Ni'(iii) species induced by the change in the metal ion coordination sphere. We also propose that transient Ni'(iii) species can rapidly attract successive electrons from the Fe4S4 proximal cluster accelerating the reduction of oxygen to water and hydroxide. The naturally occurring oxygen-tolerant [NiFe]-hydrogenases have an unusual proximal cluster that has been shown to be exceptionally plastic and capable of undergoing two successive one-electron oxidations. This double oxidation is modulated by the migration of one of the iron atoms in the

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

    PubMed

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

    2016-06-21

    Fe4Si2Sn7O16 has a unique crystal structure that contains alternating layers of Fe(2+) ions octahedrally coordinated by O (oxide layer) and Sn (stannide layer), bridged by SiO4 tetrahedra. The formula can be written as FeFe3Si2Sn7O16 to emphasise the distinction between the layers. Here, we report the changes in structure and properties as iron is selectively replaced by manganese in the oxide layer. Solid-state synthesis was used to produce polycrystalline samples of Fe(Fe3-xMnx)Si2Sn7O16 for x≤ 2.55, the structures of which were characterised using high-resolution synchrotron X-ray and neutron powder diffraction. Single-crystal samples were also grown at x = 0.35, 0.95, 2.60 and characterised by single crystal X-ray diffraction. We show that manganese is doped exclusively into the oxide layer, and that this layer contains exclusively magnetically active high-spin M(2+) transition metal cations; while the stannide layer only accommodates non-magnetic low-spin Fe(2+). All samples show clear evidence of geometrically frustrated magnetism, which we associate with the fact that the topology of the high-spin M(2+) ions in the oxide layer describes a perfect kagomé lattice. Despite this frustration, the x = 0 and x = 2.55 samples undergo long-range antiferromagnetic ordering transitions at 3.0 K and 2.5 K, respectively. PMID:27225937

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2011-09-01

    The concomitant occurrence of molecular hydrogen (H(2)) and organic acids along the alimentary canal of the earthworm is indicative of ongoing fermentation during gut passage. Fermentative H(2) production is catalyzed by [FeFe]-hydrogenases and group 4 [NiFe]-hydrogenases in obligate anaerobes (e.g., Clostridiales) and facultative aerobes (e.g., Enterobacteriaceae), respectively, functional groups that might respond differently to contrasting redox conditions. Thus, the objectives of this study were to assess the redox potentials of the alimentary canal of Lumbricus terrestris and analyze the hydrogenase transcript diversities of H(2) producers in glucose-supplemented gut content microcosms. Although redox potentials in the core of the alimentary canal were variable on an individual worm basis, average redox potentials were similar. The lowest redox potentials occurred in the foregut and midgut regions, averaging 40 and 110 mV, respectively. Correlation plots between hydrogenase amino acid sequences and 16S rRNA gene sequences indicated that closely related hydrogenases belonged to closely related taxa, whereas distantly related hydrogenases did not necessarily belong to distantly related taxa. Of 178 [FeFe]-hydrogenase gene transcripts, 177 clustered in 12 Clostridiales-affiliated operational taxonomic units, the majority of which were indicative of heretofore unknown hydrogenases. Of 86 group 4 [NiFe]-hydrogenase gene transcripts, 79% and 21% were affiliated with organisms in the Enterobacteriaceae and Aeromonadaceae, respectively. The collective results (i) suggest that fermenters must cope with variable and moderately oxidative redox conditions along the alimentary canal, (ii) demonstrate that heretofore undetected hydrogenases are present in the earthworm gut, and (iii) corroborate previous findings implicating Clostridiaceae and Enterobacteriaceae as active fermentative taxa in earthworm gut content. PMID:21784904

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

    PubMed Central

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

    2011-01-01

    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

  18. Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2 -Protected Silicon Electrode.

    PubMed

    Lee, Chong-Yong; Park, Hyun S; Fontecilla-Camps, Juan C; Reisner, Erwin

    2016-05-10

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

  19. Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

    PubMed

    Lalla-Maharajh, W V; Hall, D O; Cammack, R; Rao, K K; Le Gall, J

    1983-02-01

    The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5. PMID:6303306

  20. Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene

    SciTech Connect

    Tibelius, K.H.; Knowles, R.

    1984-10-01

    Nitrite, NO, CO, and C/sub 2/H/sub 2/ inhibited O/sub 2/-dependent H/sub 2/ uptake (H/sup 3/H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N/sub 2/O or NO/sub 3//sup -/. The apparent K/sub i/ values for inhibition of O/sub 2/-dependent H/sub 2/ uptake were 20 ..mu..M for NO/sub 2//sup -/, 0.4 ..mu..M for NO, 28 ..mu..M for CO, and 88 ..mu..M for C/sub 2/H/sub 2/. These inhibitors also affected methylene blue-dependent H/sub 2/ uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H/sub 2/ uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N/sub 2/. The C/sub 2/H/sub 2/ inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO/sub 2//sup -/ inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO/sub 2//sup -/ on H/sub 2/-dependent respiration. These results suggest that the low hydrogenase activities observed in NO/sub 3//sup -/-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO/sub 2//sup -/ and NO produced by NO/sub 3//sup -/ reduction.

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

    PubMed

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

    2012-02-01

    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

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

    PubMed

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

    2016-06-28

    [FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly. PMID:27232385

  3. Synthesis and decarbonylation reactions of the triiron phosphinidene complex [Fe3Cp3(μ-H)(μ3-PPh)(CO)4]: easy cleavage and formation of P-H and Fe-Fe bonds.

    PubMed

    Alvarez, Celedonio M; Alvarez, M Angeles; García, M Esther; González, Rocío; Ramos, Alberto; Ruiz, Miguel A

    2011-11-01

    The binuclear phosphine complex [Fe(2)Cp(2)(μ-CO)(2)(CO)(PH(2)Ph)] (Cp = η(5)-C(5)H(5)) reacted with the acetonitrile adduct [Fe(2)Cp(2)(μ-CO)(2)(CO)(NCMe)] in dichloromethane at 293 K to give the trinuclear hydride-phosphinidene derivative [Fe(3)Cp(3)(μ-H)(μ(3)-PPh)(CO)(4)] as a mixture of cis,anti and trans isomers (Fe-Fe = 2.7217(6) Å for the cis,anti isomer). In contrast, photochemical treatment of the phosphine complex with [Fe(2)Cp(2)(CO)(4)] gave the phosphide-bridged complex trans-[Fe(3)Cp(3)(μ-PHPh)(μ-CO)(2)(CO)(3)] as the major product, along with small amounts of the binuclear hydride-phosphide complexes [Fe(2)Cp(2)(μ-H)(μ-PHPh)(CO)(2)] (cis and trans isomers), which are more selectively prepared from [Fe(2)Cp(2)(CO)(4)] and PH(2)Ph at 388 K. The photochemical decarbonylation of either of the mentioned triiron compounds led reversibly to three different products depending on the reaction conditions: (a) the 48-electron phosphinidene cluster [Fe(3)Cp(3)(μ-H)(μ(3)-PPh)(μ-CO)(2)] (Fe-Fe = 2.592(2)-2.718(2) Å); (b) the 50-electron complex [Fe(3)Cp(3)(μ-H)(μ(3)-PPh)(μ-CO)(CO)(2)], also having carbonyl- and hydride-bridged metal-metal bonds (Fe-Fe = 2.6177(3) and 2.7611(4) Å, respectively); and (c) the 48-electron phosphide cluster [Fe(3)Cp(3)(μ-PHPh)(μ(3)-CO)(μ-CO)(2)], an isomer of the latter phosphinidene complex now having three intermetallic bonds (Fe-Fe = 2.5332(8)-2.6158(8) Å). PMID:21981036

  4. Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR.

    PubMed

    Chang, Jui-Jen; Chen, Wei-En; Shih, Shiou-Yun; Yu, Sian-Jhong; Lay, Jiunn-Jyi; Wen, Fu-Shyan; Huang, Chieh-Chen

    2006-05-01

    Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system. PMID:16217655

  5. The AbrB2 Autorepressor, Expressed from an Atypical Promoter, Represses the Hydrogenase Operon To Regulate Hydrogen Production in Synechocystis Strain PCC6803

    PubMed Central

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

    2012-01-01

    We have thoroughly investigated the abrB2 gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium Synechocystis strain PCC6803. We report that abrB2 is expressed from an active but atypical promoter that possesses an extended −10 element (TGTAATAT) that compensates for the absence of a −35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended −10 promoter box and the absence of a −35 element are two well-conserved features in abrB2 genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the hox operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the hox operon is weakly expressed even though it possesses the two sequences resembling canonical −10 and −35 promoter boxes. In both the AbrB2-repressed promoters of the abrB2 gene and the hox operon, we found a repeated DNA motif [TT-(N5)-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the abrB2 promoter. We think that our abrB2-deleted mutant with increased expression of the hox operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the abrB2 gene and the hox operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in Synechocystis. PMID:22865847

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

    Sode, Olaseni; Voth, Gregory A.

    2014-12-14

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

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

    SciTech Connect

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

    2015-08-10

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

  10. Rates of intra- and intermolecular electron transfers in hydrogenase deduced from steady-state activity measurements.

    PubMed

    Dementin, Sébastien; Burlat, Bénédicte; Fourmond, Vincent; Leroux, Fanny; Liebgott, Pierre-Pol; Abou Hamdan, Abbas; Léger, Christophe; Rousset, Marc; Guigliarelli, Bruno; Bertrand, Patrick

    2011-07-01

    Electrons are transferred over long distances along chains of FeS clusters in hydrogenases, mitochondrial complexes, and many other respiratory enzymes. It is usually presumed that electron transfer is fast in these systems, despite the fact that there has been no direct measurement of rates of FeS-to-FeS electron transfer in any respiratory enzyme. In this context, we propose and apply to NiFe hydrogenase an original strategy that consists of quantitatively interpreting the variations of steady-state activity that result from changing the nature of the FeS clusters which connect the active site to the redox partner, and/or the nature of the redox partner. Rates of intra- and intermolecular electron transfer are deduced from such large data sets. The mutation-induced variations of electron transfer rates cannot be explained by changes in intercenter distances and reduction potentials. This establishes that FeS-to-FeS rate constants are extremely sensitive to the nature and coordination of the centers. PMID:21615141

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

    SciTech Connect

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

    2011-01-01

    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.

  12. Fast and efficient molecular electrocatalysts for H2 production: Using hydrogenase enzymes as guides

    SciTech Connect

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

    2011-01-15

    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 potentials devices for solar generation. Studies of hydrogenase enzymes provide many import 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 include acting as relays to move protons between the metal center and solution, promoting intra- and inter-molecular 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. 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.

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

    PubMed

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

    2014-05-01

    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

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

    DOE PAGESBeta

    Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; Schilter, David; Pelmenschikov, Vladimir; van Gastel, Maurice; Neese, Frank; Rauchfuss, Thomas B.; Gee, Leland B.; Scott, Aubrey D.; et al

    2015-08-10

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

  15. Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens.

    PubMed

    Morra, Simone; Mongili, Beatrice; Maurelli, Sara; Gilardi, Gianfranco; Valetti, Francesca

    2016-05-01

    This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645 ± 16 s(-1) . The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50 °C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox -CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox ; upon reduction with H2 , vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox -CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster. PMID:25851509

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Happe, Thomas; Schütz, Kathrin; Böhme, Herbert

    2000-01-01

    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 of A. variabilis. The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N2-fixing conditions, the mutant strain exhibited significantly increased rates in H2 accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H2 developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type. PMID:10692368

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

    PubMed

    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

    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

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

    PubMed

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

    2016-07-26

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

  20. Amphiphilic polymeric micelles as microreactors: improving the photocatalytic hydrogen production of the [FeFe]-hydrogenase mimic in water.

    PubMed

    Wang, Feng; Wen, Min; Feng, Ke; Liang, Wen-Jing; Li, Xu-Bing; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-01-11

    An amphiphilic polymeric micelle is utilized as a microreactor to load a hydrophobic [FeFe]-hydrogenase mimic in water. The local concentration enhancement and strong interaction between the mimic and the photosensitizer as well as the water-mediated fast proton migration caused by the microreactor improve photocatalytic hydrogen production remarkably in water. PMID:26442776

  1. Nucleotide sequence and characterization of four additional genes of the hydrogenase structural operon from Rhizobium leguminosarum bv. viciae.

    PubMed Central

    Hidalgo, E; Palacios, J M; Murillo, J; Ruiz-Argüeso, T

    1992-01-01

    The nucleotide sequence of a 2.5-kbp region following the hydrogenase structural genes (hupSL) in the H2 uptake gene cluster from Rhizobium leguminosarum bv. viciae UPM791 was determined. Four closely linked genes encoding peptides of 27.9 (hupC), 22.1 (hupD), 19.0 (hupE), and 10.4 (hupF) kDa were identified immediately downstream of hupL. Proteins with comparable apparent molecular weights were detected by heterologous expression of these genes in Escherichia coli. The six genes, hupS to hupF, are arranged as an operon, and by mutant complementation analysis, it was shown that genes hupSLCD are cotranscribed. A transcription start site preceded by the -12 to -24 consensus sequence characteristic of NtrA-dependent promoters was identified upstream of hupS. On the basis of the lack of oxygen-dependent H2 uptake activity of a hupC::Tn5 mutant and on structural characteristics of the protein, we postulate that HupC is a b-type cytochrome involved in electron transfer from hydrogenase to oxygen. The product from hupE, which is needed for full hydrogenase activity, exhibited characteristics typical of a membrane protein. The features of HupC and HupE suggest that they form, together with the hydrogenase itself, a membrane-bound protein complex involved in hydrogen oxidation. Images PMID:1597428

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2014-03-18

    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 Δhyd23 (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 Δhyd13 (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

  4. Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans.

    PubMed Central

    Malki, S; Saimmaime, I; De Luca, G; Rousset, M; Dermoun, Z; Belaich, J P

    1995-01-01

    A genomic DNA fragment from Desulfovibrio fructosovorans, which strongly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenborough, was cloned and sequenced. This fragment was found to contain four genes, named hndA, hndB, hndC, and hndD. Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taurus complex I, the 25-kDa subunit from Paracoccus denitrificans NADH dehydrogenase type I, and the N-terminal domain of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively. HndB does not show any significant homology with any known protein. HndC shows 37 and 33% identity with the C-terminal domain of HoxF and the 51-kDa subunit from B. taurus complex I, respectively, and has the requisite structural features to be able to bind one flavin mononucleotide, one NAD, and three [4Fe-4S] clusters. HndD has 40, 42, and 48% identity with hydrogenase I from Clostridium pasteurianum and HydC and HydA from D. vulgaris Hildenborough, respectively. The 4.5-kb length of the transcripts expressed in D. fructosovorans and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit. The sizes of the four polypeptides were measured by performing heterologous expression of hndABCD in E. coli, using the T7 promoter/polymerase system. The products of hndA, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectively. One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis. Immunoblotting studies carried out on cell extracts from D. fructosovorans wild-type and SM3 strains, using antibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only. In soluble extracts from D. fructosovorans wild type, a 10-fold induction of NADP reduction was observed when H(2) was present, but no H(2)-dependent NAD reduction ever occurred. This H(2

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

    PubMed

    Ciaccafava, Alexandre; Hamon, Cyrille; Infossi, Pascale; Marchi, Valérie; Giudici-Orticoni, Marie-Thérèse; Lojou, Elisabeth

    2013-10-21

    We report the effect of UV-Vis light on the membrane-bound [Ni-Fe] hydrogenase from Aquifex aeolicus under turnover conditions. Using electrochemistry, we show a potential dependent light sensitivity and propose that a light-induced structural change of the [Ni-Fe] active site is related to an enhanced reactivation of the hydrogenase under illumination at high potentials. PMID:23999766

  6. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.

    PubMed

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

    2014-01-24

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

  7. Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

    PubMed

    Chen, Y P; Yoch, D C

    1987-10-01

    Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions. PMID:3115963

  8. Aerobic Damage to [FeFe]-Hydrogenases: Activation Barriers for the Chemical Attachment of O2**

    PubMed Central

    Kubas, Adam; De Sancho, David; Best, Robert B; Blumberger, Jochen

    2014-01-01

    [FeFe]-hydrogenases are the best natural hydrogen-producing enzymes but their biotechnological exploitation is hampered by their extreme oxygen sensitivity. The free energy profile for the chemical attachment of O2 to the enzyme active site was investigated by using a range-separated density functional re-parametrized to reproduce high-level ab initio data. An activation free-energy barrier of 13 kcal mol−1 was obtained for chemical bond formation between the di-iron active site and O2, a value in good agreement with experimental inactivation rates. The oxygen binding can be viewed as an inner-sphere electron-transfer process that is strongly influenced by Coulombic interactions with the proximal cubane cluster and the protein environment. The implications of these results for future mutation studies with the aim of increasing the oxygen tolerance of this enzyme are discussed. PMID:24615978

  9. Characterization of Hydrogenase and Reductive Dehalogenase Activities of Dehalococcoides ethenogenes Strain 195

    PubMed Central

    Nijenhuis, Ivonne; Zinder, Stephen H.

    2005-01-01

    Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride and ethene using H2 as an electron donor. PCE- and TCE-reductive dehalogenase (RD) activities were mainly membrane associated, whereas only about 20% of the hydrogenase activity was membrane associated. Experiments with methyl viologen (MV) were consistent with a periplasmic location for the RDs or a component feeding electrons to them. The protonophore uncoupler tetrachlorosalicylanilide did not inhibit reductive dechlorination in cells incubated with H2 and PCE and partially restored activity in cells incubated with the ATPase inhibitor N,N′-dicyclohexylcarbodiimide. Benzyl viologen or diquat (Eo′ ≈ −360 mV) supported reductive dechlorination of PCE or TCE at rates comparable to MV (−450 mV) in cell extracts. PMID:15746376

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-10-20

    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 (λ>420 nm). PMID:25205168

  13. An NAD(P)H-Dependent Artificial Transfer Hydrogenase for Multienzymatic Cascades.

    PubMed

    Okamoto, Yasunori; Köhler, Valentin; Ward, Thomas R

    2016-05-11

    Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines. PMID:27100673

  14. Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase

    PubMed Central

    Zerball, Maximilian; Horch, Marius; Millo, Diego; Fritsch, Johannes; Lenz, Oliver; von Klitzing, Regine; Hildebrandt, Peter; Fischer, Anna; Mroginski, Maria Andrea; Zebger, Ingo

    2015-01-01

    Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme. PMID:26580976

  15. Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase.

    PubMed

    Heidary, Nina; Utesch, Tillmann; Zerball, Maximilian; Horch, Marius; Millo, Diego; Fritsch, Johannes; Lenz, Oliver; von Klitzing, Regine; Hildebrandt, Peter; Fischer, Anna; Mroginski, Maria Andrea; Zebger, Ingo

    2015-01-01

    Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme. PMID:26580976

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

    PubMed Central

    2012-01-01

    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 γ-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 μmole 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 μmole 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

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

    PubMed Central

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

    2013-01-01

    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 = ½) [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 = ½) [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

  18. Crystal structure of a [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1.

    PubMed

    Kwon, Sunghark; Nishitani, Yuichi; Watanabe, Satoshi; Hirao, Yoshinori; Imanaka, Tadayuki; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

    2016-09-01

    A [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1 (TkHybD) is involved in the cleavage of the C-terminal residues of [NiFe] hydrogenase large subunits by Ni recognition. Here, we report the crystal structure of TkHybD at 1.82 Å resolution to better understand this process. TkHybD exhibits an α/β/α sandwich fold with conserved residues responsible for the Ni recognition. Comparisons of TkHybD with homologous proteins also reveal that they share a common overall architecture, suggesting that they have similar catalytic functions. Our results including metal binding site prediction provide insight into the substrate recognition and catalysis mechanism of TkHybD. Proteins 2016; 84:1321-1327. © 2016 Wiley Periodicals, Inc. PMID:27192667

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

    SciTech Connect

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

    2013-01-04

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

  20. Synthesis and enzymatic photo-activity of an O2 tolerant hydrogenase-CdSe@CdS quantum rod bioconjugate.

    PubMed

    Hamon, C; Ciaccafava, A; Infossi, P; Puppo, R; Even-Hernandez, P; Lojou, E; Marchi, V

    2014-05-21

    This communication reports on the preparation of stable and photo-active nano-heterostructures composed of O2 tolerant [NiFe] hydrogenase extracted from the Aquifex aeolicus bacterium grafted onto hydrophilic CdSe/CdS quantum rods in view of the development of H2/O2 biofuel cells. The resulting complex is efficient towards H2 oxidation, displays good stability and new photosensitive properties. PMID:24468861

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii.

    PubMed

    Menon, A L; Robson, R L

    1994-01-01

    H2 oxidation in Azotobacter vinelandii is catalyzed by a membrane-bound, alpha beta dimeric [NiFe] hydrogenase. Maturation of the enzyme involves cleavage of a putative N-terminal signal sequence in the beta subunit and removal of 15 amino acids from the C terminus of the alpha subunit. Cells limited for nickel exhibited low hydrogenase activities and contained an apparently large form of the alpha subunit. Addition of nickel to such cells increased hydrogenase activities fivefold over 2 h. The increase in the first hour did not require transcription and translation and correlated with processing of the large form of the alpha subunit (pre-alpha) to the small form (alpha) resembling the alpha subunit from the purified enzyme. In vivo, pre-alpha appeared soluble whereas the majority of alpha was membrane bound. Processing of pre-alpha to alpha was reproduced in vitro in membrane-depleted extracts of nickel-limited cells. Processing specifically required the addition of Ni2+, whereas Co2+, Cu2+, Ca2+, Fe2+, Mn2+, and Zn2+ were ineffective. However, Zn2+, Co2+, and Cu2+ inhibited nickel-dependent processing. Mg-ATP and Mg-GTP stimulated processing, whereas anaerobic conditions and/or the addition of dithiothreitol and sodium dithionite was unnecessary. Processing was not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, E64, and pepstatin. PMID:8288521

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

    SciTech Connect

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

    2012-01-15

    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.

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

    SciTech Connect

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

    2014-01-15

    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

  6. EPR and FTIR analysis of the mechanism of H2 activation by [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii.

    PubMed

    Mulder, David W; Ratzloff, Michael W; Shepard, Eric M; Byer, Amanda S; Noone, Seth M; Peters, John W; Broderick, Joan B; King, Paul W

    2013-05-01

    While a general model of H2 activation has been proposed for [FeFe]-hydrogenases, the structural and biophysical properties of the intermediates of the H-cluster catalytic site have not yet been discretely defined. Electron paramagnetic resonance (EPR) spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the H-cluster catalytic site, a [4Fe-4S]H subcluster linked by a cysteine thiolate to an organometallic diiron subsite with CO, CN, and dithiolate ligands, in [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii (CrHydA1). Oxidized CrHydA1 displayed a rhombic 2.1 EPR signal (g = 2.100, 2.039, 1.997) and an FTIR spectrum previously assigned to the oxidized H-cluster (Hox). Reduction of the Hox sample with 100% H2 or sodium dithionite (NaDT) nearly eliminated the 2.1 signal, which coincided with appearance of a broad 2.3-2.07 signal (g = 2.3-2.07, 1.863) and/or a rhombic 2.08 signal (g = 2.077, 1.935, 1.880). Both signals displayed relaxation properties similar to those of [4Fe-4S] clusters and are consistent with an S = 1/2 H-cluster containing a [4Fe-4S]H(+) subcluster. These EPR signals were correlated with differences in the CO and CN ligand modes in the FTIR spectra of H2- and NaDT-reduced samples compared with Hox. The results indicate that reduction of [4Fe-4S]H from the 2+ state to the 1+ state occurs during both catalytic H2 activation and proton reduction and is accompanied by structural rearrangements of the diiron subsite CO/CN ligand field. Changes in the [4Fe-4S]H oxidation state occur in electron exchange with the diiron subsite during catalysis and mediate electron transfer with either external carriers or accessory FeS clusters. PMID:23578101

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

    PubMed Central

    Wawer, C; Muyzer, G

    1995-01-01

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

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

    PubMed

    Liran, Oded; Semyatich, Rinat; Milrad, Yuval; Eilenberg, Haviva; Weiner, Iddo; Yacoby, Iftach

    2016-09-01

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

  9. Spectroscopic characterization of the key catalytic intermediate Ni-C in the O2-tolerant [NiFe] hydrogenase I from Aquifex aeolicus: evidence of a weakly bound hydride.

