Sample records for fefe hydrogenase model

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

    E-print Network

    Liu, Tianbiao

    2011-02-22

    My research primarily focuses on biomimetics of the active sites of the [FeFe]- and [Fe]-hydrogenases (H2ase) and is classified into three parts. Part A: The one-electron oxidation of asymmetrically disubstituted FeIFeI ...

  2. Site-selective X-ray spectroscopy on an asymmetric model complex of the [FeFe] hydrogenase active site.

    PubMed

    Leidel, Nils; Chernev, Petko; Havelius, Kajsa G V; Ezzaher, Salah; Ott, Sascha; Haumann, Michael

    2012-04-16

    The active site for hydrogen production in [FeFe] hydrogenase comprises a diiron unit. Bioinorganic chemistry has modeled important features of this center, aiming at mechanistic understanding and the development of novel catalysts. However, new assays are required for analyzing the effects of ligand variations at the metal ions. By high-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection (XAS/XES = XAES) and density functional theory (DFT), we studied an asymmetrically coordinated [FeFe] model complex, [(CO)(3)Fe(I)1-(bdtCl(2))-Fe(I)2(CO)(Ph(2)P-CH(2)-NCH(3)-CH(2)-PPh(2))] (1, bdt = benzene-1,2-dithiolate), in comparison to iron-carbonyl references. K? emission spectra (K?(1,3), K?') revealed the absence of unpaired spins and the low-spin character for both Fe ions in 1. In a series of low-spin iron compounds, the K?(1,3) energy did not reflect the formal iron oxidation state, but it decreases with increasing ligand field strength due to shorter iron-ligand bonds, following the spectrochemical series. The intensity of the valence-to-core transitions (K?(2,5)) decreases for increasing Fe-ligand bond length, certain emission peaks allow counting of Fe-CO bonds, and even molecular orbitals (MOs) located on the metal-bridging bdt group of 1 contribute to the spectra. As deduced from 3d ? 1s emission and 1s ? 3d absorption spectra and supported by DFT, the HOMO-LUMO gap of 1 is about 2.8 eV. K?-detected XANES spectra in agreement with DFT revealed considerable electronic asymmetry in 1; the energies and occupancies of Fe-d dominated MOs resemble a square-pyramidal Fe(0) for Fe1 and an octahedral Fe(II) for Fe2. EXAFS spectra for various K? emission energies showed considerable site-selectivity; approximate structural parameters similar to the crystal structure could be determined for the two individual iron atoms of 1 in powder samples. These results suggest that metal site- and spin-selective XAES on [FeFe] hydrogenase protein and active site models may provide a powerful tool to study intermediates under reaction conditions. PMID:22443530

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

    E-print Network

    Liu, Tianbiao

    2011-02-22

    in the FeIFeI diiron models ((mu- pdt)[Fe(CO)2L][Fe(CO)2L'] ( L, L' = CO, PPh3, or PMe3) is thermodynamically favored to produce the mu-oxo or oxidative addition product, FeII-O-FeII, nevertheless the sulfurbased HOMO-1 accounts for the experimentally...

  4. New Insights into [FeFe] Hydrogenase Activation and Maturase Function

    PubMed Central

    Kuchenreuther, Jon M.; Britt, R. David; Swartz, James R.

    2012-01-01

    [FeFe] hydrogenases catalyze H2 production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN–), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN– ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN– ligands that is a precursor to the H-cluster [2Fe]H subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe]H subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG. PMID:23049878

  5. 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

  6. Bridging-hydride influence on the electronic structure of an [FeFe] hydrogenase active-site model complex revealed by XAES-DFT.

    PubMed

    Leidel, Nils; Hsieh, Chung-Hung; Chernev, Petko; Sigfridsson, Kajsa G V; Darensbourg, Marcetta Y; Haumann, Michael

    2013-06-01

    Two crystallized [FeFe] hydrogenase model complexes, 1 = (?-pdt)[Fe(CO)(2)(PMe(3))](2) (pdt = SC1H2C2H2C3H2S), and their bridging-hydride (Hy) derivative, [1Hy](+) = [(?-H)(?-pdt)[Fe(CO)(2) (PMe(3))](2)](+) (BF(4)(?)), were studied by Fe K-edge X-ray absorption and emission spectroscopy, supported by density functional theory. Structural changes in [1Hy](+) compared to 1 involved small bond elongations (<0.03 Å) and more octahedral Fe geometries; the Fe–H bond at Fe1 (closer to pdt-C2) was ~0.03 Å longer than that at Fe2. Analyses of (1) pre-edge absorption spectra (core-to-valence transitions), (2) K?(1,3), K?', and K?(2,5) emission spectra (valence-to-core transitions), and (3) resonant inelastic X-ray scattering data (valence-to-valence transitions) for resonant and non-resonant excitation and respective spectral simulations indicated the following: (1) the mean Fe oxidation state was similar in both complexes, due to electron density transfer from the ligands to Hy in [1Hy](+). Fe 1s?3d transitions remained at similar energies whereas delocalization of carbonyl AOs onto Fe and significant Hy-contributions to MOs caused an ~0.7 eV up-shift of Fe1s?(CO)s,p transitions in [1Hy](+). Fed-levels were delocalized over Fe1 and Fe2 and degeneracies biased to O(h)–Fe1 and C(4v)–Fe2 states for 1, but to O(h)–Fe1,2 states for [1Hy](+). (2) Electron-pairing of formal Fe(d(7)) ions in low-spin states in both complexes and a higher effective spin count for [1Hy](+) were suggested by comparison with iron reference compounds. Electronic decays from Fe d and ligand s,p MOs and spectral contributions from Hys,p?1s transitions even revealed limited site-selectivity for detection of Fe1 or Fe2 in [1Hy](+). The HOMO/LUMO energy gap for 1 was estimated as 3.0 ± 0.5 eV. (3) For [1Hy](+) compared to 1, increased Fed (x(2) ? y(2)) ? (z(2)) energy differences (~0.5 eV to ~0.9 eV) and Fed?d transition energies (~2.9 eV to ~3.7 eV) were assigned. These results reveal the specific impact of Hy-binding on the electronic structure of diiron compounds and provide guidelines for a directed search of hydride species in hydrogenases. PMID:23446996

  7. 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

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

    PubMed

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

    2014-01-30

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

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

    PubMed Central

    Stapleton, James A.; Swartz, James R.

    2010-01-01

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

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

    PubMed

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

    2012-02-01

    We have completed a bioinformatic analysis of the hydrogenases encoded in the genomes of three termite gut treponeme isolates: hydrogenotrophic, homoacetogenic Treponema primitia strains ZAS-1 and ZAS-2, and the hydrogen-producing, sugar-fermenting Treponema azotonutricium ZAS-9. H(2) is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The spirochetes encoded 4, 8, and 5 [FeFe] hydrogenase-like proteins, identified by their H domains, respectively, but no other recognizable hydrogenases. The [FeFe] hydrogenases represented many sequence families previously proposed in an analysis of termite gut metagenomic data. Each strain encoded both putative [FeFe] hydrogenase enzymes and evolutionarily related hydrogen sensor/transducer proteins likely involved in phosphorelay or methylation pathways, and possibly even chemotaxis. A new family of [FeFe] hydrogenases (FDH-Linked) is proposed that may form a multimeric complex with formate dehydrogenase to provide reducing equivalents for reductive acetogenesis in T. primitia. The many and diverse [FeFe] hydrogenase-like proteins encoded within the sequenced genomes of the termite gut treponemes has enabled the discovery of a putative new class of [FeFe] hydrogenase proteins potentially involved in acetogenesis and furthered present understanding of many families, including sensory, of H domain proteins beyond what was possible through the use of fragmentary termite gut metagenome sequence data alone, from which they were initially defined. PMID:21811792

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

    PubMed

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

    2012-10-21

    Di/mono-nuclear iron(I)/(II) complexes containing conjugated and electron-withdrawing S-to-S linkers, [{(?-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(6)] (1), [{(?-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(5)(PMe(3))] (1P), and [{(?-S)(2)(C(4)N(2)H(2))}Fe(CO)(2)(PMe(3))(2)] (2) were prepared as biomimetic models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases. The N atoms in the heterocyclic pyrazines of 1 and 2 were protonated in the presence of proton acid to generate one and two hydrides, [1(NH)](+) CF(3)SO(3)(-), [2(NH)](+) CF(3)SO(3)(-), and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2), respectively. The protonation processes were evidenced by in situ IR and NMR spectroscopy. The molecular structures of the protonated species [1(NH)](+) CF(3)SO(3)(-) and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2) together with their originating complexes and , and the mono-PMe(3) substituted diiron complex were identified by X-ray crystallography. The IR and single-crystal analysis data all suggested that the electron-withdrawing bridge, pyrazine, led to decreased electron density at the Fe centers of the model complexes, which was consistent with the electrochemical studies. The cyclic voltammograms indicated that complex exhibited a low primary reduction potential at -1.17 V vs. Fc-Fc(+) with a 270 mV positive shift compared with that of the benzene-1,2-dithiolate (bdt) bridged analogue [(?-bdt)Fe(2)(CO)(6)]. Under the weak acid conditions, complexes 1 and 2 could electrochemically catalyze the proton reduction. More interestingly, the mononuclear ferrous complex 2 showed two catalytic peaks during the formation of hydrogen, confirming its potential as a catalyst for hydrogen production. PMID:22911248

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

    PubMed

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

    2015-01-01

    The reduction of the Fe(II)Fe(II) complex [Fe2(CO)2{P(OMe)3}2(?(2)-IMe-CH2-IMe)(?-CO)(?-pdt)](2+) (2P(2+); pdt = S(CH2)3S), which is a synthetic model of the H cluster of the [FeFe] hydrogenases in its inactive state, has been investigated electrochemically and theoretically (by density functional theory, DFT) in order to determine the mechanisms, intermediates, and products of the related processes. The electrochemical reduction of 2P(2+) occurs according to an ECE-type reaction where the intervening chemical step is the loss of one P(OMe)3 ligand. This outcome, which is based on cyclic voltammetric experiments, is strongly supported by DFT calculations that provide additional information on the intermediates and the energetics of the reactions involved. The electrochemical reoxidation of the neutral product of the reduction follows an EEC process where the chemical step is the binding of P(OMe)3 to a dicationic intermediate. DFT calculations reveal that this intermediate has an unusual geometry wherein one of the two C-H bonds of a side methylene from the pdt group forms an agostic interaction with one Fe center. This interaction is crucial to stabilize the 32e(-) diferrous center and concomitantly to preserve Fe(II) from binding of weakly coordinating species. Nonetheless, it could be displaced by a relatively stronger electron donor such as H2, which could be relevant for the design of new oxidation catalysts. PMID:25496017

  13. NRVS and EPR Spectroscopy of 57Fe-enriched [FeFe] Hydrogenase Indicate Stepwise Assembly of the H-cluster†

    PubMed Central

    Kuchenreuther, Jon M.; Guo, Yisong; Wang, Hongxin; Myers, William K.; George, Simon J.; Boyke, Christine A.; Yoda, Yoshitaka; Alp, E. Ercan; Zhao, Jiyong; Britt, R. David; Swartz, James R.; Cramer, Stephen P.

    2013-01-01

    The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe–S clusters that facilitate electron transfer to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H2. Here, we have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe–4S] ferredoxin as well as a model complex resembling the [2Fe]H catalytic domain of the H-cluster. In order to enrich the hydrogenase with 57Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with 56Fe-labeled Fe–S clusters was activated in vitro using 57Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe]H subcluster with 57Fe, which NRVS confirms by detecting 57Fe–CO and 57Fe–CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second 57Fe–S cluster. EPR spectroscopy indicates that this 57Fe-enriched metal center is not the [4Fe– 4S]H subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an 56Fe-enriched [4Fe–4S]H cluster during in vitro maturation, providing unambiguous evidence for stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases. PMID:23249091

  14. Patterns of [FeFe] hydrogenase diversity in the gut microbial communities of lignocellulose-feeding higher termites.

    PubMed

    Ballor, Nicholas R; Leadbetter, Jared R

    2012-08-01

    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

  15. 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

  16. Raman spectroscopy of charge transfer interactions between single wall carbon nanotubes and [FeFe] hydrogenase.

    PubMed

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

    2008-10-28

    We report a Raman spectroscopy study of charge transfer interactions in complexes formed by single-walled carbon nanotubes (SWNTs) and [FeFe] hydrogenase I (CaHydI) from Clostridium acetobutylicum. The choice of Raman excitation wavelength and sample preparation conditions allows differences to be observed for complexes involving metallic (m) and semiconducting (s) species. Adsorbed CaHydI can reversibly inject electronic charge into the LUMOs of s-SWNTs, while charge can be injected and removed from m-SWNTs at lower potentials just above the Fermi energy. Time-dependent enzymatic assays demonstrated that the reduced and oxidized forms of CaHydI are deactivated by oxygen, but at rates that varied by an order of magnitude. The time evolution of the oxidative decay of the CaHydI activity reveals different time constants when complexed with m-SWNTs and s-SWNTs. The correlation of enzymatic assays with time-dependent Raman spectroscopy provides a novel method by which the charge transfer interactions may be investigated in the various SWNT-CaHydI complexes. Surprisingly, an oxidized form of CaHydI is apparently more resistant to oxygen deactivation when complexed to m-SWNTs rather than s-SWNTs. PMID:19082027

  17. 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

  18. Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [(57)Fe2(adt)(CN)2(CO)4](2.).

    PubMed

    Gilbert-Wilson, Ryan; Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Pham, Cindy C; Reijerse, Edward; Wang, Hongxin; Cramer, Stephen P; Lubitz, Wolfgang; Rauchfuss, Thomas B

    2015-07-22

    The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively (57)Fe-labeled binuclear subsite is described. The precursor [(57)Fe2(adt)(CN)2(CO)4](2-) was synthesized from the (57)Fe metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)2[(57)Fe2(adt)(CN)2(CO)4] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]H subcluster. Complementary (57)Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with (57)FeCl3 and Na2S. The Hox-CO state of [2(57)Fe]H and [4(57)Fe-4S]H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy. PMID:26091969

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

    PubMed

    Chang, Christopher H

    2011-08-11

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

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

    PubMed

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

    2013-05-01

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

  1. The cyanide ligands of [FeFe] hydrogenase: pulse EPR studies of (13)C and (15)N-labeled H-cluster.

    PubMed

    Myers, William K; Stich, Troy A; Suess, Daniel L M; Kuchenreuther, Jon M; Swartz, James R; Britt, R David

    2014-09-01

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

  2. Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3).

    PubMed

    Akhtar, M Kalim; Jones, Patrik R

    2008-04-01

    Proteins that catalyze H2-pathways often contain iron-sulfur (Fe-S) clusters and are sensitive to O2. We tested whether deletion of the gene encoding the transcriptional negative regulator, IscR, could enhance the ability of Escherichia coli BL21 to synthesize active recombinant H2-pathway components and stimulate ferredoxin-dependent H2-accumulation in the presence or absence of oxygen. Under anoxic conditions, deletion of iscR stimulated recombinant Fe-Fe hydrogenase activity threefold, whilst plasmid-based overexpression of the isc operon had no effect on hydrogenase activity. After cultivation with 21% (v/v) O2 in the headspace, no recombinant hydrogenase activity was observed in soluble extracts of wild-type BL21, although low levels of activity could be observed in the DeltaiscR strain (700-fold lower than anoxic conditions, 180-fold greater than the limit of detection). Under closed batch conditions starting with 5% (v/v) O2, DeltaiscR strains displayed fivefold greater levels of total hydrogenase activity and recombinant ferredoxin-dependent H2-accumulation relative to the control strain. In cultures starting with 10% (v/v) O2, H2-accumulation was stimulated 35-fold relative to the control. DeltaiscR strains displayed enhanced synthesis and activity of integral H2-pathway components under all tested conditions and enhanced H2-accumulation under partially oxic conditions. Deletion of iscR is, therefore, a useful strategy to stimulate H2-production, particularly if the hydrogenase catalyzes the rate-limiting reaction. PMID:18320190

  3. 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

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

    PubMed

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

    2010-10-01

    The purpose of the present study was to evaluate the use of a non-innocent ligand as a surrogate of the anchored [4Fe4S] cubane in a synthetic mimic of the [FeFe] hydrogenase active site. Reaction of 2,3-bis(diphenylphosphino) maleic anhydride (bma) with [Fe(2)(CO)(6)(mu-pdt)] (propanedithiolate, pdt=S(CH(2))(3)S) in the presence of Me(3)NO-2H(2)O afforded the monosubstituted derivative [Fe(2)(CO)(5)(Me(2)NCH(2)PPh(2))(mu-pdt)] (1). This results from the decomposition of the bma ligand and the apparent C-H bond cleavage in the released trimethylamine. Reaction under photolytic conditions afforded [Fe(2)(CO)(4)(bma)(mu-pdt)] (2). Compounds 1 and 2 were characterized by IR, NMR and X-ray diffraction. Voltammetric study indicated that the primary reduction of 2 is centered on the bma ligand. PMID:20547420

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

    Microsoft Academic Search

    James A. Stapleton; James R. Swartz; Floyd Romesberg

    2010-01-01

    Background[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of

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

    SciTech Connect

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

    2012-01-28

    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.

  7. The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

    PubMed

    Kuchenreuther, Jon M; Myers, William K; Suess, Daniel L M; Stich, Troy A; Pelmenschikov, Vladimir; Shiigi, Stacey A; Cramer, Stephen P; Swartz, James R; Britt, R David; George, Simon J

    2014-01-24

    Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster. PMID:24458644

  8. Time-resolved vibrational spectroscopy of [FeFe]-hydrogenase model compounds.

    PubMed

    Bingaman, Jamie L; Kohnhorst, Casey L; Van Meter, Glenn A; McElroy, Brent A; Rakowski, Elizabeth A; Caplins, Benjamin W; Gutowski, Tiffany A; Stromberg, Christopher J; Webster, Charles Edwin; Heilweil, Edwin J

    2012-07-12

    Model compounds have been found to structurally mimic the catalytic hydrogen-producing active site of Fe-Fe hydrogenases and are being explored as functional models. The time-dependent behavior of Fe(2)(?-S(2)C(3)H(6))(CO)(6) and Fe(2)(?-S(2)C(2)H(4))(CO)(6) is reviewed and new ultrafast UV- and visible-excitation/IR-probe measurements of the carbonyl stretching region are presented. Ground-state and excited-state electronic and vibrational properties of Fe(2)(?-S(2)C(3)H(6))(CO)(6) were studied with density functional theory (DFT) calculations. For Fe(2)(?-S(2)C(3)H(6))(CO)(6) excited with 266 nm, long-lived signals (? = 3.7 ± 0.26 ?s) are assigned to loss of a CO ligand. For 355 and 532 nm excitation, short-lived (? = 150 ± 17 ps) bands are observed in addition to CO-loss product. Short-lived transient absorption intensities are smaller for 355 nm and much larger for 532 nm excitation and are assigned to a short-lived photoproduct resulting from excited electronic state structural reorganization of the Fe-Fe bond. Because these molecules are tethered by bridging disulfur ligands, this extended di-iron bond relaxes during the excited state decay. Interestingly, and perhaps fortuitously, the time-dependent DFT-optimized exited-state geometry of Fe(2)(?-S(2)C(3)H(6))(CO)(6) with a semibridging CO is reminiscent of the geometry of the Fe(2)S(2) subcluster of the active site observed in Fe-Fe hydrogenase X-ray crystal structures. We suggest these wavelength-dependent excitation dynamics could significantly alter potential mechanisms for light-driven catalysis. PMID:22612846

  9. Immobilized metallodithiolate ligand supports for construction of bioinorganic model complexes 

    E-print Network

    Green, Kayla Nalynn

    2009-05-15

    to the analogous NiN2S2 2- systems. As the active site of [FeFe] Hydrogenase utilizes a unique peptide-bound propane dithiolate bridge to support the FeFe organometallic unit, [FeFe]Hydrogenase models have been covalently anchored to the resin-beads via similar...

  10. Genomic Analysis Reveals Multiple [FeFe] Hydrogenases and Hydrogen Sensors Encoded by Treponemes from the H 2 Rich Termite Gut

    Microsoft Academic Search

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

    We have completed a bioinformatic analysis of the hydrogenases encoded in the genomes of three termite gut treponeme isolates:\\u000a hydrogenotrophic, homoacetogenic Treponema primitia strains ZAS-1 and ZAS-2, and the hydrogen-producing, sugar-fermenting Treponema azotonutricium ZAS-9. H2 is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations\\u000a in some species exceeding those measured for any

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

    PubMed

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

    2014-05-19

    Hydrogenase enzymes in nature use hydrogen as a fuel, but the heterolytic cleavage of H-H bonds cannot be readily observed in enzymes. Here we show that an iron complex with pendant amines in the diphosphine ligand cleaves hydrogen heterolytically. The product has a strong Fe-H???H-N dihydrogen bond. The structure was determined by single-crystal neutron diffraction, and has a remarkably short H???H distance of 1.489(10)?Å between the protic N-H(?+) and hydridic Fe-H(?-) part. The structural data for [Cp(C5F4N)FeH(P(tBu)2N(tBu)2H)](+) provide a glimpse of how the H-H bond is oxidized or generated in hydrogenase enzymes. These results now provide a full picture for the first time, illustrating structures and reactivity of the dihydrogen complex and the product of the heterolytic cleavage of H2 in a functional model of the active site of the [FeFe] hydrogenase enzyme. PMID:24757087

  12. 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

  13. Mössbauer and computational investigation of a functional [NiFe] hydrogenase model complex.

    PubMed

    Kochem, A; Bill, E; Neese, F; van Gastel, M

    2015-02-01

    Developing biomimetic complexes that model the active site of [NiFe] hydrogenase enzymes in order to catalyze the activation of H2 is a topic of major interest. A functional [NiFe] hydrogenase model complex has recently been described by Ogo et al. (Science, 2013, 339, 682-683). Here, we report a Mössbauer and computational investigation of this model complex. This study affords deeper understanding of the electronic structure, the reactivity and the mechanism of H2 activation by this complex. PMID:25535725

  14. Hydrogen generation: aromatic dithiolate-bridged metal carbonyl complexes as hydrogenase catalytic site models.

    PubMed

    Pandey, Indresh Kumar; Natarajan, Mookan; Kaur-Ghumaan, Sandeep

    2015-02-01

    The design, syntheses and characteristics of metal carbonyl complexes with aromatic dithiolate linkers reported as bioinspired hydrogenase catalytic site models are described and reviewed. Among these the complexes capable of hydrogen generation have been discussed in detail. Comparisons have been made with carbonyl complexes having alkyl dithiolates as linkers between metal centers. PMID:25528677

  15. Synthesis and reactivity of mononuclear iron models of [Fe]-hydrogenase that contain an acylmethylpyridinol ligand.

    PubMed

    Hu, Bowen; Chen, Dafa; Hu, Xile

    2014-02-01

    [Fe]-hydrogenase has a single iron-containing active site that features an acylmethylpyridinol ligand. This unique ligand environment had yet to be reproduced in synthetic models; however the synthesis and reactivity of a new class of small molecule mimics of [Fe]-hydrogenase in which a mono-iron center is ligated by an acylmethylpyridinol ligand has now been achieved. Key to the preparation of these model compounds is the successful C-O cleavage of an alkyl ether moiety to form the desired pyridinol ligand. Reaction of solvated complex [(2-CH2CO-6-HOC5H3N)Fe(CO)2(CH3CN)2](+)(BF4)(-) with thiols or thiophenols in the presence of NEt3 yielded 5-coordinate iron thiolate complexes. Further derivation produced complexes [(2-CH2CO-6-HOC5H3N)Fe(CO)2(SCH2CH2OH)] and [(2-CH2CO-6-HOC5H3N)Fe(CO)2(CH3COO)], which can be regarded as models of FeGP cofactors of [Fe]-hydrogenase extracted by 2-mercaptoethanol and acetic acid, respectively. When the derivative complexes were treated with HBF4 ?Et2O, the solvated complex was regenerated by protonation of the thiolate ligands. The reactivity of several models with CO, isocyanide, cyanide, and H2 was also investigated. PMID:24402840

  16. Combining spectroscopy and theory to evaluate structural models of metalloenzymes: a case study on the soluble [NiFe] hydrogenase from Ralstonia eutropha.

    PubMed

    Horch, Marius; Rippers, Yvonne; Mroginski, Maria A; Hildebrandt, Peter; Zebger, Ingo

    2013-01-14

    Hydrogenases catalyse the reversible cleavage of molecular hydrogen into protons and electrons. While most of these enzymes are inhibited under aerobic conditions, some hydrogenases are catalytically active even at ambient oxygen levels. In particular, the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 couples reversible hydrogen cycling to the redox conversion of NAD(H). Its insensitivity towards oxygen has been formerly ascribed to the putative presence of additional cyanide ligands at the active site, which has been, however, discussed controversially. Based on quantum chemical calculations of model compounds, we demonstrate that spectroscopic consequences of the proposed non-standard set of inorganic ligands are in contradiction to the underlying experimental findings. In this way, the previous model for structure and function of this soluble hydrogenase is disproved on a fundamental level, thereby highlighting the efficiency of computational methods for the evaluation of experimentally derived mechanistic proposals. PMID:23161555

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

    PubMed

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

    2012-12-26

    Two model compounds of the active site of [NiFe]-hydrogenases with an unusual {S(2)Ni(?-S)(?-CO)Fe(CO)(2)S}-coordination environment around the metals are reported. The neutral compound [Ni(xbsms)(?-CO)(?-S)Fe(CO)(2)('S')], (1) (H(2)xbsms = 1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) is converted to [1H][BF(4)] by reversible protonation using HBF(4)·Et(2)O. The protonation takes place at the terminal thiolate sulfur atom that is coordinated to nickel. Catalytic intermediates with a protonated terminal cysteinate were suggested for the native protein but have not yet been confirmed experimentally. [1H][BF(4)] is the first dinuclear [NiFe] model compound for such a species. Both complexes have been synthesized and characterized by X-ray crystallography, NMR-, FTIR-, and (57)Fe-Mössbauer spectroscopy as well as by electronic absorption and resonance Raman spectroscopy. The experimental results clearly show that the protonation has a significant impact on the electronic structure of the iron center, although it takes place at the nickel site. DFT calculations support the interpretation of the spectroscopic data and indicate the presence of a bonding interaction between the metal ions, which is relevant for the enzyme as well. Electrochemical experiments show that both 1 and [1H][BF(4)] are active for electrocatalytic proton reduction in aprotic solvents. PMID:23194246

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

    PubMed

    Wombwell, Claire; Reisner, Erwin

    2015-05-26

    A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe('S2 Se2 ')(CO)3 ] (H2 'S2 Se2 '=1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni('S2 Se2 ')] with [Fe(CO)3 bda] (bda=benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe('S2 Se2 ')(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('S2 Se2 ')(CO)3 ] with the previously reported thiolate analogue [NiFe('S4 ')(CO)3 ] (H2 'S4 '=H2 xbsms=1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe('S2 Se2 ')(CO)3 ] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe('S2 Se2 ')(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

  19. Insights into the structure of the active site of the O2-tolerant membrane bound [NiFe] hydrogenase of R. eutropha H16 by molecular modelling.

    PubMed

    Rippers, Yvonne; Utesch, Tillmann; Hildebrandt, Peter; Zebger, Ingo; Mroginski, Maria Andrea

    2011-09-28

    Structural models for the Ni-B state of the wild-type and C81S protein variant of the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha H16 were derived by applying the homology model technique combined with molecular simulations and a hybrid quantum mechanical/molecular mechanical approach. The active site structure was assessed by comparing calculated and experimental IR spectra, confirming the view that the active site structure is very similar to those of anaerobic standard hydrogenases. In addition, the data suggest the presence of a water molecule in the second coordination sphere of the active centre. PMID:21833416

  20. CHEMISTRY RELATED [Fe]-HYDROGENASES

    E-print Network

    Paris-Sud XI, Université de

    CHEMISTRY RELATED TO THE [Fe]-HYDROGENASES A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy Submitted July 2005 Cédric Tard Department of Biological Chemistry John of the biology and chemistry of iron-sulfur proteins and chemical models of their prosthetic groups

  1. Diiron Azadithiolates as Models for the [FeFe]-Hydrogenase Active Site and Paradigm for the Role of the Second Coordination Sphere.

    PubMed

    Rauchfuss, Thomas B

    2015-07-21

    The [FeFe] hydrogenases (H2ases) catalyze the redox reaction that interconverts protons and H2. This area of biocatalysis has attracted attention because the metal-based chemistry is unusual, and the reactions have practical implications. The active site consists of a [4Fe-4S] cluster bridged to a [Fe2(?-dithiolate)(CN)2(CO)3](z) center (z = 1- and 2-). The dithiolate cofactor is [HN(CH2S)2](2-), called the azadithiolate ([adt(H)](2-)). Although many derivatives of Fe2(SR)2(CO)6-xLx are electrocatalysts for the hydrogen evolution reaction (HER), most operate by slow nonbiomimetic pathways. Biomimetic hydrogenogenesis is thought to involve intermediates, wherein the hydride substrate is adjacent to the amine of the adt(H), being bonded to only one Fe center. Formation of terminal hydride complexes is favored when the diiron carbonyl models contain azadithiolate. Although unstable in the free state, the adt cofactor is stable once it is affixed to the Fe2 center. It can be prepared by alkylation of Fe2(SH)2(CO)6 with formaldehyde in the presence of ammonia (to give adt(H) derivatives) or amines (to give adt(R) derivatives). Weak acids protonate Fe2(adt(R))(CO)2(PR3)4 to give terminal hydrido (term-H) complexes. In contrast, protonation of the related 1,3-propanedithiolate (pdt(2-)) complexes Fe2(pdt)(CO)2(PR3)4 requires strong acids. The amine in the azadithiolate is a kinetically fast base, relaying protons to and from the iron, which is a kinetically slow base. The crystal structure of the doubly protonated model [(term-H)Fe2(Hadt(H))(CO)2(dppv)2](2+) confirms the presence of both ammonium and terminal hydrido centers, which interact through a dihydrogen bond (dppv = cis-C2H2(PPh2)2). DFT calculations indicate that this H---H interaction is sensitive to the counterions and is strengthened upon reduction of the diiron center. For the monoprotonated models, the hydride [(term-H)Fe2(adt(H))(CO)2(dppv)2](+) exists in equilibrium with the ammonium tautomer [Fe2(Hadt(H))(CO)2(dppv)2](+). Both [(term-H)Fe2(Hadt(H))(CO)2(dppv)2](2+) and [(term-H)Fe2(adt(H))(CO)2(dppv)2](+) are highly active electrocatalysts for HER. Catalysis is initiated by reduction of the diferrous center, which induces coupling of the protic ammonium center and the hydride ligand. In contrast, the propanedithiolate [(term-H)Fe2(pdt)(CO)2(dppv)2](+) is a poor electrocatalyst for HER. Oxidation of H2 has been demonstrated, starting with models for the oxidized state ("Hox"), for example, [Fe2(adt(H))(CO)3(dppv)(PMe3)](+). Featuring a distorted Fe(II)Fe(I) center, this Hox model reacts slowly with high pressures of H2 to give [(?-H)Fe2(adt(H))(CO)3(dppv)(PMe3)](+). Highlighting the role of the proton relay, the propanedithiolate [Fe2(pdt)(CO)3(dppv)(PMe3)](+) is unreactive toward H2. The Hox-model + H2 reaction is accelerated in the presence of ferrocenium salts, which simulate the role of the attached [4Fe-4S] cluster. The redox-complemented complex [Fe2(adt(Bn))(CO)3(dppv)(FcP*)](n+) catalyzes both proton reduction and hydrogen oxidation (FcP* = (C5Me5)Fe(C5Me4CH2PEt2)). PMID:26079848

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

    E-print Network

    Tye, Jesse Wayne

    2009-05-15

    background on the iron-iron hydrogenase enzyme that is relevant for understanding that chapter. Motivations for Studying H 2 Activation Why activate H 2 ? 1 Dihydrogen has the potential to act as a "clean" alternative to fossil fuels. 1 The oxidation... potential of the corresponding ? 1 -N 2 complex. 44,50,51 For ? 1 -N 2 complexes with ?(NN) values of less than 2050 cm ?1 and E 1/2 values of less than 0.5 V, the reaction with H 2 led to H-H cleavage and only the dihydride form was observed...

  3. Multiple-timescale photoreactivity of a model compound related to the active site of [FeFe]-hydrogenase.

    PubMed

    Ridley, Anna R; Stewart, A Ian; Adamczyk, Katrin; Ghosh, Hirendra N; Kerkeni, Boutheïna; Guo, Z Xiao; Nibbering, Erik T J; Pickett, Christopher J; Hunt, Neil T

    2008-09-01

    Ultraviolet (UV) photolysis of (mu-S(CH 2) 3S)Fe 2(CO) 6 ( 1), a model compound of the Fe-hydrogenase enzyme system, has been carried out. When ultrafast UV-pump infrared (IR)-probe spectroscopy, steady-state Fourier transform IR spectroscopic methods, and density functional theory simulations are employed, it has been determined that irradiation of 1 in an alkane solution at 350 nm leads to the formation of two isomers of the 16-electron complex (mu-S(CH 2) 3S)Fe 2(CO) 5 within 50 ps with evidence of a weakly associated solvent adduct complex. 1 is subsequently recovered on timescales covering several minutes. These studies constitute the first attempt to study the photochemistry and reactivity of these enzyme active site models in solution following carbonyl ligand photolysis. PMID:18665586

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

    PubMed Central

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

    2010-01-01

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

  5. Multiscale modeling of the active site of [fe] hydrogenase: the h2 binding site in open and closed protein conformations.

    PubMed

    Hedegård, Erik Donovan; Kongsted, Jacob; Ryde, Ulf

    2015-05-18

    A series of QM/MM optimizations of the full protein of [Fe] hydrogenase were performed. The FeGP cofactor has been optimized in the water-bound resting state (1), with a side-on bound dihydrogen (2), or as a hydride intermediate (3). For inclusion of H4 ?MPT in the closed structure, advanced multiscale modeling appears to be necessary, especially to obtain reliable distances between CH-H4 MPT(+) and the dihydrogen (H2 ) or hydride (H(-) ) ligand in the FeGP cofactor. Inclusion of the full protein is further important for the relative energies of the two intermediates 2 and 3. We finally find that hydride transfer from 3 has a significantly higher barrier than found in previous studies neglecting the full protein environment. PMID:25867218

  6. An iron carbonyl pyridonate complex related to the active site of the [Fe]-hydrogenase (Hmd).

    PubMed

    Obrist, Blaise V; Chen, Dafa; Ahrens, Anne; Schünemann, Volker; Scopelliti, Rosario; Hu, Xile

    2009-04-20

    A mononuclear iron bis(carbonyl) pyridonate complex (1), which exhibits several common structural features with the active site of the iron-sulfur cluster-free [Fe]-hydrogenase, was synthesized and characterized. Spectroscopic data of 1 suggests a 2+ oxidation state for the Fe ion in the [Fe]-hydrogenase. Complex 1 serves as a precursor to other hydrogenase models. PMID:19320470

  7. 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.

