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1

[FeFe] hydrogenases and their evolution: a genomic perspective  

Microsoft Academic Search

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

J. Meyer

2007-01-01

2

Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System  

PubMed Central

Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

King, Paul W.; Posewitz, Matthew C.; Ghirardi, Maria L.; Seibert, Michael

2006-01-01

3

Effect of Cyanide Ligands on the Electronic Structure of [FeFe] Hydrogenase Active-Site Model Complexes with an Azadithiolate Cofactor.  

PubMed

A detailed characterization of a close synthetic model of the [2?Fe]H subcluster in the [FeFe] hydrogenase active site is presented. It contains the full primary coordination sphere of the CO-inhibited oxidized state of the enzyme including the CN(-) ligands and the azadithiolate (adt) bridge, [((?-S?CH2 )2 NR)Fe2 (CO)4 (CN)2 ](2-) , R=CH2 CH2 SCH3 . The electronic structure of the model complex in its Fe(I) Fe(II) state was investigated by means of density functional theory (DFT) calculations and Fourier transform infrared (FTIR) spectroscopy. By using a combination of continuous-wave (CW) electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) experiments as well as DFT calculations, it is shown that, for this complex, the spin density is delocalized over both iron atoms. Interestingly, we found that the nitrogen hyperfine coupling, which represents the interaction between the unpaired electron and the nitrogen at the dithiolate bridge, is slightly larger than that in the analogous complex in which the CN(-) ligands are replaced with PMe3 ligands. This reveals, first, that the CN(-) /PMe3 ligands coordinated to the iron core are electronically coupled to the amine in the adt bridge. Second, the CN(-) ligands in this complex are somewhat stronger ?-donor ligands than the PMe3 ligand, and thereby enable more spin density to be transferred from the Fe core to the adt unit, which might in turn affect the reactivity of the bridging amine. PMID:24038239

Erdem, Ozlen F; Stein, Matthias; Kaur-Ghumaan, Sandeep; Reijerse, Edward J; Ott, Sascha; Lubitz, Wolfgang

2013-09-13

4

A unifying structural and electronic concept for Hmd and [FeFe] hydrogenase active sites.  

PubMed

The hydrogenases [FeFe] and Hmd feature at first sight rather different active sites. A closer inspection reveals striking similarities, which allow us to define swapped ligand spheres in such a way that the single active iron center of Hmd functions in a first-shell ligand environment resembling the reacting iron atom in [FeFe] hydrogenase and vice versa. These redesigned ligand environments can be conveniently studied with quantum chemical methods and point to general reactivity principles for iron centers with hydrogenase activity. PMID:20527808

Stiebritz, Martin T; Reiher, Markus

2010-07-01

5

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

PubMed Central

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

Stapleton, James A.; Swartz, James R.

2010-01-01

6

Steps along the path to dihydrogen activation at [FeFe] hydrogenase structural models: dependence of the core geometry on electrocatalytic proton reduction.  

PubMed

Differences in the rate of electrocatalytic proton reduction by Fe2(mu-PPh2)2(CO)6, DP, and the linked phosphido-bridged analogue Fe2(mu,mu-PPh(CH2)3PPh)(CO)6, 3P, suggest that dihydrogen elimination proceeds through a bridging hydride. The reaction path was examined using electrochemical, spectroscopic, and in silico studies where reduction of 3P gives a moderately stable monoanion [Kdisp(3P-) = 13] and a distorted dianion. The monomeric formulation of 3P- is supported by the form of the IR and EPR spectra. EXAFS analysis of solutions of 3P, 3P-, and 3P2- indicates a large increase in the Fe-Fe separation following reduction (from 2.63 to ca. 3.1-3.55 A). DFT calculations of the 3P, 3P-, 3P2- redox series satisfactorily reproduce the IR spectra in the nu(CO) region and the crystallographic (3P) and EXAFS-derived Fe-Fe distances. Digital simulation of the electrocatalytic response for proton reduction indicates a low rate of dihydrogen evolution from the two-electron, two-proton product of 3P (H23P), with more rapid dihydrogen evolution following further reduction of H23P. Because dihydrogen evolution is not observed upon formation of H2DP, dihydrogen evolution at the two-electron-reduced level does not involve protonation of a hydridic Fe-H ligand. The rates of dihydrogen elimination from H2DP, H23P, and H2Fe2(mu,mu-S(CH2)3S)(CO)6 (H23S) are related to the DFT-calculated H-H distances [H23S (1.880 A) < H23P (2.064 A) < H2DP (3.100 A)], and this suggests a common reaction path for the thiolato- and phosphido-bridged diiron carbonyl compounds. PMID:17256930

Cheah, Mun Hon; Borg, Stacey J; Best, Stephen P

2007-01-27

7

Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii.  

PubMed

Hydrogenases catalyze the reversible oxidation of molecular hydrogen. The active site of the [FeFe] hydrogenases (H-cluster) contains a catalytically active binuclear subcluster ([2Fe](H)) connected to a "cubane" [4Fe4S](H) subcluster. Here we present an IR spectroelectrochemical study of the [FeFe] hydrogenase HydA1 isolated from the green alga Chlamydomonas reinhardtii. The enzyme shows IR bands similar to those observed for bacterial [FeFe] hydrogenases. They are assigned to the stretching vibrations of the CN(-) and CO ligands on both irons of the [2Fe](H) subcluster. By following changes in frequencies of the IR bands during electrochemical titrations, two one-electron redox processes of the active enzyme could be distinguished. The reduction of the oxidized state (H(ox)) occurred at a midpoint potential of -400 mV vs NHE (H(ox)/H(red) transition) and relates to a change of the formal oxidation state of the binuclear subcluster. A subsequent reduction (H(red)/H(sred) transition) was determined to have a midpoint potential of -460 mV vs NHE. On the basis of the IR spectra, it is suggested that the oxidation state of the binuclear subcluster does not change in this transition. Tentatively, a reduction of the [4Fe4S](H) cluster has been proposed. In contrast to the bacterial [FeFe] hydrogenases, where the bridging CO ligand becomes terminal when going from H(ox) to H(red), in HydA1 the bridging CO is present in both the H(ox) and H(red) state. The removal of the bridging CO moiety has been observed in the H(red) to H(sred) transition. The significance of this result for the hydrogen conversion mechanism of this class of enzymes is discussed. PMID:19634879

Silakov, Alexey; Kamp, Christina; Reijerse, Eduard; Happe, Thomas; Lubitz, Wolfgang

2009-08-25

8

Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.  

PubMed

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

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

2013-01-24

9

Raman Spectroscopy of Charge Transfer Interactions Between Single Wall Carbon Nanotubes and [FeFe] Hydrogenase  

SciTech Connect

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.

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

2008-01-01

10

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

PubMed

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

Chang, Christopher H

2011-07-19

11

Advanced electron paramagnetic resonance and density functional theory study of a {2Fe3S} cluster mimicking the active site of [FeFe] hydrogenase.  

PubMed

Despite extensive investigations of the active site of the [FeFe] hydrogenases, many details concerning the properties of the "hydrogen converting cluster" are not yet fully understood. The complexity of the so-called H-cluster is one of the main difficulties in studying the properties of its components. The present study is aimed at the mixed-valence EPR active [Fe2(?-CO)(CO)3(CN)2{MeSCH2C(Me)(CH2S)2}](1-) that is structurally closely related to the redox active binuclear part of the H-cluster in its CO-inhibited oxidized state. In this work, we present a characterization of this compound by advanced pulse EPR methods. The accurate determination of the (57)Fe, (1)H, (2)H, (14)N, and (15)N electron nuclear hyperfine interactions provided a very detailed picture of the electronic structure of this complex. A theoretical study using density functional theory (DFT) calculations identified possible isomers of the compound and further refined the knowledge about its properties. It was found that upon one electron oxidation of the parent Fe(I)-Fe(I) complex, the dominant mixed-valence Fe(I)-Fe(II) species is the one in which the CN ligand of the iron center that is distal to the thioether moves from the basal to the apical position. The unpaired spin distribution of the model complex is found to be clearly different from that of the native H-cluster. These differences are discussed and provide new insight into the functional features of the [FeFe] hydrogenase active site. PMID:21082840

Silakov, Alexey; Shaw, Jennifer L; Reijerse, Eduard J; Lubitz, Wolfgang

2010-11-17

12

Homologous overexpression of [FeFe] hydrogenase in Enterobacter cloacae IIT-BT 08 to enhance hydrogen gas production from cheese whey  

Microsoft Academic Search

The present study investigated the influence of increase in intracellular [FeFe] hydrogenase levels, in Enterobacter cloacae IIT-BT 08, on the formation of molecular hydrogen. The hydA gene from E. cloacae IIT-BT 08 was successfully amplified and cloned downstream of a tac promoter in an Escherichiacoli-Enterobacter reconstructed pGEX-Kan shuttle vector and introduced into E. cloacae. Finally E. cloacae strain carrying multiple

Namita Khanna; Chitralekha Nag Dasgupta; Preeti Mishra; Debabrata Das

2011-01-01

13

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

PubMed

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

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

2013-10-25

14

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

PubMed

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

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

2010-05-26

15

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

PubMed

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

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

2008-11-15

16

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

PubMed Central

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

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

2012-01-01

17

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

Microsoft Academic Search

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

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

18

Towards [NiFe]-hydrogenase biomimetic models that couple H2 binding with functionally relevant intramolecular electron transfers: a quantum chemical study.  

PubMed

[FeFe]- and [NiFe]-hydrogenases are dihydrogen-evolving metalloenzymes that share striking structural and functional similarities, despite being phylogenetically unrelated. Most notably, they are able to combine substrate binding and redox functionalities, which has important bearings on their efficiency. Model complexes of [FeFe]-hydrogenases that are able to couple H2 binding with a substrate-dependent intramolecular electron transfer promoting dihydrogen activation were recently shown to reproduce the complex redox chemistry of the all-iron enzyme. Notably, coupling of H2 binding and intramolecular redox events was proposed to have a key role also in [NiFe]-hydrogenases, but this feature is not reproduced in currently available nickel-iron biomimetic compounds. In the present study, we exploit dedicated density functional theory approaches to show that H2 binding and activation on a NiFe core can be favored by the installment of conveniently substituted isocyanoferrocenes, thanks to their ability to undergo intramolecular reduction upon substrate binding. Our results support the concept that a unified view on hydrogenase chemistry is a key element to direct future efforts in the modeling of microbial H2 metabolism. PMID:23921968

Greco, Claudio

2013-08-06

19

Maturation of hydrogenases.  