    PubMed

    Pandelia, Maria-Eirini; Infossi, Pascale; Stein, Matthias; Giudici-Orticoni, Marie-Thérèse; Lubitz, Wolfgang

    2012-01-21

    Ni-C in the O(2)-tolerant hydrogenase I from Aquifex aeolicus binds a hydride weaker than that in O(2)-sensitive hydrogenases. This is in line with the enhanced light-sensitivity of Ni-C, greater lability of the hydride complex and increased catalytic redox potentials relevant to bio-H(2) oxidation. PMID:22143669

  10. Hydrogen evolution in [NiFe] hydrogenases and related biomimetic systems: similarities and differences.

    PubMed

    Das, Ranjita; Neese, Frank; van Gastel, Maurice

    2016-09-21

    In this work, a detailed quantum chemical study of the mechanism of [Ni(bdt)(dppf)] (Ni(II)L) catalyzed hydrogen formation [A. Gan, T. L. Groy, P. Tarakeshwar, S. K. S. Mazinani, J. Shearer, V. Mujica and A. K. Jones, J. Am. Chem. Soc., 2015, 137, 1109-1115] following an electro-chemical-electro-chemical (ECEC) pathway is reported. The complex exclusively catalyzes the reduction of protons to molecular hydrogen. The calculations suggest that the first one-electron reduction of the [Ni(II)L] catalyst is the rate limiting step of the catalytic cycle and hence, the buildup of detectable reaction intermediates is not expected. The catalytic activity of the [Ni(II)L] complex is facilitated by the flexibility of the ligand system, which allows the ligand framework to adapt to changes in the Ni oxidation state over the course of the reaction. Additionally, a comparison is made with the catalytic activity of [NiFe] hydrogenase. It is argued that the directionality of the reversible hydrogen formation reaction is controlled by the ligand field of the nickel ion and the possibility for side-on (η(2)) binding of H2: if the ligand framework does not allow for η(2) binding of H2, as is the case for [Ni(II)L], the catalyst irreversibly reduces protons. If the ligand field allows η(2) binding of H2, the catalyst can in principle work reversibly. The conditions for η(2) binding are discussed. PMID:27545687

  11. Encapsulating Subsite Analogues of the [FeFe]-Hydrogenases in Micelles Enables Direct Water Interactions.

    PubMed

    Fritzsch, Robby; Brady, Owen; Adair, Elaine; Wright, Joseph A; Pickett, Christopher J; Hunt, Neil T

    2016-07-21

    Encapsulation of subsite analogues of the [FeFe]-hydrogenase enzymes in supramolecular structures has been shown to dramatically increase their catalytic ability, but the molecular basis for this enhancement remains unclear. We report the results of experiments employing infrared absorption, ultrafast infrared pump-probe, and 2D-IR spectroscopy to investigate the molecular environment of Fe2(pdt)(CO)6 (pdt: propanedithiolate) [1] encapsulated in the dispersed alkane phase of a heptane-dodecyltrimethylammonium bromide-water microemulsion. It is demonstrated that 1 is partitioned between two molecular environments, one that closely resembles bulk heptane solution and a second that features direct hydrogen-bonding interactions with water molecules that penetrate the surfactant shell. Our results demonstrate that the extent of water access to the normally water-insoluble subsite analogue 1 can be tuned with micelle size, while IR spectroscopy provides a straightforward tool that can be used to measure and fine-tune the chemical environment of catalyst species in self-assembled structures. PMID:27396585

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

    PubMed

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

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster. PMID:19737003

  13. Metabolic Pathways for Photobiological Hydrogen Production by Nitrogenase- and Hydrogenase-containing Unicellular Cyanobacteria Cyanothece*

    PubMed Central

    Skizim, Nicholas J.; Ananyev, Gennady M.; Krishnan, Anagha; Dismukes, G. Charles

    2012-01-01

    Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H2 effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H2 production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H2 is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O2 by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO2. Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H2 production rate by 2-fold (at the expense of the dark-H2 rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement. PMID:22128188

  14. A metal-metal bond in the light-induced state of [NiFe] hydrogenases with relevance to hydrogen evolution.

    PubMed

    Kampa, Mario; Pandelia, Maria-Eirini; Lubitz, Wolfgang; van Gastel, Maurice; Neese, Frank

    2013-03-13

    The light-induced Ni-L state of [NiFe] hydrogenases is well suited to investigate the identity of the amino acid base that functions as a proton acceptor in the hydrogen turnover cycle in this important class of enzymes. Density functional theory calculations have been performed on large models that include the complete [NiFe] center and parts of the second coordination sphere. Combined with experimental data, in particular from electron paramagnetic resonance and Fourier transform infrared (FTIR) spectroscopy, the calculations indicate that the hydride ion, which is located in the bridging position between nickel and iron in the Ni-C state, dissociates upon illumination as a proton and binds to a nearby thiolate base. Moreover, the formation of a functionally relevant nickel-iron bond upon dissociation of the hydride is unequivocally observed and is in full agreement with the observed g values, ligand hyperfine coupling constants, and FTIR stretching frequencies. This metal-metal bond can be protonated and thus functions like a base. The nickel-iron bond is important for all intermediates with an empty bridge in the catalytic cycle, and the electron pair that constitutes this bond thus plays a crucial role in the hydrogen evolution catalyzed by the enzyme. PMID:23402569

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

    PubMed

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

    2015-04-01

    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 ΔzupT 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

  16. Biologically-assisted hydrogen production: attempts at optimizing the use of polymeric viologen mediators in a bioreactor based on the hydrogenase-catalyzed decomposition of dithionite

    SciTech Connect

    Williams, R.W.; Toye, B.W.; Martin, S.M.

    1981-01-01

    A small scale hydrogen-producing bioreactor has been assembled. It is based on the hydrogenase-catalyzed decomposition of sodium dithionite as mediated by a polymeric viologen. Optimization of the conditions for producing a crude, cell-free extract of hydrogenase from Clostridium pasteurianum has been investigated. Using the results obtained, a larger laboratory-scale hydrogen generator has been assembled which was shown to yield sustained hydrogen production. Incorporation of a waste-removal system into the unit was also investigated. 8 refs.

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

    PubMed Central

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

    2013-01-01

    The sulfur isotope effect produced by sulfate reducing microbes is commonly used to trace biogeochemical cycles of sulfur and carbon in aquatic and sedimentary environments. To test the contribution of intracellular coupling between carbon and sulfur metabolisms to the overall magnitude of the sulfur isotope effect, this study compared sulfur isotope fractionations by mutants of Desulfovibrio vulgaris Hildenborough. We tested mutant strains lacking one or two periplasmic (Hyd, Hyn-1, Hyn-2, and Hys) or cytoplasmic hydrogenases (Ech and CooL), and a mutant lacking type I tetraheme cytochrome (TpI-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 (ΔcycA) exhibited slower growth and sulfate reduction rates in batch culture, and produced more H2 and an approximately 50% larger sulfur isotope effect, compared to the wild type. The magnitude of sulfur isotope fractionation in the CycA deletion strain, thus, increased due to the disrupted coupling of the carbon oxidation and sulfate reduction pathways. In continuous culture, wild-type D. vulgaris and the CycA mutant produced similar sulfur isotope effects, underscoring the influence of environmental conditions on the relative contribution of hydrogen cycling to the electron transport. The large sulfur isotope effects associated with the non-ideal stoichiometry of sulfate reduction in this study imply that simultaneous fermentation and sulfate reduction may be responsible for some of the large naturally-occurring sulfur isotope effects. Overall, mutant strains provide a powerful tool to test the effect of specific redox proteins and pathways on sulfur isotope fractionation. PMID:23805134

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

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

    2015-01-01

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

  20. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

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

    2015-02-24

    [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 Brønsted 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

  1. Proton management as a design principle for hydrogenase-inspired catalysts

    SciTech Connect

    Small, Yolanda A; DuBois, Daniel L; Fujita, Estuko; Muckerman, James T.

    2011-01-01

    The properties of the hydrogenase-inspired [Ni(PNP)₂]{sup 2+} (PNP = 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 pK{sub a} values of these potential intermediates, as they control both the species in solution at a given pH and the free-energy profile of reaction pathways. Optimal relationships among these pK{sub a} values can be identified, and it is demonstrated that “proton management”, i.e., the manipulation of these pK{sub a} values (e.g., through choice of metal or substituents on ligands), can serve as a design principle for improved catalytic behavior.

  2. Unification of [FeFe]-hydrogenases into three structural and functional groups

    DOE PAGESBeta

    Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Refai, Mohammed; Schut, Gerrit J.; King, Paul W.; Maness, Pin-Ching; Adams, Michael W. W.; Peters, John W.; Bothner, Brian; et al

    2016-05-27

    [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatorymore » proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggests that they are post-translationally modified by phosphorylation. In conclusion, these results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.« less

  3. Disruption of the Operon Encoding Ehb Hydrogenase Limits Anabolic CO2 Assimilation in the Archaeon Methanococcus maripaludis

    PubMed Central

    Porat, Iris; Kim, Wonduck; Hendrickson, Erik L.; Xia, Qiangwei; Zhang, Yi; Wang, Tiansong; Taub, Fred; Moore, Brian C.; Anderson, Iain J.; Hackett, Murray; Leigh, John A.; Whitman, William B.

    2006-01-01

    Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci. PMID:16452419

  4. Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

    PubMed Central

    Adams, Michael W. W.; Holden, James F.; Menon, Angeli Lal; Schut, Gerrit J.; Grunden, Amy M.; Hou, Chun; Hutchins, Andrea M.; Jenney, Francis E.; Kim, Chulhwan; Ma, Kesen; Pan, Guangliang; Roy, Roopali; Sapra, Rajat; Story, Sherry V.; Verhagen, Marc F. J. M.

    2001-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S0). Growth rates were highest on media containing peptides and S0, with or without maltose. Growth did not occur on the peptide medium without S0. S0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S0 with or without maltose were the same, suggesting that S0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S0-reducing enzyme in this organism and the mechanism of the S0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites. PMID:11133967

  5. Radioassay for Hydrogenase Activity in Viable Cells and Documentation of Aerobic Hydrogen-Consuming Bacteria Living in Extreme Environments

    PubMed Central

    Schink, Bernhard; Lupton, F. S.; Zeikus, J. G.

    1983-01-01

    An isotopic tracer assay based on the hydrogenase-dependent formation of tritiated water from tritium gas was developed for in life analysis of microbial hydrogen transformation. This method allowed detection of bacterial hydrogen metabolism in pure cultures or in natural samples obtained from aquatic ecosystems. A differentiation between chemical-biological and aerobic-anaerobic hydrogen metabolism was established by variation of the experimental incubation temperature or by addition of selective inhibitors. Hydrogenase activity was shown to be proportional to the consumption or production of hydrogen by cultures of Desulfovibrio vulgaris, Clostridium pasteurianum, and Methanosarcina barkeri. This method was applied, in connection with measurements of free hydrogen and most-probable-number enumerations, in aerobic natural source waters to establish the activity and document the ecology of hydrogen-consuming bacteria in extreme acid, thermal, or saline environments. The utility of the assay is based in part on the ability to quantify bacterial hydrogen transformation at natural hydrogen partial pressures, without the use of artificial electron acceptors. PMID:16346288

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

    PubMed Central

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

    2012-01-01

    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

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

    PubMed

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

    2016-06-01

    The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process. PMID:27186945

  8. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

    PubMed

    Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

    2016-04-25

    [NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance. PMID:26913499

  9. Reversible oxygen-tolerant hydrogenase carried by free-living N2-fixing bacteria isolated from the rhizospheres of rice, maize, and wheat

    PubMed Central

    Roumagnac, Philippe; Richaud, Pierre; Barakat, Mohamed; Ortet, Philippe; Roncato, Marie-Anne; Heulin, Thierry; Peltier, Gilles; Achouak, Wafa; Cournac, Laurent

    2012-01-01

    Hydrogen production by microorganisms is often described as a promising sustainable and clean energy source, but still faces several obstacles, which prevent practical application. Among them, oxygen sensitivity of hydrogenases represents one of the major limitations hampering the biotechnological implementation of photobiological production processes. Here, we describe a hierarchical biodiversity-based approach, including a chemochromic screening of hydrogenase activity of hundreds of bacterial strains collected from several ecosystems, followed by mass spectrometry measurements of hydrogenase activity of a selection of the H2-oxidizing bacterial strains identified during the screen. In all, 131 of 1266 strains, isolated from cereal rhizospheres and basins containing irradiating waste, were scored as H2-oxidizing bacteria, including Pseudomonas sp., Serratia sp., Stenotrophomonas sp., Enterobacter sp., Rahnella sp., Burkholderia sp., and Ralstonia sp. isolates. Four free-living N2-fixing bacteria harbored a high and oxygen-tolerant hydrogenase activity, which was not fully inhibited within entire cells up to 150–250 μmol/L O2 concentration or within soluble protein extracts up to 25–30 μmol/L. The only hydrogenase-related genes that we could reveal in these strains were of the hyc type (subunits of formate hydrogenlyase complex). The four free-living N2-fixing bacteria were closely related to Enterobacter radicincitans based on the sequences of four genes (16S rRNA, rpoB, hsp60, and hycE genes). These results should bring interesting prospects for microbial biohydrogen production and might have ecophysiological significance for bacterial adaptation to the oxic–anoxic interfaces in the rhizosphere. PMID:23233392

  10. Bio-inspired hydrogenase models: mixed-valence triion complexes as proton reduction catalysts.

    PubMed

    Ghosh, Shishir; Hogarth, Graeme; Holt, Katherine B; Kabir, Shariff E; Rahaman, Ahibur; Unwin, David G

    2011-10-28

    Mixed-valence triiron complexes Fe(3)(CO)(7-x)(PPh(3))(x)(μ-edt)(2) (x = 0-2) have been prepared and are shown to act as proton reduction catalysts. Catalysis takes place via an ECEC mechanism with a reduced overpotential of ca. 0.45 V for Fe(3)(CO)(7)(μ-edt)(2) as compared to the corresponding diiron complex. PMID:21912795

  11. Protonation of Nickel–Iron Hydrogenase Models Proceeds after Isomerization at Nickel

    PubMed Central

    2015-01-01

    Theory and experiment indicate that the protonation of reduced NiFe dithiolates proceeds via a previously undetected isomer with enhanced basicity. In particular, it is proposed that protonation of (OC)3Fe(pdt)Ni(dppe) (1; pdt2– = –S(CH2)3S–; dppe = Ph2P(CH2)2PPh2) occurs at the Fe site of the two-electron mixed-valence Fe(0)Ni(II) species, not the Fe(I)-Ni(I) bond for the homovalence isomer of 1. The new pathway, which may have implications for protonation of other complexes and clusters, was uncovered through studies on the homologous series L(OC)2Fe(pdt)M(dppe), where M = Ni, Pd (2), and Pt (3) and L = CO, PCy3. Similar to 1, complexes 2 and 3 undergo both protonation and 1e– oxidation to afford well-characterized hydrides ([2H]+ and [3H]+) and mixed-valence derivatives ([2]+ and [3]+), respectively. Whereas the Pd site is tetrahedral in 2, the Pt site is square-planar in 3, indicating that this complex is best described as Fe(0)Pt(II). In view of the results on 2 and 3, the potential energy surface of 1 was reinvestigated with density functional theory. These calculations revealed the existence of an energetically accessible and more basic Fe(0)Ni(II) isomer with a square-planar Ni site. PMID:25094041

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

    SciTech Connect

    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

    The γ-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.

  13. The Hydrogenase Cytochrome b Heme Ligands of Azotobacter vinelandii Are Required for Full H2 Oxidation Capability

    PubMed Central

    Meek, Laura; Arp, Daniel J.

    2000-01-01

    The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H2 oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O2-dependent H2 oxidation and H2-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O2-dependent H2 oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H2 oxidation. In mutant H194A/Y, imidazole (Imd) rescued H2 oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H2-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H2 oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H2 oxidation. PMID:10852874

  14. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications

    PubMed Central

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P.

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: • Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication. • Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha. • The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry. PMID:27077052

  15. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

    PubMed

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry. PMID:27077052

  16. The weak, fluctuating, dipole moment of membrane-bound hydrogenase from Aquifex aeolicus accounts for its adaptability to charged electrodes.

    PubMed

    Oteri, Francesco; Ciaccafava, Alexandre; de Poulpiquet, Anne; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

    2014-06-21

    [NiFe] hydrogenases from Aquifex aeolicus (AaHase) and Desulfovibrio fructosovorans (DfHase) have been mainly studied to characterize physiological electron transfer processes, or to develop biotechnological devices such as biofuel cells. In this context, it remains difficult to control the orientation of AaHases on electrodes to achieve a fast interfacial electron transfer. Here, we study the electrostatic properties of these two proteins based on microsecond-long molecular dynamics simulations that we compare to voltammetry experiments. Our calculations show weak values and large fluctuations of the dipole direction in AaHase compared to DfHase, enabling the AaHase to absorb on both negatively and positively charged electrodes, with an orientation distribution that induces a spread in electron transfer rates. Moreover, we discuss the role of the transmembrane helix of AaHase and show that it does not substantially impact the general features of the dipole moment. PMID:24789038

  17. [FeFe]-Hydrogenase with Chalcogenide Substitutions at the H-Cluster Maintains Full H2 Evolution Activity.

    PubMed

    Noth, Jens; Esselborn, Julian; Güldenhaupt, Jörn; Brünje, Annika; Sawyer, Anne; Apfel, Ulf-Peter; Gerwert, Klaus; Hofmann, Eckhard; Winkler, Martin; Happe, Thomas

    2016-07-11

    The [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H-cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H-cluster into the HYDA1-apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4FeH ] cluster with selenium. The crystal structure of the reconstituted pre-mature HYDA1[4Fe4Se]H protein was determined, and a catalytically intact artificial H-cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native-like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X-ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity. PMID:27214763

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

    PubMed

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

    2014-08-18

    Reported herein is an electrode for dihydrogen (H2) oxidation, and it is based on [NiFe]Hydrogenase from Citrobacter sp. S-77 ([NiFe]S77). It has a 637 times higher mass activity than Pt (calculated based on 1 mg of [NiFe]S77 or Pt) at 50 mV in a hydrogen half-cell. The [NiFe]S77 electrode is also stable in air and, unlike Pt, can be recovered 100 % after poisoning by carbon monoxide. Following characterization of the [NiFe]S77 electrode, a fuel cell comprising a [NiFe]S77 anode and Pt cathode was constructed and shown to have a a higher power density than that achievable by Pt. PMID:24895095

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

    PubMed

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

    2013-12-17

    Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN)2CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53- and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively. PMID:24297906

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

    PubMed Central

    Boyd, Eric S.; Hamilton, Trinity L.; Swanson, Kevin D.; Howells, Alta E.; Baxter, Bonnie K.; Meuser, Jonathan E.; Posewitz, Matthew C.; Peters, John W.

    2014-01-01

    The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications. PMID:25464382

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

    PubMed

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

    2014-10-29

    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 νCO 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

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

    PubMed

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

    2014-07-11

    The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. PMID:24860091

  3. Multiscale simulation reveals multiple pathways for H2 and O2 transport in a [NiFe]-hydrogenase.

    PubMed

    Wang, Po-hung; Best, Robert B; Blumberger, Jochen

    2011-03-16

    Hydrogenases are enzymes that catalyze the reversible conversion of hydrogen molecules to protons and electrons. The mechanism by which the gas molecules reach the active site is important for understanding the function of the enzyme and may play a role in the selectivity for hydrogen over inhibitor molecules. Here, we develop a general multiscale molecular simulation approach for the calculation of diffusion rates and determination of pathways by which substrate or inhibitor gases can reach the protein active site. Combining kinetic data from both equilibrium simulations and enhanced sampling, we construct a master equation describing the movement of gas molecules within the enzyme. We find that the time-dependent gas population of the active site can be fit to the same phenomenological rate law used to interpret experiments, with corresponding diffusion rates in very good agreement with experimental data. However, in contrast to the conventional picture, in which the gases follow a well-defined hydrophobic tunnel, we find that there is a diverse network of accessible pathways by which the gas molecules can reach the active site. The previously identified tunnel accounts for only about 60% of the total flux. Our results suggest that the dramatic decrease in the diffusion rate for mutations involving the residue Val74 could be in part due to the narrowing of the passage Val74-Arg476, immediately adjacent to the binding site, explaining why mutations of Leu122 had only a negligible effect in experiment. Our method is not specific to the [NiFe]-hydrogenase and should be generally applicable to the transport of small molecules in proteins. PMID:21341658

  4. Quantification of microbial activity in subsurface environments using a hydrogenase enzyme assay

    NASA Astrophysics Data System (ADS)

    Adhikari, R. R.; Nickel, J.; Kallmeyer, J.

    2012-04-01

    The subsurface biosphere is the largest microbial ecosystem on Earth. Despite its large size and extensive industrial exploitation, very little is known about the role of microbial activity in the subsurface. Subsurface microbial activity plays a fundamental role in geochemical cycles of carbon and other biologically important elements. How the indigenous microbial communities are supplied with energy is one of the most fundamental questions in subsurface research. It is still an enigma how these communities can survive with such recalcitrant carbon over geological time scales. Despite its usually very low concentration, hydrogen is an important element in subsurface environments. Heterotrophic and chemoautotrophic microorganisms use hydrogen in their metabolic pathways; they either obtain protons from the radiolysis of water and/or cleavage of hydrogen generated by the alteration of basaltic crust, or they dispose of protons by formation of water. Hydrogenase (H2ase) is a ubiquitous intracellular enzyme that catalyzes the interconversion of molecular hydrogen and/or water into protons and electrons. The protons are used for the synthesis of ATP, thereby coupling energy-generating metabolic processes to electron acceptors such as carbon dioxide or sulfate. H2ase activity can therefore be used as a measure for total microbial activity as it targets a key metabolic compound rather than a specific turnover process. Using a highly sensitive tritium assay we measured H2ase enzyme activity in the organic-rich sediments of Lake Van, a saline, alkaline lake in eastern Turkey and in marine subsurface sediments of the Barents Sea. Additionally, sulfate reduction rates (SRRs) were measured to compare the results of the H2ase enzyme assay with the quantitatively most important electron acceptor process. H2ase activity was found at all sites, measured values and distribution of activity varied widely with depth and between sites. At the Lake Van sites H2ase activity ranged from

  5. Direct comparison of the performance of a bio-inspired synthetic nickel catalyst and a [NiFe]-hydrogenase, both covalently attached to electrodes.

    PubMed

    Rodriguez-Maciá, Patricia; Dutta, Arnab; Lubitz, Wolfgang; Shaw, Wendy J; Rüdiger, Olaf

    2015-10-12

    The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells. PMID:26140506

  6. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

    DOE PAGESBeta

    Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B.; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P.

    2014-10-30

    Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, whichmore » is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.« less

  7. Electronic structure of the unique [4Fe-3S] cluster in O2-tolerant hydrogenases characterized by 57Fe Mossbauer and EPR spectroscopy.

    PubMed

    Pandelia, Maria-Eirini; Bykov, Dmytro; Izsak, Robert; Infossi, Pascale; Giudici-Orticoni, Marie-Thérèse; Bill, Eckhard; Neese, Frank; Lubitz, Wolfgang

    2013-01-01

    Iron-sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H(2) catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O(2)-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent (57)Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron-sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron-sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher. PMID:23267108

  8. Immobilization of the [FeFe]-hydrogenase CrHydA1 on a gold electrode: design of a catalytic surface for the production of molecular hydrogen.

    PubMed

    Krassen, Henning; Stripp, Sven; von Abendroth, Gregory; Ataka, Kenichi; Happe, Thomas; Heberle, Joachim

    2009-06-01

    Hydrogenase-modified electrodes are a promising catalytic surface for the electrolysis of water with an overpotential close to zero. The [FeFe]-hydrogenase CrHydA1 from the photosynthetic green alga Chlamydomonas reinhardtii is the smallest [FeFe]-hydrogenase known and exhibits an extraordinary high hydrogen evolution activity. For the first time, we immobilized CrHydA1 on a gold surface which was modified by different carboxy-terminated self-assembled monolayers. The immobilization was in situ monitored by surface-enhanced infrared spectroscopy. In the presence of the electron mediator methyl viologen the electron transfer from the electrode to the hydrogenase was detected by cyclic voltammetry. The hydrogen evolution potential (-290 mV vs NHE, pH 6.8) of this protein modified electrode is close to the value for bare platinum (-270 mV vs NHE). The surface coverage by CrHydA1 was determined to 2.25 ng mm(-2) by surface plasmon resonance, which is consistent with the formation of a protein monolayer. Hydrogen evolution was quantified by gas chromatography and the specific hydrogen evolution activity of surface-bound CrHydA1 was calculated to 1.3 micromol H(2)min(-1)mg(-1) (or 85 mol H(2)min(-1)mol(-1)). In conclusion, a viable hydrogen-evolving surface was developed that may be employed in combination with immobilized photosystems to provide a platform for hydrogen production from water and solar energy with enzymes as catalysts. PMID:19480942

  9. Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO.