  8. 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

  9. Studies on hydrogenase.

    PubMed

    Yagi, Tatsuhiko; Higuchi, Yoshiki

    2013-01-01

    Hydrogenases are microbial enzymes which catalyze uptake and production of H(2). 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. H(2) is heterolytically cleaved on the enzyme (E) to produce EH(a)H(b), where H(a) and H(b) 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 H(2)-uptake, H(2)-production and isotopic exchange reactions.(Communicated by Shigekazu NAGATA, M.J.A.). PMID:23318679

  10. Multiple-Timescale Photoreactivity of a Model Compound Related to the Active Site of [FeFe]-Hydrogenase

    E-print Network

    Strathclyde, University of

    of iron carbonyl sulfide, Fe2(CO)6(µ- S2), in a Nujol matrix show that the two lowest-energy transitions carbonyl ligand photolysis. The Fe-only hydrogenases reversibly catalyze the reduction of protons, as well as the loss of the infrared (IR) band associated with the bridging carbonyl ligand.6 Pho- tolysis

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

    PubMed

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

    2012-07-01

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

  12. 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. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Wang, Xiaoping [Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Hoffmann, Christina [Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); DuBois, Daniel L. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Bullock, R. Morris [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

    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-H?+ and hydridic Fe-H?-. The structural data for [CpC5F4NFeH(PtBu2NtBu2H)]+ provide a glimpse of how the H-H bond is oxidized or generated in hydrogenase enzymes, with the pendant amine playing a key role as a proton relay. The iron complex [CpC5F4NFeH(PtBu2NtBu2H)]+BArF4- is an electrocatalyst for oxidation of H2 (1 atm) at 22 °C, so the structural data are obtained on a complex that is a functional model for catalysis by [FeFe]-hydrogenase enzymes. This research was supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

  13. The role of CN and CO ligands in the vibrational relaxation dynamics of model compounds of the [FeFe]-hydrogenase enzyme.

    PubMed

    Kaziannis, Spyridon; Wright, Joseph A; Candelaresi, Marco; Kania, Rafal; Greetham, Gregory M; Parker, Anthony W; Pickett, Christopher J; Hunt, Neil T

    2011-06-01

    The vibrational dynamics of (?-propanedithiolate)Fe(2)(CO)(4)(CN)(2)(2-), a model compound of the active site of the [FeFe]-hydrogenase enzyme, have been examined via ultrafast 2D-IR spectroscopy. The results indicate that the vibrational coupling between the stretching modes of the CO and CN ligands is small and restricted to certain modes but the slow growth of off-diagonal peaks is assigned to population transfer processes occurring between these modes on timescales of 30-40 ps. Analysis of the dynamics in concert with anharmonic density functional theory simulations shows that the presence of CN ligands alters the vibrational relaxation dynamics of the CO modes in comparison to all-carbonyl model systems and suggests that the presence of these ligands in the enzyme may be an important feature in terms of directing the vibrational relaxation mechanism. PMID:21537507

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

    PubMed Central

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

    1989-01-01

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

  15. Ni(I)/Ru(II) model for the Ni-L state of the [NiFe]hydrogenases: synthesis, spectroscopy, and reactivity.

    PubMed

    Chambers, Geoffrey M; Mitra, Joyee; Rauchfuss, Thomas B; Stein, Matthias

    2014-04-21

    This study describes the characterization of a mixed-valence Ru(II)/Ni(I) complex, a structural model for the Ni-L state of the [NiFe]hydrogenases. One-electron oxidation of (cymene)Ru(?-pdt)Ni(diphos) ([1](0), diphos = dppe, C2H4(PPh2)2; [2](0), diphos = dcpe, C2H4(P(C6H11)2)2] affords the mixed-valence cations [(cymene)Ru(pdt)Ni(diphos)](+) ([1](+) and [2](+)). Crystallographic and spectroscopic measurements indicate that these cations are described as Ru(II)/Ni(I). Although [1](0) and [1](+) are very similar structurally, the following changes are notable: the Ni-P distances elongate upon oxidation, and the Ru-Ni distance changes insignificantly. The molecular and electronic structures of the Ni center in [1](+) approaches that observed in the [NiFe]hydrogenases. Density functional theory calculations indicate that [1](0) is best described as Ru(II)/Ni(0), consistent with its oxidation to Ru(II)/Ni(I) in [1](+). The fast electron self-exchange rate of 10(7) M(-1) s(-1) between [1](0) and [1](+) suggests minor reorganization, more consistent with a Ni(0)/Ni(I) oxidation state change than a Ni(I)/Ni(II) couple. In solution, [1](+) slowly converts to [H1](+) and [1-H](+), with the latter being a complex of the thioaldehyde SCHCH2CH2S arising from C-H activation of the pdt backbone. Treatment of [1](+) with the H-atom abstracting reagent 2,2,6,6-tetramethylpiperidine-1-oxy also gives [1-H](+). PMID:24684697

  16. Resin-bound models of the [FeFe]-hydrogenase enzyme active site and studies of their reactivity.

    PubMed

    Green, Kayla N; Hess, Jennifer L; Thomas, Christine M; Darensbourg, Marcetta Y

    2009-06-14

    The immobilization of synthetic analogues of the [FeFe]-hydrogenase, [FeFe]H(2)ase, enzyme active site on polyethyleneglycol-rich polystyrene beads is described. Using the reactivity of the amine termini of the PEG chains with carboxylates incorporated into (mu-SRS)[Fe(CO)(3)](2) or (mu-SR)(2)[Fe(CO)(3)](2) derivative, nu(CO)IR signatures can be used to interrogate the structure and properties of the diiron carbonyl complexes once incorporated into the PEG environment of the polymer beads. Alternatively, the SRS dithiolate was first attached to the resin and the diiron unit assembled via an in situ process on the bead. PMID:19662312

  17. 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

  18. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    PubMed Central

    Wünschiers, Röbbe; Batur, Mehtap; Lindblad, Peter

    2003-01-01

    Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific. Therefore, in accordance with previous nomenclature, we propose the gene names hoxW and hupW for the bidirectional and uptake hydrogenase processing endopeptidases, respectively. Due to their constitutive expression we expect that, at least in cyanobacteria, the endopeptidases take over multiple functions. PMID:12735794

  19. 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

  20. 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

  1. Elimination of hydrogenase post-translational modification blocks H2 production and increases ethanol yield in Clostridium thermocellum

    SciTech Connect

    Biswas, Ranjita [ORNL; Zheng, Tianyong [Thayer School of Engineering at Dartmouth; Olson, Daniel G. [Thayer School of Engineering at Dartmouth; Lynd, Lee R [Thayer School of Engineering at Dartmouth; Guss, Adam M [ORNL

    2015-01-01

    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.

  2. 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

  3. BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Protein engineering of hydrogenase 3 to enhance

    E-print Network

    Wood, Thomas K.

    BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Protein engineering of hydrogenase 3 to enhance protein engineering of a hydrogenase. Keywords Protein engineering . Hydrogenase 3 . Error-prone PCR . DNA

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

    E-print Network

    Singleton, Michael Lee

    2012-02-14

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

  5. [FeFe] hydrogenase: protonation of {2Fe3S} systems and formation of super-reduced hydride states.

    PubMed

    Jablonskyt?, Aušra; Wright, Joseph A; Fairhurst, Shirley A; Webster, Lee R; Pickett, Christopher J

    2014-09-15

    The synthesis and crystallographic characterization of a complex possessing a well-defined {2Fe3S(?-H)}?core gives access to a paramagnetic bridging hydride with retention of the core geometry. Chemistry of this 35-electron species within the confines of a thin-layer FTIR spectro-electrochemistry cell provides evidence for a unprecedented super-reduced Fe(I)(?-H)Fe(I) intermediate. PMID:25079249

  6. 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

  7. Influence of Escherichia coli hydrogenases on hydrogen fermentation from glycerol

    E-print Network

    Wood, Thomas K.

    , we evaluated the effect of inactivation of each E. coli hydrogenase on cell growth, hydrogen production, but no significant effect occurred at pH 6.5 or in complex medium. Inactivation of hydrogenase 3Influence of Escherichia coli hydrogenases on hydrogen fermentation from glycerol Viviana Sanchez

  8. 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 (?1 ? 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 (?2 ? 15 s) causes a ?50% decrease of the number of ?2.7-? Fe–Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (?3 ? 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

  9. Iron hydrogenases and the evolution of anaerobic eukaryotes.

    PubMed

    Horner, D S; Foster, P G; Embley, T M

    2000-11-01

    Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain. PMID:11070057

  10. Magnetic properties and texture of sputtered Fe\\/Fe3O4 multilayer films

    Microsoft Academic Search

    Y. K. Kim; M. Oliveria

    1994-01-01

    Polycrystalline Fe\\/Fe3O4 multilayers have been fabricated with varying Fe\\/Fe3O4 ratios, period (bilayer) thickness, number of periods, and deposition sequence. The films were deposited by RF magnetron sputtering. The coercivity was found to vary in a nonlinear fashion between that of Fe and Fe3 O4. For selected multilayer geometries, the coercivity can be increased beyond that of single-layer Fe and Fe3O4

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

    Microsoft Academic Search

    C. A. Woodward; E. N. Kaufman

    1996-01-01

    Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native

  12. Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

    PubMed

    Meuser, Jonathan E; Boyd, Eric S; Ananyev, Gennady; Karns, Devin; Radakovits, Randor; Narayana Murthy, U M; Ghirardi, Maria L; Dismukes, G Charles; Peters, John W; Posewitz, Matthew C

    2011-10-01

    [FeFe]-hydrogenases (HYDA) link the production of molecular H(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(2)-photoproduction activity observed is as O(2) 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. PMID:21643991

  13. 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.

  14. Microbial hydrogenases: primary structure, classification, signatures and phylogeny.

    PubMed

    Wu, L F; Mandrand, M A

    1993-04-01

    Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16. The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8318259

  15. 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

  16. 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.

  17. Hydrogenases and H + Reduction in Primary Energy Conservation

    Microsoft Academic Search

    Paulette M. Vignais

    Hydrogenases are metalloenzymes subdivided into two classes that contain iron-sulfur clusters and\\u000a catalyze the reversible oxidation of hydrogen gas (H2???2H+?+?2e?).\\u000a Two metal atoms are present at their active center: either a Ni and an Fe atom in the [NiFe]hydrogenases,\\u000a or two Fe atoms in the [FeFe]hydrogenases. They are phylogenetically distinct classes of proteins. The\\u000a catalytic core of [NiFe]hydrogenases is a heterodimeric protein

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

    PubMed

    Tengölics, Roland; Mészáros, Lívia; Gy?ri, E; Doffkay, Zsolt; Kovács, Kornél L; Rákhely, Gábor

    2014-10-01

    Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled. PMID:25111750

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

    PubMed Central

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

    2012-01-01

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

  20. Isolation, observation, and computational modeling of proposed intermediates in catalytic proton reductions with the hydrogenase mimic Fe2(CO)6S2C6H4.

    PubMed

    Wright, Robert J; Zhang, Wei; Yang, Xinzheng; Fasulo, Meg; Tilley, T Don

    2012-01-01

    Proposed electrocatalytic proton reduction intermediates of hydrogenase mimics were synthesized, observed, and studied computationally. A new mechanism for H(2) generation appears to involve Fe(2)(CO)(6)(1,2-S(2)C(6)H(4)) (3), the dianions {[1,2-S(2)C(6)H(4)][Fe(CO)(3)(?-CO)Fe(CO)(2)](2-) (3(2-)), the bridging hydride {[1,2-S(2)C(6)H(4)][Fe(CO)(3)(?-CO)(?-H)Fe(CO)(2)]}(-), 3H(-)(bridging), and the terminal hydride 3H(-)(term-stag), {[1,2-S(2)C(6)H(4)][HFe(CO)(3)Fe(CO)(3)]}(-), as intermediates. The dimeric sodium derivative of 3(2-), {[Na(2)(THF)(OEt(2))(3)][3(2-)]}(2) (4) was isolated from reaction of Fe(2)(CO)(6)(1,2-S(2)C(6)H(4)) (3) with excess sodium and was characterized by X-ray crystallography. It possesses a bridging CO and an unsymmetrically bridging dithiolate ligand. Complex 4 reacts with 4 equiv. of triflic or benzoic acid (2 equiv. per Fe center) to generate H(2) and 3 in 75% and 60% yields, respectively. Reaction of 4 with 2 equiv. of benzoic acid generated two hydrides in a 1.7 : 1 ratio (by (1)H NMR spectroscopy). Chemical shift calculations on geometry optimized structures of possible hydride isomers strongly suggest that the main product, 3H(-)(bridging), possesses a bridging hydride ligand, while the minor product is a terminal hydride, 3H(-)(term-stag). Computational studies support a catalytic proton reduction mechanism involving a two-electron reduction of 3 that severs an Fe-S bond to generate a dangling thiolate and an electron rich Fe center. The latter iron center is the initial site of protonation, and this event is followed by protonation at the dangling thiolate to give the thiol thiolate [Fe(2)H(CO)(6)(1,2-SHSC(6)H(4))]. This species then undergoes an intramolecular acid-base reaction to form a dihydrogen complex that loses H(2) and regenerates 3. PMID:22031098

  1. A Bacterial Electron-bifurcating Hydrogenase*

    PubMed Central

    Schuchmann, Kai; Müller, Volker

    2012-01-01

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

  2. 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

  3. The hydrogenases of Geobacter sulfurreducens: a comparative genomic perspective

    E-print Network

    Lovley, Derek

    and Desulfovibrionaceae. Many Bacteria and Archaea express multiple hydrogenases, which differ in phylogenetic class encoding a multimeric Ech hydrogenase related (Ehr) complex that was similar in content to operons encoding that the G. sulfurreducens ehr cluster is part of a family of related clusters found in both the Archaea

  4. 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

  5. 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...

  6. 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

  7. 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.

  8. Hydrogenase activity in the thermophile mastigocladus laminosus

    SciTech Connect

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

    1982-06-30

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

  9. Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands.

    PubMed

    Reissmann, Stefanie; Hochleitner, Elisabeth; Wang, Haofan; Paschos, Athanasios; Lottspeich, Friedrich; Glass, Richard S; Böck, August

    2003-02-14

    NiFe-hydrogenases have an Ni-Fe site in which the iron has one CO and two CN groups as ligands. Synthesis of the CN ligands requires the activity of two hydrogenase maturation proteins: HypF and HypE. HypF is a carbamoyltransferase that transfers the carbamoyl moiety of carbamoyladenylate to the COOH-terminal cysteine of HypE and thus forms an enzyme-thiocarbamate. HypE dehydrates the S-carbamoyl moiety in an adenosine triphosphate-dependent process to yield the enzyme thiocyanate. Chemical model reactions corroborate the feasibility of this unprecedented biosynthetic route and show that thiocyanates can donate CN to iron. This finding underscores a striking parallel between biochemistry and organometallic chemistry in the formation of an iron-cyano complex. PMID:12586941

  10. 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.

  11. The influence of a S-to-S bridge in diiron dithiolate models on the oxidation reaction: a mimic of the H(air)(ox) state of [FeFe]-hydrogenases.

    PubMed

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

    2014-08-25

    Two-electron oxidation of a diiron complex (1) containing a bulky S-to-S bridge with an exocyclic carbonyl group affords [1(OH)](+), which replicates the coordination structure and electronic configuration of H(air)(ox), and the chemically reversible reaction between 1 and [1(OH)](+) mimics the bioprocess of interconversion of the inactive H(air)(ox) and the active Hred states of the [FeFe]-hydrogenases. PMID:24921710

  12. 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.

  13. 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.

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

    PubMed Central

    Yacoby, Iftach; Tegler, Lotta Tollstoy; Pochekailov, Sergii; Zhang, Shuguang; King, Paul W.

    2012-01-01

    Background 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. Principal Findings 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?1 of culture from E. coli with specific activities of 1000 U (U?=?1 µmol hydrogen evolved mg?1 min?1). Significance 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. PMID:22563413

  15. ORIGINAL ARTICLE [FeFe]-hydrogenase in Yellowstone National Park

    E-print Network

    ORIGINAL ARTICLE [FeFe]-hydrogenase in Yellowstone National Park: evidence for dispersal limitation conservatism; pH; Yellowstone National Park Introduction Hydrogen (H2) is ubiquitous in the biosphere and has

  16. Theoretical 57Fe Mössbauer spectroscopy for structure elucidation of [Fe] hydrogenase active site intermediates.

    PubMed

    Gubler, Joël; Finkelmann, Arndt R; Reiher, Markus

    2013-12-16

    [Fe] hydrogenase is a hydrogen activating enzyme that features a monoiron active site, which can be well characterized by Mössbauer spectroscopy. Mössbauer spectra have been measured of the CO and CN(-) inhibited species as well as under turnover conditions [Shima, S. et al., J. Am. Chem. Soc., 2005, 127, 10430]. This study presents calculated Mössbauer parameters for various active-site models of [Fe] hydrogenase to provide structural information about the species observed in experiment. Because theoretical Mössbauer spectroscopy requires the parametrization of observables from first-principles calculations (i.e., electric-field gradients and contact densities) to the experimental observables (i.e., quadrupole splittings and isomer shifts), nonrelativistic and relativistic density functional theory methods are parametrized against a reference set of Fe complexes specifically selected for the application to the Fe center in [Fe] hydrogenase. With this methodology, the measured parameters for the CO and CN(-) inhibited complexes can be reproduced. Evidence for the protonation states of the hydroxyl group in close proximity to the active site and for the thiolate ligand, which could participate in proton transfer, is obtained. The unknown resting state measured in the presence of the substrate and under pure H2 atmosphere is identified to be a water-coordinated complex. Consistent with previous assignments based on infrared and X-ray absorption near-edge spectroscopy, all measured Mössbauer data can be reproduced with the active site's iron atom being in oxidation state +2. PMID:24328345

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

    SciTech Connect

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

    1996-11-05

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

  18. Resonant inelastic X-ray scattering on synthetic nickel compounds and Ni-Fe hydrogenase protein

    NASA Astrophysics Data System (ADS)

    Sanganas, Oliver; Löscher, Simone; Pfirrmann, Stefan; Marinos, Nicolas; Glatzel, Pieter; Weng, Tsu-Chien; Limberg, Christian; Driess, Matthias; Dau, Holger; Haumann, Michael

    2009-11-01

    Ni-Fe hydrogenases are proteins catalyzing the oxidative cleavage of dihydrogen (H2) and proton reduction to H2 at high turnover rates. Their active site is a heterobimetallic center comprising one Ni and one Fe atom. To understand the function of the site, well resolved structural and electronic information is required. Such information is expected to become accessible by high resolution X-ray absorption and emission techniques, which are rapidly developing at third generation synchrotron radiation sources. We studied a number of synthetic Ni compounds, which mimic relevant features of the Ni site in hydrogenases, and the Ni site in the soluble, NAD-reducing hydrogenase (SH) from the bacterium Ralstonia eutropha by resonant inelastic X-ray scattering (RIXS) using a Rowland-type spectrometer at the ESRF. The SH is particularly interesting because its H2-cleavage reaction is highly resistant against inhibition by O2. K?-fluorescence detected RIXS planes in the 1s?3d region of the X-ray absorption spectrum were recorded on the protein which allow to extract L3-edge type spectra Spectral features of the protein are compared to those of the model compounds.

  19. Bioelectrocatalysis by Hydrogenase Th. Roseopersicina Immobilized on Carbon Materials

    Microsoft Academic Search

    S. V. Morozov; E. E. Karyakina; O. A. Zadvornyi; N. A. Zorin; S. D. Varfolomeev; A. A. Karyakin

    2002-01-01

    Application of various carbon materials as substrates for immobilizing hydrogenase and creating a hydrogen enzyme electrode is studied. A stable, highly-active electrocatalyst for mediatorless oxidation–evolution of hydrogen is obtained. The electrocatalyst uses hydrogenase from Th. roseopersicina immobilized on an inactive carbon material (based on carbon cloth TVSh-300M) and retains more than 50% of its initial activity after a half year

  20. 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 only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg). PMID:24287649

  1. A hydrogenosomal [Fe]-hydrogenase from the anaerobic chytrid Neocallimastix sp. L2.

    PubMed

    Voncken, Frank G J; Boxma, Brigitte; van Hoek, Angela H A M; Akhmanova, Anna S; Vogels, Godfried D; Huynen, Martijn; Veenhuis, Marten; Hackstein, Johannes H P

    2002-02-01

    The presence of a [Fe]-hydrogenase in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2 has been demonstrated by immunocytochemistry, subcellular fractionation, Western-blotting and measurements of hydrogenase activity in the presence of various concentrations of carbon monoxide (CO). Since the hydrogenosomal hydrogenase activity can be inhibited nearly completely by low concentrations of CO, it is likely that the [Fe]-hydrogenase is responsible for at least 90% of the hydrogen production in isolated hydrogenosomes. Most likely, this hydrogenase is encoded by the gene hydL2 that exhibits all the motifs that are characteristic of [Fe]-hydrogenases. The open reading frame starts with an N-terminal extension of 38 amino acids that has the potential to function as a hydrogenosomal targeting signal. The downstream sequences encode an enzyme of a calculated molecular mass of 66.4 kDa that perfectly matches the molecular mass of the mature hydrogenase in the hydrogenosome. Phylogenetic analysis revealed that the hydrogenase of Neocallimastix sp. L2. clusters together with similar ('long-type') [Fe]-hydrogenases from Trichomonas vaginalis, Nyctotherus ovalis, Desulfovibrio vulgaris and Thermotoga maritima. Phylogenetic analysis based on the H-cluster - the only module of [Fe]-hydrogenases that is shared by all types of [Fe]-hydrogenases and hydrogenase-like proteins - revealed a monophyly of all hydrogenase-like proteins of the aerobic eukaryotes. Our analysis suggests that the evolution of the various [Fe]-hydrogenases and hydrogenase-like proteins occurred by a differential loss of Fe-S clusters in the N-terminal part of the [Fe]-hydrogenase. PMID:11891051

  2. Synthetic and Structural Studies on [Fe2(SR)2(CN)x(CO)6-x]x-Active Site Models for Fe-Only Hydrogenases

    E-print Network

    Rauchfuss, Thomas B.

    61801 ReceiVed April 24, 2001 Abstract: A series of models for the active site (H-cluster) of the iron been known for over 70 years. Reihlen first prepared Fe2(SEt)2(CO)6 by the carbonylation of aqueous Fe]2-, an analogue of Roussin's red anion [Fe2(S)2(NO)4]2-,4 and showed that this carbonyl anion could be alkylated

  3. Effect of exchange interaction in dumbbell Fe-Fe pairs on the curie temperature of the rhombohedral Gd2Fe17 phase

    NASA Astrophysics Data System (ADS)

    Medvedev, M. V.; Nekrasov, I. A.

    2015-05-01

    One of important problems of improving the magnetic properties of technologically promicing intermetallics R 2Fe17 ( R is rare-earth metal) with high Fe contents is related to the increase in the Curie temperature T C. There is a hypothesis on the possibility of a marked increase in T C of the alloys, which can be achieved via the synthesis of systems characterized by pairs of atoms coupled by strong exchange interaction similar to that for Fe-Fe pairs situated in the so-called dumbbell position in the R 2Fe17 systems. In the present study, based on an example of the rhombohedral Gd2Fe17 phase, it was shown in terms of a classical nearest-neighbor spin Heisenberg model that changes in the exchange interaction for a dimer pair of dumbbell Fe-Fe atoms from zero to infinitely large lead to no more than 10% increase in T C. Thus, the creation of ferromagnetic systems characterized by dimers of magnetic atoms coupled by strong short-range exchange interaction, which, in this case, do not form an infinite magnetic cluster, cannot increase radically the Curie temperature T C.

  4. Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A

    Microsoft Academic Search

    Jonathan E. Meuser; Eric S. Boyd; Gennady Ananyev; Devin Karns; Randor Radakovits; U. M. Narayana Murthy; Maria L. Ghirardi; G. Charles Dismukes; John W. Peters; Matthew C. Posewitz

    2011-01-01

    [FeFe]-hydrogenases (HYDA) link the production of molecular H2 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

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

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

    PubMed

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

    2011-11-10

    Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5?Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(2)-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H(2) oxidation and as an electron-delivering device upon O(2) attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H(2)-converting catalysts that are capable of cycling H(2) in air. PMID:22002606

  7. 2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

    PubMed Central

    Watrous, Mary M.; Clark, Sandra; Kutty, Razia; Huang, Shouqin; Rudolph, Frederick B.; Hughes, Joseph B.; Bennett, George N.

    2003-01-01

    The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a Km of 152 ?M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R2 = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities. PMID:12620841

  8. Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

    PubMed

    Kanai, Tamotsu; Ito, Sota; Imanaka, Tadayuki

    2003-03-01

    We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames (hyhBGSL(Tk)), where the hyhS(Tk) and hyhL(Tk) gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybD(Tk)) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL(Tk) were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (beta, gamma, delta, and alpha), corresponding to the products of hyhBGSL(Tk), respectively. Each alphabetagammadelta unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95 degrees C for H(2) uptake and 90 degrees C for H(2) production with methyl viologen as the electron carrier. We found that NADP(+) and NADPH promoted high levels of uptake and evolution of H(2), respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase. PMID:12591889

  9. Partial Purification and Characterization of Two Hydrogenases from the Extreme Thermophile Methanococcus jannaschii

    PubMed Central

    Shah, Nilesh N.; Clark, Douglas S.

    1990-01-01

    F420-nonreactive and F420-reactive hydrogenases have been partially purified from Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a submarine hydrothermal vent. The molecular weights of both hydrogenases were determined by native gradient electrophoresis in 5 to 27% polyacrylamide gels. The F420-nonreactive hydrogenase produced one major band (475 kilodaltons), whereas the F420-reactive hydrogenase produced two major bands (990 and 115 kilodaltons). The F420-nonreactive hydrogenase consisted of two subunits (43 and 31 kilodaltons), and the F420-reactive hydrogenase contained three subunits (48, 32, and 25 kilodaltons). Each hydrogenase was active at very high temperatures. Methyl viologen-reducing activity of the F420-nonreactive hydrogenase was maximal at 80°C but was still detectable at 103°C. The maximum activities of F420-reactive hydrogenase for F420 and methyl viologen were measured at 80 and 90°C, respectively. Low but measureable activity toward methyl viologen was repeatedly observed at 103°C. Moreover, the half-life of the F420-nonreactive hydrogenase at 70°C was over 9 h, and that of the F420-reactive enzyme was over 3 h. Images PMID:16348172

  10. Hydrogen fuel electrode based on bioelectrocatalysis by the enzyme hydrogenase

    Microsoft Academic Search

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

    2002-01-01

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

  11. Magnetic properties of fluffy Fe@?-Fe2O3 core-shell nanowires

    PubMed Central

    2013-01-01

    Novel fluffy Fe@?-Fe2O3 core-shell nanowires have been synthesized using the chemical reaction of ferrous sulfate and sodium borohydride, as well as the post-annealing process in air. The coercivity of the as-synthesized nanowires is above 684 Oe in the temperature range of 5 to 300 K, which is significantly higher than that of the bulk Fe (approximately 0.9 Oe). Through the annealing process in air, the coercivity and the exchange field are evidently improved. Both the coercivity and the exchange field increase with increasing annealing time (TA) and reach their maximum values of 1,042 and 78 Oe, respectively, at TA?=?4 h. The magnetic measurements show that the effective anisotropy is increased with increasing the thickness of the?-Fe2O3 by annealing. The large values of coercivity and exchange field, as well as the high surface area to volume ratio, may make the fluffy Fe@?-Fe2O3 core-shell nanowire a promising candidate for the applications of the magnetic drug delivery, electrochemical energy storage, gas sensors, photocatalysis, and so forth. PMID:24134440

  12. First evidence for the presence of a hydrogenase in the sulfur-reducing bacterium Desulfuromonas acetoxidans.

    PubMed

    Brugna, M; Nitschke, W; Toci, R; Bruschi, M; Giudici-Orticoni, M T

    1999-09-01

    Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from Desulfuromonas acetoxidans. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium D. acetoxidans, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of D. acetoxidans, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium. PMID:10464227

  13. Differences in Hydrogenase Gene Expression between Methanosarcina acetivorans and Methanosarcina barkeri

    Microsoft Academic Search

    Adam M. Guss; Gargi Kulkarni; William W. Metcalf

    2009-01-01

    Methanosarcina acetivorans C2A encodes three putative hydrogenases, including one cofactor F420-linked (frh) and two methanophenazine-linked (vht) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of Methanosarcina barkeri and Methanosarcina mazei shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in M. acetivorans were

  14. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    DOEpatents

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

    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.

  17. High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases.

    PubMed

    Leidel, Nils; Popovi?-Bijeli?, Ana; Havelius, Kajsa G V; Chernev, Petko; Voevodskaya, Nina; Gräslund, Astrid; Haumann, Michael

    2012-03-01

    Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(?O)(2)Fe(III)L(4) (metal-metal distance of ~2.75Å, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(?O)(?OH)Fe(III)L(4) (~2.90Å) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55Å) with a L(4)Fe(IV)(?O)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1Å) and Mn,Fe(III)Fe(II) species (~3.3-3.4Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(?O)(?OH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1. PMID:22222354

  18. Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus.

    PubMed

    Bryant, F O; Adams, M W

    1989-03-25

    The archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative type metabolism in which H2 and CO2 are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0 contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2 evolution activity from reduced methyl viologen at 80 degrees C was 40%. The hydrogenase has a Mr value of 185,000 +/- 15,000 and is composed of three subunits of Mr 46,000 (alpha), 27,000 (beta), and 24,000 (gamma). The enzyme contains 31 +/- 3 g atoms of iron, 24 +/- 4 g atoms of acid-labile sulfide, and 0.98 +/- 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2 and is remarkably thermostable. Most of its H2 evolution activity is retained after a 1-h incubation at 100 degrees C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2 evolution is observed between 45 and 90 degrees C. The hydrogenase also catalyzes H2 oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2 oxidation and H2 evolution is greater than 95 degrees C. Arrhenius plots show two transition points at approximately 60 and approximately 80 degrees C independent of the mode of assay. That occurring at 80 degrees C is associated with a dramatic increase in H2 production activity. The enzyme preferentially catalyzes H2 production at all temperatures examined and appears to represent a new type of "evolution" hydrogenase. PMID:2538471

  19. 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

  20. Dithiomethylether As a Ligand in the Hydrogenase H-Cluster

    SciTech Connect

    Pandey, A.S.; Harris, T.V.; Giles, L.J.; Peters, J.W.; Szilagyi, R.K.

    2009-05-21

    An X-ray crystallographic refinement of the H-cluster of [FeFe]-hydrogenase from Clostridium pasteurianum has been carried out to close-to atomic resolution and is the highest resolution [FeFe]-hydrogenase presented to date. The 1.39 {angstrom}, anisotropically refined [FeFe]-hydrogenase structure provides a basis for examining the outstanding issue of the composition of the unique nonprotein dithiolate ligand of the H-cluster. In addition to influencing the electronic structure of the H-cluster, the composition of the ligand has mechanistic implications due to the potential of the bridge-head {gamma}-group participating in proton transfer during catalysis. In this work, sequential density functional theory optimizations of the dithiolate ligand embedded in a 3.5-3.9 {angstrom} protein environment provide an unbiased approach to examining the most likely composition of the ligand. Structural, conformational, and energetic considerations indicate a preference for dithiomethylether as an H-cluster ligand and strongly disfavor the dithiomethylammonium as a catalytic base for hydrogen production.