PubMed

Enzymes possessing the capacity to oxidize molecular hydrogen have developed convergently three class of enzymes leading to: [FeFe]-, [NiFe]-, and [FeS]-cluster-free hydrogenases. They differ in the composition and the structure of the active site metal centre and the sequence of the constituent structural polypeptides but they show one unifying feature, namely the existence of CN and/or CO ligands at the active site Fe. Recent developments in the analysis of the maturation of [FeFe]- and [NiFe]- hydrogenases have revealed a remarkably complex pattern of mostly novel biochemical reactions. Maturation of [FeFe]-hydrogenases requires a minimum of three auxiliary proteins, two of which belong to the class of Radical-SAM enzymes and other to the family of GTPases. They are sufficient to generate active enzyme when their genes are co-expressed with the structural genes in a heterologous host, otherwise deficient in [FeFe]-hydrogenase expression. Maturation of the large subunit of [NiFe]-hydrogenases depends on the activity of at least seven core proteins that catalyse the synthesis of the CN ligand, have a function in the coordination of the active site iron, the insertion of nickel and the proteolytic maturation of the large subunit. Whereas this core maturation machinery is sufficient to generate active hydrogenase in the cytoplasm, like that of hydrogenase 3 from Escherichia coli, additional proteins are involved in the export of the ready-assembled heterodimeric enzyme to the periplasm via the twin-arginine translocation system in the case of membrane-bound hydrogenases. A series of other gene products with intriguing putative functions indicate that the minimal pathway established for E. coli [NiFe]-hydrogenase maturation may possess even higher complexity in other organisms. PMID:17091562

Böck, August; King, Paul W; Blokesch, Melanie; Posewitz, Matthew C

2006-01-01

20

Attachment of a hydrogen-bonding carboxylate side chain to an [FeFe]-hydrogenase model complex: influence on the catalytic mechanism.  

PubMed

Azapropanedithiolate (adt)-bridged model complexes of [FeFe]-hydrogenase bearing a carboxylic acid functionality have been designed with the aim of decreasing the potential for reduction of protons to hydrogen. Protonation of the bisphosphine complexes 4-6 has been studied by in situ IR and NMR spectroscopy, which revealed that protonation with triflic acid most likely takes place first at the N-bridge for complex 4 but at the Fe-Fe bond for complexes 5 and 6. Using an excess of acid, the diprotonated species could also be observed, but none of the protonated species was sufficiently stable to be isolated in a pure state. Electrochemical studies have provided an insight into the catalytic mechanisms under strongly acidic conditions, and have also shown that complexes 3 and 6 are electro-active in aqueous solution even in the absence of acid, presumably due to hydrogen bonding. Hydrogen evolution, driven by visible light, has been observed for three-component systems consisting of [Ru(bpy)(3)](2+), complex 1, 2, or 3, and ascorbic acid in CH(3)CN/D(2)O solution by on-line mass spectrometry. PMID:20077533

Gao, Weiming; Sun, Junliang; Akermark, Torbjörn; Li, Mingrun; Eriksson, Lars; Sun, Licheng; Akermark, Björn

2010-02-22

21

Roles of HynAB and Ech, the Only Two Hydrogenases Found in the Model Sulfate Reducer Desulfovibrio gigas.  

PubMed

Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ?echBC and ?hynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism. PMID:23974026

Morais-Silva, Fabio O; Santos, Catia I; Rodrigues, Rute; Pereira, Inês A C; Rodrigues-Pousada, Claudina

2013-08-23

22

Synthesis and characterization of a series of model complexes of the active site of [Fe]-hydrogenase (Hmd).  

PubMed

A series of Fe complexes were synthesized and characterized as small molecule mimics for the active site of [Fe]-hydrogenase (Hmd). The collection includes both structurally new compounds and analogues of previously reported models. These complexes contain the essential ligands of the enzyme, namely, acyl, CO, pyridone, and sulfur ligands. They serve as IR and Mo?ssbauer spectroscopic models for the Fe center in [Fe]-hydrogenase. The field-dependent Mo?ssbauer study of representative model complexes shows that the sign and absolute value of the quadrupole splitting are sensitive to the change in the ligand environment of the Fe center. PMID:21539357

Chen, Dafa; Ahrens-Botzong, Annegret; Schünemann, Volker; Scopelliti, Rosario; Hu, Xile

2011-05-03

23

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

SciTech Connect

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

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

2009-03-13

24

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

PubMed

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

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

2012-12-12

25

Rewiring hydrogenase-dependent redox circuits in cyanobacteria  

PubMed Central

Hydrogenases catalyze the reversible reaction 2H+ + 2e-?H2 with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner. To examine the possibility of increasing the photobiological production of hydrogen within cyanobacterial cultures, we expressed the [FeFe] hydrogenase, HydA, from Clostridium acetobutylicum in the non-nitrogen-fixing cyanobacterium Synechococcus elongatus sp. 7942. We demonstrate that the heterologously expressed hydrogenase is functional in vitro and in vivo, and that the in vivo hydrogenase activity is connected to the light-dependent reactions of the electron transport chain. Under anoxic conditions, HydA activity is capable of supporting light-dependent hydrogen evolution at a rate > 500-fold greater than that supported by the endogenous [NiFe] hydrogenase. Furthermore, HydA can support limited growth solely using H2 and light as the source of reducing equivalents under conditions where Photosystem II is inactivated. Finally, we demonstrate that the addition of exogenous ferredoxins can modulate redox flux in the hydrogenase-expressing strain, allowing for greater hydrogen yields and for dark fermentation of internal energy stores into hydrogen gas.

Ducat, Daniel C.; Sachdeva, Gairik; Silver, Pamela A.

2011-01-01

26

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

PubMed Central

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

Dubini, Alexandra; Mus, Florence; Seibert, Michael; Grossman, Arthur R.; Posewitz, Matthew C.

2009-01-01

27

The mechanism of hydrogen uptake in [NiFe] hydrogenase: first-principles molecular dynamics investigation of a model compound.  

PubMed

The recent discovery of a model compounds of [NiFe] hydrogenase that catalyzes the heterolytic cleavage of the H(2) molecule into a proton and a stable hydride in water solution under room conditions opened up the possibility to understand the mechanism of H(2) uptake by this peculiar class of enzymes. The simplest model compound belongs to the class of NiRu bimetallic cationic complexes mimicking, in water solution and at room conditions, the hydrogenase active site. By using first-principles molecular dynamics computer simulations, in the Car-Parrinello scheme, we investigated models including the water solvent and nitrate counterions. Several simulations, starting from different initial configurations, provided information on the first step of the H(2) cleavage: (1) the pathway of H(2) approach towards the active site; (2) the role of the ruthenium-bonded water molecule in providing a base that extracts the proton from the activated H(2) molecule; (3) the minor role of Ni in activating the H(2) molecule and its role in stabilizing the hydride produced. PMID:21892688

Furlan, Sara; La Penna, Giovanni

2011-09-03

28

Brownian Dynamics and Molecular Dynamics Study of the Association Between Hydrogenase and Ferredoxin from the Chlamydomonas reinhardtii  

SciTech Connect

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

Long, H.; Chang, C. H.; King, P. W.; Ghirardi, M. L.; Kim, K.

2008-10-01

29

Synthesis and reactivity of iron acyl complexes modeling the active site of [Fe]-hydrogenase.  

PubMed

A [Fe(II)Fe(II)] dithiolate complex containing acyl and carbonyl ligands was synthesized. The diiron complex reacted with phosphine, cyanide, isocyanide, and CO to give monomeric Fe(II) complexes reproducing the first coordination sphere of the active site of [Fe]-hydrogenase (H(2)-forming methylene-tetrahydromethanopterin dehydrogenase, Hmd) in various states. All the acyl and carbonyl carbons in the diiron complex underwent facile isotopic exchange with (13)CO via monomeric Fe tricarbonyl intermediates. PMID:20041643

Chen, Dafa; Scopelliti, Rosario; Hu, Xile

2010-01-27

30

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

PubMed

Described are experiments demonstrating incorporation of cyanide cofactors and hydride substrate into [NiFe]-hydrogenase (H2ase) active site models. Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe) (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BAr(F)3) to give the adducts (CO)2(CNBAr(F)3)2Fe(pdt)Ni(dxpe), (1(BAr(F)3)2, 2(BAr(F)3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives [(CO)(CNBAr(F)3)2Fe(H)(pdt)Ni(dxpe)](-) (dxpe = dppe, [H3(BAr(F)3)2](-); dxpe = dcpe, [H4(BAr(F)3)2](-)). Crystallographic analysis shows that Et4N[H3(BAr(F)3)2] generally resembles the active site of the enzyme in the reduced, hydride-containing states (Ni-C/R). The Fe-H···Ni center is unsymmetrical with rFe-H = 1.51(3) Å and rNi-H = 1.71(3) Å. Both crystallographic and (19)F NMR analyses show that the CNBAr(F)3(-) ligands occupy basal and apical sites. Unlike cationic Ni-Fe hydrides, [H3(BAr(F)3)2](-) and [H4(BAr(F)3)2](-) oxidize at mild potentials, near the Fc(+/0) couple. Electrochemical measurements indicate that in the presence of base, [H3(BAr(F)3)2](-) catalyzes the oxidation of H2. NMR evidence indicates dihydrogen bonding between these anionic hydrides and R3NH(+) salts, which is relevant to the mechanism of hydrogenogenesis. In the case of Et4N[H3(BAr(F)3)2], strong acids such as HCl induce H2 release to give the chloride Et4N[(CO)(CNBAr(F)3)2Fe(Cl)(pdt)Ni(dppe)]. PMID:23899049

Manor, Brian C; Rauchfuss, Thomas B

2013-07-30

31

Absolute potential of the Fermi level of single-walled carbon nanotubes via hydrogenase complex formation  

Microsoft Academic Search

The absolute potential of the Fermi level of nanotubes as a function of nanotube type is not presently understood, and is important for many nanotube applications and sorting strategies. Here, we study complexes of recombinant [FeFe] hydrogenases and single-walled carbon nanotubes. We find evidence that novel charge-transfer complexes are formed and are stable, which enables further study and application of

Timothy McDonald; Drazenka Svedruzic; Yong-Hyun Kim; Jeffrey Blackburn; Shengbai Zhang; Paul King; Michael Heben

2007-01-01

32

The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production.  