    PubMed

    Ash, Philip A; Liu, Juan; Coutard, Nathan; Heidary, Nina; Horch, Marius; Gudim, Ingvild; Simler, Thomas; Zebger, Ingo; Lenz, Oliver; Vincent, Kylie A

    2015-10-29

    The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases. PMID:26115011

  10. Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl-Group Channeling during [NiFe]-Hydrogenase Cofactor Generation.

    PubMed

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

    2015-01-01

    [NiFe]-hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so-called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP-dependent condensation reaction, the C-terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]-hydrogenase active site cofactor. We present a FT-IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF-catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro-electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (-N=C=S) rather than thiocyanate (-S-C≡N). This has important implications for cyanyl-group channeling during [NiFe]-hydrogenase cofactor generation. PMID:26186649

  11. Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl–Group Channeling during [NiFe]–Hydrogenase Cofactor Generation

    PubMed Central

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

    2015-01-01

    [NiFe]–hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so–called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP–dependent condensation reaction, the C–terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]–hydrogenase active site cofactor. We present a FT–IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF–catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro–electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (–N=C=S) rather than thiocyanate (–S–C≡N). This has important implications for cyanyl–group channeling during [NiFe]–hydrogenase cofactor generation. PMID:26186649

  12. Fractionation of Sulfur Isotopes by Desulfovibrio vulgaris Mutants Lacking Periplasmic Hydrogenases or the Type I Tetraheme Cytochrome c3

    NASA Astrophysics Data System (ADS)

    Sim, M.; Ono, S.; Bosak, T.

    2012-12-01

    A large fraction of anaerobic mineralization of organic compounds relies on microbial sulfate reduction. Sulfur isotope fractionation by these microbes has been widely used to trace the biogeochemical cycling of sulfur and carbon, but intracellular mechanisms behind the wide range of fractionations observed in nature and cultures are not fully understood. In this study, we investigated the influence of electron transport chain components on the fractionation of sulfur isotopes by culturing Desulfovibrio vulgaris Hildenborough mutants lacking hydrogenases or type I tetraheme cytochrome c3 (Tp1-c3). The mutants were grown both in batch and continuous cultures. All tested mutants grew on lactate or pyruvate as the sole carbon and energy sources, generating sulfide. Mutants lacking cytoplasmic and periplasmic hydrogenases exhibited similar growth physiologies and sulfur isotope fractionations to their parent strains. On the other hand, a mutant lacking Tp1-c3 (ΔcycA) fractionated the 34S/32S ratio more than the wild type, evolving H2 in the headspace and exhibiting a lower specific respiration rate. In the presence of high concentrations of pyruvate, the growth of ΔcycA relied largely on fermentation rather than sulfate reduction, even when sulfate was abundant, producing the largest sulfur isotope effect observed in this study. Differences between sulfur isotope fractionation by ΔcycA and the wild type highlight the effect of electron transfer chains on the magnitude of sulfur isotope fractionation. Because Tp1-c3 is known to exclusively shuttle electrons from periplasmic hydrogenases to transmembrane complexes, electron transfers in the absence of Tp1-c3 should bypass the periplasmic hydrogen cycling, and the loss of reducing equivalents in the form of H2 can impair the flow of electrons from organic acids to sulfur, increasing isotope fractionation. Larger fractionation by ΔcycA can inform interpretations of sulfur isotope data at an environmental scale as well

  13. O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase*

    PubMed Central

    Lambertz, Camilla; Leidel, Nils; Havelius, Kajsa G. V.; Noth, Jens; Chernev, Petko; Winkler, Martin; Happe, Thomas; Haumann, Michael

    2011-01-01

    Irreversible inhibition by molecular oxygen (O2) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H2) production. Modification by O2 of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2FeH) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O2 exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O2 reactions. The first phase (τ1 ≤ 4 s) is characterized by the formation of an increased number of Fe–O,C bonds, elongation of the Fe–Fe distance in the binuclear unit (2FeH), and oxidation of one iron ion. The second phase (τ2 ≈ 15 s) causes a ∼50% decrease of the number of ∼2.7-Å Fe–Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ3 ≤ 1000 s) leads to the disappearance of most Fe–Fe and Fe–S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2FeH+ leading to the formation of a reactive oxygen species-like superoxide (O2−), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S]+ unit, leading to 3) complete O2-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O2 tolerance in [FeFe]-hydrogenase. PMID:21930709

  14. A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.

    PubMed

    Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

    2015-03-27

    The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. PMID:25666617

  15. Protonation states of intermediates in the reaction mechanism of [NiFe] hydrogenase studied by computational methods.

    PubMed

    Dong, Geng; Ryde, Ulf

    2016-06-01

    The [NiFe] hydrogenases catalyse the reversible conversion of H2 to protons and electrons. The active site consists of a Fe ion with one carbon monoxide, two cyanide, and two cysteine (Cys) ligands. The latter two bridge to a Ni ion, which has two additional terminal Cys ligands. It has been suggested that one of the Cys residues is protonated during the reaction mechanism. We have used combined quantum mechanical and molecular mechanics (QM/MM) geometry optimisations, large QM calculations with 817 atoms, and QM/MM free energy simulations, using the TPSS and B3LYP methods with basis sets extrapolated to the quadruple zeta level to determine which of the four Cys residues is more favourable to protonate for four putative states in the reaction mechanism, Ni-SIa, Ni-R, Ni-C, and Ni-L. The calculations show that for all states, the terminal Cys-546 residue is most easily protonated by 14-51 kJ/mol, owing to a more favourable hydrogen-bond pattern around this residue in the protein. PMID:26940957

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

    PubMed Central

    Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L.; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

    2015-01-01

    The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. PMID:25666617

  17. An iron-iron hydrogenase mimic with appended electron reservoir for efficient proton reduction in aqueous media.

    PubMed

    Becker, René; Amirjalayer, Saeed; Li, Ping; Woutersen, Sander; Reek, Joost N H

    2016-01-01

    The transition from a fossil-based economy to a hydrogen-based economy requires cheap and abundant, yet stable and efficient, hydrogen production catalysts. Nature shows the potential of iron-based catalysts such as the iron-iron hydrogenase (H2ase) enzyme, which catalyzes hydrogen evolution at rates similar to platinum with low overpotential. However, existing synthetic H2ase mimics generally suffer from low efficiency and oxygen sensitivity and generally operate in organic solvents. We report on a synthetic H2ase mimic that contains a redox-active phosphole ligand as an electron reservoir, a feature that is also crucial for the working of the natural enzyme. Using a combination of (spectro)electrochemistry and time-resolved infrared spectroscopy, we elucidate the unique redox behavior of the catalyst. We find that the electron reservoir actively partakes in the reduction of protons and that its electron-rich redox states are stabilized through ligand protonation. In dilute sulfuric acid, the catalyst has a turnover frequency of 7.0 × 10(4) s(-1) at an overpotential of 0.66 V. This catalyst is tolerant to the presence of oxygen, thereby paving the way for a new generation of synthetic H2ase mimics that combine the benefits of the enzyme with synthetic versatility and improved stability. PMID:26844297

  18. A hydrogen biosensor made of clay, poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode.

    PubMed

    Qian, Dong-Jin; Nakamura, Chikashi; Wenk, Stephan-Olav; Ishikawa, Hiroshi; Zorin, Nikolay; Miyake, Jun

    2002-09-01

    A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed. PMID:12191927

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

    PubMed Central

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

    2014-01-01

    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

  20. An iron-iron hydrogenase mimic with appended electron reservoir for efficient proton reduction in aqueous media

    PubMed Central

    Becker, René; Amirjalayer, Saeed; Li, Ping; Woutersen, Sander; Reek, Joost N. H.

    2016-01-01

    The transition from a fossil-based economy to a hydrogen-based economy requires cheap and abundant, yet stable and efficient, hydrogen production catalysts. Nature shows the potential of iron-based catalysts such as the iron-iron hydrogenase (H2ase) enzyme, which catalyzes hydrogen evolution at rates similar to platinum with low overpotential. However, existing synthetic H2ase mimics generally suffer from low efficiency and oxygen sensitivity and generally operate in organic solvents. We report on a synthetic H2ase mimic that contains a redox-active phosphole ligand as an electron reservoir, a feature that is also crucial for the working of the natural enzyme. Using a combination of (spectro)electrochemistry and time-resolved infrared spectroscopy, we elucidate the unique redox behavior of the catalyst. We find that the electron reservoir actively partakes in the reduction of protons and that its electron-rich redox states are stabilized through ligand protonation. In dilute sulfuric acid, the catalyst has a turnover frequency of 7.0 × 104 s−1 at an overpotential of 0.66 V. This catalyst is tolerant to the presence of oxygen, thereby paving the way for a new generation of synthetic H2ase mimics that combine the benefits of the enzyme with synthetic versatility and improved stability. PMID:26844297

  1. Novel reactions of homodinuclear Ni2 complexes [Ni(RNPyS4)]2 with Fe3(CO)12 to give heterotrinuclear NiFe2 and mononuclear Fe complexes relevant to [NiFe]- and [Fe]-hydrogenases.

    PubMed

    Song, Li-Cheng; Cao, Meng; Wang, Yong-Xiang

    2015-04-21

    The homodinuclear complexes [Ni(RNPyS4)]2 (; RNPyS4 = 2,6-bis(2-mercaptophenylthiomethyl)-4-R-pyridine; R = H, MeO, Cl, Br, i-Pr) were found to be prepared by reactions of the in situ generated Li2[Ni(1,2-S2C6H4)2] with 2,6-bis[(tosyloxy)methyl]pyridine and its substituted derivatives 2,6-bis[(tosyloxy)methyl]-4-R-pyridine. Further reactions of with Fe3(CO)12 gave both heterotrinuclear complexes NiFe2(RNPyS4)(CO)5 () and mononuclear complexes Fe(RNPyS4)(CO) (), unexpectedly. Interestingly, complexes and could be regarded as models for the active sites of [NiFe]- and [Fe]-hydrogenases, respectively. All the prepared complexes were characterized by elemental analysis, spectroscopy, and particularly for some of them, by X-ray crystallography. In addition, the electrochemical properties of and as well as the electrocatalytic H2 production catalyzed by and were investigated by CV techniques. PMID:25747808

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

    PubMed Central

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

    2011-01-01

    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

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

    PubMed

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

    2002-10-01

    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

  4. Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

    PubMed Central

    Elsen, Sylvie; Dischert, Wanda; Colbeau, Annette; Bauer, Carl E.

    2000-01-01

    Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H2 oxidation, as well as the energy-demanding processes of N2 fixation and CO2 assimilation. PMID:10781552

  5. Structure of [NiFe] Hydrogenase Maturation Protein HypE from Escherichia coli and its Interaction with HypF

    SciTech Connect

    Rangarajan,E.; Asinas, A.; Proteau, A.; Munger, C.; Baardsnes, j.; Iannuzzi, P.; Matte, A.; Cygler, m.

    2008-01-01

    Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H2 as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0- Angstroms resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a Kd (dissociation constant) of {approx}400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]2 heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.

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

    PubMed

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

    2000-11-01

    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

  7. Synthesis and Photophysical Study of a [NiFe] Hydrogenase Biomimetic Compound Covalently Linked to a Re-diimine Photosensitizer

    PubMed Central

    2015-01-01

    The synthesis, photophysics, and photochemistry of a linked dyad ([Re]-[NiFe2]) containing an analogue ([NiFe2]) of the active site of [NiFe] hydrogenase, covalently bound to a Re-diimine photosensitizer ([Re]), are described. Following excitation, the mechanisms of electron transfer involving the [Re] and [NiFe2] centers and the resulting decomposition were investigated. Excitation of the [Re] center results in the population of a diimine-based metal-to-ligand charge transfer excited state. Reductive quenching by NEt3 produces the radically reduced form of [Re], [Re]− (kq = 1.4 ± 0.1 × 107 M–1 s–1). Once formed, [Re]− reduces the [NiFe2] center to [NiFe2]−, and this reduction was followed using time-resolved infrared spectroscopy. The concentration dependence of the electron transfer rate constants suggests that both inter- and intramolecular electron transfer pathways are involved, and the rate constants for these processes have been estimated (kinter = 5.9 ± 0.7 × 108 M–1 s–1, kintra = 1.5 ± 0.1 × 105 s–1). For the analogous bimolecular system, only intermolecular electron transfer could be observed (kinter = 3.8 ± 0.5 × 109 M–1 s–1). Fourier transform infrared spectroscopic studies confirms that decomposition of the dyad occurs upon prolonged photolysis, and this appears to be a major factor for the low activity of the system toward H2 production in acidic conditions. PMID:26605700

  8. Crystal structures of the HypCD complex and the HypCDE ternary complex: transient intermediate complexes during [NiFe] hydrogenase maturation.

    PubMed

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

    2012-12-01

    [NiFe] hydrogenase maturation represents one of the most dynamic and sophisticated processes in metallocenter assembly. The Fe(CN)(2)CO moiety of [NiFe] hydrogenases is assembled via unknown transient interactions among specific maturation proteins HypC (metallochaperone), HypD (redox protein), and HypE (cyanide synthesis/donor). Here, we report the structures of the HypC-HypD and HypC-HypD-HypE complexes, providing a view of the transient interactions that take place during the maturation process. HypC binds to the conserved region of HypD through extensive hydrophobic interactions. The ternary complex formation between HypE and the HypCD complex involves both HypC and HypD, rendering the HypE conformation favorable for cyanide transfer. In the complex, the conserved cysteines of HypC and HypD form an Fe binding site. The conserved C-terminal cysteine of HypE can access the thiol redox cascade of HypD. These results provide structural insights into the Fe atom cyanation in the HypCDE complex. PMID:23123111

  9. Direct Comparison of the Performance of a Bio-inspired Synthetic Nickel Catalyst and a [NiFe]-Hydrogenase, Both Covalently Attached to Electrodes

    SciTech Connect

    Rodriguez-Macia, Patricia; Dutta, Arnab; Lubitz, Wolfgang; Shaw, Wendy J.; Rudiger, Olaf

    2015-10-12

    The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we’ve come in mimicking the impressive enzymatic performance. Here we directly compare the catalytic properties of the [Ni(PCy2NGly2)2]2+ complex with the [NiFe]-hydrogenase from Desulfobivrio vulgaris Miyazaki F (DvMF) immobilized to a functionalized electrode under identical conditions. At pH=7, the enzyme has higher performance in both activity and overpotential, and is more stable, while at low pH, the molecular catalyst outperforms the enzyme in all respects. The Ni complex also has increased tolerance to CO. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bioinspired complexes in fuel cells. AD and WJS acknowledge the Office of Science Early Career Research Program through the US Department of Energy (US DOE), Office of Science, Office of Basic Energy Sciences (BES), and Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the US DOE.

  10. Effects of Metal on the Biochemical Properties of Helicobacter pylori HypB, a Maturation Factor of [NiFe]-Hydrogenase and Urease ▿ †

    PubMed Central

    Sydor, Andrew M.; Liu, Jenny; Zamble, Deborah B.

    2011-01-01

    The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the kcat and Km of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes. PMID:21239585

  11. The Soluble NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

    PubMed Central

    Burgdorf, Tanja; van der Linden, Eddy; Bernhard, Michael; Yuan Yin, Qing; Back, Jaap W.; Hartog, Aloysius F.; Muijsers, Anton O.; de Koster, Chris G.; Albracht, Simon P. J.; Friedrich, Bärbel

    2005-01-01

    The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD+ at the expense of H2. We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI2. Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I). PMID:15838039

  12. Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells.

    PubMed

    Monsalve, Karen; Roger, Magali; Gutierrez-Sanchez, Cristina; Ilbert, Marianne; Nitsche, Serge; Byrne-Kodjabachian, Deborah; Marchi, Valérie; Lojou, Elisabeth

    2015-12-01

    For the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV-Vis spectroscopy. Two sizes around 20.0±5.3 nm and 37.2±4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidation over a large range of temperatures. A high current density up to 1.85±0.15 mA·cm(-2) was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was especially studied as a function of the AuNP size and amount, and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power density of 0.25 mW·cm(-2) was recorded. PMID:25960259

  13. Branched polyethylenimine improves hydrogen photoproduction from a CdSe quantum dot/[FeFe]-hydrogenase mimic system in neutral aqueous solutions.

    PubMed

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

    2015-02-16

    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

  14. An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure.

    PubMed

    Pfeiffer, M; Klein, A; Steinert, P; Schomburg, D

    The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism. PMID:9084873

  15. Biochemical Characterization of HydF, a Scaffolding Enzyme, in the Synthesis of the Hydrogenase Active Site Metal Center: Implications Towards the Evolution of Biocatalysts from Mineral-based Components on Early Earth

    NASA Astrophysics Data System (ADS)

    Duffus, B. R.; Shepard, E. M.; McGlynn, S. E.; Bueling, A. L.; Winslow, M. A.; Peters, J. W.; Broderick, J. B.

    2010-04-01

    [FeFe]-hydrogenase active site biosynthesis utilizes radical chemistry on a scaffold protein whose ancestor may have been one of the earliest examples of a protein that couples the chemistry of an Fe-S peptide nest with a nucleotide binding nest.

  16. Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H2

    SciTech Connect

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

    2014-03-01

    Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data is consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organism, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

  17. Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

    PubMed Central

    Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

    2011-01-01

    Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    King, Paul W

    2013-01-01

    The direct conversion of sunlight into biofuels is an intriguing alternative to a continued reliance on fossil fuels. Natural photosynthesis has long been investigated both as a potential solution, and as a model for utilizing solar energy to drive a water-to-fuel cycle. The molecules and organizational structure provide a template to inspire the design of efficient molecular systems for photocatalysis. A clear design strategy is the coordination of molecular interactions that match kinetic rates and energetic levels to control the direction and flow of energy from light harvesting to catalysis. Energy transduction and electron-transfer reactions occur through interfaces formed between complexes of donor-acceptor molecules. Although the structures of several of the key biological complexes have been solved, detailed descriptions of many electron-transfer complexes are lacking, which presents a challenge to designing and engineering biomolecular systems for solar conversion. Alternatively, it is possible to couple the catalytic power of biological enzymes to light harvesting by semiconductor nanomaterials. In these molecules, surface chemistry and structure can be designed using ligands. The passivation effect of the ligand can also dramatically affect the photophysical properties of the semiconductor, and energetics of external charge-transfer. The length, degree of bond saturation (aromaticity), and solvent exposed functional groups of ligands can be manipulated to further tune the interface to control molecular assembly, and complex stability in photocatalytic hybrids. The results of this research show how ligand selection is critical to designing molecular interfaces that promote efficient self-assembly, charge-transfer and photocatalysis. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. PMID:23541891

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

    PubMed Central

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

    2015-01-01

    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

  1. Synthetic Models for the [FeFe]-Hydrogenase: Catalytic Proton Reduction and the Structure of the Doubly Protonated Intermediate

    PubMed Central

    Carroll, Maria E.; Barton, Bryan E.; Rauchfuss, Thomas B.; Carroll, Patrick J.

    2012-01-01

    This report compares biomimetic HER catalysts with and without the amine cofactor (adtNH): Fe2(adtNH)(CO)2(dppv)2 (1NH) and Fe2(pdt)(CO)2(dppv)2 (2; (adtNH)2− = (HN(CH2S)22−, pdt2− = 1,3-(CH2)3S22−). These compounds are spectroscopically, structurally, and stereodynamically very similar but exhibit very different catalytic properties. Protonation of 1NH and 2 each give three isomeric hydrides beginning with the kinetically favored terminal hydride, which converts sequentially to sym and unsym isomers of the bridging hydrides. In the case of the amine, the corresponding ammonium-hydrides are also observed. In the case of the terminal amine hydride [t-H1NH]BF4, the ammonium/amine-hydride equilibrium is sensitive to counteranions and solvent. The species [t-H1NH2](BF4)2 represents the first example of a crystallographically characterized terminal hydride produced by protonation. The NH--HFe distance of 1.88(7) Å indicates dihydrogen bonding. The bridging hydrides [µ-H1NH]+ and [µ-H2]+ reduce near −1.8 V, about 150 mV more negative than the reductions of the terminal hydride [t-H1NH]+ and [t-H2]+ at −1.65 V. Reductions of the amine hydrides [t-H1NH]+ and [t-H1NH2]2+ are irreversible. For the pdt analog, the [t-H2]+/0 couple is unaffected by weak acids (pKaMeCN 15.3) but exhibits catalysis with HBF4•Et2O, albeit with a TOF around 4 s−1 and an overpotential greater than 1 V. The voltammetry of [t-H1NH]+ is strongly affected by relatively weak acids and proceeds at 5000 s−1 with an overpotential of 0.7 V. The ammonium-hydride [t-H1NH2]2+ is a faster catalyst with an estimated TOF of 58,000 s−1 and an overpotential of 0.5 V. PMID:23126330

  2. Active-Site Models for the Nickel-Iron Hydrogenases: Effects of Ligands on Reactivity and Catalytic Properties

    PubMed Central

    Carroll, Maria E.; Barton, Bryan E.; Gray, Danielle L.; Mack, Amanda E.; Rauchfuss, Thomas B.

    2011-01-01

    Described are new derivatives of the type [HNiFe(SR)2(diphosphine)(CO)3]+, which feature a Ni(diphosphine) group linked to a Fe(CO)3 group via two bridging thiolate ligands. Previous work had described [HNiFe(pdt)(dppe)(CO)3]+ ([1H]+) and its activity as a catalyst for the reduction of protons. Work described in this paper focused on the effects of the diphosphine attached to nickel as well as the dithiolate bridge, 1,3-propanedithiolate (pdt) vs 1,2-ethanedithiolate (edt). A new synthetic route to these Ni-Fe dithiolates is described, involving reaction of Ni(SR)2(diphosphine) with FeI2(CO)4 followed by in situ reduction with cobaltocene. Evidence is presented that this route proceeds via metastable μ-iodo derivatives. Attempted isolation of such species led to the crystallization of NiFe(Me2pdt)(dppe)I2, which features tetrahedral Fe(II) and square planar Ni(II) centers (Me2pdt = 2,2-dimethylpropanedithiol). The new tricarbonyls prepared in this work are NiFe(pdt)(dcpe)(CO)3 (2, dcpe = 1,2-bis(dicyclohexylphosphino)ethane), NiFe(edt)(dppe)(CO)3 (3), and NiFe(edt)(dcpe)(CO)3 (4). Attempted preparation of a phenylthiolate-bridged complex via the FeI2(CO)4 + Ni(SPh)2(dppe) route gave the tetrametallic species [(CO)2Fe(SPh)2Ni(CO)]2(μ-dppe)2. Crystallographic analysis of the edt-dcpe compund [2H]BF4 and the edt-dppe compound [3H]BF4 verified their close resemblance. Each features pseudo-octahedral Fe and square pyramidal Ni centers. Starting from [4H]BF4 we prepared the PPh3 derivative [HNiFe(edt)(dppe)(PPh3)(CO)2]BF4 ([5H]BF4), which was obtained as a ~2:1 mixture of unsymmetrical and symmetrical isomers. Acid-base measurements indicate that changing from Ni(dppe) to Ni(dcpe) decreases the acidity of the cationic hydride complexes by 2.5 pKaMeCN units, from ~11 to ~13.5 (previous work showed that substitution at Fe leads to more dramatic effects). The redox potentials are more strongly affected by the change from dppe to dcpe, for example the [2]0/+ couple occurs at E1/2 = −820 for [2]0/+ vs −574 mV (vs Fc+/0) for [1]0/+. Changes in the dithiolate do not affect the acidity or the reduction potentials of the hydrides. The acid-independent rate of reduction of CH2ClCO2H by [2H]+ is ca. 50 s−1 (25 °C), twice that of [1H]+. The edt-dppe complex [2H]+ proved to be the most active catalyst, with an acid-independent rate of 300 s−1. PMID:21866886

  3. Mechanism of H2 Production by Models for the [NiFe]-Hydrogenases: Role of Reduced Hydrides.

    PubMed

    Ulloa, Olbelina A; Huynh, Mioy T; Richers, Casseday P; Bertke, Jeffery A; Nilges, Mark J; Hammes-Schiffer, Sharon; Rauchfuss, Thomas B

    2016-07-27

    The intermediacy of a reduced nickel-iron hydride in hydrogen evolution catalyzed by Ni-Fe complexes was verified experimentally and computationally. In addition to catalyzing hydrogen evolution, the highly basic and bulky (dppv)Ni(μ-pdt)Fe(CO)(dppv) ([1](0); dppv = cis-C2H2(PPh2)2) and its hydride derivatives have yielded to detailed characterization in terms of spectroscopy, bonding, and reactivity. The protonation of [1](0) initially produces unsym-[H1](+), which converts by a first-order pathway to sym-[H1](+). These species have C1 (unsym) and Cs (sym) symmetries, respectively, depending on the stereochemistry of the octahedral Fe site. Both experimental and computational studies show that [H1](+) protonates at sulfur. The S = 1/2 hydride [H1](0) was generated by reduction of [H1](+) with Cp*2Co. Density functional theory (DFT) calculations indicate that [H1](0) is best described as a Ni(I)-Fe(II) derivative with significant spin density on Ni and some delocalization on S and Fe. EPR spectroscopy reveals both kinetic and thermodynamic isomers of [H1](0). Whereas [H1](+) does not evolve H2 upon protonation, treatment of [H1](0) with acids gives H2. The redox state of the "remote" metal (Ni) modulates the hydridic character of the Fe(II)-H center. As supported by DFT calculations, H2 evolution proceeds either directly from [H1](0) and external acid or from protonation of the Fe-H bond in [H1](0) to give a labile dihydrogen complex. Stoichiometric tests indicate that protonation-induced hydrogen evolution from [H1](0) initially produces [1](+), which is reduced by [H1](0). Our results reconcile the required reductive activation of a metal hydride and the resistance of metal hydrides toward reduction. This dichotomy is resolved by reduction of the remote (non-hydride) metal of the bimetallic unit. PMID:27328053

  4. The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster.

    PubMed

    Suess, Daniel L M; Pham, Cindy C; Bürstel, Ingmar; Swartz, James R; Cramer, Stephen P; Britt, R David

    2016-02-01

    Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key intermediate in H-cluster maturation. PMID:26764535

  5. Transcriptional regulation of genes encoding the selenium-free [NiFe]-hydrogenases in the archaeon Methanococcus voltae involves positive and negative control elements.