  1. THE [Fe-Fe]-HYDROGENASE MATURATION PROTEIN HydF FROM Thermotoga maritima IS A GTPase WITH AN IRON-SULFUR CLUSTER

    E-print Network

    Paris-Sud XI, Université de

    1 THE [Fe-Fe]-HYDROGENASE MATURATION PROTEIN HydF FROM Thermotoga maritima IS A GTPase WITH AN IRON-SULFUR-Fe]-hydrogenases (1), [Fe-Fe]-hydrogenases (2,3) and "Iron-Sulfur cluster- free" hydrogenase (4,5,6), which do for activity (10). Infrared spectroscopy studies have also demonstrated the presence of CO ligands in the "Iron-Sulfur

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

    PubMed

    Wu, Hao; Ai, Zhihui; Zhang, Lizhi

    2014-04-01

    In this study we comparatively investigate the removal of humic acids with Fe@Fe2O3 core-shell nanowires under anoxic and oxic conditions. The products of humic acids after reacting with Fe@Fe2O3 core-shell nanowires under anoxic and oxic conditions were carefully examined with three-dimensional excitation emission matrix fluorescence spectroscopy and gas chromatography mass spectrometry. It was found that humic acids were removed by Fe@Fe2O3 core-shell nanowires via adsorption under anoxic condition. Langmuir adsorption isotherm was applicable to describe the adsorption processes. Kinetics of humic acids adsorption onto Fe@Fe2O3 core-shell nanowires was found to follow pseudo-second-order rate equation. By contrast, the oxic removal of humic acids with Fe@Fe2O3 core-shell nanowires involved adsorption and subsequent oxidation of humic acids because Fe@Fe2O3 core-shell nanowires could activate molecular oxygen to produce reactive oxygen species to oxidize humic acids. This subsequent oxidation of humic acids could improve the oxic removal rate to 2.5 times that of anoxic removal, accompanying with about 8.4% of mineralization. This study provides a new method for humic acids removal and also sheds light on the effects of humic acids on the pollutant removal by nano zero-valent iron. PMID:24463174

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

    SciTech Connect

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

    2008-01-01

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

  4. 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

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

    Sulfur is believed to be an alloying light element in iron-rich planetary cores such as those of the Earth and Mars 1, 2. Recent studies have suggested that Mars, like the Earth, could have a liquid metallic outer core together with a solid inner core 3. Hence, it is important to investigate the evolution of the Fe-FeS phase diagram and of the structural properties of the liquid Fe-FeS alloys in respect to pressure, temperature and sulphur content. A new cell assembly has been developed to heat samples to more than 1300 K at 17 GPa using the Paris Edinburgh Press4. This allows us to conduct detailed structural investigations of the Fe-FeS eutectic liquid by in situ X-ray diffraction5 . Analysis of these data highlights an increase of the liquid compacity with increasing pressure. We also show that the eutectic liquid structure is closer to that of FeSi, explaining the closure of the miscibility gap in the Fe-S-Si system 6. The evolution of the Fe-FeS eutectic liquid structure at high pressure could have significant effect on extrapolated wave speed of metallic Fe-FeS alloy at core pressures. We have used a double-sided laser-heated diamond-anvil cell 7 to study the Fe-FeS phase diagram up to 65 GPa and 2500 K8. We used laser heated diamond anvil cell coupled with synchrotron radiation and confirm a S- solubility below 4 at% (2.3 %wt) up to 65 GPa. The eutectic temperatures present a uniform increase, with a rate of ~15K/GPa, up to 65 GPa and 2200 K. Finally, we present new constraints on the phase diagram evolution to very high pressures which provide unambiguous evidence for an upper limit of 4-8 %wt for the inner core S- content. Therefore, sulphur is not favoured to be the major light element in the Earth's core. 1. Allegre, C. J., Poirier, J. P., Humler, E. & Hofmann, A. W. The chemical composition of the Earth. Earth Planet. Sc. Lett. 134, 515-526 (1995). 2. Sohl, F. & Spohn, T. The interior structure of Mars : Implications from SNC meteorites. J. Geophys. Res. 102, 1613-1635 (1997). 3. Yoder, C. F., Konopliv, A. S., Yuan, D. N., Standish, E. M. & Folkner, W. M. Fluid core size of Mars from detection of the solar tide. Science 300, 299-303 (2003). 4. Morard, G. et al. Optimization of Paris Edinburgh cell assemblies for in situ monochromatic X-ray diffraction and X-ray absorption. High Press. Res. 27, 1-11 (2007). 5. Morard, G. et al. Structure of eutectic Fe-FeS melts up to 17 GPa: Implications for planetary cores. Earth Planet. Sc. Lett. in press (2007). 6. Sanloup, C. & Fei, Y. Closure of the Fe-S-Si liquid miscibility gap at high pressure. Phys. Earth Plan. Int. 147, 57 (2004). 7. Mezouar, M. et al. Development of a new state-of-the-art beamline optimized for monochromatic single crystal and powder X-ray diffraction under extreme conditions at the ESRF. J. Synch. Rad. 12, 659-664 (2005). 8. Morard, G. et al. Experimental constraints on the Earth's core sulphur content. Nature (Submitted).

  6. Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia.

    PubMed

    Sellstedt, A; Lindblad, P

    1990-03-01

    Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out. PMID:16667353

  7. EPR and Mössbauer spectroscopic studies on the tetrameric, NAD-linked hydrogenase of Nocardia opaca 1b and its two dimers: 1. The ?? -dimer—a prototype of a simple hydrogenase

    Microsoft Academic Search

    Christiane Zaborosch; Michael Köstert; Eckhard Bill; Klaus Schneider; Hans G. Schlegel; Alfred X. Trautweint

    1995-01-01

    The cytoplasmic, tetrameric NAD-linked hydrogenase from Nocardia opaca 1b can be separated in two dimeric substructures, an a?-dimer with NADH:electron acceptor oxidoreductase (diaphorase) activity and a ßd-dimer which displays hydrogenase activity with artificial electron carriers. These two dimers were preparatively isolated by a FPLC Mono Q procedure in the absence of nickel and at alkaline pH values. The hydrogenase-active ßd-dimer

  8. Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres.

    PubMed

    Serebryakova, L T; Medina, M; Zorin, N A; Gogotov, I N; Cammack, R

    1996-03-25

    The catalytic and spectroscopic properties of the reversible hydrogenase from the cyanobacterium Anabaena variabilis have been examined. The hydrogenase required reductive activation in order to elicit hydrogen-oxidation activity. Carbon monoxide was a weak (Ki=35 microM), reversible and competitive inhibitor. A flavin with the chromatographic properties of FMN, and nickel were detected in the purified enzyme. A. variabilis hydrogenase exhibited electron paramagnetic resonance (EPR) spectra in its hydrogen-reduced state, indicative of [2Fe-2S] and [4Fe-4S] clusters. Although no EPR signals due to nickel were detected, the results are consistent with the enzyme being a flavin-containing hydrogenase of the nickel-iron type. PMID:8612797

  9. Radical S-adenosyl-L-methionine chemistry in the synthesis of hydrogenase and nitrogenase metal cofactors.

    PubMed

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

    2015-02-13

    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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

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

    SciTech Connect

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

    2013-04-25

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

  12. A Novel Endo-Hydrogenase Activity Recycles Hydrogen Produced by Nitrogen Fixation

    PubMed Central

    Park, Angela S.; Zenad, Lounis; Ludwig, Robert A.

    2009-01-01

    Background Nitrogen (N2) fixation also yields hydrogen (H2) at 1?1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N2 as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. Methods and Findings As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH : quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by “crossover PCR”, showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium—as expected of an H2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing ?-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Conclusions Representative of aerobic N2-fixing and H2-recycling ?-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H2 respiration, and Hyq endo-hydrogenase activity recycles endogenous H2, specifically that produced by N2 fixation. To benefit human civilization, H2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such as the A. caulinodans Hyq endo-hydrogenase, offer promise as biocatalytic agents for H2 production and/or consumption. PMID:19277114

  13. Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

    PubMed Central

    2013-01-01

    Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement. PMID:23819621

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

    PubMed Central

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

    2007-01-01

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

  15. X-ray absorption spectroscopy of nickel in the hydrogenase from Desulfovibrio gigas

    SciTech Connect

    Scott, R.A.; Wallin, S.A.; Czechowski, M.; DerVartanian, D.V.; LeGall, J.; Peck, H.D. Jr.; Moura, I.

    1984-10-31

    Results of preliminary X-ray absorption spectroscopic studies of the Desulfovibrio gigas enzyme indicate that the nickel is reduced from Ni(III) to Ni(II) upon H/sub 2/ reduction of the oxidized enzyme and that the ligands are bound to the nickel through the S atoms. Hydrogenase from Desulfovibrio gigas exhibited a single bond on disc acrylamide gel electrophoresis, and hydrogenase activity was determined by H/sub 2/ evolution assay.

  16. 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.

  17. Acetylene, Not Ethylene, Inactivates the Uptake Hydrogenase of Actinorhizal Nodules during Acetylene Reduction Assays 1

    PubMed Central

    Sellstedt, Anita; Winship, Lawrence J.

    1990-01-01

    Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C2H2 and not by C2H4. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcl1 in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cell-free extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene. PMID:16667724

  18. Acetylene, Not Ethylene, Inactivates the Uptake Hydrogenase of Actinorhizal Nodules during Acetylene Reduction Assays.

    PubMed

    Sellstedt, A; Winship, L J

    1990-09-01

    Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C(2)H(2) and not by C(2)H(4). Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcl1 in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cell-free extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene. PMID:16667724

  19. 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

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

    SciTech Connect

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

    2012-10-25

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

  1. 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 ?-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 ?-hydrido complexes [CpCo(dithiolate)HFe(CO)(diphos)]+. Low-temperature NMR studies indicate that Co is the kinetic site of protonation. PMID:24803716

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

    PubMed

    Carroll, Maria E; Chen, Jinzhu; Gray, Danielle E; Lansing, James C; Rauchfuss, Thomas B; Schilter, David; Volkers, Phillip I; Wilson, Scott R

    2014-02-24

    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)2 (2-) (edt(2-)), S2(CH2)3 (2-) (pdt(2-)), S2(CH2)2(C(CH3)2)(2-) (Me2pdt(2-)) and diphos = cis-C2H2(PPh2)2 (dppv), C2H4(PPh2)2 (dppe), C6H4(PPh2)2 (dppbz), C2H4[P(C6H11)2]2 (dcpe). The incorporation of (57)Fe into such building block complexes commenced with the conversion of (57)Fe into (57)Fe2I4( (i) PrOH)4, which then was treated with K2pdt, CO, and dppe to give (57)Fe(pdt)(CO)2(dppe). NMR and IR analyses show that these complexes exist as mixtures of all-cis and trans-CO isomers, edt(2-) favoring the former and pdt(2-) 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 C s -symmetric ?-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 ?-hydrido complexes [CpCo(dithiolate)HFe(CO)(diphos)](+). Low-temperature NMR studies indicate that Co is the kinetic site of protonation. PMID:24803716

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

    NASA Astrophysics Data System (ADS)

    McIntosh, Chelsea Lee

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

  4. This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 1029510305 10295 Cite this: Phys. Chem. Chem. Phys., 2011, 13, 1029510305

    E-print Network

    Strathclyde, University of

    ligands alters the vibrational relaxation dynamics of the CO modes in comparison to all-carbonyl model of directing the vibrational relaxation mechanism. Introduction The di-iron hydrogenases ([FeFe]) have been model systems based upon the iron­sulfur cluster architecture of the enzyme active site are also able

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

    E-print Network

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

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

    PubMed Central

    Boursier, P; Hanus, F J; Papen, H; Becker, M M; Russell, S A; Evans, H J

    1988-01-01

    We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein. Images PMID:3056905

  7. Structural And Biological Analysis of the Metal Sites of Escherichia Coli Hydrogenase Accessory Protein

    SciTech Connect

    Dias, A.V.; Mulvihill, C.M.; Leach, M.R.; Pickering, I.J.; George, G.N.; Zamble, D.B.

    2009-05-12

    The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.

  8. Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Maeda, Toshinari; Vardar, Gönül; Self, William T; Wood, Thomas K

    2007-01-01

    Background Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- ? H2 (g). Results Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host. Conclusion Enhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2. PMID:17521447

  9. 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.

  10. Symbiotic Legume Nodules Employ Both Rhizobial Exo- and Endo-Hydrogenases to Recycle Hydrogen Produced by Nitrogen Fixation

    PubMed Central

    Ciccolella, Christopher O.; Raynard, Nathan A.; Mei, John H-M.; Church, Derek C.; Ludwig, Robert A.

    2010-01-01

    Background In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N2, an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H2). While in most legume nodules this H2 is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H2. Rather, endogenous H2 is efficiently respired at the expense of O2, driving oxidative phosphorylation, recouping ATP used for H2 production, and increasing the efficiency of symbiotic nodule N2 fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity. Methodology/Principal Findings Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory (exo-)hydrogenase and novel (endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or-endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H2 and thus are fully competent for endogenous H2 recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H2 quantitatively and thus suffer complete loss of H2 recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H2 may carry both exo- and endo-hydrogenase gene-sets. Conclusions/Significance In symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H2 produced by N2 fixation. Thus, H2 recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases. PMID:20838423

  11. 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

  12. [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

  13. H2 conversion in the presence of O2 as performed by the membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha.

    PubMed

    Lenz, Oliver; Ludwig, Marcus; Schubert, Torsten; Bürstel, Ingmar; Ganskow, Stefanie; Goris, Tobias; Schwarze, Alexander; Friedrich, Bärbel

    2010-04-26

    [NiFe]-hydrogenases catalyze the oxidation of H(2) to protons and electrons. This reversible reaction is based on a complex interplay of metal cofactors including the Ni-Fe active site and several [Fe-S] clusters. H(2) catalysis of most [NiFe]-hydrogenases is sensitive to dioxygen. However, some bacteria contain hydrogenases that activate H(2) even in the presence of O(2). There is now compelling evidence that O(2) affects hydrogenase on three levels: 1) H(2) catalysis, 2) hydrogenase maturation, and 3) H(2)-mediated signal transduction. Herein, we summarize the genetic, biochemical, electrochemical, and spectroscopic properties related to the O(2) tolerance of hydrogenases resident in the facultative chemolithoautotroph Ralstonia eutropha H16. A focus is given to the membrane-bound [NiFe]-hydogenase, which currently represents the best-characterized member of O(2)-tolerant hydrogenases. PMID:20186906

  14. 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:24465722

  15. 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:24465722

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

    PubMed Central

    Schäfer, Caspar; Friedrich, Bärbel

    2013-01-01

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

  17. Novel, oxygen-insensitive group 5 [NiFe]-hydrogenase in Ralstonia eutropha.

    PubMed

    Schäfer, Caspar; Friedrich, Bärbel; Lenz, Oliver

    2013-09-01

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

  18. 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

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

    PubMed

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

    2014-06-01

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

  20. A reversible electron-bifurcating ferredoxin- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Moorella thermoacetica.

    PubMed

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

    2013-03-01

    Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO(2). Depending on the growth substrate, this Gram-positive bacterium can either form H(2) or consume it. Despite the importance of H(2) in its metabolism, a hydrogenase from the organism has not yet been characterized. We report here the purification and properties of an electron-bifurcating [FeFe]-hydrogenase from M. thermoacetica and show that the cytoplasmic enzyme efficiently catalyzes both H(2) formation and H(2) uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD(+) with H(2) at 55 °C at pH 7.5 at a specific rate of about 100 ?mol min(-1) mg protein(-1) and the reverse reaction, the coupled reduction of protons to H(2) with reduced ferredoxin and NADH, at a specific rate of about 10 ?mol min(-1) mg protein(-1) in the stoichiometry Fd(ox) + NAD(+) + 2H(2) Fd(red)(2-) + NADH + 3H(+). When ferredoxin from Clostridium pasteurianum, NAD(+), and the enzyme were incubated at pH 7.0 under 100% H(2) in the gas phase (E(0)' = -414 mV), more than 95% of the ferredoxin (E(0)' = -400 mV) was reduced, which indicated that ferredoxin reduction with H(2) is driven by the exergonic reduction of NAD(+) (E(0)' = -320 mV) with H(2). In the absence of NAD(+), ferredoxin was not reduced. We identified the genes encoding HydABC within the transcriptional unit hydCBAX and mapped the transcription start site. PMID:23316038

  1. A Reversible Electron-Bifurcating Ferredoxin- and NAD-Dependent [FeFe]-Hydrogenase (HydABC) in Moorella thermoacetica

    PubMed Central

    Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2013-01-01

    Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO2. Depending on the growth substrate, this Gram-positive bacterium can either form H2 or consume it. Despite the importance of H2 in its metabolism, a hydrogenase from the organism has not yet been characterized. We report here the purification and properties of an electron-bifurcating [FeFe]-hydrogenase from M. thermoacetica and show that the cytoplasmic enzyme efficiently catalyzes both H2 formation and H2 uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD+ with H2 at 55°C at pH 7.5 at a specific rate of about 100 ?mol min?1 mg protein?1 and the reverse reaction, the coupled reduction of protons to H2 with reduced ferredoxin and NADH, at a specific rate of about 10 ?mol min?1 mg protein?1 in the stoichiometry Fdox + NAD+ + 2H2 ? Fdred2? + NADH + 3H+. When ferredoxin from Clostridium pasteurianum, NAD+, and the enzyme were incubated at pH 7.0 under 100% H2 in the gas phase (E0? = ?414 mV), more than 95% of the ferredoxin (E0? = ?400 mV) was reduced, which indicated that ferredoxin reduction with H2 is driven by the exergonic reduction of NAD+ (E0? = ?320 mV) with H2. In the absence of NAD+, ferredoxin was not reduced. We identified the genes encoding HydABC within the transcriptional unit hydCBAX and mapped the transcription start site. PMID:23316038

  2. Function of the Chloroplast Hydrogenase in the Microalga Chlamydomonas: The Role of Hydrogenase and State Transitions during Photosynthetic Activation in Anaerobiosis

    PubMed Central

    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

  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. 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

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

    PubMed

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

    2013-08-14

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

  6. Computational study of the electronic structure and magnetic properties of the Ni-C state in [NiFe] hydrogenases including the second coordination sphere.

    PubMed

    Kampa, Mario; Lubitz, Wolfgang; van Gastel, Maurice; Neese, Frank

    2012-12-01

    [NiFe] hydrogenases catalyze the reversible formation of H(2). The [NiFe] heterobimetallic active site is rich in redox states. Here, we investigate the key catalytic state Ni-C of Desulfovibrio vulgaris Miyazaki F hydrogenase using a cluster model that includes the truncated amino acids of the entire second coordination sphere of the enzyme. The optimized geometries, computed g tensors, hyperfine coupling constants, and IR stretching frequencies all agree well with experimental values. For the hydride in the bridging position, only a single minimum on the potential energy surface is found, indicating that the hydride bridges and binds to both nickel and iron. The influence of the second coordination sphere on the electronic structure is investigated by comparing results from the large cluster models with truncated models. The largest interactions of the second coordination sphere with the active site concern the hydrogen bonds with the cyanide ligands, which modulate the bond between iron and these ligands. Secondly, the electronic structure of the active site is found to be sensitive to the protonation state of His88. This residue forms a hydrogen bond with the spin-carrying sulfur atom of Cys549, which in turn tunes the spin density at the nickel and coordinating sulfur atoms. In addition, the unequal distribution of spin density over the equatorial cysteine residues results from different orientations of the cysteine side chains, which are kept in their particular orientation by the secondary structure of the protein. PMID:23053531

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

    E-print Network

    Yacoby, Iftach

    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, ...

  8. Purification and properties of a F 420 -nonreactive, membrane-bound hydrogenase from Methanosarcina strain Gö1

    Microsoft Academic Search

    U. Deppenmeier; M. Blaut; B. Schmidt; G. Gottschalk

    1992-01-01

    The distribution of the F420-reactive and F420-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F420-nonreactive enzyme. The membrane-bound F420-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 µmol H2 oxidized · min-1 · mg protein-1. The purification procedure involved dispersion of the

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

    PubMed

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

    2010-12-01

    Hydrogen (H?) has an important role in the anaerobic degradation of organic carbon and is the basis for many syntrophic interactions that commonly occur in microbial communities. Little is known, however, with regard to the biotic and/or abiotic factors that control the distribution and phylogenetic diversity of organisms which produce H? in microbial communities. In this study, we examined the [FeFe]-hydrogenase gene (hydA) as a proxy for fermentative bacterial H? production along physical and chemical gradients in various geothermal springs in Yellowstone National Park (YNP), WY, USA. The distribution of hydA in YNP geothermal springs was constrained by pH to environments co-inhabited by oxygenic phototrophs and to environments predicted to have low inputs of abiotic H?. The individual HydA asssemblages from YNP springs were more closely related when compared with randomly assembled communities, which suggests ecological filtering. Model selection approaches revealed that geographic distance was the best explanatory variable to predict the phylogenetic relatedness of HydA communities. This evinces the dispersal limitation imposed by the geothermal spring environment on HydA phylogenetic diversity even at small spatial scales. pH differences between sites is the second highest ranked explanatory variable of HydA phylogenetic relatedness, which suggests that the ecology related to pH imposes strong phylogenetic niche conservatism. Collectively, these results indicate that pH has imposed strong niche conservatism on fermentative bacteria and that, within a narrow pH realm, YNP springs are dispersal limited with respect to fermentative bacterial communities. PMID:20535223

  10. [FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis.

    PubMed

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

    2015-03-10

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

  11. Structure of [NiFe] Hydrogenase Maturation Protein HypE from Escherichia coli and Its Interaction with HypF

    Microsoft Academic Search

    Erumbi S. Rangarajan; Abdalin Asinas; Ariane Proteau; Christine Munger; Jason Baardsnes; Pietro Iannuzzi; Allan Matte; Miroslaw Cygler

    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

  12. Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha

    Microsoft Academic Search

    Antje Porthun; Michael Bernhard; Bärbel Friedrich

    2002-01-01

    The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H2:benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha

  13. A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16.

    PubMed

    Frielingsdorf, Stefan; Schubert, Torsten; Pohlmann, Anne; Lenz, Oliver; Friedrich, Bärbel

    2011-12-20

    The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit. PMID:22097922

  14. Identification and isolation of genes essential for H sub 2 oxidation in Rhodobacter capsulatus. [Hydrogenase

    SciTech Connect

    Xu, H.W.; Love, J.; Borghese, R.; Wall, J.D. (Univ. of Missouri-Columbia (USA))

    1989-02-01

    Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H{sub 2} and CO{sub 2} were isolated. Those lacking uptake hydrogenase activity as measured by H{sub 2}-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon Tn5 mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret to mean that one or both structural genes were among the genes isolated.

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

    PubMed

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

    2009-12-01

    Interconversion of water and hydrogen in unitized regenerative fuel cells is a promising energy storage framework for smoothing out the temporal fluctuations of solar and wind power. However, replacement of presently available platinum catalysts by lower-cost and more abundant materials is a requisite for this technology to become economically viable. Here, we show that the covalent attachment of a nickel bisdiphosphine-based mimic of the active site of hydrogenase enzymes onto multiwalled carbon nanotubes results in a high-surface area cathode material with high catalytic activity under the strongly acidic conditions required in proton exchange membrane technology. Hydrogen evolves from aqueous sulfuric acid solution with very low overvoltages (20 millivolts), and the catalyst exhibits exceptional stability (more than 100,000 turnovers). The same catalyst is also very efficient for hydrogen oxidation in this environment, exhibiting current densities similar to those observed for hydrogenase-based materials. PMID:19965754

  16. Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases.

    PubMed

    Paschos, A; Glass, R S; Böck, A

    2001-01-12

    The iron of the binuclear active center of [NiFe]-hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity. Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the [NiFe] cluster contains a sequence motif characteristic of O-carbamoyl-transferases it was discovered that carbamoyl phosphate is essential for formation of active [NiFe]-hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands. A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated PMID:11163786

  17. 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.

  18. Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii.

    PubMed Central

    Kow, Y W; Burris, R H

    1984-01-01

    Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors. PMID:6378882

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

    SciTech Connect

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

    2012-03-26

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

  20. Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas

    Microsoft Academic Search

    Anne Volbeda; Marie-Hélène Charon; Claudine Piras; E. Claude Hatchikian; Michel Frey; Juan C. Fontecilla-Camps

    1995-01-01

    The X-ray structure of the heterodimeric Ni-Fe hydrogenase from Desulfovibrio gigas, the enzyme responsible for the metabolism of molecular hydrogen, has been solved at 2.85 Å resolution. The active site, which appears to contain, besides nickel, a second metal ion, is buried in the 60K subunit. The 28K subunit, which coordinates one [3Fe-4S] and two [4Fe-4S] clusters, contains an amino-terminal

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

    PubMed

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

    1998-07-01

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

  2. Purification and characterization of hydrogenase from the marine green alga, Chlorococcum littorale

    Microsoft Academic Search

    Yoshiyuki Ueno; Norihide Kurano; Shigetoh Miyachi

    1999-01-01

    Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 ?mol\\/min\\/mg of protein at 25°C, using reduced methyl viologen as an electron donor. The Km value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be

  3. Cloning and Sequence Analysis of the Gene Encoding NiFe-hydrogenase from Klebsiella pneumoniae

    Microsoft Academic Search

    Fei LIU; Bai-Shan FANG

    2007-01-01

    Degenerate PCR primers were designed by multiple alignment of the protein sequences of known structural genes encoding the catalytic subunits of NiFe-hydrogenases obtained from Swiss-Prot Protein Sequence Database through CLUSTAL-W software and compared for conserved sequence motifs. A 1 kb amplified PCR product was obtained from the genomic DNA of Klebsiella pneumoniae using a set of degenerate primers, and then

  4. Bacterial Genes Involved in Incorporation of Nickel Into a Hydrogenase Enzyme

    Microsoft Academic Search

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

    1994-01-01

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

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

    PubMed

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

    2011-03-01

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

  6. Reversible [4Fe-3S] cluster morphing in an O(2)-tolerant [NiFe] hydrogenase.

    PubMed

    Frielingsdorf, Stefan; Fritsch, Johannes; Schmidt, Andrea; Hammer, Mathias; Löwenstein, Julia; Siebert, Elisabeth; Pelmenschikov, Vladimir; Jaenicke, Tina; Kalms, Jacqueline; Rippers, Yvonne; Lendzian, Friedhelm; Zebger, Ingo; Teutloff, Christian; Kaupp, Martin; Bittl, Robert; Hildebrandt, Peter; Friedrich, Bärbel; Lenz, Oliver; Scheerer, Patrick

    2014-05-01

    Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H(2) oxidation at high O(2). We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range. PMID:24705592

  7. Immunological homology between the membrane-bound uptake hydrogenases of Rhizobium japonicum and Escherichia coli.

    PubMed Central

    Harker, A R; Zuber, M; Evans, H J

    1986-01-01

    Two polypeptides present in aerobic and anaerobic cultures of Escherichia coli HB101 were shown to cross-react with antibodies to the 30- and 60-kilodalton (kDa) subunits of the uptake hydrogenase of Rhizobium japonicum. The cross-reactive polypeptides in a series of different E. coli strains are of Mrs ca. 60,000 and 30,000, and both polypeptides are present in proportion to measurable hydrogen uptake (Hup) activity (r = 0.95). The 60-kDa polypeptide from E. coli HB101 comigrated on native gels with detectable Hup activity. The exact role of the 30-kDa polypeptide in E. coli is unclear. E. coli MBM7061, a natural Hup- variant, grown anaerobically or aerobically lacked detectable Hup activity and failed to cross-react with the antisera against the hydrogenase from R. japonicum. Anaerobically cultured E. coli MBM7061, however, did express formate hydrogenlyase activity, indicating that the hydrogenases involved in the oxygen-dependent activation of hydrogen and the formate-dependent evolution of hydrogen are biochemically distinct. Images PMID:3511036

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

  9. Genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples.

    PubMed Central

    Wawer, C; Jetten, M S; Muyzer, G

    1997-01-01

    The genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples were determined in order to show in parallel the existing and active members of Desulfovibrio populations. DNA and total RNA were extracted from different anaerobic bioreactor samples; RNA was transcribed into cDNA. Subsequently, PCR was performed to amplify a ca.-440-bp fragment of the [NiFe] hydrogenase large-subunit gene and its mRNA. Denaturing gradient gel electrophoresis analysis was used to separate the PCR products according to their sequence and thereby to visualize the individual community members. Desulfovibrio strains corresponding to amplified [NiFe] hydrogenase transcripts were regarded as metabolically active, because in pure cultures transcripts were detectable in exponentially growing cells but not in cultures in the stationary phase. DNA sequencing and comparative sequence analysis were used to identify the detected organisms on the basis of their [NiFe] hydrogenase sequences. The genes of characterized Desulfovibrio spp. showed a considerable extent of divergence (ca. 30%), whereas sequences obtained from bacterial populations of the bioreactors showed a low level of variation and indicated the coexistence of closely related strains probably belonging to the species Desulfovibrio sulfodismutans. Under methanogenic conditions, all detected populations were active; under denitrifying conditions, no [NiFe] hydrogenase mRNA was visible. Changes in activity and composition of Desulfovibrio populations caused by changes in the environmental conditions could be monitored by using the approach described in this study. PMID:9361423

  10. Hydrogen Production by Termite Gut Protists: Characterization of Iron Hydrogenases of Parabasalian Symbionts of the Termite Coptotermes formosanus? †

    PubMed Central

    Inoue, Jun-Ichi; Saita, Kanako; Kudo, Toshiaki; Ui, Sadaharu; Ohkuma, Moriya

    2007-01-01

    Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H2) that is emitted by the termites; however, little is known about the physiology and biochemistry of H2 production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H2 production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H2 evolution rather than H2 uptake. H2 evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H2 evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H2 uptake activity of the endosymbiotic bacteria compensated for the strong H2 evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H2 production in termite guts has been largely underestimated. PMID:17766465

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

    PubMed Central

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

    1983-01-01

    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

  12. Metagenomic Sequencing Unravels Gene Fragments with Phylogenetic Signatures of O2-Tolerant NiFe Membrane-Bound Hydrogenases in Lacustrine Sediment.

    PubMed

    Couto, Jillian M; Ijaz, Umer Zeeshan; Phoenix, Vernon R; Schirmer, Melanie; Sloan, William T

    2015-08-01

    Many promising hydrogen technologies utilising hydrogenase enzymes have been slowed by the fact that most hydrogenases are extremely sensitive to O2. Within the group 1 membrane-bound NiFe hydrogenase, naturally occurring tolerant enzymes do exist, and O2 tolerance has been largely attributed to changes in iron-sulphur clusters coordinated by different numbers of cysteine residues in the enzyme's small subunit. Indeed, previous work has provided a robust phylogenetic signature of O2 tolerance [1], which when combined with new sequencing technologies makes bio prospecting in nature a far more viable endeavour. However, making sense of such a vast diversity is still challenging and could be simplified if known species with O2-tolerant enzymes were annotated with information on metabolism and natural environments. Here, we utilised a bioinformatics approach to compare O2-tolerant and sensitive membrane-bound NiFe hydrogenases from 177 bacterial species with fully sequenced genomes for differences in their taxonomy, O2 requirements, and natural environment. Following this, we interrogated a metagenome from lacustrine surface sediment for novel hydrogenases via high-throughput shotgun DNA sequencing using the Illumina™ MiSeq platform. We found 44 new NiFe group 1 membrane-bound hydrogenase sequence fragments, five of which segregated with the tolerant group on the phylogenetic tree of the enzyme's small subunit, and four with the large subunit, indicating de novo O2-tolerant protein sequences that could help engineer more efficient hydrogenases. PMID:26044993

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

    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

  15. Nickel-dependent reconstitution of hydrogenase apoprotein in Bradyrhizobium japonicum Hup c mutants and direct evidence for a nickel metabolism locus involved in nickel incorporation into the enzyme

    Microsoft Academic Search

    Changlin Fu; Robert J. Maier

    1992-01-01

    A double mutant (JH103K10) was created from hydrogenase constitutive mutant (JH103) by replacement of a chromosomal 0.60 kb nickel metabolism related locus with a kanamycin resistance gene. The double mutant required 10 to 20 times more nickel (Ni) to achieve near parental strain levels of hydrogenase activity. In the absence of nickel, both JH103K10 and JH103 synthesized high levels of

  16. Sequence of a 10.5 kbp fragment of Clostridium pasteurianum genomic DNA encompassing the hydrogenase I gene and two spore germination genes.

    PubMed

    Meyer, J

    1995-06-01

    The gene encoding hydrogenase I from Clostridium pasteurianum had previously been cloned and sequenced as part of a 2.3 kbp Sau3A genomic DNA fragment. The analysis of the regions surrounding the hydrogenase gene has been extended by cloning and sequencing two EcoRI fragments, both partially overlapping the 2.3 kbp Sau3A fragment. An uninterrupted genomic sequence of 10.5 kbp has thus been obtained, including 6.7 kbp upstream of the hydrogenase gene, and 2 kbp downstream. The hydrogenase gene is separated by 473 bp from the next open reading frame on its 5' side and by 366 bp from the next open reading frame on its 3' side. It is preceded by putative promoter regions, and followed by a strong transcription termination signal. Therefore the hydrogenase I gene from C. pasteurianum probably belongs to a monocistronic operon. This is consistent with previous biochemical evidence showing that the enzyme is monomeric. The sequence data also show that in the case of this Fe-hydrogenase, in contrast to the NiFe-hydrogenases, there are no accessory genes cotranscribed with, or located near, the structural genes. The sequenced region contains six open reading frames in addition to the hydrogenase gene. One of these probably encodes phosphatidylserine decarboxylase, and two others are homologous to two of the three genes of the gerA and gerB loci (spore germination) from Bacillus subtilis. These are the first spore germination gene sequences obtained from a bacterium of the genus Clostridium. PMID:16887524

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

    PubMed

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

    2014-12-11

    Picosecond time-resolved infrared spectroscopy (TRIR) was performed for the first time on a dithiolate bridged binuclear iron(I) hexacarbonyl complex ([Fe?(?-bdt)(CO)?], bdt = benzene-1,2-dithiolate) which is a structural mimic of the active site of the [FeFe]-hydrogenase enzyme. As these model active sites are increasingly being studied for their potential in photocatalytic systems for hydrogen production, understanding their excited and ground state dynamics is critical. In n-heptane, absorption of 400 nm light causes carbonyl loss with low quantum yield (<10%), while the majority (ca. 90%) of the parent complex is regenerated with biexponential kinetics (?? = 21 ps and ?? = 134 ps). In order to understand the mechanism of picosecond bleach recovery, a series of UV-pump TRIR experiments were performed in different solvents. The long time decay (??) of the transient spectra is seen to change substantially as a function of solvent, from 95 ps in THF to 262 ps in CCl?. Broadband IR-pump TRIR experiments were performed for comparison. The measured vibrational lifetimes (T?(avg)) of the carbonyl stretches were found to be in excellent correspondence to the observed ?? decays in the UV-pump experiments, signifying that vibrationally excited carbonyl stretches are responsible for the observed longtime decays. The fast spectral evolution (??) was determined to be due to vibrational cooling of low frequency modes anharmonically coupled to the carbonyl stretches that were excited after electronic internal conversion. The results show that cooling of both low and high frequency vibrational modes on the electronic ground state give rise to the observed picosecond TRIR transient spectra of this compound, without the need to invoke electronically excited states. PMID:25426927

  18. Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer.

    PubMed

    Zadvornyy, Oleg A; Lucon, Janice E; Gerlach, Robin; Zorin, Nikolay A; Douglas, Trevor; Elgren, Timothy E; Peters, John W

    2012-01-01

    The potential of hydrogen as a clean renewable fuel source and the finite reserves of platinum metal to be utilized in hydrogen production catalysts have provided the motivation for the development of non-noble metal-based solutions for catalytic hydrogen production. There are a number of microorganisms that possess highly efficient hydrogen production catalysts termed hydrogenases that generate hydrogen under certain metabolic conditions. Although hydrogenases occur in photosynthetic microorganisms, the oxygen sensitivity of these enzymes represents a significant barrier in directly coupling hydrogen production to oxygenic photosynthesis. To overcome this barrier, there has been considerable interest in identifying or engineering oxygen tolerant hydrogenases or generating mimetic systems that do not rely on oxygen producing photocatalysts. In this work, we demonstrate photo-induced hydrogen production from a stable [NiFe]-hydrogenase coupled to a [Ru(2,2'-bipyridine)(2)(5-amino-1,10-phenanthroline)](2+) photocatalyst. When the Ru(II) complex is covalently attached to the hydrogenase, photocatalytic hydrogen production occurs more efficiently in the presence of a redox mediator than if the Ru(II) complex is simply present in solution. Furthermore, sustained hydrogen production occurs even in the presence of oxygen by presumably creating a local anoxic environment through the reduction of oxygen similar to what is proposed for oxygen tolerant hydrogenases. These results provide a strong proof of concept for engineering photocatalytic hydrogen production in the presence of oxygen using biohybrid mimetic systems. PMID:22119807

  19. Comparison of N2 Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct 15N Partitioning 1

    PubMed Central

    La Favre, Jeffrey S.; Focht, Dennis D.