PubMed

The hyperthermophilic and anaerobic bacterium Thermotoga maritima ferments a wide variety of carbohydrates, producing acetate, CO(2), and H(2). Glucose is degraded through a classical Embden-Meyerhof pathway, and both NADH and reduced ferredoxin are generated. The oxidation of these electron carriers must be coupled to H(2) production, but the mechanism by which this occurs is unknown. The trimeric [FeFe]-type hydrogenase that was previously purified from T. maritima does not use either reduced ferredoxin or NADH as a sole electron donor. This problem has now been resolved by the demonstration that this hydrogenase requires the presence of both electron carriers for catalysis of H(2) production. The enzyme oxidizes NADH and ferredoxin simultaneously in an approximately 1:1 ratio and in a synergistic fashion to produce H(2). It is proposed that the enzyme represents a new class of bifurcating [FeFe] hydrogenase in which the exergonic oxidation of ferredoxin (midpoint potential, -453 mV) is used to drive the unfavorable oxidation of NADH (E(0)' = -320 mV) to produce H(2) (E(0)' = -420 mV). From genome sequence analysis, it is now clear that there are two major types of [FeFe] hydrogenases: the trimeric bifurcating enzyme and the more well-studied monomeric ferredoxin-dependent [FeFe] hydrogenase. Almost one-third of the known H(2)-producing anaerobes appear to contain homologs of the trimeric bifurcating enzyme, although many of them also harbor one or more homologs of the simpler ferredoxin-dependent hydrogenase. The discovery of the bifurcating hydrogenase gives a new perspective on our understanding of the bioenergetics and mechanism of H(2) production and of anaerobic metabolism in general. PMID:19411328

Schut, Gerrit J; Adams, Michael W W

2009-05-01

33

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

PubMed Central

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

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

2010-01-01

34

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

PubMed

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

Baffert, Carole; Artero, Vincent; Fontecave, Marc

2007-02-02

35

Catalytic Activation of H2 under Mild Conditions by an [FeFe]-Hydrogenase Model via an Active ?-Hydride Species.  

PubMed

A [FeFe]-hydrogenase model (1) containing a chelating diphosphine ligand with a pendant amine was readily oxidized by Fc(+) (Fc = Cp2Fe) to a Fe(II)Fe(I) complex ([1](+)), which was isolated at room temperature. The structure of [1](+) with a semibridging CO and a vacant apical site was determined by X-ray crystallography. Complex [1](+) catalytically activates H2 at 1 atm at 25 °C in the presence of excess Fc(+) and P(o-tol)3. More interestingly, the catalytic activity of [1](+) for H2 oxidation remains unchanged in the presence of ca. 2% CO. A computational study of the reaction mechanism showed that the most favorable activation free energy involves a rotation of the bridging CO to an apical position followed by activation of H2 with the help of the internal amine to give a bridging hydride intermediate. PMID:24001095

Wang, Ning; Wang, Mei; Wang, Ying; Zheng, Dehua; Han, Hongxian; Ahlquist, Mårten S G; Sun, Licheng

2013-09-06

36

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

PubMed Central

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

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

2012-01-01

37

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

SciTech Connect

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

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

2008-01-01

38

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

PubMed

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

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

2009-04-20

39

A theoretical study on the enhancement of functionally relevant electron transfers in biomimetic models of [FeFe]-hydrogenases.  

PubMed

Recent advances aimed at modeling the chemistry of the active site of [FeFe]-hydrogenases (the H-cluster, composed by a catalytic Fe(2)S(2) subcluster and an Fe(4)S(4) portion) have led to the synthesis of binuclear coordination compounds containing a noninnocent organophosphine ligand [2,3-bis(diphenylphosphino)maleic anhydride, bma] that is able to undergo monoelectron reduction, analogously to the tetranuclear Fe(4)S(4) subcluster portion of the H-cluster. However, such a synthetic model was shown to feature negligible electronic communication between the noninnocent ligand and the remaining portion of the cluster, at variance with the enzyme active site. Here, we report a theoretical investigation that shows why the electron transfer observed in the enzyme upon protonation of the catalytic Fe(2)S(2) subsite cannot take place in the bma-containing cluster. In addition, we show that targeted modifications of the bma ligand are sufficient to restore the electronic communication within the model, such that electron density can be more easily withdrawn from the noninnocent ligand, as a result of protonation of the iron centers. Similar results were also obtained with a ligand derived from cobaltocene. The relevance of our findings is discussed from the perspective of biomimetic reproduction of proton reduction to yield molecular hydrogen. PMID:21728321

Greco, Claudio; De Gioia, Luca

2011-07-05

40

Studies on hydrogenase  

PubMed Central

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

YAGI, Tatsuhiko; HIGUCHI, Yoshiki

2013-01-01

41

Multiple Forms of Bacterial Hydrogenases  

PubMed Central

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

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

1966-01-01

42

Iron acyl thiolato carbonyls: structural models for the active site of the [Fe]-hydrogenase (Hmd).  

PubMed

Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls, which replicate key structural features of the active site of the hydrogenase Hmd. The reaction of Ph(2)PC(6)H(4)C(O)SPh and sources of Fe(0) generates both Fe(SPh)(Ph(2)PC(6)H(4)CO)(CO)(3) (1) and the diferrous diacyl Fe(2)(SPh)(2)(CO)(3)(Ph(2)PC(6)H(4)CO)(2), which carbonylates to give 1. For the extremely bulky arylthioester Ph(2)PC(6)H(4)C(O)SC(6)H(3)-2,6-(2,4,6-trimethylphenyl)(2), oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained. Complex 1 reacts with cyanide to give Et(4)N[Fe(SPh)(Ph(2)PC(6)H(4)CO)(CN)(CO)(2)] (Et(4)N[2]). (13)C and (31)P NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2](-) in ?(CN) and ?(CO) regions very closely matches that for Hmd(CN). XANES and EXAFS measurements also indicate close structural and electronic similarity of Et(4)N[2] to the active site of wild-type Hmd. Complex 1 also stereospecifically forms a derivative with TsCH(2)NC, but the adduct is more labile than Et(4)N[2]. Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd. PMID:21062066

Royer, Aaron M; Salomone-Stagni, Marco; Rauchfuss, Thomas B; Meyer-Klaucke, Wolfram

2010-11-09

43

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

NASA Astrophysics Data System (ADS)

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

Camara, James M.; Rauchfuss, Thomas B.

2012-01-01

44

Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms.  

PubMed

The photobiological production of H2 gas, using water as the only electron donor, is a property of two types of photosynthetic microorganisms: green algae and cyanobacteria. In these organisms, photosynthetic water splitting is functionally linked to H(2) production by the activity of hydrogenase enzymes. Interestingly, each of these organisms contains only one of two major types of hydrogenases, [FeFe] or [NiFe] enzymes, which are phylogenetically distinct but perform the same catalytic reaction, suggesting convergent evolution. This idea is supported by the observation that each of the two classes of hydrogenases has a different metallo-cluster, is encoded by entirely different sets of genes (apparently under the control of different promoter elements), and exhibits different maturation pathways. The genetics, biosynthesis, structure, function, and O2 sensitivity of these enzymes have been the focus of extensive research in recent years. Some of this effort is clearly driven by the potential for using these enzymes in future biological or biohybrid systems to produce renewable fuel or in fuel cell applications. PMID:17150028

Ghirardi, Maria L; Posewitz, Matthew C; Maness, Pin-Ching; Dubini, Alexandra; Yu, Jianping; Seibert, Michael

2007-01-01

45

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

NASA Astrophysics Data System (ADS)

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

Chang, Christopher H.; Kim, Kwiseon

2007-03-01

46

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

PubMed

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

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

2012-06-08

47

Absolute potential of the Fermi level of single-walled carbon nanotubes via hydrogenase complex formation.  

NASA Astrophysics Data System (ADS)

The absolute potential of the Fermi level of nanotubes as a function of nanotube type is not presently understood, and is important for many nanotube applications and sorting strategies. Here, we study complexes of recombinant [FeFe] hydrogenases and single-walled carbon nanotubes. We find evidence that novel charge-transfer complexes are formed and are stable, which enables further study and application of this system. The hydrogenase functions as a hydrogen electrode sensitizing the nanotubes to the redox half-reaction for hydrogen. Thus the potential can be altered by changing the molecular hydrogen concentration, and this tunability is utilized to bleach various semiconducting nanotube transitions. By observing which are bleached and which remain emissive, we determine the alignment of the potential of the Fermi level of semiconducting single-walled carbon nanotubes. The experimentally determined Fermi level alignment is confirmed theoretically by the first-principles DFT-PBE method.

McDonald, Timothy; Svedruzic, Drazenka; Kim, Yong-Hyun; Blackburn, Jeffrey; Zhang, Shengbai; King, Paul; Heben, Michael

2007-03-01

48

Protein induced singlet-triplet quasidegeneracy in the active site of [NiFe]-hydrogenase  

NASA Astrophysics Data System (ADS)

Molecular hydrogen oxidation and reduction on [NiFe]-hydrogenase is an inspiring example of using abundant first row transition metals to catalyze biologically and industrially important chemical reactions. We demonstrate that by rotating terminal thiolate ligands in the active site of [NiFe]-hydrogenase, either the singlet or triplet electronic state can be made a ground state. The two states become degenerate when the ligand orientations are similar to those observed in [NiFe]-hydrogenase, where this orientation is enforced by the protein backbone. The unusual distorted coordination geometry of Ni can explain the inability of the structural models of [NiFe]-hydrogenase to bind molecular hydrogen.

Yson, Renante L.; Gilgor, Jessica L.; Guberman, Benjamin A.; Varganov, Sergey A.

2013-07-01

49

Nitrogenases and Hydrogenases in Cyanobacteria  

Microsoft Academic Search

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

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

50

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

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.

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

51

Cyanide inactivation of hydrogenase from Azotobacter vinelandii.  

PubMed Central

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

Seefeldt, L C; Arp, D J

1989-01-01

52

Synthetic and structural studies on new diiron azadithiolate (ADT)-type model compounds for active site of [FeFe]hydrogenases.  