    PubMed Central

    Noll, I; Müller, S; Klein, A

    1999-01-01

    Methanococcus voltae harbors genetic information for two pairs of homologous [NiFe]-hydrogenases. Two of the enzymes contain selenocysteine, while the other two gene groups encode apparent isoenzymes that carry cysteinyl residues in the homologous positions. The genes coding for the selenium-free enzymes, frc and vhc, are expressed only under selenium limitation. They are transcribed out of a common intergenic region. A series of deletions made in the intergenic region localized a common negative regulatory element for the vhc and frc promoters as well as two activator elements that are specific for each of the two transcription units. Repeated sequences, partially overlapping the frc promoter, were also detected. Mutations in these repeated heptanucleotide sequences led to a weak induction of a reporter gene under the control of the frc promoters in the presence of selenium. This result suggests that the heptamer repeats contribute to the negative regulation of the frc transcription unit. PMID:10430564

  6. The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster

    PubMed Central

    Suess, Daniel L. M.; Pham, Cindy C.; Bürstel, Ingmar; Swartz, James R.; Cramer, Stephen P.; Britt, R. David

    2016-01-01

    Three maturase enzymes—HydE, HydF, and HydG—synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)]− species is a key intermediate in H-cluster maturation. PMID:26764535

  7. X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly

    PubMed Central

    Dinis, Pedro; Suess, Daniel L. M.; Fox, Stephen J.; Harmer, Jenny E.; Driesener, Rebecca C.; De La Paz, Liliana; Swartz, James R.; Essex, Jonathan W.; Britt, R. David; Roach, Peter L.

    2015-01-01

    Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN− ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ2-sulfide ion to a mononuclear Fe2+ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly. PMID:25605932

  8. Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

    PubMed Central

    Watanabe, Satoshi; Kawashima, Takumi; Nishitani, Yuichi; Kanai, Tamotsu; Wada, Takehiko; Inaba, Kenji; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2015-01-01

    The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypBAT) with Ni ions. Transient association between HypA and HypBAT is controlled by the ATP hydrolysis cycle of HypBAT, which is accelerated by HypA. Only the ATP-bound form of HypBAT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypABAT complex has a nanomolar affinity (Kd = 7 nM), which is in contrast to the micromolar affinity (Kd = 4 µM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypBAT, affecting its association with HypA. These findings indicate that HypA and HypBAT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypBAT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA. PMID:26056269

  9. Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB*

    PubMed Central

    Sydor, Andrew M.; Lebrette, Hugo; Ariyakumaran, Rishikesh; Cavazza, Christine; Zamble, Deborah B.

    2014-01-01

    The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination. PMID:24338018

  10. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

    SciTech Connect

    Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B.; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P.

    2014-10-30

    Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

  11. Photoactivation of the Ni-SIr state to the Ni-SIa state in [NiFe] hydrogenase: FT-IR study on the light reactivity of the ready Ni-SIr state and as-isolated enzyme revisited.

    PubMed

    Tai, Hulin; Xu, Liyang; Inoue, Seiya; Nishikawa, Koji; Higuchi, Yoshiki; Hirota, Shun

    2016-08-10

    The Ni-SIr state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F was photoactivated to its Ni-SIa state by Ar(+) laser irradiation at 514.5 nm, whereas the Ni-SL state was light induced from a newly identified state, which was less active than any other identified state and existed in the "as-isolated" enzyme. PMID:27456760

  12. Discovery of Dark pH-Dependent H+ Migration in a [NiFe]-Hydrogenase and Its Mechanistic Relevance: Mobilizing the Hydrido Ligand of the Ni-C Intermediate

    PubMed Central

    2015-01-01

    Despite extensive studies on [NiFe]-hydrogenases, the mechanism by which these enzymes produce and activate H2 so efficiently remains unclear. A well-known EPR-active state produced under H2 and known as Ni-C is assigned as a NiIII–FeII species with a hydrido ligand in the bridging position between the two metals. It has long been known that low-temperature photolysis of Ni-C yields distinctive EPR-active states, collectively termed Ni-L, that are attributed to migration of the bridging-H species as a proton; however, Ni-L has mainly been regarded as an artifact with no mechanistic relevance. It is now demonstrated, based on EPR and infrared spectroscopic studies, that the Ni-C to Ni-L interconversion in Hydrogenase-1 (Hyd-1) from Escherichia coli is a pH-dependent process that proceeds readily in the dark—proton migration from Ni-C being favored as the pH is increased. The persistence of Ni-L in Hyd-1 must relate to unassigned differences in proton affinities of metal and adjacent amino acid sites, although the unusually high reduction potentials of the adjacent Fe–S centers in this O2-tolerant hydrogenase might also be a contributory factor, impeding elementary electron transfer off the [NiFe] site after proton departure. The results provide compelling evidence that Ni-L is a true, albeit elusive, catalytic intermediate of [NiFe]-hydrogenases. PMID:26103582

  13. Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus.

    PubMed Central

    Richaud, P; Colbeau, A; Toussaint, B; Vignais, P M

    1991-01-01

    The hupR1 gene from Rhodobacter capsulatus was cloned and sequenced. It can encode a protein of 53,843 Da which shares significant similarity with several transcriptional regulators and activates transcription of the structural hupSL genes of [NiFe]hydrogenase, as shown by the use of a translational fusion of lacZ with the hupSL promoter. A Hup- mutant having a point mutation in the hupR1 gene is described. PMID:1885559

  14. RAPD analysis and sequencing of ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase as tools for genetic classification of potentially pathogenic isolates of Trichomonas gallinae.

    PubMed

    Sansano-Maestre, José; Martínez-Herrero, María Del Carmen; Garijo-Toledo, María Magdalena; Gómez-Muñoz, María Teresa

    2016-08-01

    Trichomonas gallinae is a worldwide parasite that causes oropharyngeal avian trichomonosis. During eight years, 60 axenic isolates were obtained from different bird species and characterized by three molecular methods: RAPD analysis and PCR-sequencing of ITS1/5.8S rRNA/ITS2 fragment and Fe-hydrogenase gene. We have found two genotypes of ITS1/5.8S rRNA/ITS2 widely distributed among bird populations, a new variant and also two sequences with mixed pattern. Genotype ITS-OBT-Tg-1 was associated with the presence of gross lesions in birds. We have found eight genotypes of the Fe-hydrogenase (A1, A2, C2, C2.1, C4, C5, C6 and C7), three of them are new reports (C5, C6 and C7), and also three sequences with mixed pattern. Subtype A1 of the Fe-hydrogenase was also related with the presence of lesions. RAPD analyses included most of the strains isolated from animals with lesions in one of the sub-clusters. Potentially pathogenic isolates of T. gallinae obtained in this study fulfill the following criteria with one exception: isolated from lesions+ITS-OBT-Tg-1 genotype+FeHyd A1+RAPD sub-cluster I2. PMID:27473993

  15. Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide

    PubMed Central

    Thomas, Claudia; Muhr, Enrico

    2015-01-01

    ABSTRACT During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN)2CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that in hyp mutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN)2CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptide per se is not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2. IMPORTANCE [NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN)2CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of

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

    PubMed Central

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

    2013-01-01

    Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the

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

    PubMed

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

    2007-09-19

    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

  18. Computational Design of Iron Diphosphine Complexes with Pendant Amines for Hydrogenation of CO2 to Methanol: A Mimic of [NiFe] Hydrogenase.

    PubMed

    Chen, Xiangyang; Jing, Yuanyuan; Yang, Xinzheng

    2016-06-20

    Inspired by the active-site structure of the [NiFe] hydrogenase, we have computationally designed the iron complex [P(tBu) 2 N(tBu) 2 )Fe(CN)2 CO] by using an experimentally ready-made diphosphine ligand with pendant amines for the hydrogenation of CO2 to methanol. Density functional theory calculations indicate that the rate-determining step in the whole catalytic reaction is the direct hydride transfer from the Fe center to the carbon atom in the formic acid with a total free energy barrier of 28.4 kcal mol(-1) in aqueous solution. Such a barrier indicates that the designed iron complex is a promising low-cost catalyst for the formation of methanol from CO2 and H2 under mild conditions. The key role of the diphosphine ligand with pendent amine groups in the reaction is the assistance of the cleavage of H2 by forming a Fe-H(δ-) ⋅⋅⋅H(δ+) -N dihydrogen bond in a fashion of frustrated Lewis pairs. PMID:27225505

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

    SciTech Connect

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

    2012-06-18

    A series of dipeptide nickel complexes with the general formula, [Ni(PPh2NNNA-amino acid/ester2)2](BF4)2, have been synthesized and characterized (P2N2= 1,5-diaza-3,7-diphosphacyclooctane, amino acid/esters = glutamic acid, alanine, lysine, and aspartic acid). Each of these complexes is an efficient electrocatalyst for H2 production. The contribution of the outer-coordination sphere, specifically the impact of sterics, the ability to protonate and the pKa of amino acid side chain on the hydrogen production activity of these complexes, was investigated. The rates of all of the catalysts ranged over an order of magnitude. The amino acid containing complexes display 2-3 times higher rates of hydrogen production than the corresponding ester complexes, suggesting the significance of protonated species (side chains/backbone of amino acids) in the outer-coordination sphere. The largest had the fastest rates suggesting that catalytic activity is not hindered by sterics. However, the shapes of catalytic waves are indicative of hindered electron transfer and may suggest a competing mechanism for catalysis than that observed for the unsubstituted parent complex. These studies demonstrate the significant contribution that the outer-coordination sphere can have in tuning the catalytic activity of small molecule hydrogenase mimics.

  20. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water.

    PubMed

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-01-01

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA(-)) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts. PMID:27417065

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

    PubMed

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

    2007-09-01

    [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

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

    PubMed

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

    1998-12-01

    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

  3. Application to Photocatalytic H2 Production of a Whole-Cell Reaction by Recombinant Escherichia coli Cells Expressing [FeFe]-Hydrogenase and Maturases Genes.

    PubMed

    Honda, Yuki; Hagiwara, Hidehisa; Ida, Shintaro; Ishihara, Tatsumi

    2016-07-01

    A photocatalytic H2 production system using an inorganic-bio hybrid photocatalyst could contribute to the efficient utilization of solar energy, but would require the development of a new approach for preparing a H2 -forming biocatalyst. In the present study, we constructed a recombinant strain of Escherichia coli expressing the genes encoding the [FeFe]-hydrogenase and relevant maturases from Clostridium acetobutylicum NBRC 13948 for use as a biocatalyst. We investigated the direct application of a whole-cell of the recombinant E. coli. The combination of TiO2 , methylviologen, and the recombinant E. coli formed H2 under light irradiation, demonstrating that whole cells of the recombinant E. coli could be employed for photocatalytic H2 production without any time-consuming and costly manipulations (for example, enzyme purification). This is the first report of the direct application of a whole-cell reaction of recombinant E. coli to photocatalytic H2 production. PMID:27194524

  4. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts

    PubMed Central

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-01-01

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil. PMID:27319579

  5. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water

    PubMed Central

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-01-01

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA−) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts. PMID:27417065

  6. Secondary coordination sphere accelerates hole transfer for enhanced hydrogen photogeneration from [FeFe]-hydrogenase mimic and CdSe QDs in water

    NASA Astrophysics Data System (ADS)

    Wen, Min; Li, Xu-Bing; Jian, Jing-Xin; Wang, Xu-Zhe; Wu, Hao-Lin; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2016-07-01

    Achieving highly efficient hydrogen (H2) evolution via artificial photosynthesis is a great ambition pursued by scientists in recent decades because H2 has high specific enthalpy of combustion and benign combustion product. [FeFe]-Hydrogenase ([FeFe]-H2ase) mimics have been demonstrated to be promising catalysts for H2 photoproduction. However, the efficient photocatalytic H2 generation system, consisting of PAA-g-Fe2S2, CdSe QDs and H2A, suffered from low stability, probably due to the hole accumulation induced photooxidation of CdSe QDs and the subsequent crash of [FeFe]-H2ase mimics. In this work, we take advantage of supramolecular interaction for the first time to construct the secondary coordination sphere of electron donors (HA‑) to CdSe QDs. The generated secondary coordination sphere helps realize much faster hole removal with a ~30-fold increase, thus leading to higher stability and activity for H2 evolution. The unique photocatalytic H2 evolution system features a great increase of turnover number to 83600, which is the highest one obtained so far for photocatalytic H2 production by using [FeFe]-H2ase mimics as catalysts.

  7. Electronic Structure and Chemistry of Iron-Based Metal Oxide Nanostructured Materials: A NEXAFS Investigation of BiFeO3, Bi2Fe4O9, α-Fe2O3, γ-Fe2O3, and Fe/Fe3O4

    SciTech Connect

    Park,T.; Sambasivan, S.; Fischer, D.; Yoon, W.; Misewich, J.; Wong, S.

    2008-01-01

    We present a systematic and detailed near edge X-ray absorption fine structure (NEXAFS) experimental investigation of the electronic structure and chemistry of iron-based metal oxide nanostructured (FeMONS) materials including BiFeO3, Bi2Fe4O9, a-Fe2O3, ?-Fe2O3, and Fe/Fe3O4. Correlations of the electronic structure and structural chemistry of these intriguing nanomaterials are presented, ranging from the nano to the bulk scale. In this work, variations in the shape, position, and intensity of the O K-edge and Fe L-edge NEXAFS spectra have been analyzed in terms of electronic structure and surface chemistry of the FeMONS materials as compared with that of the bulk. We hypothesize that surface imperfection and surface strain anisotropies in nanoparticles induce distortion and site inequivalency of the oxygen Oh sites around the Fe ion located close to the surface, resulting in an increase in the degree of multiplicity as well as in nonstoichiometric effects in FeMONS materials.

  8. Binding of Iron(III) to Organic Soils: Exafs Spectroscopy And Chemical Equilibrium Modeling

    SciTech Connect

    Gustafsson, J.P.; Persson, I.; Kleja, D.B.; Schaik, J.W.J.van

    2007-07-09

    The complexation of iron(III) to soil organic matter is important for the binding of trace metals in natural environments because of competition effects. In this study, we used extended X-ray absorption fine structure (EXAFS) spectroscopy to characterize the binding mode for iron(III) in two soil samples from organic mor layers, one of which was also treated with iron(III). In most cases the EXAFS spectra had three significant contributions, inner-core Fe-O/N interactions at about 2.02(2) angstroms, Fe-C interactions in the second scattering shell at 3.00(4) angstroms, and a mean Fe-Fe distance at 3.37(3) angstroms. One untreated sample showed features typical for iron (hydr)oxides; however, after treatment of iron(III) the EXAFS spectrum was dominated by organically complexed iron. The presence of a Fe-Fe distance in all samples showed that the major part of the organically complexed iron was hydrolyzed, most likely in a mixture of complexes with an inner core of (O{sub 5}Fe){sub 2}O and (O{sub 5}Fe){sub 3}O. These results were used to constrain a model for metal-humic complexation, the Stockholm Humic Model (SHM). The model was able to describe iron(III) binding very well at low pH considering only one dimeric iron(III)-humic complex. The competition effect on trace metals was also well described.

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

    PubMed

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

    2004-06-01

    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

  10. Metagenomic and PCR-Based Diversity Surveys of [FeFe]-Hydrogenases Combined with Isolation of Alkaliphilic Hydrogen-Producing Bacteria from the Serpentinite-Hosted Prony Hydrothermal Field, New Caledonia

    PubMed Central

    Mei, Nan; Postec, Anne; Monnin, Christophe; Pelletier, Bernard; Payri, Claude E.; Ménez, Bénédicte; Frouin, Eléonore; Ollivier, Bernard; Erauso, Gaël; Quéméneur, Marianne

    2016-01-01

    High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (~11), low-temperature (< 40°C), and low-salinity fluids are discharged in both intertidal and shallow submarine environments. In this study, we investigated the diversity and distribution of potentially hydrogen-producing bacteria in Prony hyperalkaline springs by using metagenomic analyses and different PCR-amplified DNA sequencing methods. The retrieved sequences of hydA genes, encoding the catalytic subunit of [FeFe]-hydrogenases and, used as a molecular marker of hydrogen-producing bacteria, were mainly related to those of Firmicutes and clustered into two distinct groups depending on sampling locations. Intertidal samples were dominated by new hydA sequences related to uncultured Firmicutes retrieved from paddy soils, while submarine samples were dominated by diverse hydA sequences affiliated with anaerobic and/or thermophilic submarine Firmicutes pertaining to the orders Thermoanaerobacterales or Clostridiales. The novelty and diversity of these [FeFe]-hydrogenases may reflect the unique environmental conditions prevailing in the PHF (i.e., high-pH, low-salt, mesothermic fluids). In addition, novel alkaliphilic hydrogen-producing Firmicutes (Clostridiales and Bacillales) were successfully isolated from both intertidal and submarine PHF chimney samples. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce hydrogen by fermentation, potentially contributing to the molecular hydrogen balance in situ. PMID:27625634

  11. How Formaldehyde Inhibits Hydrogen Evolution by [FeFe]-Hydrogenases: Determination by ¹³C ENDOR of Direct Fe-C Coordination and Order of Electron and Proton Transfers.

    PubMed

    Bachmeier, Andreas; Esselborn, Julian; Hexter, Suzannah V; Krämer, Tobias; Klein, Kathrin; Happe, Thomas; McGrady, John E; Myers, William K; Armstrong, Fraser A

    2015-04-29

    Formaldehyde (HCHO), a strong electrophile and a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases, is used to identify the point in the catalytic cycle at which a highly reactive metal-hydrido species is formed. Investigations of the reaction of Chlamydomonas reinhardtii [FeFe]-hydrogenase with formaldehyde using pulsed-EPR techniques including electron-nuclear double resonance spectroscopy establish that formaldehyde binds close to the active site. Density functional theory calculations support an inhibited super-reduced state having a short Fe-(13)C bond in the 2Fe subsite. The adduct forms when HCHO is available to compete with H(+) transfer to a vacant, nucleophilic Fe site: had H(+) transfer already occurred, the reaction of HCHO with the Fe-hydrido species would lead to methanol, release of which is not detected. Instead, Fe-bound formaldehyde is a metal-hydrido mimic, a locked, inhibited form analogous to that in which two electrons and only one proton have transferred to the H-cluster. The results provide strong support for a mechanism in which the fastest pathway for H2 evolution involves two consecutive proton transfer steps to the H-cluster following transfer of a second electron to the active site. PMID:25871921

  12. Metagenomic and PCR-Based Diversity Surveys of [FeFe]-Hydrogenases Combined with Isolation of Alkaliphilic Hydrogen-Producing Bacteria from the Serpentinite-Hosted Prony Hydrothermal Field, New Caledonia.

    PubMed

    Mei, Nan; Postec, Anne; Monnin, Christophe; Pelletier, Bernard; Payri, Claude E; Ménez, Bénédicte; Frouin, Eléonore; Ollivier, Bernard; Erauso, Gaël; Quéméneur, Marianne

    2016-01-01

    High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (~11), low-temperature (< 40°C), and low-salinity fluids are discharged in both intertidal and shallow submarine environments. In this study, we investigated the diversity and distribution of potentially hydrogen-producing bacteria in Prony hyperalkaline springs by using metagenomic analyses and different PCR-amplified DNA sequencing methods. The retrieved sequences of hydA genes, encoding the catalytic subunit of [FeFe]-hydrogenases and, used as a molecular marker of hydrogen-producing bacteria, were mainly related to those of Firmicutes and clustered into two distinct groups depending on sampling locations. Intertidal samples were dominated by new hydA sequences related to uncultured Firmicutes retrieved from paddy soils, while submarine samples were dominated by diverse hydA sequences affiliated with anaerobic and/or thermophilic submarine Firmicutes pertaining to the orders Thermoanaerobacterales or Clostridiales. The novelty and diversity of these [FeFe]-hydrogenases may reflect the unique environmental conditions prevailing in the PHF (i.e., high-pH, low-salt, mesothermic fluids). In addition, novel alkaliphilic hydrogen-producing Firmicutes (Clostridiales and Bacillales) were successfully isolated from both intertidal and submarine PHF chimney samples. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce hydrogen by fermentation, potentially contributing to the molecular hydrogen balance in situ. PMID:27625634

  13. The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. I. Light sensitivity and magnetic hyperfine interactions as observed by electron paramagnetic resonance.

    PubMed

    Albracht, Simon P J; Roseboom, Winfried; Hatchikian, E Claude

    2006-01-01

    The hydrogen-activating cluster (H cluster) in [FeFe]-hydrogenases consists of two moieties. The [2Fe]H subcluster is a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) centre. The Cys-bound Fe is called Fe1, the other iron Fe2. The Cys-thiol forms a bridge to a [4Fe-4S] cluster, the [4Fe-4S]H subcluster. We report that electron paramagnetic resonance (EPR) spectra of the 57Fe-enriched enzyme from Desulfovibrio desulfuricans in the H(ox)-CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster. In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light. The [2Fe]H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the H(ox)-CO state giving rise to the so-called axial 2.06 EPR signal. Though not destroyed by light, the H(ox)-CO state is affected by it. As demonstrated in the accompanying paper [49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic 13CO during illumination at 2 degrees C. We found that only one of the three 13COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 0.6 mT. At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed. EPR spectra of the photolysed products are consistent with a 3d7 system of Fe with the formal oxidation state +1. The damaged enzyme shows a light-sensitive g = 5 signal which is ascribed to an S = 3/2 form of the [2Fe](H) subcluster. The light sensitivity of the enzyme explains the occurrence of the g = 5 signal and the axial 2.06 signal in published EPR spectra of nearly all preparations studied thus far. PMID:16323020

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

    PubMed Central

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

    2008-01-01

    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(μ-SPh)3Ni(μ-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(μ-StBu)3Ni(μ-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(μ-StBu)3NiBr(tmtu), 3, (CO)3Fe(StBu)(μ-StBu)2Ni{S(CH2)2SMe}, 4, and (CO)3Fe(StBu)(μ-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(μ-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)(μ-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(μ-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

  15. A computational library for multiscale modeling of material failure

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    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.

  16. Approaches to developing biological H(2)-photoproducing organisms and processes.

    PubMed

    Ghirardi, M L; King, P W; Posewitz, M C; Maness, P Ching; Fedorov, A; Kim, K; Cohen, J; Schulten, K; Seibert, M

    2005-02-01

    The development of efficient biological systems for the direct photoproduction of H(2) gas from water faces several challenges, the more serious of which is the sensitivity of the H(2)-evolving enzymes (hydrogenases) to O(2), an obligatory by-product of photosynthesis. This high sensitivity is common to both FeFe and NiFe hydrogenases, and is caused by O(2) binding to their respective metallocatalytic sites. This overview describes approaches to (i) molecular engineering of algal FeFe-hydrogenase to prevent O(2) access to its catalytic site; (ii) transform a cyanobacterium with an O(2)-tolerant bacterial NiFe hydrogenase or (c) partially inactivate algal O(2)-evolution activity to create physiologically anaerobiosis and induce hydrogenase expression. PMID:15667268

  17. Photochemical Hydrogen Generation Initiated by Oxidative Quenching of the Excited Ru(bpy)3 (2+) * by a Bio-Inspired [2Fe2S] Complex.