    1983-01-01

    Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing 15N-enriched organic matter. Seasonal N2 fixation activity was determined by periodically assaying plants for reduction of C2H2. N2 fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N2 fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of 15N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N2 fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants. Gas samples were taken from soil columns several times during the growth cycle of the plants. H2 was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H2 consumption by soil bacteria. Estimation of N2 fixation by acetylene reduction activity was closest to the direct 15N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct 15N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer. PMID:16663148

  20. 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

  1. 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 (?>420?nm). PMID:25205168

  2. Aerobic damage to [FeFe]-hydrogenases: activation barriers for the chemical attachment of O2.

    PubMed

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

    2014-04-14

    [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

  3. 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

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

    PubMed

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

    2014-06-18

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

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

    PubMed Central

    2014-01-01

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

  6. Purification, crystallization and preliminary X-ray analysis of the membrane-bound [NiFe] hydrogenase from Allochromatium vinosum

    PubMed Central

    Kellers, Petra; Ogata, Hideaki; Lubitz, Wolfgang

    2008-01-01

    The membrane-bound [NiFe] hydrogenase is a unique metalloprotein that is able to catalyze the reversible oxidation of hydrogen to protons and electrons during a complex reaction cycle. The [NiFe] hydrogenase was isolated from the photosynthetic purple sulfur bacterium Allochromatium vinosum and its crystallization and preliminary X-ray analysis are reported. It was crystallized by the hanging-drop vapour-diffusion method using sodium citrate and imidazole as crystallization agents. The crystals belong to space group P21212, with unit-cell parameters a = 205.00, b = 217.42, c = 120.44?Å. X-ray diffraction data have been collected to 2.5?Å resolution. PMID:18678940

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

    SciTech Connect

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

    2013-01-04

    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.

  8. Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function*

    PubMed Central

    Eckert, Carrie; Boehm, Marko; Carrieri, Damian; Yu, Jianping; Dubini, Alexandra; Nixon, Peter J.; Maness, Pin-Ching

    2012-01-01

    Hydrogenases are metalloenzymes that catalyze 2H+ + 2e? ? H2. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ? NAD(P)+ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions. PMID:23139416

  9. 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.

  10. A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase.

    PubMed

    Goris, Tobias; Wait, Annemarie F; Saggu, Miguel; Fritsch, Johannes; Heidary, Nina; Stein, Matthias; Zebger, Ingo; Lendzian, Friedhelm; Armstrong, Fraser A; Friedrich, Bärbel; Lenz, Oliver

    2011-05-01

    Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site. PMID:21390036

  11. 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

  12. Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08.

    PubMed

    Mishra, Jayshree; Khurana, Seema; Kumar, Narendra; Ghosh, Ananta K; Das, Debabrata

    2004-11-12

    Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster. PMID:15474481

  13. Roles of H2 uptake hydrogenases in Shigella flexneri acid tolerance.

    PubMed

    McNorton, Mykeshia M; Maier, Robert J

    2012-08-01

    Hydrogenases play many roles in bacterial physiology, and use of H(2) by the uptake-type enzymes of animal pathogens is of particular interest. Hydrogenases have never been studied in the pathogen Shigella, so targeted mutant strains were individually generated in the two Shigella flexneri H(2)-uptake enzymes (Hya and Hyb) and in the H(2)-evolving enzyme (Hyc) to address their roles. Under anaerobic fermentative conditions, a Hya mutant strain (hya) was unable to oxidize H(2), while a Hyb mutant strain oxidized H(2) like the wild-type. A hyc strain oxidized more exogenously added hydrogen than the parent. Fluorescence ratio imaging with dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) showed that the parent strain generated a membrane potential 15 times greater than hya. The hya mutant was also by far the most acid-sensitive strain, being even more acid-sensitive than a mutant strain in the known acid-combating glutamate-dependent acid-resistance pathway (GDAR pathway). In severe acid-challenge experiments, the addition of glutamate to hya restored survivability, and this ability was attributed in part to the GDAR system (removes intracellular protons) by mutant strain (e.g. hya/gadBC double mutant) analyses. However, mutant strain phenotypes indicated that a larger portion of the glutamate-rescued acid tolerance was independent of GadBC. The acid tolerance of the hya strains was aided by adding chloride ions to the growth medium. The whole-cell Hya enzyme became more active upon acid exposure (20 min), based on assays of hyc. Indeed, the very high rates of Shigella H(2) oxidation by Hya in acid can supply each cell with 2.4×10(8) protons min(-1). Electrons generated from Hya-mediated H(2) oxidation at the inner membrane likely counteract cytoplasmic positive charge stress, while abundant proton pools deposited periplasmically likely repel proton influx during severe acid stress. PMID:22628482

  14. Infrared Spectroscopy During Electrocatalytic Turnover Reveals the Ni-L Active Site State During H2 Oxidation by a NiFe Hydrogenase.

    PubMed

    Hidalgo, Ricardo; Ash, Philip A; Healy, Adam J; Vincent, Kylie A

    2015-06-01

    A novel in situ IR spectroscopic approach is demonstrated for the characterization of hydrogenase during catalytic turnover. E. coli hydrogenase?1 (Hyd-1) is adsorbed on a high surface-area carbon electrode and subjected to the same electrochemical control and efficient supply of substrate as in protein film electrochemistry during spectral acquisition. The spectra reveal that the active site state known as Ni-L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non-turnover conditions. The observation that Ni-L is present at all potentials during turnover under H2 suggests that the final steps in the catalytic cycle of H2 oxidation by Hyd-1 involve sequential proton and electron transfer via Ni-L. A broadly applicable IR spectroscopic technique is presented for addressing electrode-adsorbed redox enzymes under fast catalytic turnover. PMID:25925315

  15. Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase.

    PubMed

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

    2014-01-01

    Possible proton transport pathways in Clostridium pasteurianum (CpI) [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to evaluate the functional pathway and provide insight into the hydrogen bonding features defining an active proton transport pathway. Three pathways were evaluated, two of which consist of water wires and one of predominantly amino acid residues. Our simulations suggest that protons are not transported through water wires. Instead, the five-residue motif (Glu282, Ser319, Glu279, H2O, Cys299) was found to be the likely pathway, consistent with previously made experimental observations. The pathway was found to have a persistent hydrogen bonded core (residues Cys299 to Ser319), with less persistent hydrogen bonds at the ends of the pathway for both H2 release and H2 uptake. Single site mutations of the four residues have been shown experimentally to deactivate the enzyme. The theoretical evaluation of these mutations demonstrates redistribution of the hydrogen bonds in the pathway, resulting in enzyme deactivation. Finally, coupling between the protein dynamics near the proton transport pathway and the redox partner binding regions was also found as a function of H2 uptake and H2 release states, which may be indicative of a correlation between proton and electron movement within the enzyme. PMID:23981729

  16. 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

  17. Direct evidence of active site reduction and photo-driven catalysis in sensitized hydrogenase assemblies

    PubMed Central

    Greene, Brandon L.; Joseph, Crisjoe A.; Maroney, Michael J.; Dyer, R. Brian

    2012-01-01

    We report photo-catalytic H2 production by hydrogenase (H2ase)-quantum dot (QD) hybrid assemblies. Quenching of the CdTe exciton emission is observed, consistent with electron transfer from quantum dot to H2ase. GC analysis shows light driven H2 production in the presence of a sacrificial electron donor with an efficiency of 4%, which is likely a lower limit to these hybrid systems. FTIR was employed for direct observation of active site reduction in unprecedented detail for photo-driven H2ase catalysis with sensitivity towards both H2ase and sacrificial electron donor. Photosensitization with Ru(bpy)32+ shows distinct FTIR photo- reduction properties generating all states along the steady-state catalytic cycle with minimal H2 production indicating slow, sequential one electron reduction steps. Comparing H2ase activity and FTIR results of both systems shows that QDs bind more efficiently for electron transfer and the final enzyme state is different for the two sensitizers. The possible origins of these differences and their implications for the enzymatic mechanism are discussed. PMID:22716776

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

    PubMed Central

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

    1989-01-01

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

  19. 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

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

    PubMed

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

    2014-03-14

    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

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

    PubMed

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

    2014-05-01

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

  2. 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

  3. Effect of the protonation degree of a self-assembled monolayer on the immobilization dynamics of a [NiFe] hydrogenase.

    PubMed

    Utesch, Tillmann; Millo, Diego; Castro, Maria Ana; Hildebrandt, Peter; Zebger, Ingo; Mroginski, Maria Andrea

    2013-01-15

    Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and, in particular, for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of Desulfovibrio gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom molecular dynamics (MD) simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically modified electrode surface. While the hydrogenase undergoes a weak but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary surface-enhanced infrared absorption (SEIRA) measurements on the comparable [NiFe] standard hydrogenases from Desulfovibrio vulgaris Miyazaki F and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level. PMID:23215250

  4. Protonation Studies of the New Iron Carbonyl Cyanide trans-[Fe(CO)3(CN)2]2-: Implications with Respect to Hydrogenases

    E-print Network

    Rauchfuss, Thomas B.

    Protonation Studies of the New Iron Carbonyl Cyanide trans-[Fe(CO)3(CN)2]2-: ImplicationsVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received April 30, 2003 The new iron carbonyl cyanide by the hydrogenase enzymes, which also feature H-Fe- CN-CO centers. Iron carbonyl cyanide complexes1-7 have assumed

  5. Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1.

    PubMed

    Taketa, Midori; Nakagawa, Hanae; Habukawa, Mao; Osuka, Hisao; Kihira, Kiyohito; Komori, Hirofumi; Shibata, Naoki; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi; Yoon, Ki-Seok; Ogo, Seiji; Shomura, Yasuhito; Higuchi, Yoshiki

    2015-01-01

    NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58?Å resolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59?Å, ?=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2?Å3?Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms. PMID:25615977

  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. The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron–sulphur proteins

    PubMed Central

    Balk, Janneke; Pierik, Antonio J; Netz, Daili J Aguilar; Mühlenhoff, Ulrich; Lill, Roland

    2004-01-01

    The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron–sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes. PMID:15103330

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

    PubMed

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

    2012-12-01

    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 H(2)-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 H(2)-oxidizing bacteria, including Pseudomonas sp., Serratia sp., Stenotrophomonas sp., Enterobacter sp., Rahnella sp., Burkholderia sp., and Ralstonia sp. isolates. Four free-living N(2)-fixing bacteria harbored a high and oxygen-tolerant hydrogenase activity, which was not fully inhibited within entire cells up to 150-250 ?mol/L O(2) concentration or within soluble protein extracts up to 25-30 ?mol/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 N(2)-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

  9. The maturation factors HoxR and HoxT contribute to oxygen tolerance of membrane-bound [NiFe] hydrogenase in Ralstonia eutropha H16.

    PubMed

    Fritsch, Johannes; Lenz, Oliver; Friedrich, Bärbel

    2011-05-01

    The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O(2) and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O(2) levels. MBH-driven growth on H(2) is inhibited or retarded at high O(2) partial pressure (pO(2)) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO(2). Lack of hoxR and hoxT can phenotypically be restored by providing O(2)-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions. PMID:21441514

  10. Redox intermediates of Desulfovibrio gigas (NiFe) hydrogenase generated under hydrogen

    SciTech Connect

    Teixeira, M.; Moura, I.; Xavier, A.V.; Moura, J.J.G. (Centro de Quimica Estrutural, Lisboa (Portugal) Univ. Nova de Lisboa (Portugal)); LeGall, J.; DerVartanian, D.V.; Peck, H.D. Jr. (Univ. of Georgia, Athens (United States)); Huynh, B.H. (Emory Univ., Atlanta, GA (United States))

    1989-10-05

    The hydrogenase of Desulfovibrio gigas is a complex enzyme containing one nickel center, one (3Fe-4S) and two (4Fe-4S) clusters. Redox intermediates of this enzyme were generated under hydrogen using a redox-titration technique and were studied by EPR and Moessbauer spectroscopy. In the oxidized states, the two (4Fe-4S){sup 2+} clusters exhibit a broad quadrupole doublet with parameters typical for this type of cluster. Upon reduction, the two (4Fe-4S){sup 1+} clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential was labeled Fe-S center 1 and the other with lower potential, Fe-S center 2. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced (3Fe-4S) cluster: (1) a signal with crossover point at g {approximately} 12, labeled the g = 12 signal, and (2) a broad signal at the very weak-field region ({approximately}3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be {minus}70 {plus minus} 10 mV. At potentials below {minus}250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two (4Fe-4S) clusters indicating that the (3Fe-4S){sup 0} cluster is sensitive to the redox state of the (4Fe-4S) clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of the authors' results are discussed.

  11. Accumulation of ferrous iron in Chlamydomonas reinhardtii. Influence of CO2 and anaerobic induction of the reversible hydrogenase.

    PubMed

    Semin, Boris K; Davletshina, Lira N; Novakova, Alla A; Kiseleva, Tat'yana Y; Lanchinskaya, Victoriya Y; Aleksandrov, Anatolii Y; Seifulina, Nora; Ivanov, Il'ya I; Seibert, Michael; Rubin, Andrei B

    2003-04-01

    The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H(2) via ferredoxin and the reversible [Fe]hydrogenase enzyme under anaerobic conditions. Recently, a novel approach for sustained H(2) gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A. Melis, L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert [2000] Plant Physiol 122: 127-135). The close relationship between S and Fe in the H(2)-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase. In this study, we used Mössbauer spectroscopy to examine both the uptake of Fe by the alga at different CO(2) concentrations during growth and the influence of anaerobiosis on the accumulation of Fe. Algal cells grown in media with (57)Fe(III) at elevated (3%, v/v) CO(2) concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with Mössbauer parameters of quadrupole splitting = 0.65 mm s(-1) and isomeric shift = 0.46 mm s(-1) and (b) Fe(II) with quadrupole splitting = 3.1 mm s(-1) and isomeric shift = 1.36 mm s(-1). Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell. The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis. Growing the algae under atmospheric CO(2) (limiting) conditions, compared with 3% (v/v) CO(2), resulted in a decrease in the intracellular Fe(II) content by a factor of 3. Incubating C. reinhardtii cells, grown at atmospheric CO(2) for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO(2). This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO(2) concentration, and anaerobiosis. A comparison of Fe-uptake results with a cyanobacterium, yeast, and algae suggests that the intracellular Fe(II) pool in C. reinhardtii may reside in the cell vacuole. PMID:12692334

  12. 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

  13. [Fe-Fe]-hydrogenase Reactivated by Residue Mutations as Bridging Carbonyl Rearranges: A QM/MM Study

    PubMed Central

    Motiu, Stefan; Gogonea, Valentin

    2009-01-01

    In the current work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O2, or OH-, which metabolizes to H2O1,2. We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results3, i.e., wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fed) has been screened for residues that prevent H2O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs’ energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs’ energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs’ energy for the elimination H2O. Favorable thermodynamic results have been obtained for both single residue removal (?G?Glu374 = -1.6 kcal/mol), single substitution (?GGlu374His = -3.1 kcal/mol), and combined residue substitutions (?GArg111Glu;Thr145Val;Glu374His;Tyr375Phe = -7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, i.e., for H2O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are ?Arg111, ?Thr145, ?Ser177, ?Glu240, ?Glu374, and ?Tyr375. The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed to compensate for the electron density donated to oxygen upon the elimination of H2O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of COb towards Fed, which leads to enzyme inhibition. However, enzyme reactivation can be achieved by means of appropriate amino acid substitutions.

  14. Occurrence of H(2)-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus.

    PubMed

    Murillo, J; Villa, A; Chamber, M; Ruiz-Argüeso, T

    1989-01-01

    Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H(2) uptake by bacteroids and the relative efficiency of N(2) fixation (RE = 1 - [H(2) evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H(2)-uptake activity in symbiosis with O. compressus. The inability of these putative Hup(+) strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains. PMID:16666550

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

    PubMed Central

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

    1989-01-01

    Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H2 uptake by bacteroids and the relative efficiency of N2 fixation (RE = 1 - [H2 evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H2-uptake activity in symbiosis with O. compressus. The inability of these putative Hup+ strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains. Images Figure 1 Figure 2 PMID:16666550

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

    E-print Network

    Tye, Jesse Wayne

    2009-05-15

    to form a 3c-2eâ Fe-H-Fe bond, while Et+ reacts to form a new C-S bond. The instability of a bridging ethyl complex is attributed to the inability of the ethyl group, in contrast to a hydride, to form a stable 3c-2eâ bond with the two iron centers. Gas...

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

    PubMed

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

    2014-09-01

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

  18. Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

    PubMed

    Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

    2015-07-01

    The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. PMID:25912316

  19. X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli

    PubMed Central

    Volbeda, Anne; Amara, Patricia; Darnault, Claudine; Mouesca, Jean-Marie; Parkin, Alison; Roessler, Maxie M.; Armstrong, Fraser A.; Fontecilla-Camps, Juan C.

    2012-01-01

    The crystal structure of the membrane-bound O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H2-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O2-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one O? of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H2-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O2 to H2O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O2 reduction, located near the active site. PMID:22431599

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

    PubMed Central

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

    2010-01-01

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

  1. [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

  2. 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 ?CO modes to energies that are consistent with an increase in oxidation state of the [2Fe]H subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA1, spectra associated with Hred and Hsred states are less populated in the Cys ? Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions. PMID:25286239

  3. First-principles study of quantum-transport properties of Fe/Fe2VAl/Fe trilayers: Design for confined k-space current-perpendicular-to-plane giant-magnetoresistance

    NASA Astrophysics Data System (ADS)

    Yabuuchi, Shin; Kitagawa, Isao

    2014-09-01

    The electronic structure and quantum-transport properties of Fe/Fe2VAl/Fe trilayers were investigated by using first-principles calculations. The high-transmission area in k-space can be confined by using a semimetallic Fe2VAl layer. The confined k-space originates from the small Fermi surface of Fe2VAl. Moreover, the symmetry matching of wave functions between the leads and space of the trilayer around the Fermi level is a significant factor in regard to designing confined-k-space current-perpendicular-to-plane giant-magnetoresistance (CPP-GMR) device, which is a promising candidate to provide a resistance area (RA) product that is difficult to achieve by using conventional insulators and metals.

  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 ca. 20 mmol H2 cm-3 d-1 close to the sediment-water interface to 0.5 mmol H2 cm-3 d-1 at a depth of 0.8 m. In samples from the Barents Sea H2ase activity ranged between 0.1 to 2.5 mmol H2 cm-3 d-1 down to a depth of 1.60 m. At all sites the SRR profile followed the H2ase activity profile until SRR declined to values close to the minimum detection limit (~10 pmol cm-3 d-1). H2ase activity increased again after SRR declined, indicating that other microbial processes are becoming quantitatively more important. The H2ase and SRR data show that our assay has a potential to become a valuable tool to measure total subsurface microbial activity.

  5. Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis

    Microsoft Academic Search

    J. Balk; A. J. Pierik; U. Mühlenhoff; R. Lill

    2005-01-01

    The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. Previously, we have shown that Nar1p is an Fe-S protein and that assembly of its co-factors depends on the mitochondrial Fe-S cluster biosynthesis apparatus. Using functional studies in vivo, we demonstrated that

  6. Catalytic production of hydrogen peroxide and water by oxygen-tolerant [NiFe]-hydrogenase during H2 cycling in the presence of O2.

    PubMed

    Lauterbach, Lars; Lenz, Oliver

    2013-11-27

    Hydrogenases control the H2-related metabolism in many microbes. Most of these enzymes are prone to immediate inactivation by O2. However, a few members of the subclass of [NiFe]-hydrogenases are able to convert H2 into protons and electrons even in the presence of O2, making them attractive for biotechnological application. Recent studies on O2-tolerant membrane-bound hydrogenases indicate that the mechanism of O2 tolerance relies on their capability to completely reduce O2 with four electrons to harmless water. In order to verify this hypothesis, we probed the O2 reduction capacity of the soluble, NAD(+)-reducing [NiFe]-hydrogenase (SH) from Ralstonia eutropha H16. A newly established, homologous overexpression allowed the purification of up to 90 mg of homogeneous and highly active enzyme from 10 g of cell material. We showed that the SH produces trace amounts of superoxide in the course of H2-driven NAD(+) reduction in the presence of O2. However, the major products of the SH-mediated oxidase activity was in fact hydrogen peroxide and water as shown by the mass spectrometric detection of H2(18)O formed from H2 and isotopically labeled (18)O2. Water release was also observed when the enzyme was incubated with NADH and (18)O2, demonstrating the importance of reverse electron flow to the [NiFe] active site for O2 reduction. A comparison of the turnover rates for H2 and O2 revealed that in the presence of twice the ambient level of O2, up to 3% of the electrons generated through H2 oxidation serve as "health insurance" and are reused for O2 reduction. PMID:24180286

  7. Impact of amino acid substitutions near the catalytic site on the spectral properties of an O2-tolerant membrane-bound [NiFe] hydrogenase.

    PubMed

    Saggu, Miguel; Ludwig, Marcus; Friedrich, Bärbel; Hildebrandt, Peter; Bittl, Robert; Lendzian, Friedhelm; Lenz, Oliver; Zebger, Ingo

    2010-04-26

    [NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O(2) tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O(2)-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni(r)-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Ni(u)-A state, an irreversible inactive redox state, and the light-induced Ni(a)-L state even at room temperature. PMID:20376875

  8. Significance of hydrogen burst from growing cultures of Desulfovibrio vulgaris , Miyazaki, and the role of hydrogenase and cytochrome c 3 in energy production system

    Microsoft Academic Search

    Keiko Tsuji; Tatsuhiko Yagi

    1980-01-01

    In an early stage of the growth of Desulfovibrio vulgaris, Miyazaki, a burst of H2 occurred, and lasted for a few hours. The H2S production which paralleled the cell proliferation was very low in the H2 burst period, and began to increase thereafter. Hydrogenase (hydrogen: ferricytochrome c3 oxidoreductase, EC1. 12.2.1), cytochrome c3 and desulfoviridin also increased after the H2 burst.

  9. 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

  10. How Escherichia coli Is Equipped to Oxidize Hydrogen under Different Redox Conditions*S

    E-print Network

    Palmer, Tracy

    to their active site metal ion con- tent, and three phylogenetically distinct classes have so far been identified: di-iron [FeFe]-, nickel-iron [NiFe]-, and mono-iron [Fe]-hydrogenases (1). Nickel-iron hydrogenases accommodating one to three electron-transfer- ring iron-sulfur clusters. The active sites of [Ni

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

    PubMed

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

    2014-05-19

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  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. Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria. Key role of polyheme cytochromes c and hydrogenases.

    PubMed

    Michel, C; Brugna, M; Aubert, C; Bernadac, A; Bruschi, M

    2001-01-01

    Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 microM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c3(Mr. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c3 from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c7 from Desulfuromonas acetoxidans. Results with cytochromes c3 and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate. PMID:11234966

  16. 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

  17. Exposure studies of core-shell Fe/Fe(3)O(4) and Cu/CuO NPs to lettuce (Lactuca sativa) plants: Are they a potential physiological and nutritional hazard?

    PubMed

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

    2014-02-28

    Iron and copper nanomaterials are widely used in environmental remediation and agriculture. However, their effects on physiological parameters and nutritional quality of terrestrial plants such as lettuce (Lactuca sativa) are still unknown. In this research, 18-day-old hydroponically grown lettuce seedlings were treated for 15 days with core-shell nanoscale materials (Fe/Fe(3)O(4), Cu/CuO) at 10 and 20mg/L, and FeSO(4)·7H(2)O and CuSO(4)·5H(2)O at 10mg/L. At harvest, Fe, Cu, micro and macronutrients were determined by ICP-OES. Also, we evaluated chlorophyll content, plant growth, and catalase (CAT) and ascorbate peroxidase (APX) activities. Our results showed that iron ions/NPs did not affect the physiological parameters with respect to water control. Conversely, Cu ions/NPs reduced water content, root length, and dry biomass of the lettuce plants. ICP-OES results showed that nano-Cu/CuO treatments produced significant accumulation of Cu in roots compared to the CuSO(4)·5H(2)O treatment. In roots, all Cu treatments increased CAT activity but decreased APX activity. In addition, relative to the control, nano-Cu/CuO altered the nutritional quality of lettuce, since the treated plants had significantly more Cu, Al and S but less Mn, P, Ca, and Mg. PMID:24462971

  18. Impact of membrane-associated hydrogenases on the FOF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N'-dicyclohexylcarbodiimide in the mutants lacking hydrogenases.

    PubMed

    Blbulyan, Syuzanna; Trchounian, Armen

    2015-08-01

    Escherichia coli is able to ferment glycerol and to produce molecular hydrogen (H2) by four membrane-associated hydrogenases (Hyd) changing activity in response to different conditions. In this study, overall ATPase activity of glycerol alone and mixed carbon sources (glucose and glycerol) fermented E. coli wild type and different Hyd mutants and its inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) were first investigated. ATPase activity was higher in glycerol fermented wild type cells at pH 7.5 compared to pH 6.5 and pH 5.5; DCCD inhibited markedly ATPase activity at pH 7.5. The ATPase activity at pH 7.5, compared with wild type, was lower in selC and less in hypF single mutants, suppressed in hyaB hybC selC triple mutant. Moreover, total ATPase activity of mixed carbon fermented wild type cells was maximal at pH 7.5 and lowered at pH 5.5. The ATPase activities of hypF and hyaB hybC selC mutants were higher at pH 5.5, compared with wild type; DCCD inhibited markedly ATPase activity of hypF mutant. These results demonstrate that in E. coli during glycerol fermentation the membrane proton-translocating FOF1-ATPase has major input in overall ATPase activity and alkaline pH is more optimal for the FOF1-ATPase operation. Hyd-1 and Hyd-2 are required for the FOF1-ATPase activity upon anaerobic fermentation of glycerol. The impact of Hyd-1 and Hyd-2 on the FOF1-ATPase is more obvious during mixed carbon fermentation at slightly acidic pH. PMID:26049001

  19. Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

    PubMed

    Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A; Lenz, Oliver

    2011-01-01

    The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H?-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O?. It comprises six subunits, HoxHYFUI?, and incorporates a [NiFe] H+/H? cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K(I) values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O?, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. PMID:22016788

  20. New nitrosyl derivatives of diiron dithiolates related to the active site of the [FeFe]-hydrogenases.

    PubMed

    Olsen, Matthew T; Justice, Aaron K; Gloaguen, Frédéric; Rauchfuss, Thomas B; Wilson, Scott R

    2008-12-15

    Nitrosyl derivatives of diiron dithiolato carbonyls have been prepared starting from the precursor Fe(2)(S(2)C(n)H(2n))(dppv)(CO)(4) (dppv = cis-1,2-bis(diphenylphosphinoethylene). These studies expand the range of substituted diiron(I) dithiolato carbonyl complexes. From [Fe(2)(S(2)C(2)H(4))(CO)(3)(dppv)(NO)]BF(4) ([1(CO)(3)]BF(4)), the following compounds were prepared: [1(CO)(2)(PMe(3))]BF(4), [1(CO)(dppv)]BF(4), NEt(4)[1(CO)(CN)(2)], and 1(CO)(CN)(PMe(3)). Some of these substitution reactions occur via the addition of 2 equiv of the nucleophile followed by the dissociation of one nucleophile and decarbonylation. Such a double adduct was characterized crystallographically in the case of [Fe(2)(S(2)C(2)H(4))(CO)(3)(dppv)(NO)(PMe(3))(2)]BF(4). This result shows that the addition of two ligands causes scission of the Fe-Fe bond and one Fe-S bond. When cyanide is the nucleophile, nitrosyl migrates away from the Fe(dppv) site, yielding a Fe(CN)(2)(NO) derivative. Compounds [1(CO)(3)]BF(4), [1(CO)(2)(PMe(3))]BF(4), and [1(CO)(dppv)]BF(4) were also prepared by the addition of NO(+) to the di-, tri-, and tetrasubstituted precursors. In these cases, the NO(+) appears to form an initial 36e(-) adduct containing terminal Fe-NO, followed by decarbonylation. Several complexes were prepared by the addition of NO to the mixed-valence Fe(I)Fe(II) derivatives. The diiron nitrosyl complexes reduce at mild potentials and in certain cases form weak adducts with CO. IR and EPR spectra of 1(CO)(dppv), generated by low-temperature reduction of [1(CO)(dppv)]BF(4) with Co(C(5)Me(5))(2), indicates that the SOMO is located on the FeNO subunit. PMID:19007207

  1. 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.

  2. 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

  3. Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12

    PubMed Central

    Hayes, Everett T; Wilks, Jessica C; Sanfilippo, Piero; Yohannes, Elizabeth; Tate, Daniel P; Jones, Brian D; Radmacher, Michael D; BonDurant, Sandra S; Slonczewski, Joan L

    2006-01-01

    Background In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. Results The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2). Acid also up-regulated fimbriae (fimAC), periplasmic chaperones (hdeAB), cyclopropane fatty acid synthase (cfa), and the "constitutive" Na+/H+ antiporter (nhaB). Base up-regulated core genes for maltodextrin transport (lamB, mal), ATP synthase (atp), and DNA repair (recA, mutL). Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh) and hydrogenases (hya, hyb, hyc, hyf, hyp). A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps). Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl), and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL), but down-regulated penicillin-binding proteins (dacACD, mreBC). Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC). Conclusion pH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nhaB. Under oxygen limitation, however, pH regulation is reversed for genes encoding electron transport components and hydrogenases. Extreme-acid resistance requires yagU and hydrogenase production. Ribosome synthesis is down-regulated at low pH under oxygen limitation, possibly due to the restricted energy yield of catabolism. Under oxygen limitation, pH regulates metabolism and transport so as to maximize alternative catabolic options while minimizing acidification or alkalinization of the cytoplasm. PMID:17026754

  4. Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

    PubMed Central

    Pinske, Constanze; Jaroschinsky, Monique; Linek, Sabine; Kelly, Ciarán L.

    2014-01-01

    Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2 to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2 evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2 evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2 oxidation or H2 evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2 evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2 evolution from glycerol requires reverse electron transport. PMID:25368299

  5. X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly.

    PubMed

    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

    2015-02-01

    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 (??)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 ?2-sulfide ion to a mononuclear Fe(2+) 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

  6. Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer.

    PubMed

    Watanabe, Satoshi; Kawashima, Takumi; Nishitani, Yuichi; Kanai, Tamotsu; Wada, Takehiko; Inaba, Kenji; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2015-06-23

    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

  7. 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

  8. 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

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

    E-print Network

    Singleton, Michael Lee

    2012-02-14

    mixed-valent FeIFeII complexes. An X-ray crystal structure of one of these complexes, (?-SCH2C(CH3)2CH2S-)[Fe(CO)2PMe3]2PF6 shows both a semi-bridging carbonyl and an open site similar to the 2-Fe subsite in the Hox state of the enzyme active site...

  10. A hydrogenase model system based on the sequence of cytochrome c: photochemical hydrogen evolution in aqueous media.

    PubMed

    Sano, Yohei; Onoda, Akira; Hayashi, Takashi

    2011-08-01

    The diiron carbonyl cluster is held by a native CXXC motif, which includes Cys14 and Cys17, in the cytochrome c sequence. It is found that the diiron carbonyl complex works well as a catalyst for H(2) evolution. It has a TON of ?80 over 2 h at pH 4.7 in the presence of a Ru-photosensitizer and ascorbate as a sacrificial reagent in aqueous media. PMID:21519624

  11. 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

  12. A Reversible Proton Relay Process Mediated by Hydrogen-Bonding Interactions in [FeFe]Hydrogenase Modeling.

    PubMed

    Chu, Kai-Ti; Liu, Yu-Chiao; Huang, Yi-Lan; Hsu, Cheng-Huey; Lee, Gene-Hsiang; Chiang, Ming-Hsi

    2015-07-27

    A reversible and temperature-dependent proton-relay process is demonstrated for a Fe2 complex possessing a terminal thiolate in the presence of nitrogen-based acids. The terminal sulfur site (S(t) ) of the complex forms a hydrogen-bond interaction with N,N-dimethylanilinium acid at 183?K. The Fe2 core, instead, is protonated to generate a bridging hydride at 298?K. Reversibility is observed for the tautomerization between the hydrogen-bonded pair and the Fe-hydride species. X-ray structural analysis of the hydrogen-bonded species at 193?K reveals a short N(H)???S(t) contact. Employment of pyridinium acid also results in similar behavior, with reversible proton-hydride interconversion. DFT investigation of the proton-transfer pathways indicates that the pKa value of the hydrogen-bonded species is enhanced by 3.2 pKa units when the temperature is decreased from 298?K to 183?K. PMID:26118674

  13. 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

  14. An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli.

    PubMed

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

  15. Applications of X-ray absorption spectroscopy to biologically relevant metal-based chemistry

    NASA Astrophysics Data System (ADS)

    Best, Stephen P.; Cheah, Mun Hon

    2010-02-01

    Recent developments in the understanding of the biosynthesis of the active site of the nitrogenase enzyme, the structure of the iron centre of [Fe]-hydrogenase and the structure and biomimetic chemistry of the [FeFe] hydrogenase H-cluster as deduced by application of X-ray spectroscopy are reviewed. The techniques central to this work include X-ray absorption spectroscopy either in the form of extended X-ray absorption fine structure (EXAFS), X-ray absorption near-edge structure (XANES) and nuclear resonant vibrational spectroscopy (NRVS). Examples of the advances in the understanding of the chemistry of the system through integration of a range of spectroscopic and computational techniques with X-ray spectroscopy are highlighted. The critical role played by ab initio calculation of structural and spectroscopic properties of transition-metal compounds using density functional theory (DFT) is illustrated both by the calculation of nuclear resonance vibrational spectroscopy (NRVS) spectra and the structures and spectra of intermediates through the catalytic reactions of hydrogenase model compounds.

  16. Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis.