PubMed

A series of new diiron azadithiolate (ADT) complexes (1-8), which could be regarded as the active site models of [FeFe]hydrogenases, have been synthesized starting from parent complex [(?-SCH(2))(2)NCH(2)CH(2)OH]Fe(2)(CO)(6) (A). Treatment of A with ethyl malonyl chloride or malonyl dichloride in the presence of pyridine afforded the malonyl-containing complexes [(?-SCH(2))(2)NCH(2)CH(2)O(2)CCH(2)CO(2)Et]Fe(2)(CO)(6) (1) and [Fe(2)(CO)(6)(?-SCH(2))(2)NCH(2)CH(2)O(2)C](2)CH(2) (2). Further treatment of 1 and 2 with PPh(3) under different conditions produced the PPh(3)-substituted complexes [(?-SCH(2))(2)NCH(2)CH(2)O(2)CCH(2)CO(2)Et]Fe(2)(CO)(5)(PPh(3)) (3), [(?-SCH(2))(2)NCH(2)CH(2)O(2)CCH(2)CO(2)Et]Fe(2)(CO)(4)(PPh(3))(2) (4), and [Fe(2)(CO)(5)(PPh(3))(?-SCH(2))(2)NCH(2)CH(2)O(2)C](2)CH(2) (5). More interestingly, complexes 1-3 could react with C(60) in the presence of CBr(4) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) via Bingel-Hirsch reaction to give the C(60)-containing complexes [(?-SCH(2))(2)NCH(2)CH(2)O(2)CC(C(60))CO(2)Et]Fe(2)(CO)(6) (6), [Fe(2)(CO)(6)(?-SCH(2))(2)NCH(2)CH(2)O(2)C](2)C(C(60)) (7), and [(?-SCH(2))(2)NCH(2)CH(2)O(2)CC(C(60))CO(2)Et]Fe(2)(CO)(5)(PPh(3)) (8). The new ADT-type models 1-8 were characterized by elemental analysis and spectroscopy, whereas 2-4 were further studied by X-ray crystallography and 6-8 investigated in detail by DFT methods. PMID:21152555

Song, Li-Cheng; Xie, Zhao-Jun; Liu, Xu-Feng; Ming, Jiang-Bo; Ge, Jian-Hua; Zhang, Xiao-Guang; Yan, Tian-Ying; Gao, Peng

2010-12-09

53

Redox and Structural Properties of Mixed-Valence Models for the Active Site of the [FeFe]-Hydrogenase: Progress and Challenges  

PubMed Central

The one-electron oxidations of a series of diiron(I) dithiolato carbonyls were examined to evaluate the factors that affect the oxidation state assignments, structures, and reactivity of these low-molecular weight models for the Hox state of the [FeFe]-hydrogenases. The propanedithiolates Fe2(S2C3H6)(CO)3(L)(dppv) (L = CO, PMe3, Pi-Pr3) oxidize at potentials ~180 mV milder than the related ethanedithiolates (Angew. Chem. Int. Ed. 2007, 46, 6152). The steric clash between the central methylene of the propanedithiolate and the phosphine favors the rotated structure, which forms upon oxidation. EPR spectra for the mixed-valence cations indicate that the unpaired electron is localized on the Fe(CO)(dppv) center in both [Fe2(S2C3H6)(CO)4(dppv)]BF4 and [Fe2(S2C3H6)(CO)3(PMe3)(dppv)]BF4, as seen previously for the ethanedithiolate [Fe2(S2C2H4)(CO)3(PMe3)(dppv)]BF4. For [Fe2(S2CnH2n)(CO)3(Pi-Pr3)(dppv)]BF4, however, the spin is localized on the Fe(CO)2(Pi-Pr3) center, although the Fe(CO)(dppv) site is rotated in the crystalline state. IR and EPR spectra, as well as redox potentials and DFT-calculations, suggest, however, that the Fe(CO)2(Pi-Pr3) site is rotated in solution, driven by steric factors. Analysis of the DFT-computed partial atomic charges for the mixed-valence species shows that the Fe atom featuring a vacant apical coordination position is an electrophilic Fe(I) center. One-electron oxidation of [Fe2(S2C2H4)(CN)(CO)3(dppv)]? resulted in 2e oxidation of 0.5 equiv to give the ?-cyano derivative [FeI2(S2C2H4)(CO)3(dppv)](?-CN)[FeII2(S2C2H4)(?-CO)(CO)2(CN)(dppv)], which was characterized spectroscopically.

Justice, Aaron K.; Nilges, Mark J.; Wilson, Scott R.; Zampella, Giuseppe

2008-01-01

54

Binuclear iron-sulfur complexes with bidentate phosphine ligands as active site models of Fe-hydrogenase and their catalytic proton reduction.  

PubMed

The displacement of CO in a few simple Fe(I)-Fe(I) hydrogenase model complexes by bisphosphine ligands Ph2P-(CH2)n-PPh2 [with n = 1 (dppm) or n = 2 (dppe)] is described. The reaction of [{mu-(SCH2)2CH2}Fe2(CO)6] (1) and [{mu-(SCH2)2N(CH2CH2CH3)}Fe2(CO)6] (2) with dppe gave double butterfly complexes [{mu-(SCH2)2CH2}Fe2(CO)5(Ph2PCH2)]2 (3) and [{mu-(SCH2)2N(CH2CH2CH3)}Fe2(CO)5(Ph2PCH2)]2 (4), where two Fe2S2 units are linked by the bisphosphine. In addition, an unexpected byproduct, [{mu-(SCH2)2N(CH2CH2CH3)}Fe2(CO)5{Ph2PCH2CH2(Ph2PS)}] (5), was isolated when 2 was used as a substrate, where only one phosphorus atom of dppe is coordinated, while the other has been converted to P=S, presumably by nucleophilic attack on bridging sulfur. By contrast, the reaction of 1 and 2 with dppm under mild conditions gave only complexes [{mu-(SCH2)2CH2}Fe2(CO)5(Ph2PCH2PPh2)] (6) and [{mu-(SCH2)2N(CH2CH2CH3)}Fe2(CO)5(Ph2PCH2PPh2)] (8), where one ligand coordinated in a monodentate fashion to one Fe2S2 unit. Furthermore, under forcing conditions, the complexes [{mu-(SCH2)2CH2}Fe2(CO)4{mu-(Ph2P)2CH2}] (7) and [{mu-(SCH2)2N(CH2CH2CH3)}Fe2(CO)4{mu-(Ph2P)2CH2}] (9) were formed, where the phosphine acts as a bidentate ligand, binding to both the iron atoms in the same molecular unit. Electrochemical studies show that the complexes 3, 4, and 9 catalyze the reduction of protons to molecular hydrogen, with 4 electrolyzed already at -1.40 V versus Ag/AgNO3 (-1.0 V vs NHE). PMID:17295467

Gao, Weiming; Ekström, Jesper; Liu, Jianhui; Chen, Changneng; Eriksson, Lars; Weng, Linhong; Akermark, Björn; Sun, Licheng

2007-02-13

55

Combined spectroscopic/computational study of binuclear Fe(I)-Fe(I) complexes: implications for the fully-reduced active-site cluster of Fe-only hydrogenases.  

PubMed

The Fe(I)-Fe(I) dimer complex [Fe2(pdt)(CO)4(CN)2][Et4N]2 (2), where pdt = 1,3-propane dithiolate, serves as a model of the fully reduced [2Fe]H component of the H cluster, which is the active site for catalysis in Fe-only hydrogenases (FeHases). Electronic absorption, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopies have been employed to characterize both the ground and excited states of 2 as well as those of the related complex Fe2(pdt)(CO)6 (1). These results have been combined with density functional theory (DFT) computations to produce experimentally validated bonding descriptions of 1 and 2. It is shown that Fe(I)-S covalency is significantly reduced upon dicyano substitution (i.e., conversion of 1 --> 2), while the corresponding Fe(I)-CO/CN pi-backbonding interactions are strengthened, results that are corroborated by normal-coordinate analyses of the vibrational data. Detailed assignments of the features observed in the electronic absorption spectra of 1 and 2 have been developed on the basis of time-dependent DFT (TD-DFT) calculations, which provide remarkably accurate simulations of the experimental data. For both complexes, all bands below 32,000 cm(-1) arise from transitions involving electronic excitation within the binuclear Fe-Fe core, with the most intense feature assigned to the Fe(sigma(b)) --> Fe(sigma*) transition. Analysis of the corresponding rR excitation profiles within the framework of time-dependent Heller theory reveals that in each case the Fe-Fe bond is elongated by approximately 0.3 A in the Fe(sigma(b)) --> Fe(sigma*) excited state. Finally, building upon the insights gained from the spectroscopic/computational studies of 1 and 2, our computational methodology has been extended to the reduced enzyme active site, providing insights into the electronic structure of the [2Fe]H subcluster in the H(red) state and its relationship to catalysis. PMID:15762706

Fiedler, Adam T; Brunold, Thomas C

2005-03-21

56

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120  

PubMed Central

Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the [NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.

2009-01-01

57

Active-site models for the nickel-iron hydrogenases: effects of ligands on reactivity and catalytic properties.  

PubMed

Described are new derivatives of the type [HNiFe(SR)(2)(diphosphine)(CO)(3)](+), which feature a Ni(diphosphine) group linked to a Fe(CO)(3) group by two bridging thiolate ligands. Previous work had described [HNiFe(pdt)(dppe)(CO)(3)](+) ([1H](+)) and its activity as a catalyst for the reduction of protons (J. Am. Chem. Soc. 2010, 132, 14877). Work described in this paper focuses on the effects on properties of NiFe model complexes of the diphosphine attached to nickel as well as the dithiolate bridge, 1,3-propanedithiolate (pdt) vs 1,2-ethanedithiolate (edt). A new synthetic route to these Ni-Fe dithiolates is described, involving reaction of Ni(SR)(2)(diphosphine) with FeI(2)(CO)(4) followed by in situ reduction with cobaltocene. Evidence is presented that this route proceeds via a metastable ?-iodo derivative. Attempted isolation of such species led to the crystallization of NiFe(Me(2)pdt)(dppe)I(2), which features tetrahedral Fe(II) and square planar Ni(II) centers (H(2)Me(2)pdt = 2,2-dimethylpropanedithiol). The new tricarbonyls prepared in this work are NiFe(pdt)(dcpe)(CO)(3) (2, dcpe = 1,2-bis(dicyclohexylphosphino)ethane), NiFe(edt)(dppe)(CO)(3) (3), and NiFe(edt)(dcpe)(CO)(3) (4). Attempted preparation of a phenylthiolate-bridged complex via the FeI(2)(CO)(4) + Ni(SPh)(2)(dppe) route gave the tetrametallic species [(CO)(2)Fe(SPh)(2)Ni(CO)](2)(?-dppe)(2). Crystallographic analysis of the edt-dcpe compund [2H]BF(4) and the edt-dppe compound [3H]BF(4) verified their close resemblance. Each features pseudo-octahedral Fe and square pyramidal Ni centers. Starting from [3H]BF(4) we prepared the PPh(3) derivative [HNiFe(edt)(dppe)(PPh(3))(CO)(2)]BF(4) ([5H]BF(4)), which was obtained as a ?2:1 mixture of unsymmetrical and symmetrical isomers. Acid-base measurements indicate that changing from Ni(dppe) (dppe = Ph(2)PCH(2)CH(2)PPh(2)) to Ni(dcpe) decreases the acidity of the cationic hydride complexes by 2.5 pK(a)(PhCN) units, from ?11 to ?13.5 (previous work showed that substitution at Fe leads to more dramatic effects). The redox potentials are more strongly affected by the change from dppe to dcpe, for example the [2](0/+) couple occurs at E(1/2) = -820 for [2](0/+) vs -574 mV (vs Fc(+/0)) for [1](0/+). Changes in the dithiolate do not affect the acidity or the reduction potentials of the hydrides. The acid-independent rate of reduction of CH(2)ClCO(2)H by [2H](+) is about 50 s(-1) (25 °C), twice that of [1H](+). The edt-dppe complex [2H](+) proved to be the most active catalyst, with an acid-independent rate of 300 s(-1). PMID:21866886