    PubMed

    Na, Yong; Wei, Peicheng; Zhou, Li

    2016-07-18

    A diiron dithiolate complex 1 containing 1,8-naphthalic anhydride bridge was prepared, which possessed the lowest reduction potential for the synthetic diiron complexes modeled on the active site of [FeFe] hydrogenase reported so far. For the first time, oxidative quenching of the excited Ru(bpy)3 (2+) * through electron transfer to a bio-inspired [2Fe2S] complex was corroborated. Hydrogen evolution, driven by visible light, was successfully observed for a three-component system, consisting of Ru(bpy)3 (2+) , complex 1, and EDTA as electron donor in aqueous/organic media. These results provide a basis and also opportunity to develop a photo water splitting system employing Fe-based catalysts without sacrificial electron donors. PMID:26879325

  18. The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy.

    PubMed

    Roseboom, Winfried; De Lacey, Antonio L; Fernandez, Victor M; Hatchikian, E Claude; Albracht, Simon P J

    2006-01-01

    In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H(inact) state) and the CO-inhibited active form (H(ox)-CO state) were stable in light. Illumination of the H(ox) state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H(ox)-CO state. Illumination of active enzyme under 13CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H(ox)-CO state. Electrochemical redox titrations showed that the enzyme in the H(inact) state converts to the transition state (H(trans)) in a reversible one-electron redox step (E (m, pH 7) = -75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]H subcluster, but also the di-iron centre. Enzyme in the H(trans) state reacts with extrinsic CO, which binds to Fe2. The H(trans) state converts irreversibly into the H(ox) state in a redox-dependent reaction most likely involving two electrons (E (m, pH 7) = -261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H(red) state (E (m, pH 7) = -354 mV), where both Fe atoms of the [2Fe]H subcluster

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

    PubMed Central

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

    2008-01-01

    This report describes routes to iron dithiolato carbonyls that do not require preformed iron carbonyls. The reaction of FeCl2, Zn, and Q2S2CnH2n (Q+ = Na+, Et3NH+) under an atmosphere of CO affords Fe2(S2CnH2n)(CO)6 (n = 2, 3) in yields >70%. The method was employed to prepare Fe2(S2C2H4)(13CO)6. Treatment of these carbonylated mixtures with tertiary phosphines gave the ferrous species Fe3(S2C3H6)3(CO)4(PR3)2, for R = Et, Bu, and Ph. Like the related complex Fe3(SPh)6(CO)6, these compounds consist of a linear arrangement of three conjoined face-shared octahedral centers. Omitting the phosphine but with an excess of dithiolate, we obtained the related mixed-valence triiron species [Fe3(S2CnH2n)4(CO)4]−. The highly reducing all-ferrous species [Fe3(S2CnH2n)4(CO)4]2− is implicated as an intermediate in this transformation. Reactive forms of iron, prepared by the method of Rieke, also combined with dithiols under a CO atmosphere to give Fe2(S2CnH2n)(CO)6 in modest yields under mild conditions. Studies on the order of addition indicate that ferrous thiolates are formed prior to the onset of carbonylation. Crystallographic characterization demonstrated that the complexes Fe3(S2C3H6)3(CO)4(PEt3)2 and PBnPh3[Fe3(S2C3H6)4(CO)4] feature high spin ferrous and low spin ferric as the central metal, respectively. PMID:18610969

  20. Precursors to [FeFe]-hydrogenase models: syntheses of Fe2(SR)2(CO)6 from CO-free iron sources.

    PubMed

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

    2008-08-01

    This report describes routes to iron dithiolato carbonyls that do not require preformed iron carbonyls. The reaction of FeCl 2, Zn, and Q 2S 2C n H 2 n (Q (+) = Na (+), Et 3NH (+)) under an atmosphere of CO affords Fe 2(S 2C n H 2 n )(CO) 6 ( n = 2, 3) in yields >70%. The method was employed to prepare Fe 2(S 2C 2H 4)( (13)CO) 6. Treatment of these carbonylated mixtures with tertiary phosphines, instead of Zn, gave the ferrous species Fe 3(S 2C 3H 6) 3(CO) 4(PR 3) 2, for R = Et, Bu, and Ph. Like the related complex Fe 3(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 [Fe 3(S 2C n H 2 n ) 4(CO) 4] (-). The highly reducing all-ferrous species [Fe 3(S 2C n H 2 n ) 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 Fe 2(S 2C n H 2 n )(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 Fe 3(S 2C 3H 6) 3(CO) 4(PEt 3) 2 and PBnPh 3[Fe 3(S 2C 3H 6) 4(CO) 4] feature high-spin ferrous and low-spin ferric as the central metal, respectively. PMID:18610969

  1. The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation.

    PubMed

    Morra, Simone; Maurelli, Sara; Chiesa, Mario; Mulder, David W; Ratzloff, Michael W; Giamello, Elio; King, Paul W; Gilardi, Gianfranco; Valetti, Francesca

    2016-01-01

    A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid causes significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics. PMID:26482707

  2. Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation

    SciTech Connect

    Morra, Simone; Maurelli, Sara; Chiesa, Mario; Mulder, David W.; Ratzloff, Michael W.; Giamello, Elio; King, Paul W.; Gilardi, Gianfranco; Valettia, Francesca

    2016-01-01

    A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid caused significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.

  3. Protein environmental effects on iron-sulfur clusters: A set of rules for constructing computational models for inner and outer coordination spheres.

    PubMed

    Harris, Travis V; Szilagyi, Robert K

    2016-07-01

    The structural properties and reactivity of iron-sulfur proteins are greatly affected by interactions between the prosthetic groups and the surrounding amino acid residues. Thus, quantum chemical investigations of the structure and properties of protein-bound iron-sulfur clusters can be severely limited by truncation of computational models. The aim of this study was to identify, a priori, significant interactions that must be included in a quantum chemical model. Using the [2Fe-2S] accessory cluster of the FeFe-hydrogenase as a demonstrative example with rich electronic structural features, the electrostatic and covalent effects of the surrounding side chains, charged groups, and backbone moieties were systematically mapped through density functional theoretical calculations. Electron affinities, spin density differences, and delocalization indexes from the quantum theory of atoms in molecules were used to evaluate the importance of each interaction. Case studies for hydrogen bonding and charged side-chain interactions were used to develop selection rules regarding the significance of a given protein environmental effect. A set of general rules is proposed for constructing quantum chemical models for iron-sulfur active sites that capture all significant interactions from the protein environment. This methodology was applied to our previously used models in galactose oxidase and the 6Fe-cluster of FeFe-hydrogenase. © 2016 Wiley Periodicals, Inc. PMID:27117497

  4. Principles of sustained enzymatic hydrogen oxidation in the presence of oxygen--the crucial influence of high potential Fe-S clusters in the electron relay of [NiFe]-hydrogenases.

    PubMed

    Evans, Rhiannon M; Parkin, Alison; Roessler, Maxie M; Murphy, Bonnie J; Adamson, Hope; Lukey, Michael J; Sargent, Frank; Volbeda, Anne; Fontecilla-Camps, Juan C; Armstrong, Fraser A

    2013-02-20

    "Hyd-1", produced by Escherichia coli , exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H(2) oxidation activity in the presence of O(2)-an intruder that normally incapacitates the sulfur- and electron-rich active site. The mechanism of "O(2) tolerance" involves a critical role for the Fe-S clusters of the electron relay, which is to ensure the availability-for immediate transfer back to the active site-of all of the electrons required to reduce an attacking O(2) molecule completely to harmless H(2)O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 Å from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H(2) oxidation activity in 1% O(2). The [3Fe-4S] cluster is essential only for the first stage of complete O(2) reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state "Ni-B" or "Ready" (Ni(III)-OH). Potentiometric titrations show that Ni-B is easily reduced (E(m) ≈ +0.1 V at pH 6.0); this final stage of the O(2)-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme's response to O(2), viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H(2) oxidation activity; thus, O(2) behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe-S clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase. PMID

  5. Modelling the Thermal History of Asteroid 4 Vesta

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

    The asteroid 4 Vesta is widely thought to be the source of the HED (Howardite, Eucrite and Diogenite) meteorites, with this link supported by spectroscopic and dynamical studies. The availability of the HED meteorites for study and the new data being gained from the Dawn mission provides an excellent opportunity to investigate Vesta s history. In this study, modelling of Vesta has been undertaken to investigate its evolution from an unconsolidated chondritic body to a differentiated body with an iron core. In contrast to previous modelling, both heat and mass transfer are considered as coupled processes. This work draws on models of melt segregation in terrestrial environments to inform the evolution of Vesta into the differentiated body observed today. In order for a core to form in this body, a separation of the metallic iron from the silicates must take place. Temperatures in excess of the solidus temperatures for the Fe-FeS system and the silicates are therefore required. Thermal modelling has shown accretion before 2Myr leads to temperatures in excess of the silicate solidus.

  6. Effect of Lewis acid on the structure of a diiron dithiolate complex based on the active site of [FeFe]-hydrogenase assessed by density functional theory.

    PubMed

    Lee, Jin Woo; Jo, Won Ho

    2009-10-28

    The effect of Lewis acid on the structure and H2 productivity of a diiron dithiolate complex was investigated by using density functional theory (DFT) calculations. When a model molecule of [(CH3SH)(CO)2Fe(p)(mu-SCH2NHCH2S)Fe(d)(CO)3] was geometrically optimized, two isomers were found: one is the unrotated structure (1) with no ligand between two Fe atoms and the other is the rotated structure (1*) with one CO ligand between two Fe atoms. The energy of 1* was higher than 1 by 6.4 kcal/mol in a vacuum. DFT calculations also revealed that all Lewis acids bound to the rotated structure more strongly than to the unrotated structure, leading to the stabilization of the rotated structure. In particular, when AlCl3 is used, the rotated structure (1*/AlCl3) is more stable than the unrotated one (1/AlCl3) by 1.2 kcal/mol in a vacuum. The stabilization of the rotated structure arises from both the stronger basicity of the mu-CO ligand than the axial CO ligand and the increase of the bond strength between the mu-CO ligand and Fe(p) atom upon binding of Lewis acid to 1*. Calculation of energy barriers during electrocatalytic H2 production revealed that 1*/AlCl3 could efficiently produce H2via a chemical-electrochemical-chemical-electrochemical mechanism. The analysis of the energy level of the lowest unoccupied molecular orbital showed that 1*/AlCl3 may produce H2 at significantly lower reduction potential as compared with 1*. It is also found that the catalytic activity decreases with increasing polarity of the medium. PMID:19809728

  7. Numerical Modeling Studies of Thermospheric Metal Layers Driven by Gravity Waves

    NASA Astrophysics Data System (ADS)

    Yu, Z.; Chu, X.

    2013-12-01

    As the lower boundary of ionosphere and space weather regime, the mesosphere and thermosphere is a chemically and dynamically complex and important region. The roles of atmospheric gravity waves in transporting energy and momentum and causing atmospheric and ionospheric disturbances have been recognized by theoretical studies and observational evidence. The thermospheric neutral Fe layers discovered by the Chu lidar group at McMurdo (77.8S, 166.7E), Antarctica, exhibit well defined gravity wave signatures in the altitude range of 110~155 km. Those thermospheric Fe layers provide an excellent trace for measuring neutral temperature and winds in the thermosphere. Our theory argues that the observed Fe layers are a result of coupling of electrodynamical, neutral dynamical and chemical processes. The thermospheric Fe atoms are produced by neutralization of converged Fe+ layers driven by gravity wave wind shear. Although a qualitative understanding has been offered in a paper by Chu et al., the quantitative understanding of the neutral Fe layer formation and wave structures is unresolved. Meanwhile, the chemical process is highly coupled with the electrodynamic and thermodynamic processes in the E-F regions, which leads to more difficulties to understand the new observations. Such challenges stimulate our development of a numerical model. A time-dependent, 1-D, high-latitude Fe/Fe+ model has been developed to simulate the observed Fe profiles based on the first principles of physics and chemistry. The model solves ions' motion explicitly taking the full Fe chemistry and ambient ions chemistry in the E-F region into account. In this paper, we will demonstrate that gravity wave wind shear creates Fe+ layers of enhanced density that produce neutral Fe layers consistent with observations. Besides the wind-shear mechanism, electric field driving force is also important for transporting and converging Fe+ at high latitudes. Electric field can cause divergent or convergent ion

  8. Using Stable Isotopes to Trace Microbial Hydrogen Production Pathways

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

    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

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

    PubMed

    Williams, C R; Bees, M A

    2014-02-01

    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. PMID:24026984

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

    PubMed Central

    Williams, C R; Bees, MA

    2014-01-01

    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

  11. Iron-sulfur clusters—new features in enzymes and synthetic models

    NASA Astrophysics Data System (ADS)

    Bill, Eckhard

    2012-03-01

    Mössbauer spectroscopy is very important for the characterization of iron sulfur clusters in biological and synthetic molecules. The electric and magnetic hyperfine parameters obtained for 57Fe provide valuable information about the electronic structure of the different iron sites occurring in Fe:S clusters. Although known since more than four decades, research in this field is very active, revealing unexpected functions, structures and redox states. In this overview, new aspects of double exchange and vibronic coupling in a structurally well-characterized two-iron model compound are discussed, the electronic structure of extremely reduced clusters with all iron in ferrous or even in iron(I) state is elucidated, and an exciting new type of cubane cluster occurring in oxygen-insensitive hydrogenases is presented. The latter cluster involves structural changes during function and it supports more than one redox transition, which may be essential for oxygen protection of the enzymes.

  12. Photoactive Metal-Organic Framework and Its Film for Light-Driven Hydrogen Production and Carbon Dioxide Reduction.

    PubMed

    Wu, Pengyan; Guo, Xiangyang; Cheng, Linjuan; He, Cheng; Wang, Jian; Duan, Chunying

    2016-08-15

    The design of a new photocatalytic system and integrating the essential components in a structurally controlled manner to create artificially photosynthetic systems is high desirable. By incorporating a photoactive triphenylamine moiety to assemble a Gd-based metal-organic framework as a heterogeneous photosensitizer, new artificial systems were constructed for the proton and carbon dioxide reduction under irradiation. The assembled MOFs exhibited a one-dimensional metal-oxygen pillar that was connected together by the depronated TCA(3-) ligands to form a three-dimensional noninterpenetrating porous framework. The combining of proton reduction and/or the carbon dioxide reduction catalysts, i.e., the Fe-Fe hydrogenase active site models and the Ni(Cyclam) complexes, initiated a photoinduced single electron transfer from its excited state to the substrate. The system exhibited an initial TOF of 320 h(-1) of hydrogen per catalyst and an overall quantum yield of about 0.21% and is able to reduce carbon dioxide under irradiation. The deposit of the photoactive Gd-TCA into the film of an α-Al2O3 plate provided a platform for the practical applications through prolonging the lifetime of the artifical system and allowed the easily operated devices being recyclable as a promising photocatalytic system. PMID:27479135

  13. Lidar Observations and Numerical Modeling Studies of Thermospheric Metal Layers and Solar Effects on Mesospheric Fe Layers

    NASA Astrophysics Data System (ADS)

    Yu, Zhibin

    By blocking extreme hazards from space and regulating radio wave propagation, the space-atmosphere interaction region (SAIR) -- our window to open space -- is essential for life on Earth and modern society. However, the physical and chemical processes governing the SAIR are not sufficiently understood due to the woefully incomplete measurements of neutral properties in this region, especially between 100 and 200 km altitude. Thermospheric Fe layers extending from ~70 to 170 km discovered by the Fe Boltzmann lidar at McMurdo, Antarctica have opened a new door to observing the neutral thermosphere and mesosphere. This thesis is aimed at revealing such new discoveries, and advancing our understanding of the thermospheric Fe layer formation, through analyzing the lidar data collected by the author in Antarctic winter and developing the first thermospheric Fe/Fe+ model. A one-dimensional high-latitude Fe/Fe+ model based on physical and chemical first principles has been developed to quantitatively explore the source, formation and evolution of thermospheric Fe layers. We demonstrate that the observed Fe layers are produced by neutralization of converged Fe+, mainly through the direct electron-Fe+ recombination. We find that the polar electric field is capable of uplifting Fe+ ions from the main deposition region into the thermosphere, supplying the source of neutral Fe. Both gravity-wave-induced wind shears and the polar electric field can converge Fe+ layers. Vertical wind plays a key role in transporting Fe to form the observed wave structures, but horizontal divergence can largely offset the vertical convergence effects. These theoretical studies lay the foundation for exploring the thermosphere by resonance lidars. The diurnal variations of Fe layers in the mesopause region are characterized with our lidar observations at McMurdo. A new finding is the solar effect on the Fe layer bottomside --- daytime downward extension and nighttime upward contraction. We explain

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

    SciTech Connect

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

    2006-05-04

    Abstract Advances in DNA microarray and proteomics technologies have enabled high-throughput measurement of mRNA expression and protein abundance. Parallel profiling of mRNA and protein on a global scale and integrative analysis of these two data types could provide additional insight into the metabolic mechanisms underlying complex biological systems. However, because protein abundance and mRNA expression are affected by many cellular and physical processes, there have been conflicting results on the correlation of these two measurements. In addition, as current proteomic methods can detect only a small fraction of proteins present in cells, no correlation study of these two data types has been done thus far at the whole-genome level. In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), Zero-inflated Poisson models were used to define the correlation pattern of mRNA and protein abundance. The models assumed that there is a probability mass at zero representing some of the undetected proteins because of technical limitations. The models thus use abundance measurements of transcripts and proteins experimentally detected as input to generate predictions of protein abundances as output for all genes in the genome. We demonstrated the statistical models by comparatively analyzing D. vulgaris grown on lactate-based versus formate-based media. The increased expressions of Ech hydrogenase and alcohol dehydrogenase (Adh)-periplasmic Fe-only hydrogenase (Hyd) pathway for ATP synthesis were predicted for D. vulgaris grown on formate.

  15. Contributing factors in the improvement of cellulosic H2 production in Clostridium thermocellum/Thermoanaerobacterium co-cultures.

    PubMed

    Wang, Mingyu; Zhao, Qi; Li, Ling; Niu, Kangle; Li, Yi; Wang, Fangzhong; Jiang, Baojie; Liu, Kuimei; Jiang, Yi; Fang, Xu

    2016-10-01

    Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium. PMID:27538932

  16. Molecular Models for Conductance in Junctions and Electrochemical Electron Transfer

    NASA Astrophysics Data System (ADS)

    Mazinani, Shobeir Khezr Seddigh

    This thesis develops molecular models for electron transport in molecular junctions and intra-molecular electron transfer. The goal is to identify molecular descriptors that afford a substantial simplification of these electronic processes. First, the connection between static molecular polarizability and the molecular conductance is examined. A correlation emerges whereby the measured conductance of a tunneling junction decreases as a function of the calculated molecular polarizability for several systems, a result consistent with the idea of a molecule as a polarizable dielectric. A model based on a macroscopic extension of the Clausius-Mossotti equation to the molecular domain and Simmon's tunneling model is developed to explain this correlation. Despite the simplicity of the theory, it paves the way for further experimental, conceptual and theoretical developments in the use of molecular descriptors to describe both conductance and electron transfer. Second, the conductance of several biologically relevant, weakly bonded, hydrogen-bonded systems is systematically investigated. While there is no correlation between hydrogen bond strength and conductance, the results indicate a relation between the conductance and atomic polarizability of the hydrogen bond acceptor atom. The relevance of these results to electron transfer in biological systems is discussed. Hydrogen production and oxidation using catalysts inspired by hydrogenases provides a more sustainable alternative to the use of precious metals. To understand electrochemical and spectroscopic properties of a collection of Fe and Ni mimics of hydrogenases, high-level density functional theory calculations are described. The results, based on a detailed analysis of the energies, charges and molecular orbitals of these metal complexes, indicate the importance of geometric constraints imposed by the ligand on molecular properties such as acidity and electrocatalytic activity. Based on model calculations of

  17. Shock compression of Fe-FeS mixture up to 204 GPa

    NASA Astrophysics Data System (ADS)

    Huang, Haijun; Wu, Shijie; Hu, Xiaojun; Wang, Qingsong; Wang, Xiang; Fei, Yingwei