    PubMed

    Balk, J; Pierik, A J; Aguilar Netz, D J; Mühlenhoff, U; Lill, R

    2005-02-01

    The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. Previously, we have shown that Nar1p is an Fe-S protein and that assembly of its co-factors depends on the mitochondrial Fe-S cluster biosynthesis apparatus. Using functional studies in vivo, we demonstrated that Nar1p has an essential role in the maturation of cytosolic and nuclear, but not of mitochondrial, Fe-S proteins. Here we provide further spectroscopic evidence that Nar1p possesses two Fe-S clusters. We also show that Nar1p is required for Fe-S cluster assembly on the P-loop NTPase Nbp35p, another newly identified component of the cytosolic Fe-S protein assembly machinery. These data suggest a complex biochemical pathway of extra-mitochondrial Fe-S protein biogenesis involving unique eukaryotic proteins. PMID:15667273

  17. Role of the HoxZ subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha immobilized on electrodes.

    PubMed

    Sezer, Murat; Frielingsdorf, Stefan; Millo, Diego; Heidary, Nina; Utesch, Tillman; Mroginski, Maria-Andrea; Friedrich, Bärbel; Hildebrandt, Peter; Zebger, Ingo; Weidinger, Inez M

    2011-09-01

    The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ?-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode. PMID:21761881

  18. A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]-hydrogenase maturase HydG.

    PubMed

    Nicolet, Yvain; Martin, Lydie; Tron, Cécile; Fontecilla-Camps, Juan C

    2010-10-01

    HydG uses tyrosine to synthesize the CN(-)/CO ligands of [FeFe]-hydrogenase active site. We have mutated two of the [4Fe-4S]-cluster cysteine ligands of the HydG C-terminal domain (CTD) to serine. The double mutant can still synthesize CN(-) but not CO. In a mutant lacking the CTD both CN(-) and CO synthesis are abolished. Like in ThiH, the initial steps of CN(-) synthesis are carried out in the TIM-barrel domain of HydG but some component(s) of the CTD are later needed. The mutants indicate that CO synthesis is metal-based and occurs in the CTD. We postulate that CN(-)/CO synthesis is initiated by H(2)N-*CH-CO(2)(-). Fragmentation of this radical into H(2)N-*CH(2) and CO(2) or H(2)C=NH and *CO(2)(-) provides plausible precursors for CN(-)/CO synthesis. PMID:20837009

  19. Pd(II)-Directed Encapsulation of Hydrogenase within the Layer-by-Layer Multilayers of Carbon Nanotube Polyelectrolyte Used as a Heterogeneous Catalyst for Oxidation of Hydrogen.

    PubMed

    Liu, Jiang; Zorin, Nikolay A; Chen, Meng; Qian, Dong-Jin

    2015-06-16

    A metal-directed assembling approach has been developed to encapsulate hydrogenase (H2ase) within a layer-by-layer (LBL) multilayer of carbon nanotube polyelectrolyte (MWNT-PVPMe), which showed efficient biocatalytic oxidation of H2 gas. The MWNT-PVPMe was prepared via a diazonium process and addition reactions with poly(4-vinylpyridine) (PVP) and methyl iodide (MeI). The covalently attached polymers and organic substituents in the polyelectrolyte comprised 60-70% of the total weight. The polyelectrolyte was then used as a substrate for H2ase binding to produce MWNT-PVPMe@H2ase bionanocomposites. X-ray photoelectron spectra revealed that the bionanocomposites included the elements of Br, S, C, N, O, I, Fe, and Ni, which confirmed that they were composed of MWNT-PVPMe and H2ase. Field emission transmission electron microscope images revealed that the H2ase was adsorbed on the surface of MWNT-PVPMe with the domains ranging from 20 to 40 nm. Further, with the use of the bionanocomposites as nanolinkers and Na2PdCl4 as connectors, the (Pd/MWNT-PVPMe@H2ase)n multilayers were constructed on the quartz and gold substrate surfaces by the Pd(II)-directed LBL assembling technique. Finally, the as-prepared LBL multilayers were used as heterogeneous catalysts for hydrogen oxidation with methyl viologen (MV(2+)) as an electron carrier. The dynamic processes for the reversible color change between blue-colored MV(+) and colorless MV(2+) (catalyzed by the LBL multilayers) were video recorded, which confirmed that the H2ase encapsulated within the present LBL multilayers was of much stronger stability and higher biocatalytic activity of H2 oxidation resulting in potential applications for the development of H2 biosensors and fuel cells. PMID:26010012

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

    PubMed Central

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

    2014-01-01

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

  1. 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.

  2. Computer Modeling in Biotechnology

    NASA Astrophysics Data System (ADS)

    Aksimentiev, Aleksei; Brunner, Robert; Cohen, Jordi; Comer, Jeffrey; Cruz-Chu, Eduardo; Hardy, David; Rajan, Aruna; Shih, Amy; Sigalov, Grigori; Yin, Ying; Schulten, Klaus

    Computational modeling can be a useful partner in biotechnology, in particular, in nanodevice engineering. Such modeling guides development through nanoscale views of biomolecules and devices not available through experimental imaging methods. We illustrate the role of computational modeling, mainly of molecular dynamics, through four case studies: development of silicon bionanodevices for single molecule electrical recording, development of carbon nano-tube-biomolecular systems as in vivo sensors, development of lipoprotein nanodiscs for assays of single membrane proteins, and engineering of oxygen tolerance into the enzyme hydrogenase for photosynthetic hydrogen gas production. The four case studies show how molecular dynamics approaches were adapted to the specific technical uses through (i) multi-scale extensions, (ii) fast quantum chemical force field evaluation, (iii) coarse graining, and (iv) novel sampling methods. The adapted molecular dynamics simulations provided key information on device behavior and revealed development opportunities, arguing that the "computational microscope" is an indispensable nanoengineering tool.

  3. Protein-protein complex formation affects the Ni-Fe and Fe-S centers in the H2-sensing regulatory hydrogenase from Ralstonia eutropha H16.

    PubMed

    Löscher, Simone; Gebler, Antje; Stein, Matthias; Sanganas, Oliver; Buhrke, Thorsten; Zebger, Ingo; Dau, Holger; Friedrich, Bärbel; Lenz, Oliver; Haumann, Michael

    2010-04-26

    The regulatory Ni-Fe hydrogenase (RH) from the H(2)-oxidizing bacterium Ralstonia eutropha functions as an oxygen-resistant hydrogen sensor, which is composed of the large, active-site-containing HoxC subunit and the small subunit HoxB carrying Fe-S clusters. In vivo, the HoxBC subunits form a dimer designated as RH(wt). The RH(wt) protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH(wt) (RH(wt)-HoxJ(wt)), located in the cytoplasm. In this study, we used X-ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni-Fe active site and the Fe-S clusters. Aside from the RH(wt) protein and the RH(wt)-HoxJ(wt) complex, we investigated the RH(stop) protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RH(wt)-HoxJ(Deltakinase), in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H(2), leading to the formation of the EPR-detectable Ni(III)-C state of the active site and to the reduction of Fe-S clusters detectable by XAS, thus corroborating that H(2) cleavage is independent of the presence of the HoxJ protein. In RH(stop), presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH(stop) differed from that in RH(wt) and the RH(wt)-HoxJ complexes, in which additional Ni--O bonds were detected by XAS. The Ni--O bonds caused only very minor changes of the EPR g-values of the Ni-C and Ni-L states and of the IR vibrational frequencies of the diatomic CN(-) and CO ligands at the active-site Fe ion. Both one Fe-S cluster in HoxB and an oxygen-rich Ni coordination seem to be stabilized by RH dimerization involving the C-terminus of HoxB and by complex formation with HoxJ. PMID:20340124

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

    PubMed Central

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

    2012-01-01

    A series of mixed-valence iron-nickel dithiolates is described that exhibits structures similar to those of mixed-valence diiron dithiolates. Interaction of tricarbonyl salt [(dppe)Ni(pdt)Fe(CO)3]BF4 ([1]BF4, dppe = Ph2PCH2CH2PPh2, pdtH2 = HSCH2CH2CH2SH) with P-donor ligands (L) afforded the substituted derivatives [(dppe)Ni(pdt)Fe(CO)2L]BF4 incorporating L = PHCy2 ([1a]BF4), PPh(NEt2)2 ([1b]BF4), P(NMe2)3 ([1c]BF4), P(i-Pr)3 ([1d]BF4) and PCy3 ([1e]BF4). The related precursor [(dcpe)Ni(pdt)Fe(CO)3]BF4 ([2]BF4, dcpe = Cy2PCH2CH2PCy2) gave the more electron-rich family of compounds [(dcpe)Ni(pdt)Fe(CO)2L]BF4 for L = PPh2(2-pyridyl) ([2a]BF4), PPh3 ([2b]BF4) and PCy3 ([2c]BF4). For bulky and strongly basic monophosphorus ligands, the salts feature distorted Fe coordination geometries: crystallographic analyses of [1e]BF4 and [2c]BF4 showed they adopt ‘rotated’ Fe(I) centers, in which PCy3 occupies a basal site and one CO ligand partially bridges the Ni and Fe centers. Like the undistorted mixed-valence derivatives, the new class of complexes are described as Ni(II)Fe(I) (S = ½) systems according to EPR spectroscopy, although with attenuated 31P hyperfine interactions. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the spin for [1e]+ is localized in a Fe(I)-centered d(z2) orbital, orthogonal to the Fe-P bond. The PCy3 complexes, rare examples of species featuring ‘rotated’ Fe centers, both structurally and spectroscopically resemble mixed-valence diiron dithiolates. Also reproducing the NiS2Fe core of the [NiFe]-H2ase active site, the hybrid models incorporate key features of the two major classes of H2ase. Furthermore, cyclic voltammetry experiments suggest that the highly basic phosphine ligands enable a second oxidation corresponding to the couple [(dxpe)Ni(pdt)Fe(CO)2L]+/2+. The resulting unsaturated 32e? dications represent the closest approach to modeling the highly electrophilic Ni-SIa state. In the case of L = PPh2(2-pyridyl) chelation of this ligand accompanies the second oxidation. PMID:22838645

  5. Structural differences between the active sites of the Ni-A and Ni-B states of the [NiFe] hydrogenase: an approach by quantum chemistry and single crystal ENDOR spectroscopy.

    PubMed

    Barilone, Jessica L; Ogata, Hideaki; Lubitz, Wolfgang; van Gastel, Maurice

    2015-06-28

    The two resting forms of the active site of [NiFe] hydrogenase, Ni-A and Ni-B, have significantly different activation kinetics, but reveal nearly identical spectroscopic features which suggest the two states exhibit subtle structural differences. Previous studies have indicated that the states differ by the identity of the bridging ligand between Ni and Fe; proposals include OH(-), OOH(-), H2O, O(2-), accompanied by modified cysteine residues. In this study, we use single crystal ENDOR spectroscopy and quantum chemical calculations within the framework of density functional theory (DFT) to calculate vibrational frequencies, (1)H and (17)O hyperfine coupling constants and g values with the aim to compare these data to experimental results obtained by crystallography, FTIR and EPR/ENDOR spectroscopy. We find that the Ni-A and Ni-B states are constitutional isomers that differ in their fine structural details. Calculated vibrational frequencies for the cyano and carbonyl ligands and (1)H and (17)O hyperfine coupling constants indicate that the bridging ligand in both Ni-A and Ni-B is indeed an OH(-) ligand. The difference in the isotropic hyperfine coupling constants of the ?-CH2 protons of Cys-549 is sensitive to the orientation of Cys-549; a difference of 0.5 MHz is observed experimentally for Ni-A and 1.9 MHz for Ni-B, which results from a rotation of 7 degrees about the C?-C?-S?-Ni dihedral angle. Likewise, the difference of the intermediate g value is correlated with a rotation of Cys-546 of about 10 degrees. PMID:26035632

  6. 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

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

    PubMed

    Stiebritz, Martin T

    2015-03-30

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

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

    E-print Network

    Surawatanawong, Panida

    2010-07-14

    -addition barrier is lower on monopalladium monophosphine than dipalladium diphosphine whereas for a small phosphine, PMe3, the oxidative addition proceeds more easily via dipalladium diphosphine. Of the phosphine-free palladium complexes examined: free-Pd, Pd...

  9. Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri

    E-print Network

    the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase or topological constraints, environmental constraints or gene regulatory constraints (Price et al, 2004). One

  10. Construction of Martian Interior Model

    NASA Astrophysics Data System (ADS)

    Zharkov, V. N.; Gudkova, T. V.

    2005-09-01

    We present the results of extensive numerical modeling of the Martian interior. Yoder et al. in 2003 reported a mean moment of inertia of Mars that was somewhat smaller than the previously used value and the Love number k 2 obtained from observations of solar tides on Mars. These values of k 2 and the mean moment of inertia impose a strong new constraint on the model of the planet. The models of the Martian interior are elastic, while k 2 contains both elastic and inelastic components. We thoroughly examined the problem of partitioning the Love number k 2 into elastic and inelastic components. The information necessary to construct models of the planet (observation data, choice of a chemical model, and the cosmogonic aspect of the problem) are discussed in the introduction. The model of the planet comprises four submodels—a model of the outer porous layer, a model of the consolidated crust, a model of the silicate mantle, and a core model. We estimated the possible content of hydrogen in the core of Mars. The following parameters were varied while constructing the models: the ferric number of the mantle (Fe#) and the sulfur and hydrogen content in the core. We used experimental data concerning the pressure and temperature dependence of elastic properties of minerals and the information about the behavior of Fe(?-Fe ), FeS, FeH, and their mixtures at high P and T. The model density, pressure, temperature, and compressional and shear velocities are given as functions of the planetary radius. The trial model M13 has the following parameters: Fe#=0.20; 14 wt % of sulfur in the core; 50 mol % of hydrogen in the core; the core mass is 20.9 wt %; the core radius is 1699 km; the pressure at the mantle-core boundary is 20.4 GPa; the crust thickness is 50 km; Fe is 25.6 wt %; the Fe/Si weight ratio is 1.58, and there is no perovskite layer. The model gives a radius of the Martian core within 1600 1820 km while ?30 mol % of hydrogen is incorporated into the core. When the inelasticity of the Martian interior is taken into account, the Love number k 2 increases by several thousandths; therefore, the model radius of the planetary core increases as well. The prognostic value of the Chandler period of Mars is 199.5 days, including one day due to inelasticity. Finally, we calculated parameters of the equilibrium figure of Mars for the M13 model: J 2 0 = 1.82 × 10-3, J 4 0 = -7.79 × 10-6, e c-m D = 1/242.3 (the dynamical flattening of the core-mantle boundary).

  11. Evidence for diffractive charm production in ??Fe and ?¯?Fe scattering at the Fermilab Tevatron

    NASA Astrophysics Data System (ADS)

    Adams, T.; Alton, A.; Bolton, T.; Goldman, J.; Goncharov, M.; Naples, D.; Johnson, R. A.; Vakili, M.; Wu, V.; Conrad, J.; Fleming, B. T.; Formaggio, J.; Koutsoliotas, S.; Kim, J. H.; McNulty, C.; Romosan, A.; Shaevitz, M. H.; Spentzouris, P.; Stern, E. G.; Vaitaitis, A.; Zimmerman, E. D.; Bernstein, R. H.; Bugel, L.; Lamm, M. J.; Marsh, W.; Nienaber, P.; Yu, J.; de Barbaro, L.; Buchholz, D.; Schellman, H.; Zeller, G. P.; Brau, J.; Drucker, R. B.; Frey, R.; Mason, D.; Avvakumov, S.; de Barbaro, P.; Bodek, A.; Budd, H.; Harris, D. A.; McFarland, K. S.; Sakumoto, W. K.; Yang, U. K.

    2000-05-01

    We present evidence for the diffractive processes ??Fe-->?-D+S(D*S)Fe and ?¯?Fe-->?+D-S(D*S)Fe using the Fermilab SSQT neutrino beam and the Lab E neutrino detector. The data are consistent with standard model production of the neutrino trident reactions ??Fe-->???-?+Fe and ?¯?Fe-->?¯??+?-Fe. We see no evidence for neutral-current production of J/? via either diffractive or deep inelastic scattering mechanisms.

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

    USGS Publications Warehouse

    Sherman, David M.

    1986-01-01

    A molecular orbital description, based on spin-unrestricted X??-scattered wave calculations, is given for the electronic structures of mixed valence iron oxides and silicates. The cluster calculations show that electron hopping and optical intervalence charge-transger result from weak FeFe bonding across shared edges of FeO6 coordination polyhedra. In agreement with Zener's double exchange model, FeFe bonding is found to stabilize ferromagnetic coupling between Fe2+ and Fe3+ cations. ?? 1986.

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

    E-print Network

    Zheng, Yufeng

    of electroformed pure iron than that of pure iron produced by casting. Hermawan et al.9,10 developed a series of Fe­Mn strength and YS up to 550 and 228 MPa, respectively, with ductility up to 32%.11 Moreover, the Fe­Mn alloys

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

    SciTech Connect

    McCloy, John S.; Jiang Weilin [Pacific Northwest National Laboratory, 902 Battelle Blvd., PO Box 999, Richland, WA 99352 (United States); Sundararajan, Jennifer A.; Qiang, You [Department of Physics, University of Idaho, Moscow, ID 83844 (United States); Burks, Edward; Liu Kai [Department of Physics, University of California, Davis, CA 95616 (United States)

    2013-04-19

    A cluster deposition method was used to produce a film of loosely aggregated particles of Fe-Fe{sub 3}O{sub 4} coreshell nanoclusters with an 8 nm iron core size and 2 nm oxide shell thickness. The film of particles on a silicon substrate was irradiated with 5.5 MeV Si{sup 2+} ions to a fluence of 10{sup 16} cm{sup -2} near room temperature, and computer simulations based on the SRIM (Stopping and Range of Ions in Matter) code show that the implanted Si species stops near the filmsubstrate interface. The ion irradiation creates a structural change in the film with corresponding chemical and magnetic changes. X-ray diffraction shows that the core size and chemistry stay the same but the shell becomes FeO that grows to a thickness of 17 nm. Helium ion microscopy shows that the previously separate particles have densified into a nearly continuous film. Major loop magnetic hysteresis measurements show a decrease in saturation magnetization that we attribute to the presence of the antiferromagnetic (AFM) FeO shell. First-order reversal curve measurements on the irradiated film performed with a vibrating sample magnetometer show that the AFM shell prevents the particles from interacting magnetically, leading to low coercivity from the iron core and little bias field from the core interactions. These results, and others reported previously on different compositions (Fe{sub 3}O{sub 4} or FeO+Fe{sub 3}N nanoclusters), show that the ion irradiation behavior of nanocluster films such as these depends strongly on the initial nanostructure and chemistry.

  15. International Hydrogenases Conference 2004 105 Common principles in the biosynthesis

    E-print Network

    Palmer, Tracy

    membrane proteins TatA, TatB and TatC form the core components of the Tat translocation apparatus (or `translocon'). TatA and TatB are small sequence-related proteins each comprising an N-terminal transmembrane

  16. Survey of Hydrogenase Activity in Algae: Final Report

    SciTech Connect

    Brand, J. J.

    1982-04-01

    The capacity for hydrogen gas production was examined in nearly 100 strains of Eukaryotic algae. Each strain was assessed for rate of H2 production in darkness, at compensating light intensity and at saturating Tight intensity. Maximum H2 yield on illumination and sensitivity to molecular oxygen were also measured.

  17. First-principles study of point defects in an fcc Fe-10Ni-20Cr model alloy

    NASA Astrophysics Data System (ADS)

    Piochaud, J. B.; Klaver, T. P. C.; Adjanor, G.; Olsson, P.; Domain, C.; Becquart, C. S.

    2014-01-01

    The influence of the local environment on vacancy and self-interstitial formation energies has been investigated in a face-centered-cubic (fcc) Fe-10Ni-20Cr model alloy by analyzing an extensive set of first-principle calculations based on density functional theory. Chemical disorder has been considered by designing special quasirandom structures and four different collinear magnetic structures have been investigated in order to determine a relevant reference state to perform point defect calculations at 0 K. Two different convergence methods have also been used to characterize the importance of the method on the results. Although our fcc Fe-10Ni-20Cr would be better represented in terms of applications by the paramagnetic state, we found that the antiferromagnetic single-layer magnetic structure was the most stable at 0 K and we chose it as a reference state to determine the point defect properties. Point defects have been introduced in this reference state, i.e., vacancies and Fe-Fe, Fe-Ni, Fe-Cr, Cr-Cr, Ni-Ni, and Ni-Cr dumbbell interstitials oriented either parallel or perpendicular to the single layer antiferromagnetic planes. Each point defect studied was introduced at different lattice sites to consider a sufficient variety of local environments and analyze its influence on the formation energy values. We have estimated the point defect formation energies with linear regressions using variables which describe the local environment surrounding the point defects. The number and the position of Ni and Cr first nearest neighbors to the point defects were found to drive the evolution of the formation energies. In particular, Ni is found to decrease and Cr to increase the vacancy formation energy of the model alloy, while the opposite trends are found for the dumbbell interstitials. This study suggested that, to a first approximation, the first nearest atoms to point defects can provide reliable estimates of point defect formation energies.

  18. Geochemical modeling of low melt-fraction anatexis in a peraluminous system: The Pena Negra complex (central Spain)

    SciTech Connect

    Bea, F. (Univ. of Salamanca (Spain))

    1991-07-01

    A study was made of the chemical fractionation associated with four cases of anatectic segregation of low melt-fraction cordieritic granites from migmatized meta-greywackes. The aims of the study were to (1) reveal the fractionation patterns of major and trace elements, (2) compare the major element chemistry of leucogranites and the quantitative behavior of source minerals during anatexis - inferred by mass-balance adjustment - with available experimental data for peraluminous systems, and (3) discuss the behavior of trace elements in crustal melting by comparing the chemically determined composition of leucogranites with the results of three fractionation models. Two of these assume a perfect diffusive behavior of trace elements within residual solids, but they use a different set of distribution coefficients. The third assumes a perfect nondiffusive behavior. In relation to their source rocks, the leucogranites are strongly depleted in Li, Transition Elements, and Light Rare Earth Elements, but enriched in K{sub 2}O, SiO{sub 2}, and Ba. Mass balance analysis using the Anatexis Mixing Model shows that the chemistry of cordierite leucogranites is compatible with its having originated by closed-system, water-undersaturated anatexis on previously migmatized meta-greywackes, leaving a residue enriched in cordierite plus biotite and exhausted in K-feldspar. Biotite melts congruently unless important amounts of sillimanite were also present in the source. Compared with experimental metals obtained from sources with the same chemical composition but with a different femic mineralogy (biotite + sillimanite, instead of cordierite + biotite), the Pena Negra leucogranites are richer in K{sub 2}O and MgO with a lower Fe/(Fe + Mg) ratio. The differences in magnesium are believed to result from the changes in the mineral assemblage of the source rocks.

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

    SciTech Connect

    Chhabra, S.R.; Joachimiak, M.P.; Petzold, C.J.; Zane, G.M.; Price, M.N.; Gaucher, S.; Reveco, S.A.; Fok, V.; Johanson, A.R.; Batth, T.S.; Singer, M.; Chandonia, J.M.; Joyner, D.; Hazen, T.C.; Arkin, A.P.; Wall, J.D.; Singh, A.K.; Keasling, J.D.

    2011-05-01

    Protein–protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study E. coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio 5 vulgaris Hildenborough, a model anaerobe and sulfate reducer. In this paper we present the first attempt to identify protein-protein interactions in an obligate anaerobic bacterium. We used suicide vector-assisted chromosomal modification of 12 open reading frames encoded by this sulfate reducer to append an eight amino acid affinity tag to the carboxy-terminus of the chosen proteins. Three biological replicates of the 10 ‘pulled-down’ proteins were separated and analyzed using liquid chromatography-mass spectrometry. Replicate agreement ranged between 35% and 69%. An interaction network among 12 bait and 90 prey proteins was reconstructed based on 134 bait-prey interactions computationally identified to be of high confidence. We discuss the biological significance of several unique metabolic features of D. vulgaris revealed by this protein-protein interaction data 15 and protein modifications that were observed. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.

  20. Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus.

    PubMed

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

    2014-01-01

    We report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO2 and H2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H2 in support of cell growth, and a regulatory hydrogenase that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth. PMID:25479613

  1. Large inverse magnetoresistance in fully epitaxial Fe\\/Fe3O4\\/MgO\\/Co magnetic tunnel junctions

    Microsoft Academic Search

    F. Greullet; E. Snoeck; C. Tiusan; M. Hehn; D. Lacour; O. Lenoble; C. Magen; L. Calmels

    2008-01-01

    Fully epitaxial Fe(001)\\/Fe3O4(001)\\/MgO(001)\\/Co micron-sized magnetic tunnel junctions have been elaborated on MgO(001) substrates. X-ray reflectivity and high-resolution transmission electron microscopy revealed a good quality and epitaxial growth of the stack with abrupt interfaces. The magnetotransport measurements exhibit a large negative tunneling magnetoresistance (TMR) value for magnetic tunnel junctions including an Fe3O4 layer and a MgO tunnel barrier (-8.5% at 300

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

    SciTech Connect

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

    2013-08-23

    A cluster deposition method was used to produce films of loosely aggregated nanoclusters (NC) of Fe core-Fe3O4 shell or fully oxidized Fe3O4. Films of these NC on Si(100) or MgO(100)/Fe3O4(100) were irradiated to 1016 Si2+/cm2 near room temperature using an ion accelerator. Ion irradiation creates structural change in the NC film with corresponding chemical and magnetic changes which depend on the initial oxidation state of the cluster. Films were characterized using magnetometry (hysteresis, first order reversal curves), microscopy (transmission electron, helium ion), and x-ray diffraction. In all cases, the particle sizes increased due to ion irradiation, and when a core of Fe is present, irradiation reduces the oxide shells to lower valent Fe species. These results show that ion irradiated behavior of the nanocluster films depends strongly on the initial nanostructure and chemistry, but in general saturation magnetization decreases slightly.

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

    SciTech Connect

    McCloy, John S.; Jiang, Weilin; Droubay, Timothy C.; Varga, Tamas; Kovarik, Libor [Pacific Northwest National Laboratory, 902 Battelle Blvd., PO Box 999, Richland, Washington 99352 (United States)] [Pacific Northwest National Laboratory, 902 Battelle Blvd., PO Box 999, Richland, Washington 99352 (United States); Sundararajan, Jennifer A.; Kaur, Maninder; Qiang, You [Department of Physics, University of Idaho, Moscow, Idaho 83844 (United States)] [Department of Physics, University of Idaho, Moscow, Idaho 83844 (United States); Burks, Edward C.; Liu, Kai [Department of Physics, University of California, Davis, California 95616 (United States)] [Department of Physics, University of California, Davis, California 95616 (United States)

    2013-08-28

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

    A cluster deposition method was used to produce films of loosely aggregated nanoclusters (NCs) of Fe core-Fe3O4 shell or fully oxidized Fe3O4. Films of these NC on Si(100) or MgO(100)/Fe3O4(100) were irradiated to 1016 Si2+/cm2 near room temperature using an ion accelerator. Ion irradiation creates structural change in the NC film with corresponding chemical and magnetic changes which depend on the initial oxidation state of the cluster. Films were characterized using magnetometry (hysteresis, first order reversal curves), microscopy (transmission electron, helium ion), and x-ray diffraction. In all cases, the particle sizes increased due to ion irradiation, and when a core of Fe is present, irradiation reduces the oxide shells to lower valent Fe species. These results show that ion irradiated behavior of the NC films depends strongly on the initial nanostructure and chemistry, but in general saturation magnetization decreases slightly.

  5. Magnetic properties of sputtered Fe1?xO and Fe+Fe3O4 thin films

    Microsoft Academic Search

    T. S. Chin; N. C. Chiang

    1997-01-01

    An attempt was made to increase saturation magnetization of Fe3O4 films by incorporating ?-Fe phase through the decomposition of as-deposited Fe1?xO films, which were dc-sputtered onto Si(100) substrate by controlling the oxygen flow rates to a thickness of 70–400 nm. The as-prepared Fe1?xO films, being ferrimagnetic, show a thickness dependent microstructure and properties. The formation region of the single phase

  6. Bioinspired Hydrogenase Models: The Mixed-Valence Triiron Complex [Fe3(CO)7(?-edt)2] and Phosphine Derivatives [Fe3(CO)7-x (PPh3) x (?-edt)2] (x = 1, 2) and [Fe3(CO)5(?(2)-diphosphine)(?-edt)2] as Proton Reduction Catalysts.

    PubMed

    Rahaman, Ahibur; Ghosh, Shishir; Unwin, David G; Basak-Modi, Sucharita; Holt, Katherine B; Kabir, Shariff E; Nordlander, Ebbe; Richmond, Michael G; Hogarth, Graeme

    2014-03-24

    The mixed-valence triiron complexes [Fe3(CO)7-x (PPh3) x (?-edt)2] (x = 0-2; edt = SCH2CH2S) and [Fe3(CO)5(?(2)-diphosphine)(?-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(?-edt)2](+) and [Fe3(CO)5(?(2)-diphosphine)(?-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(?-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(?-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(?-edt)], [Fe3(CO)7(?-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

  7. A Synthetic Nickel Electrocatalyst With a Turnover Frequency Above 100,000 s-1 for H2 Production

    SciTech Connect

    Helm, Monte L.; Stewart, Michael P.; Bullock, R. Morris; Rakowski DuBois, Mary; DuBois, Daniel L.

    2011-08-12

    Increased worldwide energy demand will require greater use of carbon-neutral sustainable energy sources. The intermittent nature of solar and wind power requires storage of energy, so electrocatalysts that convert electrical energy to chemical bonds in fuels are needed. Platinum is an excellent catalyst, but it is of low abundance and high cost. Hydrogenase enzymes in Nature catalyze the evolution of H2 and use earth-abundant metals such as nickel and iron. We report that a synthetic nickel catalyst, [Ni(7PPh2NPh)2](BF4)2, (7PPh2NPh = 1,3,6-triphenyl-1-aza-3,6-diphosphacycloheptane) catalyzes the production of H2 using [(DMF)H]+OTf as the proton source, with turnover frequencies of 31,000 s-1 in dry acetonitrile and 108,000 s-1 in the presence of H2O (1.2 M), at a potential of -1.13 V (vs. the ferrocenium/ferrocene couple). These turnover frequencies exceed those reported for the [FeFe] hydrogenase enzyme by more than an order of magnitude, and are the fastest reported for any molecular catalyst for H2 production. This material is based upon work supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.

  8. Models, figures, and gravitational moments of Jupiter's satellites Io and Europa

    NASA Astrophysics Data System (ADS)

    Zharkov, V. N.; Karamurzov, B. S.

    2006-07-01

    Two types of trial three-layer models have been constructed for the satellites Io and Europa. In the models of the first type (Io1 and E1), the cores are assumed to consist of eutectic Fe-FeS melt with the densities ? 1 = 5.15 g cm-3 (Io1) and 5.2 g cm-3 (E1). In the models of the second type (Io3 and E3), the cores consist of FeS with an admixture of nickel and have the density ? 1 = 4.6 g cm-3. The approach used here differs from that used previously both in chosen model chemical composition of these satellites and in boundary conditions imposed on the models. The most important question to be answered by modeling the internal structure of the Galilean satellites is that of the condensate composition at the formation epoch of Jupiter's system. Jupiter's core and the Galilean satellites were formed from the condensate. Ganymede and Callisto were formed fairly far from Jupiter in zones with temperatures below the water condensation temperature, water was entirely incorporated into their bodies, and their modeling showed the mass ratio of the icy (I) component to the rock (R) component in them to be I/R ˜ 1. The R composition must be clarified by modeling Io and Europa. The models of the second type (Io3 and E3), in which the satellite cores consist of FeS, yield 25.2 (Io3) and 22.8 (E3) for the core masses (in weight %). In discussing the R composition, we note that, theoretically, the material of which the FeS+Ni core can consist in the R accounts for ˜25.4% of the satellite mass. In this case, such an important parameter as the mantle silicate iron saturation is Fe# = 0.265. The Io3 and E3 models agree well with this theoretical prediction. The models of the first and second types differ markedly in core radius; thus, in principle, the R composition in the formation zone of Jupiter's system can be clarified by geophysical studies. Another problem studied here is that of the error made in modeling Io and Europa using the Radau-Darvin formula when passing from the Love number k 2 to the nondimensional polar moment of inertia bar C. For Io, the Radau-Darvin formula underestimates the true value of bar C by one and a half units in the third decimal digit. For Europa, this effect is approximately a factor of 3 smaller, which roughly corresponds to a ratio of the small parameters for the satellites under consideration ? Io/? Europa ˜ 3.4. In modeling the internal structure of the satellites, the core radius depends strongly on both the mean moment of inertia I* and k 2. Therefore, the above discrepancy in bar C for Io is appreciable.

  9. Insights into the P-to-Q conversion in the catalytic cycle of methane monooxygenase from a synthetic model system

    PubMed Central

    Xue, Genqiang; Fiedler, Adam T.; Martinho, Marlène; Münck, Eckard; Que, Lawrence

    2008-01-01

    For the catalytic cycle of soluble methane monooxygenase (sMMO), it has been proposed that cleavage of the O–O bond in the (?-peroxo)diiron(III) intermediate P gives rise to the diiron(IV) intermediate Q with an Fe2(?–O)2 diamond core, which oxidizes methane to methanol. As a model for this conversion, (?–oxo) diiron(III) complex 1 ([FeIII2(?–O)(?–O2H3)(L)2]3+, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) has been treated consecutively with one eq of H2O2 and one eq of HClO4 to form 3 ([FeIV2(?–O)2(L)2]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 {[Fe2O3L2H](OTf)2}+ ion in which 1 oxygen atom derives from 1 and the other two originate from H2O2. Mössbauer 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 ? and an Fe–Fe distance of 3.32 ?. Taken together, the spectroscopic data point to an HO-FeIV-O-FeIV = O core for 2. Protonation of 2 results in the loss of H2O and the formation of 3. Isotope labeling experiments show that the [FeIV2(?–O)2] core of 3 can incorporate both oxygen atoms from H2O2. 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 [FeIV2(?–O)2] core.

  10. Analysis of the cyanobacterial hydrogen photoproduction process via model identification and process simulation

    E-print Network

    Zhang, Dongda; Dechatiwongse, Pongsathorn; Del-Rio-Chanona, Ehecatl Antonio; Hellgardt, Klaus; Maitland, Geoffrey C.; Vassiliadis, Vassilios S.

    2015-02-02

    of sulphur, oxygen production rate is significantly reduced and even drops lower than algal respiration rate. Hence, oxygen produced by oxygenic photosynthesis is totally consumed by algae respira- tion and anaerobic conditions are achieved. In addition... by water through photosynthesis with the generation of oxygen. As the activity of hydrogenase is completely inhibited by oxygen, C. reinhardtii can only produce hydrogen in anaerobic conditions (Antal et al., 2011). Different methods have been used...