Carroll, Maria E; Barton, Bryan E; Gray, Danielle L; Mack, Amanda E; Rauchfuss, Thomas B

2011-08-25

58

Reversible hydrogenase in Anabaena variabilis ATCC 29413  

Microsoft Academic Search

The presence and localization of a reversible hydrogenase in non-N2-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE\\/activity stain, SDS-PAGE\\/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO2 for 20–26 h. Native-PAGE\\/activity stain demonstrated the presence

Larissa Serebriakova; Nikolaj A. Zorin; Peter Lindblad

1994-01-01

59

Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina?  

PubMed Central

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

Maroti, Gergely; Tong, Yingkai; Yooseph, Shibu; Baden-Tillson, Holly; Smith, Hamilton O.; Kovacs, Kornel L.; Frazier, Marvin; Venter, J. Craig; Xu, Qing

2009-01-01

60

Molecular biology of microbial hydrogenases.  

PubMed

Hydrogenases (H2ases) are metalloproteins. The great majority of them contain iron-sulfur clusters and two metal atoms at their active center, either a Ni and an Fe atom, the [NiFe]-H2ases, or two Fe atoms, the [FeFe]-H2ases. Enzymes of these two classes catalyze the reversible oxidation of hydrogen gas (H2 <--> 2 H+ + 2 e-) and play a central role in microbial energy metabolism; in addition to their role in fermentation and H2 respiration, H2ases may interact with membrane-bound electron transport systems in order to maintain redox poise, particularly in some photosynthetic microorganisms such as cyanobacteria. Recent work has revealed that some H2ases, by acting as H2-sensors, participate in the regulation of gene expression and that H2-evolving H2ases, thought to be involved in purely fermentative processes, play a role in membrane-linked energy conservation through the generation of a protonmotive force. The Hmd hydrogenases of some methanogenic archaea constitute a third class of H2ases, characterized by the absence of Fe-S cluster and the presence of an iron-containing cofactor with catalytic properties different from those of [NiFe]- and [FeFe]-H2ases. In this review, we emphasise recent advances that have greatly increased our knowledge of microbial H2ases, their diversity, the structure of their active site, how the metallocenters are synthesized and assembled, how they function, how the synthesis of these enzymes is controlled by external signals, and their potential use in biological H2 production. PMID:15119826

Vignais, P M; Colbeau, A

2004-07-01

61

O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase.  

PubMed

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) 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 O(2) 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 O(2) 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 (2Fe(H)), 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 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), 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 O(2)-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 O(2) tolerance in [FeFe]-hydrogenase. PMID:21930709

Lambertz, Camilla; Leidel, Nils; Havelius, Kajsa G V; Noth, Jens; Chernev, Petko; Winkler, Martin; Happe, Thomas; Haumann, Michael

2011-09-19

62

Purification of Hydrogenase from Chlamydomonas reinhardtii1  

PubMed Central

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H2 produced per milligram protein per minute. The molecular weight was determined to be 4.5 × 104 by gel filtration and 4.75 × 104 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H2 production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55°C. The Km of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag. Images Fig. 2

Roessler, Paul G.; Lien, Stephen

1984-01-01

63

Hydrogenases and H(+)-reduction in primary energy conservation.  

PubMed

Hydrogenases are metalloenzymes subdivided into two classes that contain iron-sulfur clusters and catalyze the reversible oxidation of hydrogen gas (H(2)[Symbol: see text]left arrow over right arrow[Symbol: see text]2H(+)[Symbol: see text]+[Symbol: see text]2e(-)). Two metal atoms are present at their active center: either a Ni and an Fe atom in the [NiFe]hydrogenases, or two Fe atoms in the [FeFe]hydrogenases. They are phylogenetically distinct classes of proteins. The catalytic core of [NiFe]hydrogenases is a heterodimeric protein associated with additional subunits in many of these enzymes. The catalytic core of [FeFe]hydrogenases is a domain of about 350 residues that accommodates the active site (H cluster). Many [FeFe]hydrogenases are monomeric but possess additional domains that contain redox centers, mostly Fe-S clusters. A third class of hydrogenase, characterized by a specific iron-containing cofactor and by the absence of Fe-S cluster, is found in some methanogenic archaea; this Hmd hydrogenase has catalytic properties different from those of [NiFe]- and [FeFe]hydrogenases. The [NiFe]hydrogenases can be subdivided into four subgroups: (1) the H(2) uptake [NiFe]hydrogenases (group 1); (2) the cyanobacterial uptake hydrogenases and the cytoplasmic H(2) sensors (group 2); (3) the bidirectional cytoplasmic hydrogenases able to bind soluble cofactors (group 3); and (4) the membrane-associated, energy-converting, H(2) evolving hydrogenases (group 4). Unlike the [NiFe]hydrogenases, the [FeFe]hydrogenases form a homogeneous group and are primarily involved in H(2) evolution. This review recapitulates the classification of hydrogenases based on phylogenetic analysis and the correlation with hydrogenase function of the different phylogenetic groupings, discusses the possible role of the [FeFe]hydrogenases in the genesis of the eukaryotic cell, and emphasizes the structural and functional relationships of hydrogenase subunits with those of complex I of the respiratory electron transport chain. PMID:18500479

Vignais, Paulette M

2008-01-01

64

A model for the CO-inhibited form of [NiFe] hydrogenase: synthesis of CO3Fe(micro-StBu)3Ni{SC6H3-2,6-(mesityl)2} and reversible CO addition at the Ni site.  

PubMed

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

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

2010-02-10

65

Action of inhibitors on hydrogenase in Azotobacter  

Microsoft Academic Search

The inhibitors usually associated with the activity of the cytochrome oxidase system - cyanide and carbon monoxide - are also effective in reducing the oxidation of Hâ by intact cells of Azotobacter vinclandii. The hydrogenase system is more sensitive to CO than is the respiratory system. Oxidation of a carbon source and of hydrogen by Azotobacter cells is inhibited in

J. B. Wilson; P. W. Wilson

1943-01-01

66

Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton Transfer Pathway in [NiFe] Hydrogenases  

PubMed Central

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis.

Szori-Doroghazi, Emma; Maroti, Gergely; Szori, Milan; Nyilasi, Andrea; Rakhely, Gabor; Kovacs, Kornel L.

2012-01-01

67

Biomimetic model featuring the NH proton and bridging hydride related to a proposed intermediate in enzymatic H(2) production by Fe-only hydrogenase.  

PubMed

Iron azadithiolate phosphine-substituted complex and its protonated species featuring the NH proton and/or bridging Fe hydride, [Fe(2)(mu-S(CH(2))(2)N(n)Pr(H)(m)(CH(2))(2)S)(mu-H)(n)(CO)(4)(PMe(3))(2)](2)((2m+2n)+) (1, m = n = 0; [1-2H(N)](2+), m = 1, n = 0; [1-2H(N)2H(Fe)](4+), m = n = 1), are prepared to mimic the active site of Fe-only hydrogenase. X-ray crystallographic analyses of these three complexes reveal that two diiron subunits are linked by two azadiethylenethiolate bridges to construct a dimer-of-dimer structure. (31)P NMR spectroscopy confirms two trimethylphosphine ligands within the diiron moiety are arranged in the apical/basal configuration, which is consistent with the solid-state structural characterization. Deprotonation of the NH proton in [1-2H(N)](2+) and [1-2H(N)2H(Fe)](4+) occurs in the presence of triethanolamine (TEOA), which generates 1 and [1-2H(Fe)](2+), respectively. Deprotonation of the Fe hydride is accomplished by addition of bistriphenylphosphineimminium chloride ([PPN]Cl). It is observed that the Fe hydride species, [1-2H(Fe)](2+), is a kinetic product which converts to its thermodynamically stable tautomer, [1-2H(N)](2+), in solution, as evidenced by IR and NMR spectroscopy. The pK(a) values of the aza nitrogen and the diiron sites are estimated to be 8.9-15.9 and <8.9, respectively. [1-2H(N)2H(Fe)](4+) has been observed to evolve H(2) electrocatalytically at a mild potential (-1.42 V vs Fc/Fc(+)) in CH(3)CN solution. Catalysis of [1-2H(N)2H(Fe)](4+) is found to be as efficient as that of the related diiron azadithiolate complexes. In the absence of a proton source, [1-2H(N)2H(Fe)](4+) undergoes four irreversible reduction processes at -1.26, -1.42, -1.82, and -2.43 V, which are attributed to the reduction events from [1-2H(N)2H(Fe)](4+), [1-2H(Fe)](2+), [1-2H(N)](2+), and 1, respectively, according to bulk electrolysis and voltammetry in combination of titration experiments with acids. PMID:19601586

Chiang, Ming-Hsi; Liu, Yu-Chiao; Yang, Shu-Ting; Lee, Gene-Hsiang

2009-08-17

68

A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases  

PubMed Central

In biology, rapid oxidation and evolution of H2 is catalyzed by metalloenzymes known as hydrogenases. These enzymes have unusual active sites, consisting of iron complexed by carbonyl, cyanide, and thiolate ligands, often together with nickel, and are typically inhibited or irreversibly damaged by O2. The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H2 as an energy source with O2 as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an “O2-tolerant” hydrogenase. The mechanism of O2 tolerance of Re MBH has been probed by measuring H2 oxidation activity in the presence of O2 over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O2 and subsequent reactivation of the active site. Most significantly, O2 tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H2 affinity or the kinetics of O2 attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O2 tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases.