    2013-02-01

    AbstractUsing a two-stage light gas gun, we obtained new shock wave Hugoniot data for an iron-sulfur alloy (Fe-11.8wt%S) over the pressure range of 94-204 GPa. A least-squares fit to the Hugoniot data yields a linear relationship between shock velocity DS and particle velocity u, DS (km/s) =3.60(0.14) +1.57(0.05) u. The measured Hugoniot data for Fe-11.8wt%S agree well with the calculated results based on the thermodynamic parameters of Fe and FeS using the additive law. By comparing the calculated densities along the adiabatic core temperature with the PREM density profile, an iron core with 10 wt.% sulfur (S) provides the best solution for the composition of the Earth's outer core.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2014PEPI..228...92W','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2014PEPI..228...92W"><span id="translatedtitle">Elastic properties of iron-bearing wadsleyite to 17.7 GPa: Implications for mantle mineral <span class="hlt">models</span></span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Wang, Jingyun; Bass, Jay D.; Kastura, Tomoo</p> <p>2014-03-01</p> <p>The sound velocities and single-crystal elastic moduli of iron-bearing wadsleyite with [<span class="hlt">Fe]/[Fe</span> + Mg] molar ratio of 0.075 have been measured by Brillouin scattering experiments at high pressures up to 17.7 GPa. Pressure derivatives for the adiabatic bulk modulus (KS0) and shear modulus (μ0) are 4.1(1) and 1.45(4), respectively. A comparison of our results with previous Brillouin scattering results on the Mg end-member wadsleyite shows that incorporating 7.5 mol% iron in wadsleyite at high-pressure conditions decreases the shear moduli by ∼4-5%, but does not have a discernable effect on the bulk modulus. The effects of iron on the elastic moduli of wadsleyite at ambient pressure persist to high-pressure conditions at a relatively constant level. Using our results on iron-bearing wadsleyite at high pressure, we conclude that less olivine than in the pyrolite <span class="hlt">model</span> of mantle composition provides a satisfactory explanation for 410 km seismic discontinuity at the top of the transition zone.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1828657','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1828657"><span id="translatedtitle">Hydrogen Metabolism in Shewanella oneidensis MR-1▿</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Meshulam-Simon, Galit; Behrens, Sebastian; Choo, Alexander D.; Spormann, Alfred M.</p> <p>2007-01-01</p> <p>Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 <span class="hlt">hydrogenases</span>, hydA, encoding a periplasmic [<span class="hlt">Fe-Fe</span>] <span class="hlt">hydrogenase</span>, and hyaB, encoding a periplasmic [Ni-Fe] <span class="hlt">hydrogenase</span>, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming <span class="hlt">hydrogenase</span> while HyaB has a bifunctional role and represents the dominant <span class="hlt">hydrogenase</span> activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA <span class="hlt">hydrogenase</span> and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions. PMID:17189435</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/913611','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/913611"><span id="translatedtitle"><span class="hlt">Modeling</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Loth, E.; Tryggvason, G.; Tsuji, Y.; Elghobashi, S. E.; Crowe, Clayton T.; Berlemont, A.; Reeks, M.; Simonin, O.; Frank, Th; Onishi, Yasuo; Van Wachem, B.</p> <p>2005-09-01</p> <p>Slurry flows occur in many circumstances, including chemical manufacturing processes, pipeline transfer of coal, sand, and minerals; mud flows; and disposal of dredged materials. In this section we discuss slurry flow applications related to radioactive waste management. The Hanford tank waste solids and interstitial liquids will be mixed to form a slurry so it can be pumped out for retrieval and treatment. The waste is very complex chemically and physically. The ARIEL code is used to <span class="hlt">model</span> the chemical interactions and fluid dynamics of the waste.</p> </li> </ol> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_19");'>19</a></li> <li><a href="#" onclick='return showDiv("page_20");'>20</a></li> <li class="active"><span>21</span></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div><!-- col-sm-12 --> </div><!-- row --> </div><!-- page_21 --> <div id="page_22" class="hiddenDiv"> <div class="row"> <div class="col-sm-12"> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_20");'>20</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li class="active"><span>22</span></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li><a href="#" onclick='return showDiv("page_24");'>24</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div> </div> <div class="row"> <div class="col-sm-12"> <ol class="result-class" start="421"> <li> <p><a target="_blank" onclick="trackOutboundLink('http://pubs.er.usgs.gov/publication/70015588','USGSPUBS'); return false;" href="http://pubs.er.usgs.gov/publication/70015588"><span id="translatedtitle">Cluster molecular orbital description of the electronic structures of mixed-valence iron oxides and silicates</span></a></p> <p><a target="_blank" href="http://pubs.er.usgs.gov/pubs/index.jsp?view=adv">USGS Publications Warehouse</a></p> <p>Sherman, David M.</p> <p>1986-01-01</p> <p>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 <span class="hlt">FeFe</span> bonding across shared edges of FeO6 coordination polyhedra. In agreement with Zener's double exchange <span class="hlt">model</span>, <span class="hlt">FeFe</span> bonding is found to stabilize ferromagnetic coupling between Fe2+ and Fe3+ cations. ?? 1986.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/1175631','DOE-PATENT-XML'); return false;" href="http://www.osti.gov/scitech/servlets/purl/1175631"><span id="translatedtitle">Hydrogen production using <span class="hlt">hydrogenase</span>-containing oxygenic photosynthetic organisms</span></a></p> <p><a target="_blank" href="http://www.osti.gov/doepatents">DOEpatents</a></p> <p>Melis, Anastasios; Zhang, Liping; Benemann, John R.; Forestier, Marc; Ghirardi, Maria; Seibert, Michael</p> <p>2006-01-24</p> <p>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.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/894277','DOE-PATENT-XML'); return false;" href="http://www.osti.gov/scitech/biblio/894277"><span id="translatedtitle">Hydrogen Production Using <span class="hlt">Hydrogenase</span>-Containing Oxygenic Photosynthetic Organisms</span></a></p> <p><a target="_blank" href="http://www.osti.gov/doepatents">DOEpatents</a></p> <p>Melis, A.; Zhang, L.; Benemann, J. R.; Forestier, M.; Ghirardi, M.; Seibert, M.</p> <p>2006-01-24</p> <p>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.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/1068602','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/1068602"><span id="translatedtitle">Survey of <span class="hlt">Hydrogenase</span> Activity in Algae: Final Report</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Brand, J. J.</p> <p>1982-04-01</p> <p>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.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/cgi-bin/nph-data_query?bibcode=2014PhRvB..89b4101P&link_type=ABSTRACT','NASAADS'); return false;" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?bibcode=2014PhRvB..89b4101P&link_type=ABSTRACT"><span id="translatedtitle">First-principles study of point defects in an fcc Fe-10Ni-20Cr <span class="hlt">model</span> alloy</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Piochaud, J. B.; Klaver, T. P. C.; Adjanor, G.; Olsson, P.; Domain, C.; Becquart, C. S.</p> <p>2014-01-01</p> <p>The influence of the local environment on vacancy and self-interstitial formation energies has been investigated in a face-centered-cubic (fcc) Fe-10Ni-20Cr <span class="hlt">model</span> alloy by analyzing an extensive set of first-principle calculations based on density functional theory. Chemical disorder has been considered by designing special quasirandom structures and four different collinear magnetic structures have been investigated in order to determine a relevant reference state to perform point defect calculations at 0 K. Two different convergence methods have also been used to characterize the importance of the method on the results. Although our fcc Fe-10Ni-20Cr would be better represented in terms of applications by the paramagnetic state, we found that the antiferromagnetic single-layer magnetic structure was the most stable at 0 K and we chose it as a reference state to determine the point defect properties. Point defects have been introduced in this reference state, i.e., vacancies and <span class="hlt">Fe-Fe</span>, Fe-Ni, Fe-Cr, Cr-Cr, Ni-Ni, and Ni-Cr dumbbell interstitials oriented either parallel or perpendicular to the single layer antiferromagnetic planes. Each point defect studied was introduced at different lattice sites to consider a sufficient variety of local environments and analyze its influence on the formation energy values. We have estimated the point defect formation energies with linear regressions using variables which describe the local environment surrounding the point defects. The number and the position of Ni and Cr first nearest neighbors to the point defects were found to drive the evolution of the formation energies. In particular, Ni is found to decrease and Cr to increase the vacancy formation energy of the <span class="hlt">model</span> alloy, while the opposite trends are found for the dumbbell interstitials. This study suggested that, to a first approximation, the first nearest atoms to point defects can provide reliable estimates of point defect formation energies.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/5694589','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/5694589"><span id="translatedtitle">Geochemical <span class="hlt">modeling</span> of low melt-fraction anatexis in a peraluminous system: The Pena Negra complex (central Spain)</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Bea, F. )</p> <p>1991-07-01</p> <p>A study was made of the chemical fractionation associated with four cases of anatectic segregation of low melt-fraction cordieritic granites from migmatized meta-greywackes. The aims of the study were to (1) reveal the fractionation patterns of major and trace elements, (2) compare the major element chemistry of leucogranites and the quantitative behavior of source minerals during anatexis - inferred by mass-balance adjustment - with available experimental data for peraluminous systems, and (3) discuss the behavior of trace elements in crustal melting by comparing the chemically determined composition of leucogranites with the results of three fractionation <span class="hlt">models</span>. Two of these assume a perfect diffusive behavior of trace elements within residual solids, but they use a different set of distribution coefficients. The third assumes a perfect nondiffusive behavior. In relation to their source rocks, the leucogranites are strongly depleted in Li, Transition Elements, and Light Rare Earth Elements, but enriched in K{sub 2}O, SiO{sub 2}, and Ba. Mass balance analysis using the Anatexis Mixing <span class="hlt">Model</span> shows that the chemistry of cordierite leucogranites is compatible with its having originated by closed-system, water-undersaturated anatexis on previously migmatized meta-greywackes, leaving a residue enriched in cordierite plus biotite and exhausted in K-feldspar. Biotite melts congruently unless important amounts of sillimanite were also present in the source. Compared with experimental metals obtained from sources with the same chemical composition but with a different femic mineralogy (biotite + sillimanite, instead of cordierite + biotite), the Pena Negra leucogranites are richer in K{sub 2}O and MgO with a lower <span class="hlt">Fe/(Fe</span> + Mg) ratio. The differences in magnesium are believed to result from the changes in the mineral assemblage of the source rocks.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://ntrs.nasa.gov/search.jsp?R=19870064649&hterms=donation&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DAll%26N%3D0%26No%3D10%26Ntt%3Ddonation','NASA-TRS'); return false;" href="http://ntrs.nasa.gov/search.jsp?R=19870064649&hterms=donation&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DAll%26N%3D0%26No%3D10%26Ntt%3Ddonation"><span id="translatedtitle">The bonding of FeN2, FeCO, and Fe2N2 - <span class="hlt">Model</span> systems for side-on bonding of CO and N2</span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Bauschlicher, Charles W., Jr.; Pettersson, Lars G. M.; Siegbahn, Per E. M.</p> <p>1987-01-01</p> <p>Qualitative calculations are performed to elucidate the nature of the side-on interaction of both N2 and CO with a single Fe atom. The systems are found to be quite similar, with bonding leading to an increase in the CO or N2 bond length and a decrease in the vibrational frequency. The CO or N2 stretching modes lead to a large dipole derivative along the metal-ligand bond axis. The populations show an almost identical, large donation from the Fe 3d orbitals into the CO or N2 Pi-asterisk. The larger system Fe2N2 is then considered, with the N2 bridging the Fe2, both parallel and perpendicular to the Fe2 bond axis for two different <span class="hlt">Fe-Fe</span> distances. For FeN2, the shift in the observed N2 frequency is smaller than observed for the alpha state of N2/Fe(111). The shift in the N2 vibrational frequency increases when the N2 interacts with two Fe atoms, either at the <span class="hlt">Fe-Fe</span> nearest neighbor distance or at the first layer <span class="hlt">Fe-Fe</span> distance, when the side-on N2 axis is oriented perpendicular to an <span class="hlt">Fe-Fe</span> bond.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2014AGUFM.B23B0198Y','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2014AGUFM.B23B0198Y"><span id="translatedtitle">A Tale of Two Gases: Isotope Effects Associated with the Enzymatic Production of H2 and N2O</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Yang, H.; Gandhi, H.; Kreuzer, H. W.; Moran, J.; Hill, E. A.; McQuarters, A.; Lehnert, N.; Ostrom, N. E.; Hegg, E. L.</p> <p>2014-12-01</p> <p>Stable isotopes can provide considerable insight into enzymatic mechanisms and fluxes in various biological processes. In our studies, we used stable isotopes to characterize both enzyme-catalyzed H2 and N2O production. H2 is a potential alternative clean energy source and also a key metabolite in many microbial communities. Biological H2 production is generally catalyzed by <span class="hlt">hydrogenases</span>, enzymes that combine protons and electrons to produce H2 under anaerobic conditions. In our study, H isotopes and fractionation factors (α) were used to characterize two types of <span class="hlt">hydrogenases</span>: [<span class="hlt">FeFe</span>]- and [NiFe]-<span class="hlt">hydrogenases</span>. Due to differences in the active site, the α associated with H2 production for [<span class="hlt">FeFe</span>]- and [NiFe]-<span class="hlt">hydrogenases</span> separated into two distinct clusters (αFeFe > αNiFe). The calculated kinetic isotope effects indicate that <span class="hlt">hydrogenase</span>-catalyzed H2 production has a preference for light isotopes, consistent with the relative bond strengths of O-H and H-H bonds. Interestingly, the isotope effects associated with H2 consumption and H2-H2O exchange reactions were also characterized, but in this case no specific difference was observed between the different enzymes. N2O is a potent greenhouse gas with a global warming potential 300 times that of CO2, and the concentration of N2O is currently increasing at a rate of ~0.25% per year. Thus far, bacterial and fungal denitrification processes have been identified as two of the major sources of biologically generated N2O. In this study, we measured the δ15N, δ18O, δ15Nα (central N atom in N2O), and δ15Nβ (terminal N atom in N2O) of N2O generated by purified fungal P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum. We observed normal isotope effects for δ18O and δ15Nα, and inverse isotope effects for bulk δ15N (the average of Nα and Nβ) and δ15Nβ. The observed isotope effects have been used in conjunction with DFT calculations to provide important insight into the mechanism of P450nor. Similar</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/22218100','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/22218100"><span id="translatedtitle">Ion irradiation of <span class="hlt">Fe-Fe</span> oxide core-shell nanocluster films: Effect of interface on stability of magnetic properties</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>McCloy, John S.; Jiang, Weilin; Droubay, Timothy C.; Varga, Tamas; Kovarik, Libor; Sundararajan, Jennifer A.; Kaur, Maninder; Qiang, You; Burks, Edward C.; Liu, Kai</p> <p>2013-08-28</p> <p>A cluster deposition method was used to produce films of loosely aggregated nanoclusters (NCs) of Fe core-Fe{sub 3}O{sub 4} shell or fully oxidized Fe{sub 3}O{sub 4}. Films of these NC on Si(100) or MgO(100)/Fe{sub 3}O{sub 4}(100) were irradiated to 10{sup 16} Si{sup 2+}/cm{sup 2} near room temperature using an ion accelerator. Ion irradiation creates structural change in the NC film with corresponding chemical and magnetic changes which depend on the initial oxidation state of the cluster. Films were characterized using magnetometry (hysteresis, first order reversal curves), microscopy (transmission electron, helium ion), and x-ray diffraction. In all cases, the particle sizes increased due to ion irradiation, and when a core of Fe is present, irradiation reduces the oxide shells to lower valent Fe species. These results show that ion irradiated behavior of the NC films depends strongly on the initial nanostructure and chemistry, but in general saturation magnetization decreases slightly.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2015PhPro..75.1396L','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2015PhPro..75.1396L"><span id="translatedtitle">Study of AC Magnetic Properties and Core Losses of <span class="hlt">Fe/Fe</span>3O4-epoxy Resin Soft Magnetic Composite</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Laxminarayana, T. A.; Manna, Subhendu Kumar; Fernandes, B. G.; Venkataramani, N.</p> <p></p> <p>Soft Magnetic Composites (SMC) were prepared by coating of nanocrystalline Fe3O4 particles, synthesized by co-precipitation method, on atomized iron powder of particle size less than 53 μm in size using epoxy resin as a binder between iron and Fe3O4. Fe3O4 was chosen, for its high electric resistivity and suitable magnetic properties, to keep the coating layer magnetic and seek improvement to the magnetic properties of SMC. SEM images and XRD patterns were recorded in order to investigate the coatings on the surface of iron powder. A toroid was prepared by cold compaction of coated iron powder at 1050 MPa and subsequently cured at 150˚C for 1 hr in argon atmosphere. For comparison of properties, a toroid of uncoated iron powder was also compacted at 1050 MPa and annealed at 600˚C for 2 hr in argon atmosphere. The coated iron powder composite has a resistivity of greater than 200 μΩm, measured by four probe method. A comparison of Magnetic Hysteresis loops and core losses using B-H Loop tracer in the frequency range 0 to 1500 Hz on the coated and uncoated iron powder is reported.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/24826090','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/24826090"><span id="translatedtitle">Hepta-carbonyl-bis-(μ-propane-1,3-di-thiol-ato)triiron(I,II)(2 <span class="hlt">Fe-Fe</span>).</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Hu, Mingqiang; Ma, Chengbing; Wen, Huimin; Cui, Honghua; Chen, Changneng</p> <p>2014-04-01</p> <p>The trinuclear title compound, [Fe3(C3H6S2)2(CO)7], is a mixed-valent Fe(I)/Fe(II) complex and crystallizes with two mol-ecules of similar configuration in the asymmetric unit. The three Fe atoms in each mol-ecule display a bent arrangement [Fe-Fe-Fe = 156.22 (4) and 157.06 (3)°]. Both outer Fe(I) atoms are six-coordinated in a distorted ocahedral coordination geometry defined by the bridging Fe(II) atom, three carbonyl C atoms and two bridging S atoms. The coordination number of the central Fe(II) atom is seven and includes bonding to the two outer Fe(I) atoms, four bridging S atoms and one carbonyl C atom. The resulting coordination polyhedron might be described as a highly distorted monocapped trigonal prism. In the crystal packing, the mol-ecules exhibit a chain-like arrangement parallel to [100] and [001], and the resulting layers are stacked along [010]. The cohesion of the structure is dominated by van der Waals inter-actions. PMID:24826090</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/1051787','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/1051787"><span id="translatedtitle">Toward a rigorous network of protein-protein interactions of the <span class="hlt">model</span> sulfate reducer Desulfovibrio vulgaris Hildenborough</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>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.</p> <p>2011-05-01</p> <p>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 <span class="hlt">model</span> 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 <span class="hlt">hydrogenase</span>, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/1214968','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/1214968"><span id="translatedtitle">Hydrogen Production by Water Biophotolysis</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Ghirardi, Maria L.; King, Paul W.; Mulder, David W.; Eckert, Carrie; Dubini, Alexandra; Maness, Pin-Ching; Yu, Jianping</p> <p>2014-01-22</p> <p>The use of microalgae for production of hydrogen gas from water photolysis has been studied for many years, but its commercialization is still limited by multiple challenges. Most of the barriers to commercialization are attributed to the existence of biological regulatory mechanisms that, under anaerobic conditions, quench the absorbed light energy, down-regulate linear electron transfer, inactivate the H2-producing enzyme, and compete for electrons with the <span class="hlt">hydrogenase</span>. Consequently, the conversion efficiency of absorbed photons into H2 is significantly lower than its estimated potential of 12–13 %. However, extensive research continues towards addressing these barriers by either trying to understand and circumvent intracellular regulatory mechanisms at the enzyme and metabolic level or by developing biological systems that achieve prolonged H2 production albeit under lower than 12–13 % solar conversion efficiency. This chapter describes the metabolic pathways involved in biological H2 photoproduction from water photolysis, the attributes of the two <span class="hlt">hydrogenases</span>, [<span class="hlt">FeFe</span>] and [NiFe], that catalyze biological H2 production, and highlights research related to addressing the barriers described above. These highlights include: (a) recent advances in improving our understanding of the O2 inactivation mechanism in different classes of <span class="hlt">hydrogenases</span>; (b) progress made in preventing competitive pathways from diverting electrons from H2 photoproduction; and (c) new developments in bypassing the non-dissipated proton gradient from down-regulating photosynthetic electron transfer. As an example of a major success story, we mention the generation of truncated-antenna mutants in Chlamydomonas and Synechocystis that address the inherent low-light saturation of photosynthesis. In addition, we highlight the rationale and progress towards coupling biological <span class="hlt">hydrogenases</span> to non-biological, photochemical charge-separation as a means to bypass the barriers of photobiological</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3985925','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3985925"><span id="translatedtitle">Bioinspired <span class="hlt">Hydrogenase</span> <span class="hlt">Models</span>: The Mixed-Valence Triiron Complex [Fe3(CO)7(μ-edt)2] and Phosphine Derivatives [Fe3(CO)7–x(PPh3)x(μ-edt)2] (x = 1, 2) and [Fe3(CO)5(κ2-diphosphine)(μ-edt)2] as Proton Reduction Catalysts</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p></p> <p>2014-01-01</p> <p>The mixed-valence triiron complexes [Fe3(CO)7–x(PPh3)x(μ-edt)2] (x = 0–2; edt = SCH2CH2S) and [Fe3(CO)5(κ2-diphosphine)(μ-edt)2] (diphosphine = dppv, dppe, dppb, dppn) have been prepared and structurally characterized. All adopt an anti arrangement of the dithiolate bridges, and PPh3 substitution occurs at the apical positions of the outer iron atoms, while the diphosphine complexes exist only in the dibasal form in both the solid state and solution. The carbonyl on the central iron atom is semibridging, and this leads to a rotated structure between the bridged diiron center. IR studies reveal that all complexes are inert to protonation by HBF4·Et2O, but addition of acid to the pentacarbonyl complexes results in one-electron oxidation to yield the moderately stable cations [Fe3(CO)5(PPh3)2(μ-edt)2]+ and [Fe3(CO)5(κ2-diphosphine)(μ-edt)2]+, species which also result upon oxidation by [Cp2Fe][PF6]. The electrochemistry of the formally Fe(I)–Fe(II)–Fe(I) complexes has been investigated. Each undergoes a quasi-reversible oxidation, the potential of which is sensitive to phosphine substitution, generally occurring between 0.15 and 0.50 V, although [Fe3(CO)5(PPh3)2(μ-edt)2] is oxidized at −0.05 V. Reduction of all complexes is irreversible and is again sensitive to phosphine substitution, varying between −1.47 V for [Fe3(CO)7(μ-edt)2] and around −1.7 V for phosphine-substituted complexes. In their one-electron-reduced states, all complexes are catalysts for the reduction of protons to hydrogen, the catalytic overpotential being increased upon successive phosphine substitution. In comparison to the diiron complex [Fe2(CO)6(μ-edt)], [Fe3(CO)7(μ-edt)2] catalyzes proton reduction at 0.36 V less negative potentials. Electronic structure calculations have been carried out in order to fully elucidate the nature of the oxidation and reduction processes. In all complexes, the HOMO comprises an iron–iron bonding orbital localized between the two iron atoms not ligated by the semibridging carbonyl, while the LUMO is highly delocalized in nature and is antibonding between both pairs of iron atoms but also contains an antibonding dithiolate interaction. PMID:24748710</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/24748710','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/24748710"><span id="translatedtitle">Bioinspired <span class="hlt">Hydrogenase</span> <span class="hlt">Models</span>: The Mixed-Valence Triiron Complex [Fe3(CO)7(μ-edt)2] and Phosphine Derivatives [Fe3(CO)7-x (PPh3) x (μ-edt)2] (x = 1, 2) and [Fe3(CO)5(κ(2)-diphosphine)(μ-edt)2] as Proton Reduction Catalysts.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Rahaman, Ahibur; Ghosh, Shishir; Unwin, David G; Basak-Modi, Sucharita; Holt, Katherine B; Kabir, Shariff E; Nordlander, Ebbe; Richmond, Michael G; Hogarth, Graeme</p> <p>2014-03-24</p> <p>The mixed-valence triiron complexes [Fe3(CO)7-x (PPh3) x (μ-edt)2] (x = 0-2; edt = SCH2CH2S) and [Fe3(CO)5(κ(2)-diphosphine)(μ-edt)2] (diphosphine = dppv, dppe, dppb, dppn) have been prepared and structurally characterized. All adopt an anti arrangement of the dithiolate bridges, and PPh3 substitution occurs at the apical positions of the outer iron atoms, while the diphosphine complexes exist only in the dibasal form in both the solid state and solution. The carbonyl on the central iron atom is semibridging, and this leads to a rotated structure between the bridged diiron center. IR studies reveal that all complexes are inert to protonation by HBF4·Et2O, but addition of acid to the pentacarbonyl complexes results in one-electron oxidation to yield the moderately stable cations [Fe3(CO)5(PPh3)2(μ-edt)2](+) and [Fe3(CO)5(κ(2)-diphosphine)(μ-edt)2](+), species which also result upon oxidation by [Cp2Fe][PF6]. The electrochemistry of the formally Fe(I)-Fe(II)-Fe(I) complexes has been investigated. Each undergoes a quasi-reversible oxidation, the potential of which is sensitive to phosphine substitution, generally occurring between 0.15 and 0.50 V, although [Fe3(CO)5(PPh3)2(μ-edt)2] is oxidized at -0.05 V. Reduction of all complexes is irreversible and is again sensitive to phosphine substitution, varying between -1.47 V for [Fe3(CO)7(μ-edt)2] and around -1.7 V for phosphine-substituted complexes. In their one-electron-reduced states, all complexes are catalysts for the reduction of protons to hydrogen, the catalytic overpotential being increased upon successive phosphine substitution. In comparison to the diiron complex [Fe2(CO)6(μ-edt)], [Fe3(CO)7(μ-edt)2] catalyzes proton reduction at 0.36 V less negative potentials. Electronic structure calculations have been carried out in order to fully elucidate the nature of the oxidation and reduction processes. In all complexes, the HOMO comprises an iron-iron bonding orbital localized between the two iron atoms not ligated by the semibridging carbonyl, while the LUMO is highly delocalized in nature and is antibonding between both pairs of iron atoms but also contains an antibonding dithiolate interaction. PMID:24748710</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4257681','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4257681"><span id="translatedtitle">Genome Annotation Provides Insight into Carbon Monoxide and Hydrogen Metabolism in Rubrivivax gelatinosus</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Wawrousek, Karen; Noble, Scott; Korlach, Jonas; Chen, Jin; Eckert, Carrie; Yu, Jianping; Maness, Pin-Ching</p> <p>2014-01-01</p> <p>We report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a <span class="hlt">model</span> 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 <span class="hlt">hydrogenase</span>. 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 <span class="hlt">hydrogenases</span>, an uptake <span class="hlt">hydrogenase</span> that liberates the electrons in H2 in support of cell growth, and a regulatory <span class="hlt">hydrogenase</span> that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake <span class="hlt">hydrogenase</span>. The genome also contains two sets of <span class="hlt">hydrogenase</span> maturation genes which are known to assemble the catalytic metallocluster of the <span class="hlt">hydrogenase</span> 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4287179','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4287179"><span id="translatedtitle">Genome sequence of the <span class="hlt">model</span> sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus*</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>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</p> <p>2014-01-01</p> <p>Desulfovibrio gigas is a <span class="hlt">model</span> 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 <span class="hlt">hydrogenases</span> 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/1024072','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/1024072"><span id="translatedtitle">A Synthetic Nickel Electrocatalyst With a Turnover Frequency Above 100,000 s-1 for H2 Production</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Helm, Monte L.; Stewart, Michael P.; Bullock, R. Morris; Rakowski DuBois, Mary; DuBois, Daniel L.