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

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

    PubMed

    Ahmed, Asma; Lewis, Randy S

    2007-08-01

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

  13. Modeling 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.

    PubMed

    Friedle, Simone; Kodanko, Jeremy J; Morys, Anna J; Hayashi, Takahiro; Moënne-Loccoz, Pierre; Lippard, Stephen J

    2009-10-14

    In order to model 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 Fe-Fe 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 contrast to hydrogen peroxide reactions of diiron(III) complexes that lack a dinucleating ligand, the intermediates generated here could be re-formed in significant quantities after a second addition of H(2)O(2), as observed spectroscopically and by mass spectrometry. PMID:19757795

  14. Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes

    PubMed Central

    2015-01-01

    Conspectus A [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5?-deoxyadenosyl radical (5?-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5?-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5?-dA• to the product radical l-?-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding. PMID:24991701

  15. Paramagnetic intermediates generated by radical S-adenosylmethionine (SAM) enzymes.

    PubMed

    Stich, Troy A; Myers, William K; Britt, R David

    2014-08-19

    A [4Fe-4S](+) cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5'-deoxyadenosyl radical (5'-dA(•)). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5'-dA(•) is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, L-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5'-dA(•) to the product radical L-?-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the L-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN(-) ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp(2)-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding. PMID:24991701

  16. Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water-splitting

    E-print Network

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

    2015-06-05

    Subscriber access provided by UNIV OF CAMBRIDGE Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American... Carbolite furnace (ELF 11/14B/301) was used to anneal the electrodes. An Agilent 7890A Series gas chromatograph equipped with a 5 Å molecular sieve column and N2 carrier gas was employed for the quantification of H2. Quantification of O2 was performed...

  17. Classification of the uptake hydrogenase-positive (Hup+) bean rhizobia as Rhizobium tropici.

    PubMed Central

    van Berkum, P; Navarro, R B; Vargas, A A

    1994-01-01

    Phenotypic and genetic characterization indicated that Hup+ bean rhizobial strains are type IIA and type IIB Rhizobium tropici. The Hup+ strain USDA 2840, which did not cluster with either of the two types of R. tropici in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences similar to those of three reference strains of R. tropici type IIA. The Hup+ strain USDA 2738, which clustered with the reference strain of R. tropici IIB in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences more closely resembling those of the reference strains of R. tropici type IIA than those of type IIB. DNA amplification with the Y1 and Y2 primers to generate a portion of the 16S rDNA operon was useful to distinguish R. tropici type IIA strains from other bean rhizobial strains. The phylogenetic position of the type IIA strain of R. tropici USDA 2840, determined from the partial 16S rDNA sequence, indicated a more distant relationship with the type IIB strain of R. tropici CIAT899 than with the as yet unnamed rhizobial species of Leucaena leucocephala, TAL 1145. Therefore, we suggest that it may be appropriate either to separate R. tropici types IIA and IIB into two different species or to identify TAL 1145 to the species level as a third type of R. tropici. Images PMID:8135515

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

    E-print Network

    fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results of the Tricarboxylic Acid Cycle with fermentation when the rate of respiratory O2 consumption exceeds the rate

  19. Design Model Design Model

    E-print Network

    van Sinderen, Marten

    Design Model 123 Chapter 6 Design Model This chapter presents a design model that allows refinement types are identified, and their relevance to design steps in the application protocol design, interaction and causality relation are the elementary design, or architectural, concepts of our design model

  20. Input modeling: input modeling

    Microsoft Academic Search

    Lawrence Leemis

    2003-01-01

    Most discrete-event simulation models have stochastic elements that mimic the probabilistic nature of the system under consideration. A close match between the input model and the true underlying probabilistic mechanism associated with the system is required for successful input modeling. The general question considered here is how to model an element (e.g., arrival process, service times) in a discrete-event simulation

  1. Models and Modeling.

    ERIC Educational Resources Information Center

    Lesh, Richard; Carmona, Guadalupe; Post, Thomas

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

  2. Genome-guided analysis of physiological capacities of Tepidanaerobacter acetatoxydans provides insights into environmental adaptations and syntrophic acetate oxidation.

    PubMed

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

    2015-01-01

    This paper describes the genome-based analysis of Tepidanaerobacter acetatoxydans strain Re1, a syntrophic acetate-oxidising bacterium (SAOB). Principal issues such as environmental adaptations, metabolic capacities, and energy conserving systems have been investigated and the potential consequences for syntrophic acetate oxidation discussed. Briefly, in pure culture, T. acetatoxydans grows with different organic compounds and produces acetate as the main product. In a syntrophic consortium with a hydrogenotrophic methanogen, it can also reverse its metabolism and instead convert acetate to formate/H2 and CO2. It can only proceed if the product formed is continuously removed. This process generates a very small amount of energy that is scarcely enough for growth, which makes this particular syntrophy of special interest. As a crucial member of the biogas-producing community in ammonium-rich engineered AD processes, genomic features conferring ammonium resistance, bacterial defense, oxygen and temperature tolerance were found, as well as attributes related to biofilm formation and flocculation. It is likely that T. acetatoxydans can form an electrochemical gradient by putative electron-bifurcating Rnf complex and [Fe-Fe] hydrogenases, as observed in other acetogens. However, genomic deficiencies related to acetogenic metabolism and anaerobic respiration were discovered, such as the lack of formate dehydrogenase and F1F0 ATP synthase. This has potential consequences for the metabolic pathways used under SAO and non-SAO conditions. The two complete sets of bacteriophage genomes, which were found to be encoded in the genome, are also worthy of mention. PMID:25811859

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

    PubMed Central

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

    2015-01-01

    This paper describes the genome-based analysis of Tepidanaerobacter acetatoxydans strain Re1, a syntrophic acetate-oxidising bacterium (SAOB). Principal issues such as environmental adaptations, metabolic capacities, and energy conserving systems have been investigated and the potential consequences for syntrophic acetate oxidation discussed. Briefly, in pure culture, T. acetatoxydans grows with different organic compounds and produces acetate as the main product. In a syntrophic consortium with a hydrogenotrophic methanogen, it can also reverse its metabolism and instead convert acetate to formate/H2 and CO2. It can only proceed if the product formed is continuously removed. This process generates a very small amount of energy that is scarcely enough for growth, which makes this particular syntrophy of special interest. As a crucial member of the biogas-producing community in ammonium-rich engineered AD processes, genomic features conferring ammonium resistance, bacterial defense, oxygen and temperature tolerance were found, as well as attributes related to biofilm formation and flocculation. It is likely that T. acetatoxydans can form an electrochemical gradient by putative electron-bifurcating Rnf complex and [Fe-Fe] hydrogenases, as observed in other acetogens. However, genomic deficiencies related to acetogenic metabolism and anaerobic respiration were discovered, such as the lack of formate dehydrogenase and F1F0 ATP synthase. This has potential consequences for the metabolic pathways used under SAO and non-SAO conditions. The two complete sets of bacteriophage genomes, which were found to be encoded in the genome, are also worthy of mention. PMID:25811859

  4. Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes.

    PubMed

    Stechmann, Alexandra; Hamblin, Karleigh; Pérez-Brocal, Vicente; Gaston, Daniel; Richmond, Gregory S; van der Giezen, Mark; Clark, C Graham; Roger, Andrew J

    2008-04-22

    Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes. PMID:18403202

  5. Organelles in Blastocystis that Blur the Distinction between Mitochondria and Hydrogenosomes

    PubMed Central

    Stechmann, Alexandra; Hamblin, Karleigh; Pérez-Brocal, Vicente; Gaston, Daniel; Richmond, Gregory S.; van der Giezen, Mark; Clark, C. Graham; Roger, Andrew J.

    2008-01-01

    Summary Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans [1, 2]. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA [2–4]. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria [5, 6]. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes [7, 8]. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis[9]. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes. PMID:18403202

  6. Mental Models, Conceptual Models, and Modelling.

    ERIC Educational Resources Information Center

    Greca, Ileana Maria; Moreira, Marco Antonio

    2000-01-01

    Reviews science education research into representations constructed by students in their interactions with the world, its phenomena, and artefacts. Features discussions of mental models, conceptual models, and the activity of modeling. (Contains 30 references.) (Author/WRM)

  7. MODEL DEVELOPMENT - DOSE MODELS

    EPA Science Inventory

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

  8. Modeling Malaria

    NSDL National Science Digital Library

    Angela B. Shiflet

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

  9. Fair Model

    NSDL National Science Digital Library

    Betty Blecha

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

  10. Sloppy Modeling

    Microsoft Academic Search

    Katharina Morik

    1987-01-01

    In this paper, I would like to present a unifying view on knowledge acquisition and machine learning. In this view, knowledge acquisition systems should support the user in doing the modeling of a domain, and machine learning systems are those which perform part of the modeling autonomously. Taking the notion of modeling as the central point, some aspects of modeling

  11. Architectural Models

    ERIC Educational Resources Information Center

    Levenson, Harold E.; Hurni, Andre

    1978-01-01

    Suggests building models as a way to reinforce and enhance related subjects such as architectural drafting, structural carpentry, etc., and discusses time, materials, scales, tools or equipment needed, how to achieve realistic special effects, and the types of projects that can be built (model of complete building, a panoramic model, and model

  12. Understanding Models

    NSDL National Science Digital Library

    Shirley Watt Ireton

    2003-01-01

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

  13. MODEL ABSTRACTION IN HYDROLOGIC MODELING

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Iron Carbonyl Sulfides, Formaldehyde, and Amines Condense To Give the Proposed Azadithiolate Cofactor of the Fe-Only Hydrogenases

    E-print Network

    Rauchfuss, Thomas B.

    to develop a fundamentally new approach to the azadithiolates, an approach that entails the unprecedented and formaldehyde would condense to give imine derivatives, and this idea led to a revised synthetic protocol and hexamethylenetetramine, (CH2)6N4 (the condensation product of ammonia and formaldehyde). The latter method involves

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

    Microsoft Academic Search

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

    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(mu-SPh)3Ni(mu-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(mu-StBu)3Ni(mu-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

  16. [FeFe]?Hydrogenase: Protonation of {2Fe3S} Systems and Formation of Super-reduced Hydride States**

    PubMed Central

    Jablonskyt?, Aušra; Wright, Joseph A; Fairhurst, Shirley A; Webster, Lee R; Pickett, Christopher J

    2014-01-01

    The synthesis and crystallographic characterization of a complex possessing a well-defined {2Fe3S(?-H)}?core gives access to a paramagnetic bridging hydride with retention of the core geometry. Chemistry of this 35-electron species within the confines of a thin-layer FTIR spectro-electrochemistry cell provides evidence for a unprecedented super-reduced FeI(?-H)FeI intermediate. PMID:25079249

  17. Synthesis of alanine and leucine by reductive amination of 2-oxoic acid with combination of hydrogenase and dehydrogenase

    Microsoft Academic Search

    Fumihiko Hasuni; Katsunori Fukuoka; Shuichi Adachi; Yasumitsu Miyamoto; Ichiro Okura

    1996-01-01

    Alanine synthesis by reductive amination of pyruvate was performed by the combination of NADH regeneration system and alanine\\u000a dehydrogenase (AlaDH). The conversion of pyruvate to alanine was 99% after 1 h. Leucine synthesis was also carried out by\\u000a the combination of NADH regeneration system and leucine dehydrogenase (LeuDH). The conversion of 4-methyl-2-oxovalerate to\\u000a leucine was 60% after 1.5 h.

  18. Supergravity Models

    E-print Network

    R. Arnowitt; Pran Nath

    1993-11-24

    Theoretical and experimental motivations behind supergravity grand unified models are described. The basic ideas of supergravity, and the origin of the soft breaking terms are reviewed. Effects of GUT thresholds and predictions arising from models possessing proton decay are discussed. Speculations as to which aspects of the Standard Model might be explained by supergravity models and which may require Planck scale physics to understand are mentioned.

  19. Input modeling

    Microsoft Academic Search

    Lawrence Leemis

    2000-01-01

    Discrete-event simulation models typically have stochastic elements that mimic the probabilistic nature of the system under consideration. Successful input modeling requires a close match between the input model and the true underlying probabilistic mechanism associated with the system. The general question considered here is how to model an element (e.g., arrival process, service times) in a discrete-event simulation given a

  20. Modeling Convection

    NSDL National Science Digital Library

    Amanda Schulz

    2004-09-01

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

  1. Phoenix model

    EPA Science Inventory

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

  2. Radiation Models

    ERIC Educational Resources Information Center

    James, W. G. G.

    1970-01-01

    Discusses the historical development of both the wave and the corpuscular photon model of light. Suggests that students should be informed that the two models are complementary and that each model successfully describes a wide range of radiation phenomena. Cites 19 references which might be of interest to physics teachers and students. (LC)

  3. Animal models.

    PubMed

    Coppola, Antonietta; Moshé, Solomon L

    2012-01-01

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

  4. Model Reduction in Groundwater Modeling

    NASA Astrophysics Data System (ADS)

    Yeh, W. W. G.

    2014-12-01

    Model reduction has been shown to be a very effective method for reducing the computational burden of large-scale simulations. Model reduction techniques preserve much of the physical knowledge of the system and primarily seek to remove components from the model that do not provide significant information of interest. Proper Orthogonal Decomposition (POD) is a model 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 model 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 model, the reduced model can be two to three orders of magnitude smaller than the original model and runs 1,000 faster. More importantly, the reduced model captures the dominating characteristics of the full model and produces sufficiently accurate solutions. One of the major tasks in the development of the reduced model 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 model to parameter estimation, Monte Carlo simulation and experimental design in groundwater modeling.

  5. Station Models

    NSDL National Science Digital Library

    Mr. Ertl

    2007-11-03

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

  6. Computer Models

    NSDL National Science Digital Library

    John Nielsen-Gammon

    1996-01-01

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

  7. Model Selection in Acoustic Modeling

    Microsoft Academic Search

    S. S. Chen; R. A. Gopinath

    2001-01-01

    Recently several classes of models have been suggested for use in continuousdensity HMMs for speech recognition. This paper proposes tochoose both the model type and model size (number of parameters) byoptimizing the Bayesian information criterion. Specically we apply thisto Gaussian mixture density estimation to determine both the numberof Gaussians and the covariance structure of each Gaussian, and decisiontree clustering of

  8. Functions and Models: Mathematical Models

    NSDL National Science Digital Library

    Michael Freeze

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

  9. Ventilation Model

    SciTech Connect

    H. Yang

    1999-11-04

    The purpose of this analysis and model report (AMR) for the Ventilation Model 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 Model. The objective of the analysis is to develop, describe, and apply calculation methods and models 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 modeling 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 modeling 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 modeling in this document will be limited to heat-only modeling and calculations. Only a preliminary assessment of the heat/moisture ventilation effects and modeling method will be performed in this revision. Modeling of moisture effects on heat removal and emplacement drift temperature may be performed in the future.

  10. Climate Models

    NASA Technical Reports Server (NTRS)

    Druyan, Leonard M.

    2012-01-01

    Climate models is a very broad topic, so a single volume can only offer a small sampling of relevant research activities. This volume of 14 chapters includes descriptions of a variety of modeling studies for a variety of geographic regions by an international roster of authors. The climate research community generally uses the rubric climate models to refer to organized sets of computer instructions that produce simulations of climate evolution. The code is based on physical relationships that describe the shared variability of meteorological parameters such as temperature, humidity, precipitation rate, circulation, radiation fluxes, etc. Three-dimensional climate models are integrated over time in order to compute the temporal and spatial variations of these parameters. Model domains can be global or regional and the horizontal and vertical resolutions of the computational grid vary from model to model. Considering the entire climate system requires accounting for interactions between solar insolation, atmospheric, oceanic and continental processes, the latter including land hydrology and vegetation. Model simulations may concentrate on one or more of these components, but the most sophisticated models will estimate the mutual interactions of all of these environments. Advances in computer technology have prompted investments in more complex model configurations that consider more phenomena interactions than were possible with yesterday s computers. However, not every attempt to add to the computational layers is rewarded by better model performance. Extensive research is required to test and document any advantages gained by greater sophistication in model formulation. One purpose for publishing climate model research results is to present purported advances for evaluation by the scientific community.

  11. Model Cheking

    Microsoft Academic Search

    Edmund M. Clarke

    1997-01-01

    Model checking is an automatic technique for verifying finite-state reactive systems, such as sequential circuit designs and\\u000a communication protocols. Specifications are expressed in temporal logic, and the reactive system is modeled as a statetransition\\u000a graph. An efficient search procedure is used to determine whether or not the state-transition graph satisfies the specifications.\\u000a \\u000a We describe the basic model checking algorithm and

  12. SCARP Model

    NSDL National Science Digital Library

    Bill Locke

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

  13. Ventilation Model

    SciTech Connect

    V. Chipman

    2002-10-05

    The purpose of the Ventilation Model is to simulate the heat transfer processes in and around waste emplacement drifts during periods of forced ventilation. The model evaluates the effects of emplacement drift ventilation on the thermal conditions in the emplacement drifts and surrounding rock mass, and calculates the heat removal by ventilation as a measure of the viability of ventilation to delay the onset of peak repository temperature and reduce its magnitude. The heat removal by ventilation is temporally and spatially dependent, and is expressed as the fraction of heat carried away by the ventilation air compared to the fraction of heat produced by radionuclide decay. One minus the heat removal is called the wall heat fraction, or the remaining amount of heat that is transferred via conduction to the surrounding rock mass. Downstream models, such as the ''Multiscale Thermohydrologic Model'' (BSC 2001), use the wall heat fractions as outputted from the Ventilation Model to initialize their post-closure analyses. The Ventilation Model report was initially developed to analyze the effects of preclosure continuous ventilation in the Engineered Barrier System (EBS) emplacement drifts, and to provide heat removal data to support EBS design. Revision 00 of the Ventilation Model included documentation of the modeling results from the ANSYS-based heat transfer model. The purposes of Revision 01 of the Ventilation Model are: (1) To validate the conceptual model for preclosure ventilation of emplacement drifts and verify its numerical application in accordance with new procedural requirements as outlined in AP-SIII-10Q, Models (Section 7.0). (2) To satisfy technical issues posed in KTI agreement RDTME 3.14 (Reamer and Williams 2001a). Specifically to demonstrate, with respect to the ANSYS ventilation model, the adequacy of the discretization (Section 6.2.3.1), and the downstream applicability of the model results (i.e. wall heat fractions) to initialize post-closure thermal models (Section 6.6). (3) To satisfy the remainder of KTI agreement TEF 2.07 (Reamer and Williams 2001b). Specifically to provide the results of post-test ANSYS modeling of the Atlas Facility forced convection tests (Section 7.1.2). This portion of the model report also serves as a validation exercise per AP-SIII.10Q, Models, for the ANSYS ventilation model. (4) To further satisfy KTI agreements RDTME 3.01 and 3.14 (Reamer and Williams 2001a) by providing the source documentation referred to in the KTI Letter Report, ''Effect of Forced Ventilation on Thermal-Hydrologic Conditions in the Engineered Barrier System and Near Field Environment'' (Williams 2002). Specifically to provide the results of the MULTIFLUX model which simulates the coupled processes of heat and mass transfer in and around waste emplacement drifts during periods of forced ventilation. This portion of the model report is presented as an Alternative Conceptual Model with a numerical application, and also provides corroborative results used for model validation purposes (Section 6.3 and 6.4).

  14. Model Selection for Geostatistical Models

    SciTech Connect

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

    2006-02-01

    We consider the problem of model selection for geospatial data. Spatial correlation is typically ignored in the selection of explanatory variables and this can influence model selection results. For example, the inclusion or exclusion of particular explanatory variables may not be apparent when spatial correlation is ignored. To address this problem, we consider the Akaike Information Criterion (AIC) as applied to a geostatistical model. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical model is superior to the often used approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a model for lizard abundance. We also employ the principle of minimum description length (MDL) to variable selection for the geostatistical model. The effect of sampling design on the selection of explanatory covariates is also explored.

  15. Local structures of Sr{sub 2}FeMnO{sub 5+y} (y=0, 0.5) and Sr{sub 2}Fe{sub 1.5}Cr{sub 0.5}O{sub 5} from reverse Monte Carlo modeling of pair distribution function data and implications for magnetic order

    SciTech Connect

    King, Graham, E-mail: gking@lanl.gov [Lujan Neutron Scattering Center, Los Alamos National Laboratory, MS H805, Los Alamos, NM 87545 (United States)] [Lujan Neutron Scattering Center, Los Alamos National Laboratory, MS H805, Los Alamos, NM 87545 (United States); Ramezanipour, Farshid [Department of Chemistry and Brockhouse Institute for Materials Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 (Canada)] [Department of Chemistry and Brockhouse Institute for Materials Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 (Canada); Llobet, Anna [Lujan Neutron Scattering Center, Los Alamos National Laboratory, MS H805, Los Alamos, NM 87545 (United States)] [Lujan Neutron Scattering Center, Los Alamos National Laboratory, MS H805, Los Alamos, NM 87545 (United States); Greedan, John E. [Department of Chemistry and Brockhouse Institute for Materials Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 (Canada)] [Department of Chemistry and Brockhouse Institute for Materials Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 (Canada)

    2013-02-15

    The local structures of the oxygen deficient perovskites Sr{sub 2}FeMnO{sub 5}, Sr{sub 2}FeMnO{sub 5.5}, and Sr{sub 2}Fe{sub 1.5}Cr{sub 0.5}O{sub 5} have been analyzed using neutron pair distribution function data. The results show that locally all three structures are more complex than implied by their average cubic structures and that the distributions of oxygen vacancies are not completely random on a local level. For both Sr{sub 2}FeMnO{sub 5+y} compounds it is found that there is no short range ordering of the Fe and Mn cations. For Sr{sub 2}Fe{sub 1.5}Cr{sub 0.5}O{sub 5} there is evidence to suggest that the Fe/Cr distribution is not completely random and is locally ordered such that there are fewer Fe--Fe nearest neighbor pairs than in a random distribution. Reverse Monte Carlo modeling of the pair distribution function data has provided the Fe--O, Mn--O, and Cr--O bond length distributions and information on the coordination numbers of the Fe, Mn, and Cr cations. In Sr{sub 2}FeMnO{sub 5} it is found that the Fe{sup 3+} cations are most often in 4-fold coordination but there is also a large amount of Fe{sup 3+} in 5-fold coordination and a small amount in 6-fold coordination. The Mn{sup 3+} is split between 5-fold and 6-fold coordination. The Mn--O bond length distributions indicate that the Mn{sup 3+}O{sub 6} octahedra and Mn{sup 3+}O{sub 5} square pyramids are locally Jahn-Teller distorted. In Sr{sub 2}FeMnO{sub 5.5} the Fe{sup 3+} is almost entirely 5 coordinate while the Mn{sup 4+} is almost entirely 6 coordinate. The Cr{sup 3+} in Sr{sub 2}Fe{sub 1.5}Cr{sub 0.5}O{sub 5} is almost entirely 6-fold coordinated, giving the Fe{sup 3+} an average coordination number of 4.67. In Sr{sub 2}FeMnO{sub 5} and Sr{sub 2}Fe{sub 1.5}Cr{sub 0.5}O{sub 5} the Fe{sup 3+} and Sr{sup 2+} cations undergo local displacements which are driven by the oxygen vacancies, while the Mn{sup 3+} and Cr{sup 3+} cations remain near their positions in the average structures. In Sr{sub 2}FeMnO{sub 5.5} these cations are not significantly displaced. The local coordination geometries are used to explain previously observed but yet poorly understood magnetic properties of these materials. - Graphical abstract: The actual bond angle distributions in the cubic perovskite Sr{sub 2}FeMnO{sub 5} obtained from reverse Monte Carlo modeling of the local structure using neutron pair distribution function data. Highlights: Black-Right-Pointing-Pointer No long range ordering of oxygen vacancies, but short range order is present. Black-Right-Pointing-Pointer No short range Fe/Mn order but short range Fe/Cr order is present. Black-Right-Pointing-Pointer Fe tends to have lower coordination numbers while Mn and Cr have higher ones. Black-Right-Pointing-Pointer Local bond distances and bond angles have been determined. Black-Right-Pointing-Pointer Local structures can help explain long range magnetic ordering behavior.

  16. Turbulence modeling

    NASA Technical Reports Server (NTRS)

    Bardina, Jorge E.

    1995-01-01

    The objective of this work is to develop, verify, and incorporate the baseline two-equation turbulence models which account for the effects of compressibility into the three-dimensional Reynolds averaged Navier-Stokes (RANS) code and to provide documented descriptions of the models and their numerical procedures so that they can be implemented into 3-D CFD codes for engineering applications.

  17. Modeling Daisyworld

    NSDL National Science Digital Library

    David Bice

    Daisyworld is a classic model of complex feedbacks in a simple climate system; this activity guides students through the construction of a STELLA model that can be used to experiment with the system, exploring the somewhat surprising dynamics that arise from the interplay of positive and negative feedbacks between daisies and the temperature of their environment.

  18. PREDICTIVE MODELS

    Microsoft Academic Search

    1988-01-01

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1) chemical flooding; 2) carbon dioxide miscible flooding; 3)

  19. PREDICTIVE MODELS

    Microsoft Academic Search

    1986-01-01

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1) chemical flooding, where soap-like surfactants are injected into

  20. Minibeast Models

    NSDL National Science Digital Library

    2012-06-26

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

  1. Dispersion Modeling.

    ERIC Educational Resources Information Center

    Budiansky, Stephen

    1980-01-01

    This article discusses the need for more accurate and complete input data and field verification of the various models of air pollutant dispension. Consideration should be given to changing the form of air quality standards based on enhanced dispersion modeling techniques. (Author/RE)

  2. Daisyworld Model

    NSDL National Science Digital Library

    James Lovelock

    The simulation exercise uses a STELLA-based model called Daisyworld to explore concepts associated with Earth's energy balance and climate change. Students examine the evolution of a simplified model of an imaginary planet with only two species of life on its surface -- white and black daisies -- with different albedos. The daisies can alter the temperature of the surface where they are growing.

  3. GLACPRO Model

    NSDL National Science Digital Library

    Bill Locke

    In the GLACPRO exercise student teams (1-3 members) use a numerical model to reconstruct a former glacial flowline from moraines to source. They must interact with teams studying adjacent flowlines to accurately place ice divides. They can calculate average thicknesses, volumes, ice loading, and sea level equivalent from the class model.

  4. Scale Models

    NSDL National Science Digital Library

    2012-06-26

    In this activity, learners explore the relative sizes and distances of objects in the solar system. Without being informed of the expected product, learners will make a Play-doh model of the Earth-Moon system, scaled to size and distance. The facilitator reveals the true identity of the system at the conclusion of the activity. During the construction phase, learners try to guess what members of the solar system their model represents. Each group receives different amounts of Play-doh, with each group assigned a color (red, blue, yellow, white). At the end, groups set up their models and inspect the models of other groups. They report patterns of scale that they notice; as the amount of Play-doh increases, for example, so do the size and distance of the model. This resource guide includes background information about the Earth to Moon ratio and solar eclipses.

  5. OSPREY Model

    SciTech Connect

    Veronica J. Rutledge

    2013-01-01

    The absence of industrial scale nuclear fuel reprocessing in the U.S. has precluded the necessary driver for developing the advanced simulation capability now prevalent in so many other countries. Thus, it is essential to model complex series of unit operations to simulate, understand, and predict inherent transient behavior and feedback loops. A capability of accurately simulating the dynamic behavior of advanced fuel cycle separation processes will provide substantial cost savings and many technical benefits. The specific fuel cycle separation process discussed in this report is the off-gas treatment system. The off-gas separation consists of a series of scrubbers and adsorption beds to capture constituents of interest. Dynamic models are being developed to simulate each unit operation involved so each unit operation can be used as a stand-alone model and in series with multiple others. Currently, an adsorption model has been developed within Multi-physics Object Oriented Simulation Environment (MOOSE) developed at the Idaho National Laboratory (INL). Off-gas Separation and REcoverY (OSPREY) models the adsorption of off-gas constituents for dispersed plug flow in a packed bed under non-isothermal and non-isobaric conditions. Inputs to the model include gas, sorbent, and column properties, equilibrium and kinetic data, and inlet conditions. The simulation outputs component concentrations along the column length as a function of time from which breakthrough data is obtained. The breakthrough data can be used to determine bed capacity, which in turn can be used to size columns. It also outputs temperature along the column length as a function of time and pressure drop along the column length. Experimental data and parameters were input into the adsorption model to develop models specific for krypton adsorption. The same can be done for iodine, xenon, and tritium. The model will be validated with experimental breakthrough curves. Customers will be given access to OSPREY to used and evaluate the model.

  6. Modular Modeling System Model Builder

    SciTech Connect

    McKim, C.S.; Matthews, M.T. [Framatome Technologies, Lynchburg, VA (United States)

    1996-12-31

    The latest release of the Modular Modeling System (MMS) Model Builder adds still more time-saving features to an already powerful MMS dynamic-simulation tool set. The Model Builder takes advantage of 32-bit architecture within the Microsoft Windows 95/NT{trademark} Operating Systems to better integrate a mature library of power-plant components. In addition, the MMS Library of components can now be modified and extended with a new tool named MMS CompGen{trademark}. The MMS Model Builder allows the user to quickly build a graphical schematic representation for a plant by selecting from a library of predefined power plant components to dynamically simulate their operation. In addition, each component has a calculation subroutine stored in a dynamic-link library (DLL), which facilitates the determination of a steady-state condition and performance of routine calculations for the component. These calculations, termed auto-parameterization, help avoid repetitive and often tedious hand calculations for model initialization. In striving to meet the needs for large models and increase user productivity, the MMS Model Builder has been completely revamped to make power plant model creation and maintainability easier and more efficient.

  7. Hierarchical Dynamic Models

    E-print Network

    Penny, Will

    Hierarchical Dynamic Models Will Penny OU Processes Embedding OU(2) process Dynamic Models Model State Equation Observation Equation Generative Model Energies and Actions Linear Convolution Model Generative Model Generated Data Filtering Triple Estimation Hierarchical Dynamic Models References

  8. Anchor Modeling

    NASA Astrophysics Data System (ADS)

    Regardt, Olle; Rönnbäck, Lars; Bergholtz, Maria; Johannesson, Paul; Wohed, Petia

    Maintaining and evolving data warehouses is a complex, error prone, and time consuming activity. The main reason for this state of affairs is that the environment of a data warehouse is in constant change, while the warehouse itself needs to provide a stable and consistent interface to information spanning extended periods of time. In this paper, we propose a modeling technique for data warehousing, called anchor modeling, that offers non-destructive extensibility mechanisms, thereby enabling robust and flexible management of changes in source systems. A key benefit of anchor modeling is that changes in a data warehouse environment only require extensions, not modifications, to the data warehouse. This ensures that existing data warehouse applications will remain unaffected by the evolution of the data warehouse, i.e. existing views and functions will not have to be modified as a result of changes in the warehouse model.

  9. Modeling Arcs

    SciTech Connect

    Insepov, Z.; Norem, J. [Argonne National Lab, Argonne, IL 60439 (United States); Vetizer, S.; Mahalingam, S. [Tech-X Corp., Boulder, CO (United States)

    2011-12-23

    Although vacuum arcs were first identified over 110 years ago, they are not yet well understood. We have since developed a model of breakdown and gradient limits that tries to explain, in a self-consistent way: arc triggering, plasma initiation, plasma evolution, surface damage and gradient limits. We use simple PIC codes for modeling plasmas, molecular dynamics for modeling surface breakdown, and surface damage, and mesoscale surface thermodynamics and finite element electrostatic codes for to evaluate surface properties. Since any given experiment seems to have more variables than data points, we have tried to consider a wide variety of arcing (rf structures, e beam welding, laser ablation, etc.) to help constrain the problem, and concentrate on common mechanisms. While the mechanisms can be comparatively simple, modeling can be challenging.

  10. Model Center

    NSDL National Science Digital Library

    2009-04-14

    A human is a complicated organism, and it is considered unethical to do many kinds of experiments on human subjects. For these reasons, biologists often use simpler 'model' organisms that are easy to keep and manipulate in the laboratory. Despite obvious differences, model organisms share with humans many key biochemical and physiological functions that have been conserved (maintained) by evolution. Each of the following model organisms has its advantages and disadvantages in different research applications. This tool allows you to examine the similarities between different systems by comparing the proteins they share and the proportion of DNA they have in common. Choose a gene from the drop-down menu and select the species you want to compare. Rolling over the images will give you a more detailed description of each model. Clicking on a gene�s name will take you to the National Center for Biological Information, where you can explore the latest relevant scientific literature.

  11. Programming models

    SciTech Connect

    Daniel, David J [Los Alamos National Laboratory; Mc Pherson, Allen [Los Alamos National Laboratory; Thorp, John R [Los Alamos National Laboratory; Barrett, Richard [SNL; Clay, Robert [SNL; De Supinski, Bronis [LLNL; Dube, Evi [LLNL; Heroux, Mike [SNL; Janssen, Curtis [SNL; Langer, Steve [LLNL; Laros, Jim [SNL

    2011-01-14

    A programming model is a set of software technologies that support the expression of algorithms and provide applications with an abstract representation of the capabilities of the underlying hardware architecture. The primary goals are productivity, portability and performance.

  12. Noise Models

    Microsoft Academic Search

    Alper Demir; Alberto Sangiovanni-Vincentelli

    \\u000a To reach the final goal of simulating and characterizing the effect of noise on the performance of an electronic circuit or\\u000a system, we first need to investigate the actual noise sources in the system and develop models for these noise sources in\\u000a the framework of the theory of signals and systems we will be operating with. The models we are

  13. Model selection for geostatistical models.

    PubMed

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

    2006-02-01

    We consider the problem of model selection for geospatial data. Spatial correlation is often ignored in the selection of explanatory variables, and this can influence model selection results. For example, the importance of particular explanatory variables may not be apparent when spatial correlation is ignored. To address this problem, we consider the Akaike Information Criterion (AIC) as applied to a geostatistical model. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical model is superior to the often-used traditional approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a model for lizard abundance. We also apply the principle of minimum description length (MDL) to variable selection for the geostatistical model. The effect of sampling design on the selection of explanatory covariates is also explored. R software to implement the geostatistical model selection methods described in this paper is available in the Supplement. PMID:16705963

  14. The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

    Microsoft Academic Search

    Rekha Seshadri; Shelley A Haveman; Christopher L Hemme; Ian T Paulsen; James F Kolonay; Jonathan A Eisen; Barbara Methe; Lauren M Brinkac; Sean C Daugherty; Robert T Deboy; Robert J Dodson; A Scott Durkin; Ramana Madupu; William C Nelson; Steven A Sullivan; Derrick Fouts; Daniel H Haft; Jeremy Selengut; Jeremy D Peterson; Tanja M Davidsen; Nikhat Zafar; Liwei Zhou; Diana Radune; George Dimitrov; Mark Hance; Kevin Tran; Hoda Khouri; John Gill; Terry R Utterback; Tamara V Feldblyum; Judy D Wall; Gerrit Voordouw; Claire M Fraser; John F Heidelberg

    2004-01-01

    Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature

  15. Nanocomposite Fe1-xO/Fe3O4, Fe/Fe1-xO thin films prepared by RF sputtering and revealed by magnetic coupling effects

    NASA Astrophysics Data System (ADS)

    Mauvernay, B.; Presmanes, L.; Bonningue, C.; Tailhades, Ph.