Cracknell, James A.; Wait, Annemarie F.; Lenz, Oliver; Friedrich, Barbel; Armstrong, Fraser A.

2009-01-01

69

Hydrogenases and H + Reduction in Primary Energy Conservation  

Microsoft Academic Search

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

Paulette M. Vignais

70

Hydrogenase activity in the thermophile mastigocladus laminosus  

Microsoft Academic Search

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. Hâ-supported acetylene reduction in reductant-limited cultures was a light-dependent, but Oâ-independent reaction. In vitro hydrogen evolution was unaffected by carbon

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

1982-01-01

71

A bacterial electron-bifurcating hydrogenase.  

PubMed

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

Schuchmann, Kai; Müller, Volker

2012-07-18

72

A Bacterial Electron-bifurcating Hydrogenase*  

PubMed Central

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

Schuchmann, Kai; Muller, Volker

2012-01-01

73

Regulation of hydrogenase biosynthesis by nickel in Bradyrhizobium japonicum  

Microsoft Academic Search

The addition of 5 µM nickel to heterotropically growing hydrogen uptake constitutive mutants resulted in up to a 10-fold increase in hydrogen uptake activity. Strain SR wild type cells required nickel for the derepression of hydrogenase in media treated to remove contaminating nickel and produced increasing levels of hydrogenase activity as a function of increasing nickel concentration. Immunoblots of SR

L. W. Stults; W. A. Sray; R. J. Maier

1986-01-01

74

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

NASA Astrophysics Data System (ADS)

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.

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

2008-08-01

75

Electron transfer between hydrogenase and 316L stainless steel: identification of a hydrogenase-catalyzed cathodic reaction in anaerobic mic  

Microsoft Academic Search

Electron transfer between the NAD-dependent hydrogenase from Ralstonia eutropha and AISI 316L stainless steel was studied with a view to identifying a possible role of hydrogenase in anaerobic microbially influenced corrosion (mic), as already suspected. A thin spectroelectrochemical cell was designed and qualified, taking great care to obtain a reproducible surface state of the stainless steel grid electrode. Successive electrolyses

S Da Silva; R Basséguy; A Bergel

2004-01-01

76

Characterization of a Cytosolic NiFe-Hydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1  

Microsoft Academic Search

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 (hyhBGSLTk), where the hyhSTk and hyhLTk gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific

Tamotsu Kanai; Sota Ito; Tadayuki Imanaka

2003-01-01

77

Enhanced decomposition of dimethyl phthalate via molecular oxygen activated by Fe@Fe2O3/AC under microwave irradiation.  

PubMed

In this study, we demonstrate that the decomposition of dimethyl phthalate under microwave irradiation could be greatly enhanced over Fe@Fe(2)O(3) nanowires supported on activated carbon (Fe@Fe(2)O(3)/AC). The great enhanced decomposition of dimethyl phthalate could be attributed to a unique microwave induced molecular oxygen activation process. Upon microwave irradiation, electrons could be transferred from activated carbon to zero-valent iron, and then react with molecular oxygen to form O(2)(-) and OH radicals for the decomposition of dimethyl phthalate. The deactivation and the regeneration of Fe@Fe(2)O(3)/AC catalyst were systematically studied. We also found that microwave heating could accelerate the electron transferring from AC to Fe@Fe(2)O(3) to generate more reactive oxygen species for the decomposition of DMP than conventional oil bath heating. This novel molecular oxygen activation approach may find applications for wastewater treatment and drinking water purification. PMID:22883705

Chen, Yiling; Ai, Zhihui; Zhang, Lizhi

2012-07-24

78

Hydrogenase in actinorhizal root nodules and root nodule homogenates.  

PubMed Central

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

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

1980-01-01

79

Force Field Development and Molecular Dynamics of [NiFe] Hydrogenase  

SciTech Connect

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

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

2012-05-09

80

Photosensitized Production of Hydrogen by Hydrogenase in Reversed Micelles  

NASA Astrophysics Data System (ADS)

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

Hilhorst, Riet; Laane, Colja; Veeger, Cees

1982-06-01

81

Photosensitized production of hydrogen by hydrogenase in reversed micelles  

PubMed Central

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

Hilhorst, Riet; Laane, Colja; Veeger, Cees

1982-01-01

82

Synthesis of the H-cluster framework of iron-only hydrogenase.  

PubMed

The metal-sulphur active sites of hydrogenases catalyse hydrogen evolution or uptake at rapid rates. Understanding the structure and function of these active sites--through mechanistic studies of hydrogenases, synthetic assemblies and in silico models--will help guide the design of new materials for hydrogen production or uptake. Here we report the assembly of the iron-sulphur framework of the active site of iron-only hydrogenase (the H-cluster), and show that it functions as an electrocatalyst for proton reduction. Through linking of a di-iron subsite to a {4Fe4S} cluster, we achieve the first synthesis of a metallosulphur cluster core involved in small-molecule catalysis. In addition to advancing our understanding of the natural biological system, the availability of an active, free-standing analogue of the H-cluster may enable us to develop useful electrocatalytic materials for application in, for example, reversible hydrogen fuel cells. (Platinum is currently the preferred electrocatalyst for such applications, but is expensive, limited in availability and, in the long term, unsustainable.). PMID:15703741

Tard, Cédric; Liu, Xiaoming; Ibrahim, Saad K; Bruschi, Maurizio; De Gioia, Luca; Davies, Siân C; Yang, Xin; Wang, Lai-Sheng; Sawers, Gary; Pickett, Christopher J

2005-02-10

83

Involvement of Ech hydrogenase in energy conservation of Methanosarcina mazei.  

PubMed

Methanosarcina mazei belongs to the group of aceticlastic methanogens and converts acetate into the potent greenhouse gases CO(2) and CH(4). The aceticlastic respiratory chain involved in methane formation comprises the three transmembrane proteins Ech hydrogenase, F(420) nonreducing hydrogenase and heterodisulfide reductase. It has been shown that the latter two contribute to the proton motive force. The data presented here clearly demonstrate that Ech hydrogenase is also involved in energy conservation. ATP synthesis was observed in a cytoplasm-free vesicular system of Ms. mazei that was dependent on the oxidation of reduced ferredoxin and the formation of molecular hydrogen (as catalysed by Ech hydrogenase). Such an ATP formation was not observed in a Deltaech mutant strain. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) led to complete inhibition of ATP formation in the Ms. mazei wild-type without inhibiting hydrogen production by Ech hydrogenase, whereas the sodium ion ionophore ETH157 did not affect ATP formation in this system. Thus, we conclude that Ech hydrogenase acts as primary proton pump in a ferredoxin-dependent electron transport system. PMID:20629748

Welte, Cornelia; Krätzer, Christian; Deppenmeier, Uwe

2010-07-12

84

Chitosan confinement enhances hydrogen photogeneration from a mimic of the diiron subsite of [FeFe]-hydrogenase.  

PubMed

Nature has created [FeFe]-hydrogenase enzyme as a hydrogen-forming catalyst with a high turnover rate. However, it does not meet the demands of economically usable catalytic agents because of its limited stability and the cost of its production and purification. Synthetic chemistry has allowed the preparation of remarkably close mimics of [FeFe]-hydrogenase but so far failed to reproduce its catalytic activity. Most models of the active site represent mimics of the inorganic cofactor only, and the enzyme-like reaction that proceeds within restricted environments is less well understood. Here we report that chitosan, a natural polysaccharide, improves the efficiency and durability of a typical mimic of the diiron subsite of [FeFe]-hydrogenase for photocatalytic hydrogen evolution. The turnover number of the self-assembling system increases ~4,000-fold compared with the same system in the absence of chitosan. Such significant improvements to the activity and stability of artificial [FeFe]-hydrogenase-like systems have, to our knowledge, not been reported to date. PMID:24158139

Jian, Jing-Xin; Liu, Qiang; Li, Zhi-Jun; Wang, Feng; Li, Xu-Bing; Li, Cheng-Bo; Liu, Bin; Meng, Qing-Yuan; Chen, Bin; Feng, Ke; Tung, Chen-Ho; Wu, Li-Zhu

2013-10-25

85

Identification of a Locus Upstream from the Hydrogenase Structural Genes That Is Involved in Hydrogenase Expression in Bradyrhizobium japonicum†  

PubMed Central

A locus involved in the expression of the uptake hydrogenase system of Bradyrhizobium japonicum was identified adjacent to genes encoding the hydrogenase subunits. A cloned fragment of DNA was used to complement to autotrophy a Hup? putative regulatory mutant of B. japonicum. The mutant strain lacked hydrogenase activity and synthesized low levels of the large subunit of hydrogenase as determined by Western gels. Tn5-induced mutagenesis located the region within the fragment which was necessary for complementation of the mutant phenotype. The locus identified is adjacent to that encoding the small subunit of hydrogenase; its right border is <0.5 kilobase upstream from the hydrogenase transcriptional start site, and its left border is between 1 and 2.5 kilobases from that start site. However, the locus is outside the region previously shown to contain hup-related genes of B. japonicum. Thus, the localization of this gene describes a previously unidentified hup-related gene on a region of DNA not previously shown to contain hup-specific DNA. Images

Novak, Patricia D.; Maier, Robert J.

1989-01-01

86

Catalytic mechanism of hydrogenase from aerobic N2-fixing microorganisms. Progress report.  

National Technical Information Service (NTIS)

Hydrogenases are enzymes which catalyze reactions involving dihydrogen. They serve integral roles in a number of microbial metabolic pathways. Our research is focussed on investigations of the catalytic mechanism of the hydrogenases found in aerobic, N(su...

D. J. Arp

1990-01-01

87

Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans  

Microsoft Academic Search

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase\\u000a has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing\\u000a hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the

Souria Malki; Gilles De Luca; Marie Laure Fardeau; M. Rousset; Jean Pierre Belaich; Z. Dermoun

1997-01-01

88

Bioaccumulation of Palladium by Desulfovibrio fructosivorans Wild-Type and Hydrogenase-Deficient Strains?  