</p> <p>2011-08-12</p> <p>Increased worldwide energy demand will require greater use of carbon-neutral sustainable energy sources. The intermittent nature of solar and wind power requires storage of energy, so electrocatalysts that convert electrical energy to chemical bonds in fuels are needed. Platinum is an excellent catalyst, but it is of low abundance and high cost. <span class="hlt">Hydrogenase</span> enzymes in Nature catalyze the evolution of H2 and use earth-abundant metals such as nickel and iron. We report that a synthetic nickel catalyst, [Ni(7PPh2NPh)2](BF4)2, (7PPh2NPh = 1,3,6-triphenyl-1-aza-3,6-diphosphacycloheptane) catalyzes the production of H2 using [(DMF)H]+OTf as the proton source, with turnover frequencies of 31,000 s-1 in dry acetonitrile and 108,000 s-1 in the presence of H2O (1.2 M), at a potential of -1.13 V (vs. the ferrocenium/ferrocene couple). These turnover frequencies exceed those reported for the [<span class="hlt">FeFe</span>] <span class="hlt">hydrogenase</span> enzyme by more than an order of magnitude, and are the fastest reported for any molecular catalyst for H2 production. This material is based upon work supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3933540','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3933540"><span id="translatedtitle">Engineering Synechocystis PCC6803 for Hydrogen Production: Influence on the Tolerance to Oxidative and Sugar Stresses</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Ortega-Ramos, Marcia; Jittawuttipoka, Thichakorn; Saenkham, Panatda; Czarnecka-Kwasiborski, Aurelia; Bottin, Hervé; Cassier-Chauvat, Corinne; Chauvat, Franck</p> <p>2014-01-01</p> <p>In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the <span class="hlt">model</span> unicellular strain Synechocystis PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature). We demonstrate that the <span class="hlt">hydrogenase</span>-encoding hoxEFUYH operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H2O2) and sugar (glucose and glycerol) stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of <span class="hlt">hydrogenase</span>), combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active <span class="hlt">hydrogenase</span>. These novel results and mutants have major implications for those interested in <span class="hlt">hydrogenase</span>, hydrogen production and redox metabolism, and their connections with environmental conditions. PMID:24586727</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/953511','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/953511"><span id="translatedtitle">Insights Into the P-To-Q Conversion in the Catalytic Cycle of Methane Monooxygenase From a Synthetic <span class="hlt">Model</span> System</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Xue, G.; Fiedler, A.T.; Martinho, M.; Munck, E.; Que, L.; Jr.</p> <p>2009-05-28</p> <p>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 <span class="hlt">model</span> 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 <span class="hlt">Fe-Fe</span> 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.</p> </li> </ol> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_20");'>20</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li class="active"><span>22</span></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li><a href="#" onclick='return showDiv("page_24");'>24</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div><!-- col-sm-12 --> </div><!-- row --> </div><!-- page_22 --> <div id="page_23" class="hiddenDiv"> <div class="row"> <div class="col-sm-12"> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li class="active"><span>23</span></li> <li><a href="#" onclick='return showDiv("page_24");'>24</a></li> <li><a href="#" onclick='return showDiv("page_25");'>25</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div> </div> <div class="row"> <div class="col-sm-12"> <ol class="result-class" start="441"> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3232296','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3232296"><span id="translatedtitle">Metal-Metal Bonds in Biology</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Lindahl, Paul A.</p> <p>2011-01-01</p> <p>Nickel-containing carbon monoxide dehydrogenases, acetyl-CoA synthases, nickel-iron <span class="hlt">hydrogenases</span>, and diron <span class="hlt">hydrogenases</span> 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 <span class="hlt">hydrogenases</span> 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 <span class="hlt">Fe-Fe</span> bonds in the nitrogenase FeMoco active site is discussed. PMID:22119810</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://ntrs.nasa.gov/search.jsp?R=19930040727&hterms=eutectic&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DTitle%26N%3D0%26No%3D20%26Ntt%3Deutectic','NASA-TRS'); return false;" href="http://ntrs.nasa.gov/search.jsp?R=19930040727&hterms=eutectic&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DTitle%26N%3D0%26No%3D20%26Ntt%3Deutectic"><span id="translatedtitle">Experimental partitioning studies near the <span class="hlt">Fe-FeS</span> eutectic, with an emphasis on elements important to iron meteorite chronologies (Pb, Ag, Pd, and Tl)</span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Jones, J. H.; Hart, S. R.; Benjamin, T. M.</p> <p>1993-01-01</p> <p>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.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/8508801','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/8508801"><span id="translatedtitle">Zn-exchange and Mössbauer studies on the [<span class="hlt">Fe-Fe</span>] derivatives of the purple acid Fe(III)-Zn(II)-phosphatase from kidney beans.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Suerbaum, H; Körner, M; Witzel, H; Althaus, E; Mosel, B D; Müller-Warmuth, W</p> <p>1993-05-15</p> <p>In order to perform Mössbauer studies, Zn(II) in the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)-57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)-57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, but not the mixed valent Fe(III)-Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III). The Mössbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with delta of 0.51 mm/s and 0.53 mm/s and delta E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a mu-oxo bridge. The mixed-valent Fe(III)-Fe(II) species shows two quadrupole doublets with delta values of 0.55 mm/s and 1.14 mm/s and delta E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half-integer spin system with antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with delta values of 1.18 mm/s and 1.22 mm/s and with delta E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)-Fe(II) system. Interaction with O2 is discussed. PMID:8508801</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/19757795','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/19757795"><span id="translatedtitle"><span class="hlt">Modeling</span> the syn disposition of nitrogen donors in non-heme diiron enzymes. Synthesis, characterization, and hydrogen peroxide reactivity of diiron(III) complexes with the syn N-donor ligand H2BPG2DEV.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Friedle, Simone; Kodanko, Jeremy J; Morys, Anna J; Hayashi, Takahiro; Moënne-Loccoz, Pierre; Lippard, Stephen J</p> <p>2009-10-14</p> <p>In order to <span class="hlt">model</span> the syn disposition of histidine residues in carboxylate-bridged non-heme diiron enzymes, we prepared a new dinucleating ligand, H(2)BPG(2)DEV, that provides this geometric feature. The ligand incorporates biologically relevant carboxylate functionalities, which have not been explored as extensively as nitrogen-only analogues. Three novel oxo-bridged diiron(III) complexes, [Fe(2)(mu-O)(H(2)O)(2)(BPG(2)DEV)](ClO(4))(2) (6), [Fe(2)(mu-O)(mu-O(2)CAr(iPrO))(BPG(2)DEV)](ClO(4)) (7), and [Fe(2)(mu-O)(mu-CO(3))(BPG(2)DEV)] (8), were prepared. Single-crystal X-ray structural characterization confirms that two pyridyl groups are bound syn with respect to the <span class="hlt">Fe-Fe</span> vector in these compounds. The carbonato-bridged complex 8 forms quantitatively from 6 in a rapid reaction with gaseous CO(2) in organic solvents. A common maroon-colored intermediate (lambda(max) = 490 nm; epsilon = 1500 M(-1) cm(-1)) forms in reactions of 6, 7, or 8 with H(2)O(2) and NEt(3) in CH(3)CN/H(2)O solutions. Mass spectrometric analyses of this species, formed using (18)O-labeled H(2)O(2), indicate the presence of a peroxide ligand bound to the oxo-bridged diiron(III) center. The Mossbauer spectrum at 90 K of the EPR-silent intermediate exhibits a quadrupole doublet with delta = 0.58 mm/s and DeltaE(Q) = 0.58 mm/s. The isomer shift is typical for a peroxodiiron(III) species, but the quadrupole splitting parameter is unusually small compared to those of related complexes. These Mossbauer parameters are comparable to those observed for a peroxo intermediate formed in the reaction of reduced toluene/o-xylene monooxygenase hydroxylase with dioxygen. Resonance Raman studies reveal an unusually low-energy O-O stretching mode in the peroxo intermediate that is consistent with a short diiron distance. Although peroxodiiron(III) intermediates generated from 6, 7, and 8 are poor O-atom-transfer catalysts, they display highly efficient catalase activity, with turnover numbers up to 10,000. In</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://files.eric.ed.gov/fulltext/ED116270.pdf','ERIC'); return false;" href="http://files.eric.ed.gov/fulltext/ED116270.pdf"><span id="translatedtitle">Leadership <span class="hlt">Models</span>.</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Freeman, Thomas J.</p> <p></p> <p>This paper discusses six different <span class="hlt">models</span> of organizational structure and leadership, including the scalar chain or pyramid <span class="hlt">model</span>, the continuum <span class="hlt">model</span>, the grid <span class="hlt">model</span>, the linking pin <span class="hlt">model</span>, the contingency <span class="hlt">model</span>, and the circle or democratic <span class="hlt">model</span>. Each <span class="hlt">model</span> is examined in a separate section that describes the <span class="hlt">model</span> and its development, lists…</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/pages/biblio/1169210-genome-annotation-provides-insight-carbon-monoxide-hydrogen-metabolism-rubrivivax-gelatinosus','SCIGOV-DOEP'); return false;" href="http://www.osti.gov/pages/biblio/1169210-genome-annotation-provides-insight-carbon-monoxide-hydrogen-metabolism-rubrivivax-gelatinosus"><span id="translatedtitle">Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus</span></a></p> <p><a target="_blank" href="http://www.osti.gov/pages">DOE PAGESBeta</a></p> <p>Wawrousek, Karen; Noble, Scott; Korlach, Jonas; Chen, Jin; Eckert, Carrie; Yu, Jianping; Maness, Pin -Ching</p> <p>2014-12-05</p> <p>In this article, we report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a <span class="hlt">model</span> 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 <span class="hlt">hydrogenase</span>. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzymemore » complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional <span class="hlt">hydrogenases</span>, an uptake <span class="hlt">hydrogenase</span> that liberates the electrons in H2 in support of cell growth, and a regulatory <span class="hlt">hydrogenase</span> that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake <span class="hlt">hydrogenase</span>. The genome also contains two sets of <span class="hlt">hydrogenase</span> maturation genes which are known to assemble the catalytic metallocluster of the <span class="hlt">hydrogenase</span> NiFe active site. Finally and collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth.« less</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/1169210','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/1169210"><span id="translatedtitle">Genome annotation provides insight into carbon monoxide and hydrogen metabolism in <i>Rubrivivax gelatinosus</i></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Wawrousek, Karen; Noble, Scott; Korlach, Jonas; Eckert, Carrie; Yu, Jianping; Maness, Pin -Ching</p> <p>2014-12-05</p> <p>In this article, we report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium <i>Rubrivivax gelatinosus</i> CBS. This microbe is a <span class="hlt">model</span> 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 CO<sub>2</sub> and H<sub>2</sub> as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and <span class="hlt">hydrogenase</span>. 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 <span class="hlt">hydrogenases</span>, an uptake <span class="hlt">hydrogenase</span> that liberates the electrons in H<sub>2</sub> in support of cell growth, and a regulatory <span class="hlt">hydrogenase</span> that senses H<sub>2</sub> and relays the signal to a two-component system that ultimately controls synthesis of the uptake <span class="hlt">hydrogenase</span>. The genome also contains two sets of <span class="hlt">hydrogenase</span> maturation genes which are known to assemble the catalytic metallocluster of the <span class="hlt">hydrogenase</span> NiFe active site. Finally and collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H<sub>2</sub> in support of cell growth.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/26375327','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/26375327"><span id="translatedtitle">Reengineering cyt b562 for hydrogen production: A facile route to artificial <span class="hlt">hydrogenases</span>.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Sommer, Dayn Joseph; Vaughn, Michael David; Clark, Brett Colby; Tomlin, John; Roy, Anindya; Ghirlanda, Giovanna</p> <p>2016-05-01</p> <p>Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. This article is part of a Special Issue entitled Biodesign for Bioenergetics - the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. PMID:26375327</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/17565990','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/17565990"><span id="translatedtitle">Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, <span class="hlt">hydrogenase</span> induction, and metabolic pathways.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Mus, Florence; Dubini, Alexandra; Seibert, Michael; Posewitz, Matthew C; Grossman, Arthur R</p> <p>2007-08-31</p> <p>Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production. PMID:17565990</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3861653','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3861653"><span id="translatedtitle">The <span class="hlt">Hydrogenase</span> Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans*</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Wilcoxen, Jarett; Hille, Russ</p> <p>2013-01-01</p> <p>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 μm; 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/25871413','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/25871413"><span id="translatedtitle"><span class="hlt">Models</span> and role <span class="hlt">models</span>.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>ten Cate, Jacob M</p> <p>2015-01-01</p> <p>Developing experimental <span class="hlt">models</span> to understand dental caries has been the theme in our research group. Our first, the pH-cycling <span class="hlt">model</span>, was developed to investigate the chemical reactions in enamel or dentine, which lead to dental caries. It aimed to leverage our understanding of the fluoride mode of action and was also utilized for the formulation of oral care products. In addition, we made use of intra-oral (in situ) <span class="hlt">models</span> to study other features of the oral environment that drive the de/remineralization balance in individual patients. This <span class="hlt">model</span> addressed basic questions, such as how enamel and dentine are affected by challenges in the oral cavity, as well as practical issues related to fluoride toothpaste efficacy. The observation that perhaps fluoride is not sufficiently potent to reduce dental caries in the present-day society triggered us to expand our knowledge in the bacterial aetiology of dental caries. For this we developed the Amsterdam Active Attachment biofilm <span class="hlt">model</span>. Different from studies on planktonic ('single') bacteria, this biofilm <span class="hlt">model</span> captures bacteria in a habitat similar to dental plaque. With data from the combination of these <span class="hlt">models</span>, it should be possible to study separate processes which together may lead to dental caries. Also products and novel agents could be evaluated that interfere with either of the processes. Having these separate <span class="hlt">models</span> in place, a suggestion is made to design computer <span class="hlt">models</span> to encompass the available information. <span class="hlt">Models</span> but also role <span class="hlt">models</span> are of the utmost importance in bringing and guiding research and researchers. PMID:25871413</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4139163','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4139163"><span id="translatedtitle">Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p></p> <p>2015-01-01</p> <p>Conspectus A [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5′-deoxyadenosyl radical (5′-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5′-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [<span class="hlt">FeFe</span>] <span class="hlt">hydrogenase</span> maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5′-dA• to the product radical l-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [<span class="hlt">FeFe</span>] cluster of <span class="hlt">hydrogenase</span>. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=137225&keyword=art+AND+history&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=76641695&CFTOKEN=79432102','EPA-EIMS'); return false;" href="http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=137225&keyword=art+AND+history&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=76641695&CFTOKEN=79432102"><span id="translatedtitle"><span class="hlt">MODEL</span> DEVELOPMENT - DOSE <span class="hlt">MODELS</span></span></a></p> <p><a target="_blank" href="http://oaspub.epa.gov/eims/query.page">EPA Science Inventory</a></p> <p></p> <p></p> <p><span class="hlt">Model</span> Development<br><br>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...</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/19298055','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/19298055"><span id="translatedtitle">Correcting for electrocatalyst desorption and inactivation in chronoamperometry experiments.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Fourmond, Vincent; Lautier, Thomas; Baffert, Carole; Leroux, Fanny; Liebgott, Pierre-Pol; Dementin, Sébastien; Rousset, Marc; Arnoux, Pascal; Pignol, David; Meynial-Salles, Isabelle; Soucaille, Phillippe; Bertrand, Patrick; Léger, Christophe</p> <p>2009-04-15</p> <p>Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe <span class="hlt">hydrogenase</span>, and <span class="hlt">FeFe</span> <span class="hlt">hydrogenase</span>. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst. PMID:19298055</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/23974031','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/23974031"><span id="translatedtitle">Flexibility of syntrophic enzyme systems in Desulfovibrio species ensures their adaptation capability to environmental changes.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Meyer, Birte; Kuehl, Jennifer V; Deutschbauer, Adam M; Arkin, Adam P; Stahl, David A</p> <p>2013-11-01</p> <p>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 <span class="hlt">hydrogenases</span>) and the ratio shift in electron carriers used for interspecies metabolite exchange (hydrogen/formate). A putative monomeric [<span class="hlt">Fe-Fe</span>] <span class="hlt">hydrogenase</span> 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, DsrC(red) and Fd(red), 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3807489','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3807489"><span id="translatedtitle">Flexibility of Syntrophic Enzyme Systems in Desulfovibrio Species Ensures Their Adaptation Capability to Environmental Changes</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Meyer, Birte; Kuehl, Jennifer V.; Deutschbauer, Adam M.; Arkin, Adam P.</p> <p>2013-01-01</p> <p>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 <span class="hlt">hydrogenases</span>) and the ratio shift in electron carriers used for interspecies metabolite exchange (hydrogen/formate). A putative monomeric [<span class="hlt">Fe-Fe</span>] <span class="hlt">hydrogenase</span> 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3119410','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3119410"><span id="translatedtitle">A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Pereira, Inês A. Cardoso; Ramos, Ana Raquel; Grein, Fabian; Marques, Marta Coimbra; da Silva, Sofia Marques; Venceslau, Sofia Santos</p> <p>2011-01-01</p> <p>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 <span class="hlt">hydrogenases</span>. 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 [<span class="hlt">FeFe</span>] <span class="hlt">hydrogenases</span>, 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, β-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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2010LNCS.6445..234L','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2010LNCS.6445..234L"><span id="translatedtitle">Promoting <span class="hlt">Models</span></span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Li, Qin; Zhao, Yongxin; Wu, Xiaofeng; Liu, Si</p> <p></p> <p>There can be multitudinous <span class="hlt">models</span> specifying aspects of the same system. Each <span class="hlt">model</span> has a bias towards one aspect. These <span class="hlt">models</span> often override in specific aspects though they have different expressions. A specification written in one <span class="hlt">model</span> can be refined by introducing additional information from other <span class="hlt">models</span>. The paper proposes a concept of promoting <span class="hlt">models</span> which is a methodology to obtain refinements with support from cooperating <span class="hlt">models</span>. It refines a primary <span class="hlt">model</span> by integrating the information from a secondary <span class="hlt">model</span>. The promotion principle is not merely an academic point, but also a reliable and robust engineering technique which can be used to develop software and hardware systems. It can also check the consistency between two specifications from different <span class="hlt">models</span>. A case of <span class="hlt">modeling</span> a simple online shopping system with the cooperation of the guarded design <span class="hlt">model</span> and CSP <span class="hlt">model</span> illustrates the practicability of the promotion principle.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4992972','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4992972"><span id="translatedtitle">Hydrogen Metabolism in Helicobacter pylori Plays a Role in Gastric Carcinogenesis through Facilitating CagA Translocation</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Wang, Ge; Romero-Gallo, Judith; Benoit, Stéphane L.; Piazuelo, M. Blanca; Dominguez, Ricardo L.; Morgan, Douglas R.; Peek, Richard M.</p> <p>2016-01-01</p> <p>ABSTRACT A known virulence factor of Helicobacter pylori that augments gastric cancer risk is the CagA cytotoxin. A carcinogenic derivative strain, 7.13, that has a greater ability to translocate CagA exhibits much higher <span class="hlt">hydrogenase</span> activity than its parent noncarcinogenic strain, B128. A Δhyd mutant strain with deletion of <span class="hlt">hydrogenase</span> genes was ineffective in CagA translocation into human gastric epithelial AGS cells, while no significant attenuation of cell adhesion was observed. The quinone reductase inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) was used to specifically inhibit the H2-utilizing respiratory chain of outer membrane-permeabilized bacterial cells; that level of inhibitor also greatly attenuated CagA translocation into AGS cells, indicating the H2-generated transmembrane potential is a contributor to toxin translocation. The Δhyd strain showed a decreased frequency of DNA transformation, suggesting that H. pylori <span class="hlt">hydrogenase</span> is also involved in energizing the DNA uptake apparatus. In a gerbil <span class="hlt">model</span> of infection, the ability of the Δhyd strain to induce inflammation was significantly attenuated (at 12 weeks postinoculation), while all of the gerbils infected with the parent strain (7.13) exhibited a high level of inflammation. Gastric cancer developed in 50% of gerbils infected with the wild-type strain 7.13 but in none of the animals infected with the Δhyd strain. By examining the <span class="hlt">hydrogenase</span> activities from well-defined clinical H. pylori isolates, we observed that strains isolated from cancer patients (n = 6) have a significantly higher <span class="hlt">hydrogenase</span> (H2/O2) activity than the strains isolated from gastritis patients (n = 6), further supporting an association between H. pylori <span class="hlt">hydrogenase</span> activity and gastric carcinogenesis in humans. PMID:27531909</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://eric.ed.gov/?q=big+AND+internet&pg=7&id=EJ663143','ERIC'); return false;" href="http://eric.ed.gov/?q=big+AND+internet&pg=7&id=EJ663143"><span id="translatedtitle"><span class="hlt">Models</span>, Part IV: Inquiry <span class="hlt">Models</span>.</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Callison, Daniel</p> <p>2002-01-01</p> <p>Discusses <span class="hlt">models</span> for information skills that include inquiry-oriented activities. Highlights include WebQuest, which uses Internet resources supplemented with videoconferencing; Minnesota's Inquiry Process based on the Big Six <span class="hlt">model</span> for information problem-solving; Indiana's Student Inquiry <span class="hlt">Model</span>; constructivist learning <span class="hlt">models</span> for inquiry; and…</p> </li> </ol> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li class="active"><span>23</span></li> <li><a href="#" onclick='return showDiv("page_24");'>24</a></li> <li><a href="#" onclick='return showDiv("page_25");'>25</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div><!-- col-sm-12 --> </div><!-- row --> </div><!-- page_23 --> <div id="page_24" class="hiddenDiv"> <div class="row"> <div class="col-sm-12"> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li class="active"><span>24</span></li> <li><a href="#" onclick='return showDiv("page_25");'>25</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div> </div> <div class="row"> <div class="col-sm-12"> <ol class="result-class" start="461"> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/25811859','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/25811859"><span id="translatedtitle">Genome-guided analysis of physiological capacities of Tepidanaerobacter acetatoxydans provides insights into environmental adaptations and syntrophic acetate oxidation.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Müller, Bettina; Manzoor, Shahid; Niazi, Adnan; Bongcam-Rudloff, Erik; Schnürer, Anna</p> <p>2015-01-01</p> <p>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 [<span class="hlt">Fe-Fe</span>] <span class="hlt">hydrogenases</span>, 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4374699','PMC'); return false;" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4374699"><span id="translatedtitle">Genome-Guided Analysis of Physiological Capacities of Tepidanaerobacter acetatoxydans Provides Insights into Environmental Adaptations and Syntrophic Acetate Oxidation</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Niazi, Adnan; Bongcam-Rudloff, Erik; Schnürer, Anna</p> <p>2015-01-01</p> <p>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 [<span class="hlt">Fe-Fe</span>] <span class="hlt">hydrogenases</span>, 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</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/26999575','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/26999575"><span id="translatedtitle">Formate-derived H2 , a driver of hydrogenotrophic processes in the root-zone of a methane-emitting fen.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Hunger, Sindy; Schmidt, Oliver; Gößner, Anita S; Drake, Harold L</p> <p>2016-09-01</p> <p>Wetlands are important sources of globally emitted methane. Plants mediate much of that emission by releasing root-derived organic carbon, including formate, a direct precursor of methane. Thus, the objective of this study was to resolve formate-driven processes potentially linked to methanogenesis in the fen root-zone. Although, formate was anticipated to directly trigger methanogenesis, the rapid anaerobic consumption of formate by Carex roots unexpectedly yielded H2 and CO2 via enzymes such as formate-H2 -lyase (FHL), and likewise appeared to enhance the utilization of organic carbon. Collectively, 57 [<span class="hlt">FeFe</span>]- and [NiFe]-<span class="hlt">hydrogenase</span>-containing family level phylotypes potentially linked to FHL activity were detected. Under anoxic conditions, root-derived fermentative Citrobacter and Hafnia isolates produced H2 from formate via FHL. Formate-derived H2 fueled methanogenesis and acetogenesis, and methanogenic (Methanoregula, Methanobacterium, Methanocella) and acetogenic (Acetonema, Clostridum, Sporomusa) genera potentially linked to these hydrogenotrophic activities were identified. The findings (i) provide novel insights on highly diverse root-associated FHL-containing taxa that can augment secondary hydrogenotrophic processes via the production of formate-derived H2 , (ii) demonstrate that formate can have a 'priming' effect on the utilization of organic carbon, and (iii) raise questions regarding the fate of formate-derived H2 when it diffuses away from the root-zone. PMID:26999575</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/18167542','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/18167542"><span id="translatedtitle">Genetic evidence for a mitochondriate ancestry in the 'amitochondriate' flagellate Trimastix pyriformis.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Hampl, Vladimir; Silberman, Jeffrey D; Stechmann, Alexandra; Diaz-Triviño, Sara; Johnson, Patricia J; Roger, Andrew J</p> <p>2008-01-01</p> <p>Most modern eukaryotes diverged from a common ancestor that contained the alpha-proteobacterial endosymbiont that gave rise to mitochondria. The 'amitochondriate' anaerobic protist parasites that have been studied to date, such as Giardia and Trichomonas harbor mitochondrion-related organelles, such as mitosomes or hydrogenosomes. Yet there is one remaining group of mitochondrion-lacking flagellates known as the Preaxostyla that could represent a primitive 'pre-mitochondrial' lineage of eukaryotes. To test this hypothesis, we conducted an expressed sequence tag (EST) survey on the preaxostylid flagellate Trimastix pyriformis, a poorly-studied free-living anaerobe. Among the ESTs we detected 19 proteins that, in other eukaryotes, typically function in mitochondria, hydrogenosomes or mitosomes, 12 of which are found exclusively within these organelles. Interestingly, one of the proteins, aconitase, functions in the tricarboxylic acid cycle typical of aerobic mitochondria, whereas others, such as pyruvate:ferredoxin oxidoreductase and [<span class="hlt">FeFe</span>] <span class="hlt">hydrogenase</span>, are characteristic of anaerobic hydrogenosomes. Since Trimastix retains genetic evidence of a mitochondriate ancestry, we can now say definitively that all known living eukaryote lineages descend from a common ancestor that had mitochondria. PMID:18167542</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/5813096','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/5813096"><span id="translatedtitle">Supermatrix <span class="hlt">models</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Yost, S.A.</p> <p>1991-05-01</p> <p>Radom matrix <span class="hlt">models</span> based on an integral over supermatrices are proposed as a natural extension of bosonic matrix <span class="hlt">models</span>. The subtle nature of superspace integration allows these <span class="hlt">models</span> to have very different properties from the analogous bosonic <span class="hlt">models</span>. 