    Magnetic and semi-conducting nanocomposite iron oxide thin films have been prepared under bias polarization, by radio-frequency sputtering of a magnetite target. The nature of the phases obtained in the thin films depends on the bias power density. The increase in power density, from 0 to 110 mW/cm2, allows the preparation of magnetite, magnetite/wustite and wustite/ ?-iron nanocomposites successively. Magnetic measurements at low temperature show exchange bias for two-phases films even though the minor phase is not detected by grazing angle X-ray diffraction. The exchange bias can reach very high values of about 4300 Oe. Electrical properties at room temperature are interpreted taking into account both the modifications of the film compactness, and the nature of the phases from which they are made.

  16. Modeling reality

    NASA Technical Reports Server (NTRS)

    Denning, Peter J.

    1990-01-01

    Although powerful computers have allowed complex physical and manmade hardware systems to be modeled successfully, we have encountered persistent problems with the reliability of computer models for systems involving human learning, human action, and human organizations. This is not a misfortune; unlike physical and manmade systems, human systems do not operate under a fixed set of laws. The rules governing the actions allowable in the system can be changed without warning at any moment, and can evolve over time. That the governing laws are inherently unpredictable raises serious questions about the reliability of models when applied to human situations. In these domains, computers are better used, not for prediction and planning, but for aiding humans. Examples are systems that help humans speculate about possible futures, offer advice about possible actions in a domain, systems that gather information from the networks, and systems that track and support work flows in organizations.

  17. Smashnova Model

    E-print Network

    C Sivaram

    2007-07-07

    An alternate model for gamma ray bursts is suggested. For a white dwarf (WD) and neutron star (NS) very close binary system, the WD (close to Mch) can detonate due to tidal heating, leading to a SN. Material falling on to the NS at relativistic velocities can cause its collapse to a magnetar or quark star or black hole leading to a GRB. As the material smashes on to the NS, it is dubbed the Smashnova model. Here the SN is followed by a GRB. NS impacting a RG (or RSG) (like in Thorne-Zytkow objects) can also cause a SN outburst followed by a GRB. Other variations are explored.

  18. Transducer Models

    NASA Astrophysics Data System (ADS)

    Backman, Juha Reinhold

    This chapter discusses the basic models with emphasis on audio applications. Loudspeakers are most commonly used as an example of electroacoustic transducers yet, from a modelling point of view, they present the broadest range of challenges to the theoreticians. The fundamental principles are, however, applicable to all transducer problems (microphones, hydrophones, ultrasonics). The reader is assumed to be reasonably familiar with the fundamental concepts of electroacoustics; introductory summaries have been presented by, e.g., Poldy (1994) and Hickson and Busch-Vishniac (1997).

  19. Network epistemology Discrete models

    E-print Network

    Zollman, Kevin

    Network epistemology Discrete models Continuous models Social Structure and Social Influence Structure and Social Influence #12;Network epistemology Discrete models Continuous models Network and Social Influence #12;Network epistemology Discrete models Continuous models Network epistemology

  20. Modeling Muscles

    ERIC Educational Resources Information Center

    Goodwyn, Lauren; Salm, Sarah

    2007-01-01

    Teaching the anatomy of the muscle system to high school students can be challenging. Students often learn about muscle anatomy by memorizing information from textbooks or by observing plastic, inflexible models. Although these mediums help students learn about muscle placement, the mediums do not facilitate understanding regarding integration of…

  1. Diffusion Models

    NSDL National Science Digital Library

    Alexei Sharov

    Web-based intructional material describing the use of diffusion models in population ecology. This page is part of a set of on-line lectures on Quantitative Population Ecology produced by Alexei Sharov in the Department of Entomology at Virginia Tech.

  2. Modeling Lessons

    ERIC Educational Resources Information Center

    Casey, Katherine

    2011-01-01

    As teachers learn new pedagogical strategies, they crave explicit demonstrations that show them how the new strategies will work with their students in their classrooms. Successful instructional coaches, therefore, understand the importance of modeling lessons to help teachers develop a vision of effective instruction. The author, an experienced…

  3. Atmospheric Modeling

    EPA Science Inventory

    Although air quality models have been applied historically to address issues specific to ambient air quality standards (i.e., one criteria pollutant at a time) or welfare (e.g.. acid deposition or visibility impairment). they are inherently multipollutant based. Therefore. in pri...

  4. Groundwater Model

    NSDL National Science Digital Library

    In this activity, students build a model to demonstrate how aquifers are formed and ground water becomes polluted. For younger students, the teacher can perform this activity as a demonstration, or older students can perform it themselves. A materials list, instructions, and extension activities are provided.

  5. Modeling Convection

    ERIC Educational Resources Information Center

    Ebert, James R.; Elliott, Nancy A.; Hurteau, Laura; Schulz, Amanda

    2004-01-01

    Students must understand the fundamental process of convection before they can grasp a wide variety of Earth processes, many of which may seem abstract because of the scales on which they operate. Presentation of a very visual, concrete model prior to instruction on these topics may facilitate students' understanding of processes that are largely…

  6. Daisyworld Model

    NSDL National Science Digital Library

    Kirsten Menking

    The Daisyworld model created by Andrew Watson and James Lovelock (1983, Tellus, v. 35B, p. 284-289) is a wonderful example of a self-regulating system incorporating positive and negative feedbacks. The model consists of a planet on which black and white daisies are growing. The growth of these daisies is governed by a parabolic shaped growth function regulated by planetary temperature and is set to zero for temperatures less than 5 ºC or greater than 40 ºC and optimized at 22.5 ºC. The model explores the effect of a steadily increasing solar luminosity on the growth of daisies and the resulting planetary temperature. The growth function for the daisies allows them to modulate the planet's temperature for many years, warming it early on as black daisies grow, and cooling it later as white daisies grow. Eventually, the solar luminosity increases beyond the daisies' capability to modulate the temperature and they die out, leading to a rapid rise in the planetary temperature. Students read Watson and Lovelock's original paper, and then use STELLA to create their own Daisyworld model with which they can experiment. Experiments include changing the albedos of the daisies, changing their death rates, and changing the rate at which energy is conducted from one part of the planet to another. In all cases, students keep track of daisy populations and of planetary temperature over time.

  7. Graphical models, causal inference, and econometric models

    E-print Network

    Spirtes, Peter

    Graphical models, causal inference, and econometric models Peter Spirtes Abstract A graphical model modeling has historical ties to causal modeling in econometrics and other social sciences, there have been in graphical causal modeling, and their relevance to econometrics and other social sciences. The use of graphs

  8. ATMOSPHERIC MODELING: MODEL AND ACCURACY

    EPA Science Inventory

    The development of models to assess the emission control requirements of primary precursor pollutants in the production of photochemical oxidants has been underway for approximately 20 years. Over the period there has been a considerable increase in our understanding of the basic...

  9. Students' Models of Curve Fitting: A Models and Modeling Perspective

    ERIC Educational Resources Information Center

    Gupta, Shweta

    2010-01-01

    The Models and Modeling Perspectives (MMP) has evolved out of research that began 26 years ago. MMP researchers use Model Eliciting Activities (MEAs) to elicit students' mental models. In this study MMP was used as the conceptual framework to investigate the nature of students' models of curve fitting in a problem-solving environment consisting of…

  10. 10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: 1' = 400' HORIZONTAL, 1' = 100' VERTICAL), AND GREENVILLE BRIDGE MODEL (MODEL SCALE: 1' = 360' HORIZONTAL, 1' = 100' VERTICAL). - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  11. Micrometer Model

    NSDL National Science Digital Library

    2009-09-10

    This interactive simulation gives students practice in the operation and the physical parts of a real micrometer, a measuring device that employs a screw to amplify distances that are too small to measure easily. The accuracy of a micrometer derives from the accuracy of the thread that is at its heart. The basic operating principle of a micrometer is that the rotation of an accurately made screw can be directly and precisely correlated to a certain amount of axial movement (and vice-versa), through the constant known as the screw's lead. The Micrometer model was created using the Easy Java Simulations (EJS) modeling tool. It is distributed as a ready-to-run (compiled) Java archive. Double click the ejs_ntnu_Micrometer.jar file to run the program (Java must be installed).

  12. Modeling Molecules

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The molecule modeling method known as Multibody Order (N) Dynamics, or MBO(N)D, was developed by Moldyn, Inc. at Goddard Space Flight Center through funding provided by the SBIR program. The software can model the dynamics of molecules through technology which stimulates low-frequency molecular motions and properties, such as movements among a molecule's constituent parts. With MBO(N)D, a molecule is substructured into a set of interconnected rigid and flexible bodies. These bodies replace the computation burden of mapping individual atoms. Moldyn's technology cuts computation time while increasing accuracy. The MBO(N)D technology is available as Insight II 97.0 from Molecular Simulations, Inc. Currently the technology is used to account for forces on spacecraft parts and to perform molecular analyses for pharmaceutical purposes. It permits the solution of molecular dynamics problems on a moderate workstation, as opposed to on a supercomputer.

  13. Model Well

    NSDL National Science Digital Library

    2012-07-12

    In this quick activity about pollutants and groundwater (page 2 of PDF), learners build a model well with a toilet paper tube. Learners use food coloring to simulate pollutants and observe how they can be carried by groundwater and eventually enter water sources such as wells, rivers, and streams. This activity is associated with nanotechnology and relates to linked video, DragonflyTV Nano: Water Clean-up.

  14. Modeling biomembranes.

    SciTech Connect

    Plimpton, Steven James; Heffernan, Julieanne; Sasaki, Darryl Yoshio; Frischknecht, Amalie Lucile; Stevens, Mark Jackson; Frink, Laura J. Douglas

    2005-11-01

    Understanding the properties and behavior of biomembranes is fundamental to many biological processes and technologies. Microdomains in biomembranes or ''lipid rafts'' are now known to be an integral part of cell signaling, vesicle formation, fusion processes, protein trafficking, and viral and toxin infection processes. Understanding how microdomains form, how they depend on membrane constituents, and how they act not only has biological implications, but also will impact Sandia's effort in development of membranes that structurally adapt to their environment in a controlled manner. To provide such understanding, we created physically-based models of biomembranes. Molecular dynamics (MD) simulations and classical density functional theory (DFT) calculations using these models were applied to phenomena such as microdomain formation, membrane fusion, pattern formation, and protein insertion. Because lipid dynamics and self-organization in membranes occur on length and time scales beyond atomistic MD, we used coarse-grained models of double tail lipid molecules that spontaneously self-assemble into bilayers. DFT provided equilibrium information on membrane structure. Experimental work was performed to further help elucidate the fundamental membrane organization principles.

  15. Hierarchical Reflexion Models

    Microsoft Academic Search

    Rainer Koschke; Daniel Simon

    2003-01-01

    The reexion model originally proposed by Murphy and Notkin allows one to structurally validate a de- scriptive or prescriptive architecture model against a source model. First, the entities in the source model are mapped onto the architectural model, then discrep- ancies between the architecture model and source model are computed automatically. The original reexion model allows an analyst to specify

  16. Biomimetic modelling.

    PubMed Central

    Vincent, Julian F V

    2003-01-01

    Biomimetics is seen as a path from biology to engineering. The only path from engineering to biology in current use is the application of engineering concepts and models to biological systems. However, there is another pathway: the verification of biological mechanisms by manufacture, leading to an iterative process between biology and engineering in which the new understanding that the engineering implementation of a biological system can bring is fed back into biology, allowing a more complete and certain understanding and the possibility of further revelations for application in engineering. This is a pathway as yet unformalized, and one that offers the possibility that engineers can also be scientists. PMID:14561351

  17. The Standard Model Beyond the Standard Model

    E-print Network

    The Standard Model Beyond the Standard Model New physics with top quark Search for Extra, January 13, 2010 Ritesh Singh New physics at LHC #12;The Standard Model Beyond the Standard Model New physics with top quark Search for Extra-dimensions Conclusions 1 The Standard Model Building block

  18. Technological Forecasting---Model Selection, Model Stability, and Combining Models

    Microsoft Academic Search

    Nigel Meade; Towhidul Islam

    1998-01-01

    The paper identifies 29 models that the literature suggests are appropriate for technological forecasting. These models are divided into three classes according to the timing of the point of inflexion in the innovation or substitution process. Faced with a given data set and such a choice, the issue of model selection needs to be addressed. Evidence used to aid model

  19. Modelling intonational structure using hidden markov models

    E-print Network

    Wright, Helen; Taylor, Paul A

    1997-01-01

    A method is introduced for using hidden Markov models (HMMs) to model intonational structure. HMMs are probabilistic and can capture the variability in structure which previous finite state network models lack. We show ...

  20. ECOBAS — modelling and documentation

    Microsoft Academic Search

    J Benz; R Hoch; T Legovi?

    2001-01-01

    Until now modelling and model documentation were two different processes. As a consequence, model documentation was prone to error. It was rarely possible to run larger models from their documentation. Model exchange was limited to simple models due to different languages in which they were created. To facilitate more efficient model creation, documentation and exchange we are introducing ECOBAS system.

  1. Modeling fatigue.

    PubMed Central

    Sumner, Walton; Xu, Jin Zhong

    2002-01-01

    The American Board of Family Practice is developing a patient simulation program to evaluate diagnostic and management skills. The simulator must give temporally and physiologically reasonable answers to symptom questions such as "Have you been tired?" A three-step process generates symptom histories. In the first step, the simulator determines points in time where it should calculate instantaneous symptom status. In the second step, a Bayesian network implementing a roughly physiologic model of the symptom generates a value on a severity scale at each sampling time. Positive, zero, and negative values represent increased, normal, and decreased status, as applicable. The simulator plots these values over time. In the third step, another Bayesian network inspects this plot and reports how the symptom changed over time. This mechanism handles major trends, multiple and concurrent symptom causes, and gradually effective treatments. Other temporal insights, such as observations about short-term symptom relief, require complimentary mechanisms. PMID:12463924

  2. CISNET lung models: Comparison of model assumptions and model structures

    PubMed Central

    McMahon, Pamela M.; Hazelton, William; Kimmel, Marek; Clarke, Lauren

    2012-01-01

    Sophisticated modeling techniques can be powerful tools to help us understand the effects of cancer control interventions on population trends in cancer incidence and mortality. Readers of journal articles are however rarely supplied with modeling details. Six modeling groups collaborated as part of the National Cancer Institute’s Cancer Intervention and Surveillance Modeling Network (CISNET) to investigate the contribution of US tobacco control efforts towards reducing lung cancer deaths over the period 1975 to 2000. The models included in this monograph were developed independently and use distinct, complementary approaches towards modeling the natural history of lung cancer. The models used the same data for inputs and agreed on the design of the analysis and the outcome measures. This article highlights aspects of the models that are most relevant to similarities of or differences between the results. Structured comparisons can increase the transparency of these complex models. PMID:22882887

  3. Multivariate Receptor Models and Model Uncertainty

    E-print Network

    Washington at Seattle, University of

    that are unobservable (latent variables or factors), P is the unknown q×p factor loading matrix, and q is the unknown pollution data are presented. Key words: Latent variable models; Factor analysis models; Model uncertainty. INTRODUCTION Multivariate receptor modeling aims to identify the pollution sources and assess the amounts

  4. Rossignac & Requicha Solid Modeling 1 Solid Modeling

    E-print Network

    Rossignac, Jarek

    Rossignac & Requicha Solid Modeling 1 Solid Modeling Jarek R. Rossignac GVU Center, College University of Southern California at Los Angeles 1 Introduction A solid model is a digital representation of the geometry of an existing or envisioned physical object. Solid models are used in many industries, from

  5. CISNET: Standardized Model Documents

    Cancer.gov

    Modeling is a complex endeavor, and often it is very difficult to reconcile results from different models. To aid in this process of model description and comparison, CISNET has developed and implemented standardized model documentation. Model profiles are standardized descriptions that facilitate the comparison of models and their results. Users can read documentation about a single model or read side-by-side descriptions that contrast how models address different components of the process.

  6. Comparative Protein Structure Modeling Using Modeller

    PubMed Central

    Eswar, Narayanan; Marti-Renom, Marc A.; Madhusudhan, M.S.; Eramian, David; Shen, Min-yi; Pieper, Ursula

    2014-01-01

    Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:18428767

  7. Modelling project management performance

    Microsoft Academic Search

    David James Bryde

    2003-01-01

    This paper presents an argument that it is appropriate to develop a model of project management (PM) performance from models for assessing quality management. The paper presents a model, labelled the project management performance assessment (PMPA) model, based upon the EFQM business excellence model. The model proposes six criteria for assessing PM performance: project management leadership; project management staff; project

  8. Standard Solar Model

    SciTech Connect

    Loong, Lim Yaw; Yusof, Norhasliza; Kassim, Hasan Abu [Physics Department, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2008-05-20

    Solar models are important in our understanding of stars and stellar evolution. Solar models have been constructed using different methods. In this work, a solar model will be built using the fitting method. The model will incorporate the most recent input data. The model will be evolved to the current epoch starting from the zero-age main sequence model.

  9. A shock-metamorphic model for silicate darkening and compositionally variable plagioclase in CK and ordinary chondrites

    SciTech Connect

    Rubin, A.E. (Univ. of California, Los Angeles (United States))

    1992-04-01

    Silicate darkening in ordinary chondrites (OC) is caused by tiny grains of metallic Fe-Ni and troilite occurring mainly within curvilinear trails that traverse silicate interiors and decorate or, in some cases, cut across silicate grain boundaries. Highly shocked OC tend to have greater degrees of silicate darkening than lightly shocked OC; this indicates that silicate darkening is probably a result of shock metamorphism. The low Fe-FeS eutectic temperature (988C) renders metal and troilite susceptible to melting and mobilization during shock heating. Unshocked OC tend to have plagioclase with uniform compositions; shocked OC tend to have plagioclase with more variable (albeit still stoichiometric) compositions. The low impedance of plagioclase to shock compression makes it particularly susceptible to melting and mobilization; this is consistent with the molten appearance of plagioclase in highly shocked OC (e.g., Rose City and Paragould). CK chondrites also have compositionally variable plagioclase. The common association of silicate darkening with compositionally variable plagioclase is consistent with the hypothesis that both are products of shock metamorphism. Some CK and OC chondrites exhibit light shock effects in olivine that are consistent with equilibrium peak shock pressures that are too low to account for the silicate darkening or opaque shock veins in these meteorites. Therefore, the olivine in these chondrites may have been annealed after intense shock produced these effects. A few CK chondrites that contain olivine with undulose or mosaic extinction (e.g., LEW87009 and EET83311) may have been shocked again, after annealing.

  10. Modeling Mercury

    NASA Astrophysics Data System (ADS)

    Burger, M. H.; Killen, R. M.; M, N.; Sarantos, M.; Crider, D. H.; Vervak, R. J.

    2009-04-01

    Mercury has a tenuous exosphere created by the combined effects of solar radiation and micrometeoroid bombardment on the surface and the interaction of the solar wind with Mercury's magnetic field and surface. Observations of this exosphere provide essential data necessary for understanding the composition and evolution of Mercury's surface, as well as the interaction between Mercury's magnetosphere with the solar wind. The sodium component of the exosphere has been well observed from the ground (see review by Killen et al., 2007). These observations have revealed a highly variable and inhomogeneous exosphere with emission often peaking in the polar regions. Radiation acceleration drives exospheric escape producing a sodium tail pointing away from the sun which has been detected up to 1400 Mercury radii from the planet (Potter et al. 2002; Baumgardner et al. 2008). Calcium has also been observed in Mercury's exosphere showing a distribution distinct from sodium, although also variable (Killen et al. 2005). During the first two encounters with Mercury by MESSENGER, observations of the exosphere were made by the UltraViolet and Visible Spectrometer (UVVS) channel of the Mercury Atmospheric and Surface Composition Spectrometer (MASCS). Sodium and calcium emission were detected during both flybys, and magnesium was detected for the first time in Mercury's exosphere during the second flyby. The spatial distributions of these species showed significant, unexpected differences which suggest differences in the mechanisms responsible for releasing them from the surface. We present a Monte-Carlo model of sodium, magnesium, and calcium in Mercury's exosphere. The important source mechanisms for ejecting these species from the surface are sputtering by solar wind ions, photon-stimulated desorption, and micrometeoroid impact vaporization. Thermal desorption on the dayside does not supply enough energy to significantly populate the exosphere, although it does play a role in redistributing volatiles over the surface. In addition, atomic calcium can be produced from the dissociation of Ca-bearing molecules, such as CaO, which can be formed in impact vapors. The primary loss processes are the escape of neutrals ejected with sufficient energy and photoionization. The former process is supplemented by radiation pressure which accelerates neutrals anti-sunward such that escaping neutrals form a tail pointing away from the sun. Because Mercury's heliocentric distance and radial velocity vary during its orbit, both loss processes are functions of Mercury's true anomaly. We also consider the spatial distribution of the surface source. Impact vaporization is roughly isotropic over the surface, although there may be a leading/trailing asymmetry in the impact rate due to Mercury's orbital motion. Sputtering is confined to regions where the solar wind can impact the surface, which is shielded somewhat by the internal magnetic field. The surface regions vulnerable depend on the solar wind conditions. References: Baumgardner et al., GRL, 35, L03201, 2008. Killen, R.M. et al., Space Sci. Rev. 132, 433-509, 2007. Killen, R.M. et al., Icarus, 173, 300-311, 2005. Potter et al., Meteoritics & Planetary Sci., 37, 1165, 2002.

  11. Modeling cholera outbreaks

    PubMed Central

    Longini, Ira M.; Morris, J. Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating models and the need to tailor models for different outbreak scenarios. PMID:23412687

  12. Protein Structure Modeling With MODELLER Narayanan Eswar$

    E-print Network

    Sali, Andrej

    sequences. Key Words: Comparative modeling, fold assignment, sequence-structure alignment, model assessment resonance (NMR) spectroscopy. In the absence of experimentally determined structures, computationally template structure (see Section on Materials for definitions of these terms); (ii) alignment of the target

  13. Model selection for logistic regression models

    NASA Astrophysics Data System (ADS)

    Duller, Christine

    2012-09-01

    Model selection for logistic regression models decides which of some given potential regressors have an effect and hence should be included in the final model. The second interesting question is whether a certain factor is heterogeneous among some subsets, i.e. whether the model should include a random intercept or not. In this paper these questions will be answered with classical as well as with Bayesian methods. The application show some results of recent research projects in medicine and business administration.

  14. Multilevel Model Prediction

    ERIC Educational Resources Information Center

    Frees, Edward W.; Kim, Jee-Seon

    2006-01-01

    Multilevel models are proven tools in social research for modeling complex, hierarchical systems. In multilevel modeling, statistical inference is based largely on quantification of random variables. This paper distinguishes among three types of random variables in multilevel modeling--model disturbances, random coefficients, and future response…

  15. Programming Definitions Model

    E-print Network

    Browne, James C.

    1 9/11/01 Naming Models and Parallel Programming Overview of Name Model Lectures 1. Roles of Name An Instance of the Taxonomy 3. Associative Broadcast Model of Parallel Programming 2 9/11/01 Naming Models. 8. Propagation ­ Algorithm for moving names among objects. 3 9/11/01 Naming Models and Parallel

  16. Anisotropic reflection models

    Microsoft Academic Search

    James T. Kajiya

    1985-01-01

    We present a new set of lighting models derived from the questions of electromagnetism. These models describe the reflection and refraction of light from surfaces which exhibit anisotropy---surfaces with preferred directions. The model allows a new mapping technique, which we call . We also discuss the general relationship between geometric models, surface mapping of all types, and lighting models in

  17. Fire models and design

    Microsoft Academic Search

    Alan N. Beard

    1997-01-01

    Over the last 20 years there has been a great increase in the construction of computer-based models related to fire risk. Both probabilistic and deterministic models have been produced. Many existing models are in a state of development and new models are being created continually. However, how such models are to be efficaciously employed as part of the design process

  18. Modeling transient rootzone salinity (SWS Model)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The combined, water quality criteria for irrigation, water and ion processes in soils, and plant and soil response is sufficiently complex that adequate analysis requires computer models. Models for management are also needed but these models must consider that the input requirements must be reasona...

  19. Cosmological Models Generalising Robertson-Walker Models

    E-print Network

    Abdussattar

    2003-08-07

    Considering the physical 3-space t = constant of the spacetime metrics as spheroidal and pseudo spheroidal, cosmological models which are generalizations of Robertson-Walker models are obtained. Specific forms of these general models as solutions of Einstein's field equations are also discussed in the radiation- and the matter-dominated eras of the universe.

  20. Bayesian Model Averaging for Linear Regression Models

    Microsoft Academic Search

    Adrian E. Raftery; David Madigan; Jennifer A. Hoeting

    1998-01-01

    We consider the problem of accounting for model uncertainty in linear regressionmodels. Conditioning on a single selected model ignores model uncertainty, and thusleads to the underestimation of uncertainty when making inferences about quantitiesof interest. A Bayesian solution to this problem involves averaging over all possiblemodels (i.e., combinations of predictors) when making inferences about quantities ofAdrian E. Raftery is Professor of

  1. Cognitive Modeling Cognitive Modelling -The nature of

    E-print Network

    Bremen, Universität

    Cognitive Modeling Cognitive Modelling - The nature of Connectionism and notes on computability Mathias Hinz Universität Bremen November 17, 2014 November 17, 2014 1 #12;Cognitive Modeling topic · Comparing PDP and nature · properties of PDP · computability · discussion November 17, 2014 2 #12;Cognitive

  2. Iron/iron oxide core/shell nanoparticles for magnetic targeting MRI and near-infrared photothermal therapy.

    PubMed

    Zhou, Zhiguo; Sun, Yanan; Shen, Jinchao; Wei, Jie; Yu, Chao; Kong, Bin; Liu, Wei; Yang, Hong; Yang, Shiping; Wang, Wei

    2014-08-01

    The development of photothermal agents (PTAs) with good stability, low toxicity, highly targeting ability and photothermal conversion efficiency is an essential pre-requisite to near-infrared photothermal therapy (PTT) in vivo. Herein, we report the readily available PEGylated Fe@Fe3O4 NPs, which possess triple functional properties in one entity - targeting, PTT, and imaging. Compared to Au nanorods, they exhibit comparable photothermal conversion efficiency (?20%), and much higher photothermal stability. They also show a high magnetization value and transverse relaxivity (?156 mm(-1) s(-1)), which should be applied for magnetic targeting MRI. With the Nd-Fe-B magnet (0.5 T) beside the tumour for 12 h on the xenograft HeLa tumour model, PEGylated Fe@Fe3O4 NPs exhibit an obvious accumulation. In tumour, the intensity of MRI signal is ? three folds and the increased temperature is ? two times than those without magnetic targeting, indicating the good magnetic targeting ability. Notably, the intrinsic high photothermal conversion efficiency and selective magnetic targeting effect of the NPs in tumour play synergistically in highly efficient ablation of cancer cells in vitro and in vivo. PMID:24881997

  3. The origin and chemical composition of the earth's core

    NASA Technical Reports Server (NTRS)

    Murthy, V. R.; Hall, T.

    1972-01-01

    The process of core formation in the earth is subject to the constraints that it be nearly simultaneous with accretion and yet occur in a manner that the mantle retains radiogenic Xe-129 produced from the extinct radioactivity of I-129 with a half life of 17.6 million y. From these constraints, it appears that the only feasible mechanism is the segregation of an Fe-FeS melt. Trace element abundances in major classes of meteorites and the silicate fractions of the earth show that not only there is a high depletion of sulphur in the crust and the mantle, but that it is even more highly depleted than the rare gases, water and the halogens. From the nature of this depletion pattern and the fact that any model of accretion of the earth will necessarily produce an Fe-FeS melt, it is concluded that the light element in the core is largely sulphur with minor amounts of carbon. A consequence of this mode of core formation is found to be the availability of K-40 radioactive heat production in the liquid core, estimated at about 10 to the 19th power erg/s at the present time.

  4. Modeling input processes

    SciTech Connect

    Iman, R.L.

    1986-01-01

    Computer models for various applications are closely scrutinized both from the standpoint of questioning the correctness of the underlying mathematical model with respect to the process it is attempting to model, and from the standpoint of verifying that the computer model correctly implements the underlying mathematical model. A process that receives less scrutiny, but is nonetheless of equal importance, concerns the individual and joint modeling of the inputs. This modeling effort clearly has a great impact on the credibility of results obtained from simulation studies. Model characteristics are reviewed that have a direct bearing on the model input process and reasons are given for using probabilistic based modeling with the inputs. Discussions are presented on how to model distributions for individual inputs and how to model multivariate input structures when dependence and other constraints may be present. 12 refs.

  5. MODEL CONSERVATION STANDARD INTRODUCTION

    E-print Network

    MODEL CONSERVATION STANDARD INTRODUCTION As directed by the Northwest Power Act, the Council has designed model conservation standards to produce all electricity savings that are cost believes the measures used to achieve the model conservation standards should provide reliable savings

  6. Editor's Roundtable: Model behavior

    NSDL National Science Digital Library

    Inez Liftig

    2010-11-01

    Models are manageable representations of objects, concepts, and phenomena, and are everywhere in science. Models are "thinking tools" for scientists and have always played a key role in the development of scientific knowledge. Models of the solar system,

  7. Educating with Aircraft Models

    ERIC Educational Resources Information Center

    Steele, Hobie

    1976-01-01

    Described is utilization of aircraft models, model aircraft clubs, and model aircraft magazines to promote student interest in aerospace education. The addresses for clubs and magazines are included. (SL)

  8. Modelling hot air balloons

    Microsoft Academic Search

    N. W. Brimicombe

    1991-01-01

    Hot air balloons can be modelled in a number of different ways. The most satisfactory, but least useful model is at a microscopic level. Macroscopic models are easier to use but can be very misleading.

  9. Nonlinear models Nonlinear Regression

    E-print Network

    Penny, Will

    Nonlinear models Will Penny Nonlinear Regression Nonlinear Regression Priors Energies Posterior Metropolis-Hasting Proposal density References Nonlinear models Will Penny Bayesian Inference Course, WTCN, UCL, March 2013 #12;Nonlinear models Will Penny Nonlinear Regression Nonlinear Regression Priors

  10. Modeling Natural Selection

    NSDL National Science Digital Library

    Christine Lotter

    2011-02-01

    In their research, scientists generate, test, and modify scientific models. These models can be shared with others and demonstrate a scientist's understanding of how the natural world works. Similarly, students can generate and modify models to gain a bet

  11. Maintenance & reliability models

    Microsoft Academic Search

    Richard E. Barlow; Carsten Boe; Tor Heimly; Tor-Chr. Mathliesen; Aridaman K. Jain; V. P. Sobczynski; C. J. Pearson

    1973-01-01

    Three models are presented in which stochastic simulation models are used to construct simulations of systems composed of unreliable components. The releability of the resultant systems is inferred from behavior of these simulation models.

  12. Mathematics and Statistics Models

    NSDL National Science Digital Library

    Developed by Bob MacKay, Clark College. What are Mathematical and Statistical Models These types of models are obviously related, but there are also real differences between them. Mathematical Models: grow out of ...

  13. Orbital Debris Modeling

    NASA Technical Reports Server (NTRS)

    Liou, J. C.

    2012-01-01

    Presentation outlne: (1) The NASA Orbital Debris (OD) Engineering Model -- A mathematical model capable of predicting OD impact risks for the ISS and other critical space assets (2) The NASA OD Evolutionary Model -- A physical model capable of predicting future debris environment based on user-specified scenarios (3) The NASA Standard Satellite Breakup Model -- A model describing the outcome of a satellite breakup (explosion or collision)

  14. Modeling of geothermal systems

    SciTech Connect

    Bodvarsson, G.S.; Pruess, K.; Lippmann, M.J.

    1985-03-01

    During the last decade the use of numerical modeling for geothermal resource evaluation has grown significantly, and new modeling approaches have been developed. In this paper we present a summary of the present status in numerical modeling of geothermal systems, emphasizing recent developments. Different modeling approaches are described and their applicability discussed. The various modeling tasks, including natural-state, exploitation, injection, multi-component and subsidence modeling, are illustrated with geothermal field examples. 99 refs., 14 figs.

  15. Continuous system modeling

    NASA Technical Reports Server (NTRS)

    Cellier, Francois E.

    1991-01-01

    A comprehensive and systematic introduction is presented for the concepts associated with 'modeling', involving the transition from a physical system down to an abstract description of that system in the form of a set of differential and/or difference equations, and basing its treatment of modeling on the mathematics of dynamical systems. Attention is given to the principles of passive electrical circuit modeling, planar mechanical systems modeling, hierarchical modular modeling of continuous systems, and bond-graph modeling. Also discussed are modeling in equilibrium thermodynamics, population dynamics, and system dynamics, inductive reasoning, artificial neural networks, and automated model synthesis.

  16. The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes 

    E-print Network

    Bethel, Ryan D

    2014-08-20

    )(CO)2]+, synthesized through CO substitution by opposing nucleophilic (PMe3) and electrophilic (NO+) ligands provide insight into the reactivity of both irons as a function of their ?-acidity. The intramolecular fluxional processes of a series of (?...

  17. Comparative amperometric study of uptake hydrogenase and hydrogen photoproduction activities between heterocystous cyanobacterium Anabaena cylindrica B629 and nonheterocystous cyanobacterium Oscillatoria sp. strain Miami BG7

    SciTech Connect

    Kumazawa, S.; Mitsui, A.

    1985-08-01

    Heterocystous filamentous cyanobacterium Anabaena cylindrica B629 and nonheterocystous filamentous cyanobacterium Oscillatoria sp. strain Miami BG7 were cultured in media with N/sub 2/ as the sole nitrogen source; and activities of oxygen-dependent hydrogen uptake, photohydrogen production photooxygen evolution, and respiration were compared amperometrically under the same or similar experimental conditions for both strains. Distinct differences in these activities were observed in both strains. The rates of hydrogen photoproduction and hydrogen accumulation were significantly higher in Oscillatoria sp. strain BG7 than in A. cylindrica B629 at every light intensity tested. The major reason for the difference was attributable to the fact that the heterocystous cyanobacterium had a high rate of oxygen-dependent hydrogen consumption activity and the nonheterocystous cyanobacterium did not. The activity of oxygen photoevolution and respiration also contributed to the difference. Oscillatoria sp. strain BG7 had lower O/sub 2/ evolution and higher respiration than did A. cylindrica B629. Thus, the effect of O/sub 2/ on hydrogen photoproduction was minimized in Oscillatoria sp. strain BG7. 32 references, 5 figures.