PubMed Central

Wild-type Desulfovibrio fructosivorans and three hydrogenase-negative mutants reduced Pd(II) to Pd(0). The location of Pd(0) nanoparticles on the cytoplasmic membrane of the mutant retaining only cytoplasmic membrane-bound hydrogenase was strong evidence for the role of hydrogenases in Pd(0) deposition. Hydrogenase activity was retained at acidic pH, shown previously to favor Pd(0) deposition.

Mikheenko, I. P.; Rousset, M.; Dementin, S.; Macaskie, L. E.

2008-01-01

89

Desulfovibrio vulgaris Hydrogenase: A Nonheme Iron Enzyme Lacking Nickel That Exhibits Anomalous EPR and Mossbauer Spectra  

Microsoft Academic Search

A purification procedure for the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough, National Collection of Industrial Bacteria 8303) is reported. The purified hydrogenase has a specific activity of 4800 units per mg of protein. Plasma emission studies reveal that this highly active hydrogenase is free of nickel and contains 11 (± 1) non-heme iron atoms per molecule. A combined EPR and

Boi Hanh Huynh; Melvin H. Czechowski; Hans-Jorg Kruger; Daniel V. Dervartanian; Harry D. Peck; Jean Legall

1984-01-01

90

[FeFe]-hydrogenases and photobiological hydrogen production  

NASA Astrophysics Data System (ADS)

The promise of efficient, economic and renewable H2 photoproduction from water can potentially be met by green algae. These organisms are able to functionally link photosynthetic water oxidation to the catalytic recombination of protons and electrons to generate H2 gas through the activity of the hydrogenase enzyme. Green algal hydrogenases contain a unique metallo-catalytic H-cluster that performs the reversible H2 oxidation /evolution reactions. The H-cluster, located in the interior of the protein structure is irreversibly inactivated by O2, the by-product of water oxidation. We developed an Escherichi coli expression system to produce [FeFe]-hydrogenases from different biological sources and demonstrated that clostridial [FeFe]-hydrogenases have higher tolerance to O2 inactivation compared to their algal counterparts. We have been using computational simulations of gas diffusion within the Clostridium pasteurianum CpI hydrogenase to identify the pathways through which O2 can reach its catalytic site. Subsequently, we modify the protein structure at specific sites along the O2 pathways (identified by the computational simulations) by site-directed mutagenesis with the goal of generating recombinant enzymes with higher O2 tolerance. In this paper, we review the computational simulation work and report on preliminary results obtained through this strategy.

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

2006-09-01

91

Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography.  

PubMed Central

We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits. Images

Stults, L W; Moshiri, F; Maier, R J

1986-01-01

92

Transcription and regulation of the hydrogenase(s) accessory genes, hypFCDEAB , in the cyanobacterium Lyngbya majuscula CCAP 1446\\/4  

Microsoft Academic Search

Lyngbya majuscula CCAP 1446\\/4 is a filamentous cyanobacterium possessing both an uptake and a bi-directional hydrogenase. The presence of a\\u000a single copy of the hyp operon in the cyanobacterial genomes suggests that these accessory genes might be responsible for the maturation of both\\u000a hydrogenases. We investigated the concomitant transcription of hypFCDEAB with the hydrogenases structural genes—hup and hox. RT-PCRs performed

Daniela Ferreira; Elsa Leitão; Johannes Sjöholm; Paulo Oliveira; Peter Lindblad; Pedro Moradas-Ferreira; Paula Tamagnini

2007-01-01

93

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum  

SciTech Connect

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

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

1990-08-01

94

Fabrication of Fe/Fe3C-functionalized carbon nanotubes and their electromagnetic and microwave absorbing properties  

NASA Astrophysics Data System (ADS)

Fe/Fe3C-functionalized carbon nanotubes (CNTs) have been prepared by the floating catalyst chemical vapor-deposition method. It is demonstrated that the Fe and Fe3C nanostructures are both encapsulated in the CNTs or decorated on the surface of CNTs. The Fe/Fe3C content in the composites can easily be adjusted by changing the ferrocene concentration in the preparation. The electromagnetic properties of the CNTs have been evaluated in the frequency range of 2-18 GHz, and the nanocomposites exhibit excellent microwave absorbing performance. The CNT composites with higher Fe/Fe3C content show enhanced microwave reflection losses. The significant influence of the Fe/Fe3C nanostructures on the microwave absorption is realized by tuning the characteristic impedance of the nanocomposites. With increasing thickness, the maximum reflection loss peak shifts to lower frequency. The microwave absorbing performance of the composites is mainly caused by dielectric loss, resulting from the continuous CNT networks with excellent electrical conductivity.

Su, Qingmei; Zhong, Guo; Li, Jie; Du, Gaohui; Xu, Bingshe

2012-01-01

95

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

Microsoft Academic Search

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

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

2008-01-01

96

Development of Fe/Fe3O4 Core-Shell Nanocubes as a Promising Magnetic Resonance Imaging Contrast Agent.  

PubMed

Here, we report the synthesis, characterization, and properties of Fe/Fe3O4 core-shell nanocubes prepared via a simple route. It includes NaBH4 reduction of FeCl3 in an ethylene glycol solution in the presence of 2-mercaptopropionic acid (surfactant) and trisodium citrate (cosurfactant) followed by surface oxidation with trimethylamine N-oxide. The morphology and structure of Fe/Fe3O4 core-shell nanocubes were characterized using transmission electron microscopy (TEM), high-resolution TEM, selected area electron diffraction, X-ray powder diffraction, and X-ray photoelectron spectroscopy. All of the methods confirm a Fe/Fe3O4 core-shell structure of nanocubes. Magnetic measurements revealed that the Fe/Fe3O4 core/shell nanocubes are superparamagnetic at 300 K with a saturation magnetization of 129 emu/g. The T2 weighted imaging and the T2 relaxation time showed high MRI contrast and sensitivity, making these nanocubes viable candidates as enhanced MRI contrast agents. PMID:24079275

Mahmoud, Waleed E; Bronstein, Lyudmila M; Al-Hazmi, Faten; Al-Noaiser, Fowzia; Al-Ghamdi, A A

2013-10-10

97

Switchable Transport Strategy to Deposit Active Fe/Fe3 C Cores into Hollow Microporous Carbons for Efficient Chromium Removal.  

PubMed

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

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

2013-06-10

98

Hydrogenase-catalysed deposition of vivianite on mild steel  

Microsoft Academic Search

A simple device was designed with two mild steel electrodes placed face to face in the same phosphate solution and coupled through an external electrical circuit. A dialysis membrane retained hydrogenase from Ralstonia eutropha in contact with one electrode only. The simultaneous measurements of the electron flux in the electrical circuit and of nicotinamide adenine dinucleotide (NADH) production catalysed by

S. Da Silva; R. Basséguy; A. Bergel

2004-01-01

99

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

PubMed Central

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.

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

2003-01-01

100

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

PubMed Central

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

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

1985-01-01

101

Hydrogenases from methanogenic archaea, nickel, a novel cofactor, and H2 storage.  

PubMed

Most methanogenic archaea reduce CO(2) with H(2) to CH(4). For the activation of H(2), they use different [NiFe]-hydrogenases, namely energy-converting [NiFe]-hydrogenases, heterodisulfide reductase-associated [NiFe]-hydrogenase or methanophenazine-reducing [NiFe]-hydrogenase, and F(420)-reducing [NiFe]-hydrogenase. The energy-converting [NiFe]-hydrogenases are phylogenetically related to complex I of the respiratory chain. Under conditions of nickel limitation, some methanogens synthesize a nickel-independent [Fe]-hydrogenase (instead of F(420)-reducing [NiFe]-hydrogenase) and by that reduce their nickel requirement. The [Fe]-hydrogenase harbors a unique iron-guanylylpyridinol cofactor (FeGP cofactor), in which a low-spin iron is ligated by two CO, one C(O)CH(2)-, one S-CH(2)-, and a sp(2)-hybridized pyridinol nitrogen. Ligation of the iron is thus similar to that of the low-spin iron in the binuclear active-site metal center of [NiFe]- and [FeFe]-hydrogenases. Putative genes for the synthesis of the FeGP cofactor have been identified. The formation of methane from 4 H(2) and CO(2) catalyzed by methanogenic archaea is being discussed as an efficient means to store H(2). PMID:20235826

Thauer, Rudolf K; Kaster, Anne-Kristin; Goenrich, Meike; Schick, Michael; Hiromoto, Takeshi; Shima, Seigo

2010-01-01

102

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

SciTech Connect

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

De Hatten, Xavier [University of Bordeaux; Cournia, Zoe [Yale University; Smith, Jeremy C [ORNL; Huc, I [University of Bochum, Germany; Metzler-Nolte, Nils [University of Bochum, Germany

2007-08-01

103

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

SciTech Connect

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

De Hatten, Xavier [University of Bordeaux; Cournia, Zoe [Yale University; Smith, Jeremy C [ORNL; Metzler-Nolte, Nils [University of Bochum, Germany

2007-08-01

104

Crystal Structure of HydF Scaffold Protein Provides Insights into [FeFe]-Hydrogenase Maturation*  

PubMed Central

[FeFe]-hydrogenases catalyze the reversible production of H2 in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 ? resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous ?-sheet comprising eight ?-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.

Cendron, Laura; Berto, Paola; D'Adamo, Sarah; Vallese, Francesca; Govoni, Chiara; Posewitz, Matthew C.; Giacometti, Giorgio M.; Costantini, Paola; Zanotti, Giuseppe

2011-01-01

105

Oxygen-resistant hydrogenases and methods for designing and making same  

DOEpatents

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

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

2009-03-10

106

The effect of aging time and calcination temperature on the magnetic properties of ?-Fe\\/Fe 3O 4 composite  

Microsoft Academic Search

Composites of ?-Fe\\/Fe3O4 having dimensions in the range of 100–150nm have been prepared by disproportion method. The structure and morphology are investigated by XRD and TEM. XRD shows that the metal has got the BCC structure. TEM shows balls of metallic iron about 100-nm-wide stuck to magnetite grains. Magnetic measurement shows that the sample aged for 3h and calcined at

Lijun Zhao; Hua Yang; Shuiming Li; Lianxiang Yu; Yuming Cui; Xueping Zhao; Shouhua Feng

2006-01-01

107

Dithiomethylether As a Ligand in the Hydrogenase H-Cluster  

SciTech Connect

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.