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 <span class="hlt">models</span>. Another leads to an eigenvalue reduction which can be described by a two component plasma in one dimension. A stationary point of the <span class="hlt">model</span> is described.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/6937054','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/6937054"><span id="translatedtitle">Supermatrix <span class="hlt">models</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Yost, S.A. . Dept. of Physics and Astronomy)</p> <p>1992-09-30</p> <p>In this paper, random matrix <span class="hlt">models</span> based on an integral over supermatrices are proposed as a natural extension of bosonic matrix <span class="hlt">models</span>. The subtle nature of superspace integration allows these <span class="hlt">models</span> to have very different properties from the analogous bosonic <span class="hlt">models</span>. 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 <span class="hlt">models</span>. Another leads to an eigenvalue reduction which can be described by a two-component plasma in one dimension. A stationary point of the <span class="hlt">model</span> is described.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=2808&keyword=fields+AND+scientists&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=67529609&CFTOKEN=11861924','EPA-EIMS'); return false;" href="http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=2808&keyword=fields+AND+scientists&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=67529609&CFTOKEN=11861924"><span id="translatedtitle"><span class="hlt">MODELS</span> - 3</span></a></p> <p><a target="_blank" href="http://oaspub.epa.gov/eims/query.page">EPA Science Inventory</a></p> <p></p> <p></p> <p><span class="hlt">Models</span>-3 is a third generation air quality <span class="hlt">modeling</span> system that contains a variety of tools to perform research and analysis of critical environmental questions and problems. These tools provide regulatory analysts and scientists with quicker results, greater scientific accuracy ...</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=151530&keyword=objective+AND+Visual+AND+treatment&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=59257661&CFTOKEN=65283034','EPA-EIMS'); return false;" href="http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=151530&keyword=objective+AND+Visual+AND+treatment&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=59257661&CFTOKEN=65283034"><span id="translatedtitle">ENTRAINMENT <span class="hlt">MODELS</span></span></a></p> <p><a target="_blank" href="http://oaspub.epa.gov/eims/query.page">EPA Science Inventory</a></p> <p></p> <p></p> <p>This presentation presented information on entrainment <span class="hlt">models</span>. Entrainment <span class="hlt">models</span> use entrainment hypotheses to express the continuity equation. The advantage is that plume boundaries are known. A major disadvantage is that the problems that can be solved are rather simple. The ...</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://hdl.handle.net/2060/19870016585','NASA-TRS'); return false;" href="http://hdl.handle.net/2060/19870016585"><span id="translatedtitle">Turbulence <span class="hlt">modeling</span></span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Rubesin, Morris W.</p> <p>1987-01-01</p> <p>Recent developments at several levels of statistical turbulence <span class="hlt">modeling</span> applicable to aerodynamics are briefly surveyed. Emphasis is on examples of <span class="hlt">model</span> improvements for transonic, two-dimensional flows. Experience with the development of these improved <span class="hlt">models</span> is cited to suggest methods of accelerating the <span class="hlt">modeling</span> process necessary to keep abreast of the rapid movement of computational fluid dynamics into the computation of complex three-dimensional flows.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://ntrs.nasa.gov/search.jsp?R=19810011804&hterms=Barberton&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DAll%26N%3D0%26No%3D40%26Ntt%3DBarberton','NASA-TRS'); return false;" href="http://ntrs.nasa.gov/search.jsp?R=19810011804&hterms=Barberton&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DAll%26N%3D0%26No%3D40%26Ntt%3DBarberton"><span id="translatedtitle">Waveguide <span class="hlt">model</span></span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p></p> <p>1981-01-01</p> <p>A <span class="hlt">model</span> is presented which quantifies the electromagnetic modes (field configurations) in the immediate vicinity of the rectenna element. Specifically, the waveguide <span class="hlt">model</span> characterizes the electromagnetic modes generated by planar waves normal to the array. The <span class="hlt">model</span> applies only to incidence normal to the array.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=240424&keyword=Geothermal&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=77066026&CFTOKEN=65736685','EPA-EIMS'); return false;" href="http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=240424&keyword=Geothermal&actType=&TIMSType=+&TIMSSubTypeID=&DEID=&epaNumber=&ntisID=&archiveStatus=Both&ombCat=Any&dateBeginCreated=&dateEndCreated=&dateBeginPublishedPresented=&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&dateBeginCompleted=&dateEndCompleted=&personID=&role=Any&journalID=&publisherID=&sortBy=revisionDate&count=50&CFID=77066026&CFTOKEN=65736685"><span id="translatedtitle">Phoenix <span class="hlt">model</span></span></a></p> <p><a target="_blank" href="http://oaspub.epa.gov/eims/query.page">EPA Science Inventory</a></p> <p></p> <p></p> <p>Phoenix (formerly referred to as the Second Generation <span class="hlt">Model</span> or SGM) is a global general equilibrium <span class="hlt">model</span> designed to analyze energy-economy-climate related questions and policy implications in the medium- to long-term. This <span class="hlt">model</span> disaggregates the global economy into 26 industr...</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://eric.ed.gov/?q=Two+AND+photon&pg=2&id=EJ020652','ERIC'); return false;" href="http://eric.ed.gov/?q=Two+AND+photon&pg=2&id=EJ020652"><span id="translatedtitle">Radiation <span class="hlt">Models</span></span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>James, W. G. G.</p> <p>1970-01-01</p> <p>Discusses the historical development of both the wave and the corpuscular photon <span class="hlt">model</span> of light. Suggests that students should be informed that the two <span class="hlt">models</span> are complementary and that each <span class="hlt">model</span> successfully describes a wide range of radiation phenomena. Cites 19 references which might be of interest to physics teachers and students. (LC)</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2013HESSD..1010535B','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2013HESSD..1010535B"><span id="translatedtitle">Hydrological <span class="hlt">models</span> are mediating <span class="hlt">models</span></span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Babel, L. V.; Karssenberg, D.</p> <p>2013-08-01</p> <p>Despite the increasing role of <span class="hlt">models</span> in hydrological research and decision-making processes, only few accounts of the nature and function of <span class="hlt">models</span> exist in hydrology. Earlier considerations have traditionally been conducted while making a clear distinction between physically-based and conceptual <span class="hlt">models</span>. A new philosophical account, primarily based on the fields of physics and economics, transcends classes of <span class="hlt">models</span> and scientific disciplines by considering <span class="hlt">models</span> as "mediators" between theory and observations. The core of this approach lies in identifying <span class="hlt">models</span> 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 <span class="hlt">models</span>, each showing a different degree of apparent physicality, are confronted to the main characteristics of the "mediating <span class="hlt">models</span>" concept. We argue that irrespective of their kind, hydrological <span class="hlt">models</span> 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, <span class="hlt">model</span> objectives, knowledge and preferences of the <span class="hlt">modeller</span>, as well as hardware and software resources. We show that hydrological <span class="hlt">models</span> 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 <span class="hlt">models</span> is necessarily too simplistic and refers at best to the stage at which theory and observations are steering <span class="hlt">model</span> construction. The large variety of ingredients involved in <span class="hlt">model</span> construction would deserve closer attention, for being rarely explicitly presented in peer-reviewed literature. We believe that devoting</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://ntrs.nasa.gov/search.jsp?R=20000024740&hterms=chlorofluorocarbon&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DAll%26N%3D0%26No%3D40%26Ntt%3Dchlorofluorocarbon','NASA-TRS'); return false;" href="http://ntrs.nasa.gov/search.jsp?R=20000024740&hterms=chlorofluorocarbon&qs=Ntx%3Dmode%2Bmatchall%26Ntk%3DAll%26N%3D0%26No%3D40%26Ntt%3Dchlorofluorocarbon"><span id="translatedtitle"><span class="hlt">Model</span> Experiments and <span class="hlt">Model</span> Descriptions</span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Jackman, Charles H.; Ko, Malcolm K. W.; Weisenstein, Debra; Scott, Courtney J.; Shia, Run-Lie; Rodriguez, Jose; Sze, N. D.; Vohralik, Peter; Randeniya, Lakshman; Plumb, Ian</p> <p>1999-01-01</p> <p>The Second Workshop on Stratospheric <span class="hlt">Models</span> and Measurements Workshop (M&M II) is the continuation of the effort previously started in the first Workshop (M&M I, Prather and Remsberg [1993]) held in 1992. As originally stated, the aim of M&M is to provide a foundation for establishing the credibility of stratospheric <span class="hlt">models</span> used in environmental assessments of the ozone response to chlorofluorocarbons, aircraft emissions, and other climate-chemistry interactions. To accomplish this, a set of measurements of the present day atmosphere was selected. The intent was that successful simulations of the set of measurements should become the prerequisite for the acceptance of these <span class="hlt">models</span> as having a reliable prediction for future ozone behavior. This section is divided into two: <span class="hlt">model</span> experiment and <span class="hlt">model</span> descriptions. In the <span class="hlt">model</span> experiment, participant were given the charge to design a number of experiments that would use observations to test whether <span class="hlt">models</span> are using the correct mechanisms to simulate the distributions of ozone and other trace gases in the atmosphere. The purpose is closely tied to the needs to reduce the uncertainties in the <span class="hlt">model</span> predicted responses of stratospheric ozone to perturbations. The specifications for the experiments were sent out to the <span class="hlt">modeling</span> community in June 1997. Twenty eight <span class="hlt">modeling</span> groups responded to the requests for input. The first part of this section discusses the different <span class="hlt">modeling</span> group, along with the experiments performed. Part two of this section, gives brief descriptions of each <span class="hlt">model</span> as provided by the individual <span class="hlt">modeling</span> groups.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2014AGUFMIN43B3684Y','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2014AGUFMIN43B3684Y"><span id="translatedtitle"><span class="hlt">Model</span> Reduction in Groundwater <span class="hlt">Modeling</span></span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Yeh, W. W. G.</p> <p>2014-12-01</p> <p><span class="hlt">Model</span> reduction has been shown to be a very effective method for reducing the computational burden of large-scale simulations. <span class="hlt">Model</span> reduction techniques preserve much of the physical knowledge of the system and primarily seek to remove components from the <span class="hlt">model</span> that do not provide significant information of interest. Proper Orthogonal Decomposition (POD) is a <span class="hlt">model</span> reduction technique by which a system of ordinary equations is projected onto a much smaller subspace in such a way that the span of the subspace is equal to the span of the original full <span class="hlt">model</span> space. Basically, the POD technique selects a small number of orthonormal basis functions (principal components) that span the spatial variability of the solutions. In this way the state variable (head) is approximated by a linear combination of these basis functions and, using a Galerkin projection, the dimension of the problem is significantly reduced. It has been shown that for a highly discritized <span class="hlt">model</span>, the reduced <span class="hlt">model</span> can be two to three orders of magnitude smaller than the original <span class="hlt">model</span> and runs 1,000 faster. More importantly, the reduced <span class="hlt">model</span> captures the dominating characteristics of the full <span class="hlt">model</span> and produces sufficiently accurate solutions. One of the major tasks in the development of the reduced <span class="hlt">model</span> is the selection of snapshots which are used to determine the dominant eigenvectors. This paper discusses ways to optimize the snapshot selection. Additionally, the paper also discusses applications of the reduced <span class="hlt">model</span> to parameter estimation, Monte Carlo simulation and experimental design in groundwater <span class="hlt">modeling</span>.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://eric.ed.gov/?q=Iron&pg=2&id=EJ770219','ERIC'); return false;" href="http://eric.ed.gov/?q=Iron&pg=2&id=EJ770219"><span id="translatedtitle">Synthesis, Purification, and Characterization of a [mu]-(1,3-Propanedithiolato)-Hexacarbonyldiiron</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Works, Carmen F.</p> <p>2007-01-01</p> <p>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 <span class="hlt">model</span> for an iron-only <span class="hlt">hydro-genase</span>. The project provides the students with experience of Schlenk line techniques, purification,…</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/27311461','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/27311461"><span id="translatedtitle"><span class="hlt">Modeling</span> Pharmacokinetics.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Bois, Frederic Y; Brochot, Céline</p> <p>2016-01-01</p> <p>Pharmacokinetics is the study of the fate of xenobiotics in a living organism. Physiologically based pharmacokinetic (PBPK) <span class="hlt">models</span> provide realistic descriptions of xenobiotics' absorption, distribution, metabolism, and excretion processes. They <span class="hlt">model</span> the body as a set of homogeneous compartments representing organs, and their parameters refer to anatomical, physiological, biochemical, and physicochemical entities. They offer a quantitative mechanistic framework to understand and simulate the time-course of the concentration of a substance in various organs and body fluids. These <span class="hlt">models</span> are well suited for performing extrapolations inherent to toxicology and pharmacology (e.g., between species or doses) and for integrating data obtained from various sources (e.g., in vitro or in vivo experiments, structure-activity <span class="hlt">models</span>). In this chapter, we describe the practical development and basic use of a PBPK <span class="hlt">model</span> from <span class="hlt">model</span> building to <span class="hlt">model</span> simulations, through implementation with an easily accessible free software. PMID:27311461</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/828102','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/828102"><span id="translatedtitle">Ventilation <span class="hlt">Model</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>H. Yang</p> <p>1999-11-04</p> <p>The purpose of this analysis and <span class="hlt">model</span> report (AMR) for the Ventilation <span class="hlt">Model</span> is to analyze the effects of pre-closure continuous ventilation in the Engineered Barrier System (EBS) emplacement drifts and provide heat removal data to support EBS design. It will also provide input data (initial conditions, and time varying boundary conditions) for the EBS post-closure performance assessment and the EBS Water Distribution and Removal Process <span class="hlt">Model</span>. The objective of the analysis is to develop, describe, and apply calculation methods and <span class="hlt">models</span> that can be used to predict thermal conditions within emplacement drifts under forced ventilation during the pre-closure period. The scope of this analysis includes: (1) Provide a general description of effects and heat transfer process of emplacement drift ventilation. (2) Develop a <span class="hlt">modeling</span> approach to simulate the impacts of pre-closure ventilation on the thermal conditions in emplacement drifts. (3) Identify and document inputs to be used for <span class="hlt">modeling</span> emplacement ventilation. (4) Perform calculations of temperatures and heat removal in the emplacement drift. (5) Address general considerations of the effect of water/moisture removal by ventilation on the repository thermal conditions. The numerical <span class="hlt">modeling</span> in this document will be limited to heat-only <span class="hlt">modeling</span> and calculations. Only a preliminary assessment of the heat/moisture ventilation effects and <span class="hlt">modeling</span> method will be performed in this revision. <span class="hlt">Modeling</span> of moisture effects on heat removal and emplacement drift temperature may be performed in the future.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://hdl.handle.net/2060/20120013440','NASA-TRS'); return false;" href="http://hdl.handle.net/2060/20120013440"><span id="translatedtitle">Climate <span class="hlt">Models</span></span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Druyan, Leonard M.</p> <p>2012-01-01</p> <p>Climate <span class="hlt">models</span> 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 <span class="hlt">modeling</span> studies for a variety of geographic regions by an international roster of authors. The climate research community generally uses the rubric climate <span class="hlt">models</span> 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 <span class="hlt">models</span> are integrated over time in order to compute the temporal and spatial variations of these parameters. <span class="hlt">Model</span> domains can be global or regional and the horizontal and vertical resolutions of the computational grid vary from <span class="hlt">model</span> to <span class="hlt">model</span>. Considering the entire climate system requires accounting for interactions between solar insolation, atmospheric, oceanic and continental processes, the latter including land hydrology and vegetation. <span class="hlt">Model</span> simulations may concentrate on one or more of these components, but the most sophisticated <span class="hlt">models</span> will estimate the mutual interactions of all of these environments. Advances in computer technology have prompted investments in more complex <span class="hlt">model</span> 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 <span class="hlt">model</span> performance. Extensive research is required to test and document any advantages gained by greater sophistication in <span class="hlt">model</span> formulation. One purpose for publishing climate <span class="hlt">model</span> research results is to present purported advances for evaluation by the scientific community.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/6365078','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/6365078"><span id="translatedtitle">Phenomenological <span class="hlt">models</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Braby, L.A.</p> <p>1990-09-01</p> <p>The biological effects of ionizing radiation exposure are the result of a complex sequence of physical, chemical, biochemical, and physiological interactions. One way to begin a search for an understanding of health effects of radiation is through the development of phenomenological <span class="hlt">models</span> of the response. Many <span class="hlt">models</span> have been presented and tested in the slowly evolving process of characterizing cellular response. A range of <span class="hlt">models</span> covering different endpoints and phenomena has developed in parallel. Many of these <span class="hlt">models</span> employ similar assumptions about some underlying processes while differing about the nature of others. An attempt is made to organize many of the <span class="hlt">models</span> into groups with similar features and to compare the consequences of those features with the actual experimental observations. It is assumed that by showing that some assumptions are inconsistent with experimental observations, the job of devising and testing mechanistic <span class="hlt">models</span> can be simplified. 43 refs., 13 figs.</p> </li> </ol> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li class="active"><span>24</span></li> <li><a href="#" onclick='return showDiv("page_25");'>25</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div><!-- col-sm-12 --> </div><!-- row --> </div><!-- page_24 --> <div id="page_25" class="hiddenDiv"> <div class="row"> <div class="col-sm-12"> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li><a href="#" onclick='return showDiv("page_24");'>24</a></li> <li class="active"><span>25</span></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div> </div> <div class="row"> <div class="col-sm-12"> <ol class="result-class" start="481"> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/96357','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/96357"><span id="translatedtitle">Building <span class="hlt">models</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Burr, M.T.</p> <p>1995-04-01</p> <p>As developers make progress on independent power projects around the world, <span class="hlt">models</span> for success are beginning to emerge. Different <span class="hlt">models</span> are evolving to create ownership structures that accomoate a complex system of regulatory requirements. Other frameworks make use of previously untapped fuel resources, or establish new sources of financing; however, not all <span class="hlt">models</span> may be applied to a given project. This article explores how developers are finding new alternatives for overcoming development challenges that are common to projects in many countries.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/837038','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/837038"><span id="translatedtitle">Ventilation <span class="hlt">Model</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>V. Chipman</p> <p>2002-10-05</p> <p>The purpose of the Ventilation <span class="hlt">Model</span> is to simulate the heat transfer processes in and around waste emplacement drifts during periods of forced ventilation. The <span class="hlt">model</span> 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 <span class="hlt">models</span>, such as the ''Multiscale Thermohydrologic <span class="hlt">Model</span>'' (BSC 2001), use the wall heat fractions as outputted from the Ventilation <span class="hlt">Model</span> to initialize their post-closure analyses. The Ventilation <span class="hlt">Model</span> 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 <span class="hlt">Model</span> included documentation of the <span class="hlt">modeling</span> results from the ANSYS-based heat transfer <span class="hlt">model</span>. The purposes of Revision 01 of the Ventilation <span class="hlt">Model</span> are: (1) To validate the conceptual <span class="hlt">model</span> for preclosure ventilation of emplacement drifts and verify its numerical application in accordance with new procedural requirements as outlined in AP-SIII-10Q, <span class="hlt">Models</span> (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 <span class="hlt">model</span>, the adequacy of the discretization (Section 6.2.3.1), and the downstream applicability of the <span class="hlt">model</span> results (i.e. wall heat fractions) to initialize post</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/878658','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/878658"><span id="translatedtitle"><span class="hlt">Model</span> Selection for Geostatistical <span class="hlt">Models</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Hoeting, Jennifer A.; Davis, Richard A.; Merton, Andrew A.; Thompson, Sandra E.</p> <p>2006-02-01</p> <p>We consider the problem of <span class="hlt">model</span> selection for geospatial data. Spatial correlation is typically ignored in the selection of explanatory variables and this can influence <span class="hlt">model</span> 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 <span class="hlt">model</span>. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical <span class="hlt">model</span> is superior to the often used approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a <span class="hlt">model</span> for lizard abundance. We also employ the principle of minimum description length (MDL) to variable selection for the geostatistical <span class="hlt">model</span>. The effect of sampling design on the selection of explanatory covariates is also explored.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://hdl.handle.net/2060/19950020245','NASA-TRS'); return false;" href="http://hdl.handle.net/2060/19950020245"><span id="translatedtitle">Turbulence <span class="hlt">modeling</span></span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Bardina, Jorge E.</p> <p>1995-01-01</p> <p>The objective of this work is to develop, verify, and incorporate the baseline two-equation turbulence <span class="hlt">models</span> which account for the effects of compressibility into the three-dimensional Reynolds averaged Navier-Stokes (RANS) code and to provide documented descriptions of the <span class="hlt">models</span> and their numerical procedures so that they can be implemented into 3-D CFD codes for engineering applications.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://eric.ed.gov/?q=beyond+AND+budgeting&pg=7&id=ED165814','ERIC'); return false;" href="http://eric.ed.gov/?q=beyond+AND+budgeting&pg=7&id=ED165814"><span id="translatedtitle">Budget <span class="hlt">Model</span>.</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Washington State Board for Community Coll. Education, Olympia.</p> <p></p> <p>Computerized formula-driven budget <span class="hlt">models</span> 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 <span class="hlt">model</span> and illustrates the principles on which…</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://eric.ed.gov/?q=Dispersion+AND+pollutants&id=EJ222510','ERIC'); return false;" href="http://eric.ed.gov/?q=Dispersion+AND+pollutants&id=EJ222510"><span id="translatedtitle">Dispersion <span class="hlt">Modeling</span>.</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Budiansky, Stephen</p> <p>1980-01-01</p> <p>This article discusses the need for more accurate and complete input data and field verification of the various <span class="hlt">models</span> of air pollutant dispension. Consideration should be given to changing the form of air quality standards based on enhanced dispersion <span class="hlt">modeling</span> techniques. (Author/RE)</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://eric.ed.gov/?q=star&pg=4&id=EJ1046112','ERIC'); return false;" href="http://eric.ed.gov/?q=star&pg=4&id=EJ1046112"><span id="translatedtitle"><span class="hlt">Modeling</span> Sunspots</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Oh, Phil Seok; Oh, Sung Jin</p> <p>2013-01-01</p> <p><span class="hlt">Modeling</span> 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 <span class="hlt">modeling</span> activities in an extracurricular science club in a high…</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://files.eric.ed.gov/fulltext/ED024022.pdf','ERIC'); return false;" href="http://files.eric.ed.gov/fulltext/ED024022.pdf"><span id="translatedtitle">Phonological <span class="hlt">Models</span>.</span></a></p> <p><a target="_blank" href="http://www.eric.ed.gov/ERICWebPortal/search/extended.jsp?_pageLabel=advanced">ERIC Educational Resources Information Center</a></p> <p>Ballard, W.L.</p> <p>1968-01-01</p> <p>The article discusses <span class="hlt">models</span> of synchronic and diachronic phonology and suggests changes in them. The basic generative <span class="hlt">model</span> of phonology is outlined with the author's reinterpretations. The systematic phonemic level is questioned in terms of its unreality with respect to linguistic performance and its lack of validity with respect to historical…</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/biblio/6176091','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/biblio/6176091"><span id="translatedtitle">Zitterbewegung <span class="hlt">modeling</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Hestenes, D. )</p> <p>1993-03-01</p> <p>Guidelines for constructing point particle <span class="hlt">models</span> of the electron with [ital zitterbewegung] and other features of the Dirac theory are discussed. Such <span class="hlt">models</span> may at least be useful approximations to the Dirac theory, but the more exciting possibility is that this approach may lead to a more fundamental reality. 6 refs.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/1062195','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/1062195"><span id="translatedtitle">OSPREY <span class="hlt">Model</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Veronica J. Rutledge</p> <p>2013-01-01</p> <p>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 <span class="hlt">model</span> 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 <span class="hlt">models</span> are being developed to simulate each unit operation involved so each unit operation can be used as a stand-alone <span class="hlt">model</span> and in series with multiple others. Currently, an adsorption <span class="hlt">model</span> has been developed within Multi-physics Object Oriented Simulation Environment (MOOSE) developed at the Idaho National Laboratory (INL). Off-gas Separation and REcoverY (OSPREY) <span class="hlt">models</span> the adsorption of off-gas constituents for dispersed plug flow in a packed bed under non-isothermal and non-isobaric conditions. Inputs to the <span class="hlt">model</span> 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 <span class="hlt">model</span> to develop <span class="hlt">models</span> specific for krypton adsorption. The same can be done for iodine, xenon, and tritium. The <span class="hlt">model</span> will be validated with experimental breakthrough curves. Customers will be given access to</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.ncbi.nlm.nih.gov/pubmed/1811477','PUBMED'); return false;" href="http://www.ncbi.nlm.nih.gov/pubmed/1811477"><span id="translatedtitle">Phenomenological <span class="hlt">models</span>.</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Braby, L A</p> <p>1991-01-01</p> <p>The biological effects of ionizing radiation exposure are the result of a complex sequence of physical, chemical, biochemical, and physiological interactions which are modified by characteristics of the radiation, the timing of its administration, the chemical and physical environm