  18. First Generation Analogues of the Binuclear Site in the Fe-Only Hydrogenases: Fe2(-SR)2(CO)4(CN)2

    E-print Network

    Rauchfuss, Thomas B.

    the series Fe2(SMe)2- (CO)6-n(CN)n n- . Solutions of Fe2(SMe)2(CO)6 9 in methanol or acetonitrile react in minutes with Et4NCN to give exclusively Fe2(SMe)2(CO)4(CN)2 2- (1, eq 1). Analytically pure Et4N+ (1a to the formation of higher cyanides, i.e., Fe2(SMe)2(CO)6-n(CN)n n- (n > 2). Furthermore, the substitution

  19. Effects of biomass-generated producer gas constituents on cell growth, product distribution and hydrogenase activity of Clostridium carboxidivorans P7 T

    Microsoft Academic Search

    Asma Ahmed; Bruno G. Cateni; Raymond L. Huhnke; Randy S. Lewis

    2006-01-01

    In our previous work, we demonstrated that biomass-generated producer gas can be converted to ethanol and acetic acid using a microbial catalyst Clostridium carboxidivorans P7T. Results showed that the producer gas (1) induced cell dormancy, (2) inhibited H2 consumption, and (3) affected the acetic acid\\/ethanol product distribution. Results of this work showed that tars were the likely cause of cell

  20. Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12

    Microsoft Academic Search

    Everett T Hayes; Jessica C Wilks; Piero Sanfilippo; Elizabeth Yohannes; Daniel P Tate; Brian D Jones; Michael D Radmacher; Sandra S BonDurant; Joan L Slonczewski

    2006-01-01

    Background  InEscherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins.\\u000a Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation\\u000a has not been investigated.\\u000a \\u000a \\u000a \\u000a \\u000a Results  The pH dependence of gene expression was analyzed in oxygen-limited cultures ofE. coli K-12 strain W3110.E. coli K-12 strain W3110 was

  1. Fire Model Matrix

    NSDL National Science Digital Library

    COMET

    2008-02-05

    The Fire Model Matrix is an on-line resource that presents four fire community models in a matrix that facilitates the exploration of the characteristics of each model. As part of the Advanced Fire Weather Forecasters Course, this matrix is meant to sensitize forecasters to the use of weather data in these fire models to forecast potential fire activity.

  2. Connectionist models of development

    Microsoft Academic Search

    Yuko Munakata; James L. McClelland

    2003-01-01

    How have connectionist models informed the study of development? This paper considers three contributions from specific models. First, connectionist models have proven useful for exploring nonlinear dynamics and emergent properties, and their role in non- linear developmental trajectories, critical periods and developmental disorders. Second, connectionist models have informed the study of the representations that lead to behavioral dissociations. Third, connectionist

  3. Fair stateless model checking

    Microsoft Academic Search

    Madanlal Musuvathi; Shaz Qadeer

    2008-01-01

    Stateless model checking is a useful state-space exploration tech- nique for systematically testing complex real-world software. Ex- isting stateless model checkers are limited to the verification of safety properties on terminating programs. However, realistic con- current programs are nonterminating, a property that significantly reduces the efficacy of stateless model checking in testing them. Moreover, existing stateless model checkers are unable

  4. Generalized additive mixed models

    Microsoft Academic Search

    Colin Chen

    2000-01-01

    Following the extension from linear mixed models to additive mixed models, extension from generalized linear mixed models to generalized additive mixed models is made, Algorithms are developed to compute the MLE's of the nonlinear effects and the covariance structures based on the penalized marginal likelihood. Convergence of the algorithms and selection of the smooth param¬eters are discussed.

  5. Building Credible Input Models

    Microsoft Academic Search

    Lawrence M. Leemis

    2004-01-01

    Most discrete-event simulation models have stochastic el- ements that mimic the probabilistic nature of the system under consideration. A close match between the input model and the true underlying probabilistic mechanism associated with the system is required for successful input modeling. The general question considered here is how to model an element (e.g., arrival process, service times) in a discrete-

  6. Generalized Weibull Linear Models

    Microsoft Academic Search

    Andrea A. Prudente; Gauss M. Cordeiro

    2010-01-01

    For the first time, a new class of generalized Weibull linear models is introduced to be competitive to the well-known generalized (gamma and inverse Gaussian) linear models which are adequate for the analysis of positive continuous data. The proposed models have a constant coefficient of variation for all observations similar to the gamma models and may be suitable for a

  7. Generative Models of Disfluency

    ERIC Educational Resources Information Center

    Miller, Timothy A.

    2010-01-01

    This thesis describes a generative model for representing disfluent phenomena in human speech. This model makes use of observed syntactic structure present in disfluent speech, and uses a right-corner transform on syntax trees to model this structure in a very natural way. Specifically, the phenomenon of speech repair is modeled by explicitly…

  8. PREDICTIVE MODELS. Enhanced Oil Recovery Model

    SciTech Connect

    Ray, R.M. [DOE Bartlesville Energy Technology Center, Bartlesville, OK (United States)

    1992-02-26

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1 chemical flooding; 2 carbon dioxide miscible flooding; 3 in-situ combustion; 4 polymer flooding; and 5 steamflood. CFPM, the Chemical Flood Predictive Model, models micellar (surfactant)-polymer floods in reservoirs, which have been previously waterflooded to residual oil saturation. Thus, only true tertiary floods are considered. An option allows a rough estimate of oil recovery by caustic or caustic-polymer processes. CO2PM, the Carbon Dioxide miscible flooding Predictive Model, is applicable to both secondary (mobile oil) and tertiary (residual oil) floods, and to either continuous CO2 injection or water-alternating gas processes. ICPM, the In-situ Combustion Predictive Model, computes the recovery and profitability of an in-situ combustion project from generalized performance predictive algorithms. PFPM, the Polymer Flood Predictive Model, is switch-selectable for either polymer or waterflooding, and an option allows the calculation of the incremental oil recovery and economics of polymer relative to waterflooding. SFPM, the Steamflood Predictive Model, is applicable to the steam drive process, but not to cyclic steam injection (steam soak) processes. The IBM PC/AT version includes a plotting capability to produces a graphic picture of the predictive model results.

  9. Nearshore Wave Modeling

    NSDL National Science Digital Library

    2014-09-14

    Ocean waves near shore impact public safety, commerce, navigation, and, of course recreation. Predicting these waves has driven efforts to model them for more than two decades. This module introduces forecasters to different nearshore wave models, including phase-resolving and 1- and 2-dimensional spectral models. It describes the processes that wave models simulate, the assumptions they make, the initial and boundary conditions required to run the models, and potential sources of error in model forecasts. While focusing on SWAN, the module also examines the Navy Standard surf Model and Bouss-2D.

  10. Models of Loudness

    Microsoft Academic Search

    Jeremy Marozeau

    \\u000a Any chapter dedicated to reviewing models should first try to define what a model is. The question is more difficult than\\u000a it first appears. Whereas most scientists agree that a model should represent a real-world phenomenon, many disagree on the\\u000a level of complexity that a model should have. In his chapter on pitch models, De Cheveigné (2004) cites Norbert Wiener:

  11. Stable Models of superacceleration

    E-print Network

    Manoj Kaplinghat; Arvind Rajaraman

    2007-05-16

    We discuss an instability in a large class of models where dark energy is coupled to matter. In these models the mass of the scalar field is much larger than the expansion rate of the Universe. We find models in which this instability is absent, and show that these models generically predict an apparent equation of state for dark energy smaller than -1, i.e., superacceleration. These models have no acausal behavior or ghosts.

  12. Modernizing Our Cognitive Model

    Microsoft Academic Search

    David J. Bryant

    Although still popular, the Observe-Orient-Decide-Act (OODA) Loop is outdated as a model of human cognition. Based on advances in the cognitive sciences since the 1950s, the Critique- Explore-Compare-Adapt (CECA) Loop is proposed as a better descriptive model. The model puts two mental representations, the conceptual model established through operational planning, and the situation model, which represents the state of the

  13. 14. Quark model 1 14. QUARK MODEL

    E-print Network

    Krusche, Bernd

    14. Quark model 1 14. QUARK MODEL Revised December 2005 by C. Amsler (University of Z¨urich), T. DeGrand (University of Colorado, Boulder) and B. Krusche (University of Basel). 14.1. Quantum numbers of the quarks Quarks are strongly interacting fermions with spin 1/2 and, by convention, positive parity

  14. 14. Quark model 1 14. QUARK MODEL

    E-print Network

    14. Quark model 1 14. QUARK MODEL Revised August 2011 by C. Amsler (University of Z¨urich), T. DeGrand (University of Colorado, Boulder), and B. Krusche (University of Basel). 14.1. Quantum numbers of the quarks and its constituents are a set of fermions, the quarks, and gauge bosons, the gluons. Strongly interacting

  15. 1. Quark model 1 1. QUARK MODEL

    E-print Network

    Krusche, Bernd

    1. Quark model 1 1. QUARK MODEL Revised December 2005 by C. Amsler (University of Z¨urich), T. DeGrand (University of Colorado, Boulder) and B. Krusche (University of Basel). 1.1. Quantum numbers of the quarks Quarks are strongly interacting fermions with spin 1/2 and, by convention, positive parity

  16. 14. Quark model 1 14. QUARK MODEL

    E-print Network

    14. Quark model 1 14. QUARK MODEL Revised September 2009 by C. Amsler (University of Z¨urich), T numbers of the quarks Quarks are strongly interacting fermions with spin 1/2 and, by convention, positive parity. Antiquarks have negative parity. Quarks have the additive baryon number 1/3, antiquarks -1

  17. PREDICTIVE MODELS. Enhanced Oil Recovery Model

    Microsoft Academic Search

    1992-01-01

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1 chemical flooding, where soap-like surfactants are injected into

  18. PREDICTIVE MODELS. Enhanced Oil Recovery Model

    Microsoft Academic Search

    1992-01-01

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1 chemical flooding; 2 carbon dioxide miscible flooding; 3

  19. WASP TRANSPORT MODELING AND WASP ECOLOGICAL MODELING

    EPA Science Inventory

    A combination of lectures, demonstrations, and hands-on excercises will be used to introduce pollutant transport modeling with the U.S. EPA's general water quality model, WASP (Water Quality Analysis Simulation Program). WASP features include a user-friendly Windows-based interfa...

  20. Efficient Model Determination for Discrete Graphical Models

    Microsoft Academic Search

    Paolo Giudici; Peter Green; Claudia Tarantola

    2000-01-01

    We present a novel methodology for bayesian model determination in discretedecomposable graphical models. We assign, for each given graph, a Hyper Dirichletdistribution on the matrix of cell probabilities. To ensure compatibility acrossmodels such prior distributions are obtained by marginalisation from the prior conditionalon the complete graph. This leads to a prior distribution automaticallysatisfying the hyperconsistency criterion. Our contribution is twofold.

  1. Geochemistry Model Validation Report: External Accumulation Model

    SciTech Connect

    K. Zarrabi

    2001-09-27

    The purpose of this Analysis and Modeling Report (AMR) is to validate the External Accumulation Model that predicts accumulation of fissile materials in fractures and lithophysae in the rock beneath a degrading waste package (WP) in the potential monitored geologic repository at Yucca Mountain. (Lithophysae are voids in the rock having concentric shells of finely crystalline alkali feldspar, quartz, and other materials that were formed due to entrapped gas that later escaped, DOE 1998, p. A-25.) The intended use of this model is to estimate the quantities of external accumulation of fissile material for use in external criticality risk assessments for different types of degrading WPs: U.S. Department of Energy (DOE) Spent Nuclear Fuel (SNF) codisposed with High Level Waste (HLW) glass, commercial SNF, and Immobilized Plutonium Ceramic (Pu-ceramic) codisposed with HLW glass. The scope of the model validation is to (1) describe the model and the parameters used to develop the model, (2) provide rationale for selection of the parameters by comparisons with measured values, and (3) demonstrate that the parameters chosen are the most conservative selection for external criticality risk calculations. To demonstrate the applicability of the model, a Pu-ceramic WP is used as an example. The model begins with a source term from separately documented EQ6 calculations; where the source term is defined as the composition versus time of the water flowing out of a breached waste package (WP). Next, PHREEQC, is used to simulate the transport and interaction of the source term with the resident water and fractured tuff below the repository. In these simulations the primary mechanism for accumulation is mixing of the high pH, actinide-laden source term with resident water; thus lowering the pH values sufficiently for fissile minerals to become insoluble and precipitate. In the final section of the model, the outputs from PHREEQC, are processed to produce mass of accumulation, density of accumulation, and the geometry of the accumulation zone. The density of accumulation and the geometry of the accumulation zone are calculated using a characterization of the fracture system based on field measurements made in the proposed repository (BSC 2001k). The model predicts that accumulation would spread out in a conical accumulation volume. The accumulation volume is represented with layers as shown in Figure 1. This model does not directly feed the assessment of system performance. The output from this model is used by several other models, such as the configuration generator, criticality, and criticality consequence models, prior to the evaluation of system performance.

  2. jModelTest: phylogenetic model averaging.

    PubMed

    Posada, David

    2008-07-01

    jModelTest is a new program for the statistical selection of models of nucleotide substitution based on "Phyml" (Guindon and Gascuel 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 52:696-704.). It implements 5 different selection strategies, including "hierarchical and dynamical likelihood ratio tests," the "Akaike information criterion," the "Bayesian information criterion," and a "decision-theoretic performance-based" approach. This program also calculates the relative importance and model-averaged estimates of substitution parameters, including a model-averaged estimate of the phylogeny. jModelTest is written in Java and runs under Mac OSX, Windows, and Unix systems with a Java Runtime Environment installed. The program, including documentation, can be freely downloaded from the software section at http://darwin.uvigo.es. PMID:18397919

  3. Trapped Radiation Model Uncertainties: Model-Data and Model-Model Comparisons

    NASA Technical Reports Server (NTRS)

    Armstrong, T. W.; Colborn, B. L.

    2000-01-01

    The standard AP8 and AE8 models for predicting trapped proton and electron environments have been compared with several sets of flight data to evaluate model uncertainties. Model comparisons are made with flux and dose measurements made on various U.S. low-Earth orbit satellites (APEX, CRRES, DMSP. LDEF, NOAA) and Space Shuttle flights, on Russian satellites (Photon-8, Cosmos-1887, Cosmos-2044), and on the Russian Mir space station. This report gives the details of the model-data comparisons -- summary results in terms of empirical model uncertainty factors that can be applied for spacecraft design applications are given in a companion report. The results of model-model comparisons are also presented from standard AP8 and AE8 model predictions compared with the European Space Agency versions of AP8 and AE8 and with Russian trapped radiation models.

  4. Trapped Radiation Model Uncertainties: Model-Data and Model-Model Comparisons

    NASA Technical Reports Server (NTRS)

    Armstrong, T. W.; Colborn, B. L.

    2000-01-01

    The standard AP8 and AE8 models for predicting trapped proton and electron environments have been compared with several sets of flight data to evaluate model uncertainties. Model comparisons are made with flux and dose measurements made on various U.S. low-Earth orbit satellites (APEX, CRRES, DMSP, LDEF, NOAA) and Space Shuttle flights, on Russian satellites (Photon-8, Cosmos-1887, Cosmos-2044), and on the Russian Mir Space Station. This report gives the details of the model-data comparisons-summary results in terms of empirical model uncertainty factors that can be applied for spacecraft design applications are given in a combination report. The results of model-model comparisons are also presented from standard AP8 and AE8 model predictions compared with the European Space Agency versions of AP8 and AE8 and with Russian-trapped radiation models.

  5. Model Validation Status Review

    SciTech Connect

    E.L. Hardin

    2001-11-28

    The primary objective for the Model Validation Status Review was to perform a one-time evaluation of model validation associated with the analysis/model reports (AMRs) containing model input to total-system performance assessment (TSPA) for the Yucca Mountain site recommendation (SR). This review was performed in response to Corrective Action Request BSC-01-C-01 (Clark 2001, Krisha 2001) pursuant to Quality Assurance review findings of an adverse trend in model validation deficiency. The review findings in this report provide the following information which defines the extent of model validation deficiency and the corrective action needed: (1) AMRs that contain or support models are identified, and conversely, for each model the supporting documentation is identified. (2) The use for each model is determined based on whether the output is used directly for TSPA-SR, or for screening (exclusion) of features, events, and processes (FEPs), and the nature of the model output. (3) Two approaches are used to evaluate the extent to which the validation for each model is compliant with AP-3.10Q (Analyses and Models). The approaches differ in regard to whether model validation is achieved within individual AMRs as originally intended, or whether model validation could be readily achieved by incorporating information from other sources. (4) Recommendations are presented for changes to the AMRs, and additional model development activities or data collection, that will remedy model validation review findings, in support of licensing activities. The Model Validation Status Review emphasized those AMRs that support TSPA-SR (CRWMS M&O 2000bl and 2000bm). A series of workshops and teleconferences was held to discuss and integrate the review findings. The review encompassed 125 AMRs (Table 1) plus certain other supporting documents and data needed to assess model validity. The AMRs were grouped in 21 model areas representing the modeling of processes affecting the natural and engineered barriers, plus the TSPA model itself Description of the model areas is provided in Section 3, and the documents reviewed are described in Section 4. The responsible manager for the Model Validation Status Review was the Chief Science Officer (CSO) for Bechtel-SAIC Co. (BSC). The team lead was assigned by the CSO. A total of 32 technical specialists were engaged to evaluate model validation status in the 21 model areas. The technical specialists were generally independent of the work reviewed, meeting technical qualifications as discussed in Section 5.

  6. Inflation models and observation

    E-print Network

    Laila Alabidi; David Lyth

    2005-12-01

    We consider small-field models which invoke the usual framework for the effective field theory, and large-field models which go beyond that. Present and future possibilities for discriminating between the models are assessed, on the assumption that the primordial curvature perturbation is generated during inflation. With PLANCK data, the theoretical and observational uncertainties on the spectral index will be comparable, providing useful discrimination between small-field models. Further discrimination between models may come later through the tensor fraction, the running of the spectral index and non-gaussianity. The prediction for the trispectrum in a generic multi-field inflation model is given for the first time.

  7. Marine Wave Model Matrix

    NSDL National Science Digital Library

    COMET

    2006-05-16

    The Marine Wave Model Matrix provides information on the formulation of wave models developed by the National Centers for Environmental Prediction (NCEP) and other modeling centers, including how these models forecast the generation, propagation, and dissipation of ocean waves using NWP model forecasts for winds and near-surface temperature and stability. Additionally, information is provided on data assimilation, post-processing of data, and verfication of wave models currently in operation. Within the post-processing pages are links to forecast output both in graphical and raw form, including links for data downloads. Links to COMET training on wave processes are also provided.

  8. SAM EMP (SEMP) model

    SciTech Connect

    Thatcher, R.M.

    1984-05-01

    The Surface-To-Air Missile (SAM) Electro-Magnetic-Pulse (EMP) (SEMP) model simulates the illumination of an entire SAM brigade with an EMP weapon. It computes probability distributions of SAM brigade performance levels after an EMP attack has occurred. Brigade performance is determined by the combination of components that survive the EMP. Accordingly, the SEMP model is separated into the component failure model and the condition model. The component failure model computes the failure probability of each component in the brigade from data supplied by two input data files. The condition model converts component failure probabilities into brigade performance in the form of missile availability probability tables.

  9. Mathematical model of retina

    NASA Astrophysics Data System (ADS)

    Cendrowski, S. K.

    1996-12-01

    The paper presented describes the mathematical model of light stimulus transforming by the neural layers of eye retina. The model contains the descriptions of photoreceptors, horizontal and bipolar cells which belong to outer plexiform layer of retina. There were physical mechanisms of neurons exciting which were in the base of model. The model allows to predict the set of well-known visual phenomena, for example, the Broca-Sulzer temporal and spatial effects, the Mach's bands effect. The quantitative validation of the model have been made. The model may be taken as a principle of designing sensory layer of networks.

  10. Holographic Twin Higgs Model

    NASA Astrophysics Data System (ADS)

    Geller, Michael; Telem, Ofri

    2015-05-01

    We present the first realization of a "twin Higgs" model as a holographic composite Higgs model. Uniquely among composite Higgs models, the Higgs potential is protected by a new standard model (SM) singlet elementary "mirror" sector at the sigma model scale f and not by the composite states at mKK , naturally allowing for mKK beyond the LHC reach. As a result, naturalness in our model cannot be constrained by the LHC, but may be probed by precision Higgs measurements at future lepton colliders, and by direct searches for Kaluza-Klein excitations at a 100 TeV collider.

  11. Energy-consumption modelling

    SciTech Connect

    Reiter, E.R.

    1980-01-01

    A highly sophisticated and accurate approach is described to compute on an hourly or daily basis the energy consumption for space heating by individual buildings, urban sectors, and whole cities. The need for models and specifically weather-sensitive models, composite models, and space-heating models are discussed. Development of the Colorado State University Model, based on heat-transfer equations and on a heuristic, adaptive, self-organizing computation learning approach, is described. Results of modeling energy consumption by the city of Minneapolis and Cheyenne are given. Some data on energy consumption in individual buildings are included.

  12. Marine Modeling and Analysis

    NSDL National Science Digital Library

    National Centers for Environmental Prediction, National Oceanic and Atmospheric Administration

    The Marine Modeling and Analysis Branch (MMAB) of the Environmental Modeling Center is responsible for the development of improved numerical weather and marine prediction modeling systems. These models provide analysis and real-time forecast guidance on marine meteorological, oceanographic, and cryospheric parameters over the global oceans and coastal areas of the US. This site provides access to MMAB modeling tools for ocean waves (including an interactive presentation,) sea ice, marine meteorology, sea surface temperature and more. The site also features a mailing list, bibliography of publications, and information about modeling products still in the experimental and development phases.

  13. Biosphere Model Report

    SciTech Connect

    D.W. Wu; A.J. Smith

    2004-11-08

    The purpose of this report is to document the biosphere model, the Environmental Radiation Model for Yucca Mountain, Nevada (ERMYN), which describes radionuclide transport processes in the biosphere and associated human exposure that may arise as the result of radionuclide release from the geologic repository at Yucca Mountain. The biosphere model is one of the process models that support the Yucca Mountain Project (YMP) Total System Performance Assessment (TSPA) for the license application (LA), TSPA-LA. The ERMYN provides the capability of performing human radiation dose assessments. This report documents the biosphere model, which includes: (1) Describing the reference biosphere, human receptor, exposure scenarios, and primary radionuclides for each exposure scenario (Section 6.1); (2) Developing a biosphere conceptual model using site-specific features, events, and processes (FEPs) (Section 6.2), the reference biosphere (Section 6.1.1), the human receptor (Section 6.1.2), and approximations (Sections 6.3.1.4 and 6.3.2.4); (3) Building a mathematical model using the biosphere conceptual model (Section 6.3) and published biosphere models (Sections 6.4 and 6.5); (4) Summarizing input parameters for the mathematical model, including the uncertainty associated with input values (Section 6.6); (5) Identifying improvements in the ERMYN compared with the model used in previous biosphere modeling (Section 6.7); (6) Constructing an ERMYN implementation tool (model) based on the biosphere mathematical model using GoldSim stochastic simulation software (Sections 6.8 and 6.9); (7) Verifying the ERMYN by comparing output from the software with hand calculations to ensure that the GoldSim implementation is correct (Section 6.10); (8) Validating the ERMYN by corroborating it with published biosphere models; comparing conceptual models, mathematical models, and numerical results (Section 7).

  14. Introduction Discriminative Language Modeling (DLM)

    E-print Network

    Mohri, Mehryar

    Introduction Discriminative Language Modeling (DLM) Discriminative Training of Acoustic Models Discriminative Language and Acoustic Modeling for Large Vocabulary Continuous Speech Recognition Murat Sarac Language and Acoustic Modeling for LVCSR #12;Introduction Discriminative Language Modeling (DLM

  15. Modeling error in Approximate Deconvolution Models

    E-print Network

    Adrian Dunca; Roger Lewandowski

    2012-10-09

    We investigate the assymptotic behaviour of the modeling error in approximate deconvolution model in the 3D periodic case, when the order $N$ of deconvolution goes to $\\infty$. We consider successively the generalised Helmholz filters of order $p$ and the Gaussian filter. For Helmholz filters, we estimate the rate of convergence to zero thanks to energy budgets, Gronwall's Lemma and sharp inequalities about Fouriers coefficients of the residual stress. We next show why the same analysis does not allow to conclude convergence to zero of the error modeling in the case of Gaussian filter, leaving open issues.

  16. Volumetric particle modeling 

    E-print Network

    Dingle, Brent Michael

    2007-09-17

    This dissertation presents a robust method of modeling objects and forces for computer animation. Within this method objects and forces are represented as particles. As in most modeling systems, the movement of objects is driven by physically based...

  17. Osteoporotic fracture models.

    PubMed

    Simpson, A Hamish; Murray, Iain R

    2015-02-01

    Animal models are widely used to investigate the pathogenesis of osteoporosis and for the clinical testing of anti-resorptive drugs. However, osteoporotic fracture models designed to investigate novel ways to treat fractures of osteoporotic bone must fulfil requirements distinct from those of pharmacological testing. Bone strength and toughness, implant fixation and osteointegration and fracture repair are of particular interest. Osteoporotic models should reflect the underlying clinical scenario be that primary type 1 (post-menopausal) osteoporosis, primary type 2 (senile) osteoporosis or secondary osteoporosis. In each scenario, small and large animal models have been developed. While rodent models facilitate the study of fractures in strains specifically established to facilitate understanding of the pathologic basis of disease, concerns remain about the relevance of small animal fracture models to the human situation. There is currently no all-encompassing model, and the choice of species and model must be individualized to the scientific question being addressed. PMID:25388154

  18. Monte Carlo Modeling

    NSDL National Science Digital Library

    David Joiner

    Monte Carlo modeling refers to the solution of mathematical problems with the use of random numbers. This can include both function integration and the modeling of stochastic phenomena using random processes.

  19. Dahl friction modeling

    E-print Network

    Chou, Danielle, 1981-

    2004-01-01

    The drive behind improved friction models has been better prediction and control of dynamic systems. The earliest model was of classical Coulomb friction; however, the discontinuity during force reversal of the Coulomb ...

  20. Aircraft collision models

    E-print Network

    Endoh, Shinsuke

    1982-01-01

    Introduction: The threat of midair collisions is one of the most serious problems facing the air traffic control system and has been studied by many researchers. The gas model is one of the models which describe the expected ...

  1. Make a DNA Model

    NSDL National Science Digital Library

    American Museum of Natural History

    2012-06-26

    In this activity, learners make a 3-D model of DNA using paper and toothpicks. While constructing this model, learners will explore the composition and structure of DNA. The activity also gives suggestions for alternate materials and challenges to explore.

  2. Models of scientific explanation

    E-print Network

    Sutton, Peter Andrew

    2005-08-29

    Ever since Hempel and Oppenheim's development of the Deductive Nomological model of scientific explanation in 1948, a great deal of philosophical energy has been dedicated to constructing a viable model of explanation that concurs both with our...

  3. X-33 RCS model

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Model support system and instumentation cabling of the 1% scale X-33 reaction control system model. Installed in the Unitary Plan Wind Tunnel for supersonic testing. In building 1251, test section #2.

  4. Bounding Species Distribution Models

    NASA Technical Reports Server (NTRS)

    Stohlgren, Thomas J.; Jarnevich, Cahterine S.; Morisette, Jeffrey T.; Esaias, Wayne E.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5): 642-647, 2011].

  5. Bounding species distribution models

    USGS Publications Warehouse

    Stohlgren, T.J.; Jarnevich, C.S.; Esaias, W.E.; Morisette, J.T.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used. ?? 2011 Current Zoology.

  6. TMDL RUSLE MODEL

    EPA Science Inventory

    We developed a simplified spreadsheet modeling approach for characterizing and prioritizing sources of sediment loadings from watersheds in the United States. A simplified modeling approach was developed to evaluate sediment loadings from watersheds and selected land segments. ...

  7. Exposure Analysis Modeling System

    EPA Science Inventory

    The Exposure Analysis Modeling System (EXAMS) is an interactive software application for formulating aquatic ecosystem models and evaluating the fate, transport, and exposure concentrations of synthetic organic chemicals including pesticides, industrial materials, and leachates f...

  8. How Mesoscale Models Work

    NSDL National Science Digital Library

    2014-09-14

    The goal of this training module is to help you increase your understanding of how mesoscale models work. Such understanding, in turn, can help you more efficiently and accurately evaluate model-generated forecast products.

  9. METEOROLOGICAL AND TRANSPORT MODELING

    EPA Science Inventory

    Advanced air quality simulation models, such as CMAQ, as well as other transport and dispersion models, require accurate and detailed meteorology fields. These meteorology fields include primary 3-dimensional dynamical and thermodynamical variables (e.g., winds, temperature, mo...

  10. Of Molecules and Models.

    ERIC Educational Resources Information Center

    Brinner, Bonnie

    1992-01-01

    Presents an activity in which models help students visualize both the DNA process and transcription. After constructing DNA, RNA messenger, and RNA transfer molecules; students model cells, protein synthesis, codons, and RNA movement. (MDH)

  11. Visualization of Model Output

    NSDL National Science Digital Library

    Visualization of output from mathematical or statistical models is one of the best ways to introduce introductory geoscience students to the results and behavior of sophisticated models. Example of good sites ...

  12. Viscous Universe Models

    NASA Astrophysics Data System (ADS)

    Grøn, Øyvind; Darian, Diako

    We give a review of viscous relativistic universe models that have been presented during the period from 1990 until the present time. In particular we discuss the properties of isotropic and homogeneous universe models, and of anisotropic and homogeneous Bianchi type I models. We consider these types of models both in the context of the non-causal Eckhart theory and the causal Israel-Stewart theory.

  13. ACES terminal model enhancement

    Microsoft Academic Search

    George J. Couluris; Paul C. Davis; Nathan C. Mittler; Aditya P. Saraf; Sebastian D. Timar

    2009-01-01

    Terminal model enhancement is an advanced modeling capability for simulating terminal area airport and airspace traffic operations. But, more importantly, TME is a platform for testing advanced air traffic management concepts using plug-and-play modeling. TME augments the existing airport surface and terminal airspace modeling capabilities of NASA's airspace concept evaluation system, an agent-based fast-time National Airspace System simulation. TME supports

  14. Modeling Complex Calorimeters

    NASA Technical Reports Server (NTRS)

    Figueroa-Feliciano, Enectali

    2004-01-01

    We have developed a software suite that models complex calorimeters in the time and frequency domain. These models can reproduce all measurements that we currently do in a lab setting, like IV curves, impedance measurements, noise measurements, and pulse generation. Since all these measurements are modeled from one set of parameters, we can fully describe a detector and characterize its behavior. This leads to a model than can be used effectively for engineering and design of detectors for particular applications.

  15. Hierarchical Bass model

    E-print Network

    Tashiro, Tohru

    2013-01-01

    We propose a new model about diffusion of a product which includes a memory of how many adopters or advertisements a non-adopter met, where (non-)adopters mean people (not) possessing the product. This effect is lacking in the Bass model. As an application, we utilize the model to fit the iPod sales data, and so the better agreement is obtained than the Bass model.

  16. Modelling of cells bioenergetics.

    PubMed

    Kasperski, Andrzej

    2008-09-01

    This paper presents an integrated model describing the control of Saccharomyces cerevisiae yeast cells bioenergetics. This model describes the oxidative and respirofermentative metabolism. The model assumes that the mitochondria of the Saccharomyces cerevisiae cells are charged with NADH during the tricarboxylic acid cycle, and NADH is discharged from mitochondria later in the electron transport system. Selected effects observed in the Saccharomyces cerevisiae eucaryotic cells, including the Pasteur's and Crabtree effects, are also modeled. PMID:18379882

  17. Introduction to Ocean Models

    NSDL National Science Digital Library

    2014-09-14

    Oceans cover over 70% of the surface of the earth, yet many details of their workings are not fully understood. To better understand and forecast the state of the ocean, we rely on numerical ocean models. Ocean models combine observations and physics to predict the ocean temperature, salinity, and currents at any time and any place across the ocean basins. This module will discuss what goes into numerical ocean models, including model physics, coordinate systems, parameterization, initialization, and boundary conditions.

  18. Generalized Additive Models

    Microsoft Academic Search

    Trevor Hastie; Robert Tibshirani

    1986-01-01

    Likelihood-based regression models such as the normal linear regression model and the linear logistic model, assume a linear (or some other parametric) form for the covariates $X_1, X_2, \\\\cdots, X_p$. We introduce the class of generalized additive models which replaces the linear form $\\\\sum \\\\beta_jX_j$ by a sum of smooth functions $\\\\sum s_j(X_j)$. The $s_j(\\\\cdot)$'s are unspecified functions that are

  19. Aerosol Modeling for the Global Model Initiative

    NASA Technical Reports Server (NTRS)

    Weisenstein, Debra K.; Ko, Malcolm K. W.

    2001-01-01

    The goal of this project is to develop an aerosol module to be used within the framework of the Global Modeling Initiative (GMI). The model development work will be preformed jointly by the University of Michigan and AER, using existing aerosol models at the two institutions as starting points. The GMI aerosol model will be tested, evaluated against observations, and then applied to assessment of the effects of aircraft sulfur emissions as needed by the NASA Subsonic Assessment in 2001. The work includes the following tasks: 1. Implementation of the sulfur cycle within GMI, including sources, sinks, and aqueous conversion of sulfur. Aerosol modules will be added as they are developed and the GMI schedule permits. 2. Addition of aerosol types other than sulfate particles, including dust, soot, organic carbon, and black carbon. 3. Development of new and more efficient parameterizations for treating sulfate aerosol nucleation, condensation, and coagulation among different particle sizes and types.

  20. 3 Human vs. model 2 Salience model

    E-print Network

    Peters, Rob

    4 Contour model Robert J. Peters (1), T. Nathan Mundhenk (2), Laurent Itti (2), and Christof Koch (1 Winner-take-all Inhibition of return Attended location adapted from Itti&Koch (2001), Nat. Rev. Neurosci