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

2009-05-21

108

HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria  

Microsoft Academic Search

NAD(P)+-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase

Oliver Schmitz; Gudrun Boison; Heike Salzmann; Hermann Bothe; Kathrin Schütz; Shu-hua Wang; Thomas Happe

2002-01-01

109

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

PubMed Central

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

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

2010-01-01

110

Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation  

PubMed Central

The active site of [NiFe]-hydrogenase contains nickel and iron coordinated by cysteine residues, cyanide and carbon monoxide. Metal chaperone proteins HypA and HypB are required for the nickel insertion step of [NiFe]-hydrogenase maturation. How HypA and HypB work together to deliver nickel to the catalytic core remains elusive. Here we demonstrated that HypA and HypB from Archaeoglobus fulgidus form 1?1 heterodimer in solution and HypA does not interact with HypB dimer preloaded with GMPPNP and Ni. Based on the crystal structure of A. fulgidus HypB, mutants were designed to map the HypA binding site on HypB. Our results showed that two conserved residues, Tyr-4 and Leu-6, of A. fulgidus HypB are required for the interaction with HypA. Consistent with this observation, we demonstrated that the corresponding residues, Leu-78 and Val-80, located at the N-terminus of the GTPase domain of Escherichia coli HypB were required for HypA/HypB interaction. We further showed that L78A and V80A mutants of HypB failed to reactivate hydrogenase in an E. coli ?hypB strain. Our results suggest that the formation of the HypA/HypB complex is essential to the maturation process of hydrogenase. The HypA binding site is in proximity to the metal binding site of HypB, suggesting that the HypA/HypB interaction may facilitate nickel transfer between the two proteins.

Chan, Kwok-Ho; Lee, Ka-Man; Wong, Kam-Bo

2012-01-01

111

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli  

PubMed Central

Background Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H2 production involves consumption of 2H+, hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms. Methods and Principal Findings We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H2 to 2H+. Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H2 production and consumption was tested using a H2-specific Clark-type electrode. Hyd-3-dependent H2 production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H2 consumption was maximal at alkaline pH. H2 production, was unaffected by a shift in external or internal pH. H2 production was associated with hycE expression levels as a function of external pH. Conclusions Anaerobic growing cultures of E. coli generate H2 via Hyd-3 at low external pH, and consume H2 via Hyd-2 at high external pH. Hyd-3 proton conversion to H2 is required for acid resistance in anaerobic cultures of E. coli.

Noguchi, Ken; Riggins, Daniel P.; Eldahan, Khalid C.; Kitko, Ryan D.; Slonczewski, Joan L.

2010-01-01

112

Differences in hydrogenase gene expression between Methanosarcina acetivorans and Methanosarcina barkeri.  

PubMed

Methanosarcina acetivorans C2A encodes three putative hydrogenases, including one cofactor F(420)-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 aligned with those in other Methanosarcina species to identify conserved transcription and translation signals. The M. acetivorans vht promoter region is well conserved among the sequenced Methanosarcina species, while the second vht-type homolog (here called vhx) and frh promoters have only limited similarity. To experimentally determine whether these promoters are functional in vivo, we constructed and characterized both M. acetivorans and M. barkeri strains carrying reporter gene fusions to each of the M. acetivorans and M. barkeri hydrogenase promoters. Generally, the M. acetivorans gene fusions are not expressed in either organism, suggesting that cis-acting mutations inactivated the M. acetivorans promoters. The M. barkeri hydrogenase gene fusions, on the other hand, are expressed in both organisms, indicating that M. acetivorans possesses the machinery to express hydrogenases, although it does not express its own hydrogenases. These data are consistent with specific inactivation of the M. acetivorans hydrogenase promoters and highlight the importance of testing hypotheses generated by using genomic data. PMID:19201801

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

2009-02-06

113

Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus  

PubMed Central

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H2 evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

Weyman, Philip D.; Vargas, Walter A.; Tong, Yingkai; Yu, Jianping; Maness, Pin-Ching; Smith, Hamilton O.; Xu, Qing

2011-01-01

114

Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation.  

PubMed Central

The presumed beneficial effect of hydrogenase on growth of diazotrophic bacteria was reinvestigated with carbon-limited chemostat cultures of the hydrogenase-deficient mutant hoxKG of Azotobacter vinelandii and its parent. The results revealed that hydrogen recycling was too low to benefit the cellular energy metabolism or activities of nitrogenase and respiration.

Linkerhagner, K; Oelze, J

1995-01-01

115

Acetylene inhibition of Azotobacter vinelandii hydrogenase: Acetylene binds tightly to the large subunit  

SciTech Connect

Acetylene is a slow-binding inhibitor of the Ni- and Fe-containing dimeric hydrogenase isolated from Azotobacter vinelandii. Acetylene was released from hydrogenase during the recovery from inhibition. This indicates that no transformation of acetylene to another compound occurred as a result of the interaction with hydrogenase. However, the release of C{sub 2}H{sub 2} proceeds more rapidly than the recovery of activity, which indicates that release of C{sub 2}H{sub 2} is not sufficient for recovery of activity. Acetylene binds tightly to native hydrogenase; hydrogenase and radioactivity coelute from a gel permeation column following inhibition with {sup 14}C{sub 2}H{sub 2}. Acetylene, or a derivative, remains bound to the large 65,000 MW subunit (and not to the small 35,000 MW subunit) of hydrogenase following denaturation as evidence by SDS-PAGE and fluorography of {sup 14}C{sub 2}H{sub 2}-inhibited hydrogenase. This result suggests that C{sub 2}H{sub 2}, and by analogy H{sub 2}, binds to and is activated by the large subunit of this dimeric hydrogenase. Radioactivity is lost from {sup 14}C{sub 2}H{sub 2}-inhibited protein during recovery. The inhibition is remarkably specific for C{sub 2}H{sub 2}; propyne, butyne, and ethylene are not inhibitors.

Jinhua Sun; Hyman, M.R.; Arp, D.J. (Oregon State Univ., Corvallis (United States))

1992-03-31

116

Iron-Dependent Hydrogenases of Entamoeba histolytica and Giardia lamblia: Activity of the Recombinant Entamoebic Enzyme and Evidence for Lateral Gene Transfer  

Microsoft Academic Search

Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes. To understand better the biochemis- try of the protist Fe-hydrogenases, we prepared a recombi- nant E. histolytica short Fe-hydrogenase and measured its activity in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic se- quences, and

JULIE E. J. NIXON; JESSICA FIELD; ANDREW G. MCARTHUR; MITCHELL L. SOGIN; NIGEL YARLETT; BRENDAN J. LOFTUS; JOHN SAMUELSON

117

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120  

Microsoft Academic Search

BACKGROUND: The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene

Ellenor Devine; Marie Holmqvist; Karin Stensjö; Peter Lindblad

2009-01-01

118

Structure-Function Relationships in [FeFe]-Hydrogenase Active Site Maturation*  

PubMed Central

Since the discovery that, despite the active site complexity, only three gene products suffice to obtain active recombinant [FeFe]-hydrogenase, significant light has been shed on this process. Both the source of the CO and CN? ligands to iron and the assembly site of the catalytic subcluster are known, and an apo structure of HydF has been published recently. However, the nature of the substrate(s) for the synthesis of the bridging dithiolate ligand to the subcluster remains to be established. From both spectroscopy and model chemistry, it is predicted that an amine function in this ligand plays a central role in catalysis, acting as a base in the heterolytic cleavage of hydrogen.

Nicolet, Yvain; Fontecilla-Camps, Juan C.

2012-01-01

119

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

NASA Astrophysics Data System (ADS)

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.

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

2013-04-01

120

The Role of Frozen Spins in the Exchange Anisotropy of Core-Shell Fe@Fe3O4 Nanoparticles  

PubMed Central

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

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

2011-01-01

121

Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12.  

PubMed Central

Two membrane-bound hydrogenase isoenzymes present in Escherichia coli during anaerobic growth have been resolved. The isoenzymes are immunologically and electrophoretically distinct. The physically more abundant isoenzyme (hydrogenase 1) contains a subunit of Mr 64,000 and is not released from the membrane by exposure to either trypsin or pancreatin. The second isoenzyme (hydrogenase 2) apparently contributes the greater part of the membrane-bound hydrogen:benzyl viologen oxidoreductase activity and exists in two electrophoretic forms revealed by nondenaturing polyacrylamide gel analysis. This isoenzyme is irreversibly inactivated at alkaline pH and gives rise to an active, soluble derivative when the membrane-bound enzyme is exposed to either trypsin or pancreatin. Both hydrogenase isoenzymes contain nickel. Images

Ballantine, S P; Boxer, D H

1985-01-01

122

Effect of hydrogenase and mixed sulfate-reducing bacterial populations on the corrosion of steel  

SciTech Connect

The importance of hydrogenase activity to corrosion of steel was assessed by using mixed populations of sulfate-reducing bacteria isolated from corroded and noncorroded oil pipelines. Biofilms which developed on the steel studs contained detectable numbers of sulfate-reducing bacteria (10{sup 4} increasing to 10{sup 7}/0.5 cm{sup 2}). However, the biofilm with active hydrogenase activity (i.e., corrosion pipeline organisms), as measured by a semiquantitative commercial kit, was associated with a significantly higher corrosion rate (7.79 mm/year) relative to noncorrosive biofilm (0.48 mm/year) with 10{sup 5} sulfate-reducing bacteria per 0.5 cm{sup 2} but no measurable hydrogenase activity. The importance of hydrogenase and the microbial sulfate-reducing bacterial population making up the biofilm are discussed relative to biocorrosion.

Bryant, R.D.; Jansen, W.; Boivin, J.; Laishley, E.J.; Costerton, J.W. (Univ. of Calgary, Alberta (Canada))

1991-10-01

123

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

PubMed Central

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.

2013-01-01

124

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

SciTech Connect

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

Arp, D.J.

1997-10-01

125

Inhibition by acetylene of conventional hydrogenase in nitrogen-fixing bacteria  

Microsoft Academic Search

ONE of the earliest observations associated with nitrogen fixation was that all nitrogen-fixing organisms contained hydrogenase and nitrogenase1. We show here that acetylene, which is widely used as a substrate for nitrogenase, inhibits conventional hydrogenase activity and thus prevents the uptake of H2 formed by nitrogenase in the presence of CO. Our evidence also suggests that azotobacter nitrogenase forms H2

Linda A. Smith; Susan Hill; M. G. Yates

1976-01-01

126

Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446\\/4  

Microsoft Academic Search

BACKGROUND: Lyngbya majuscula CCAP 1446\\/4 is a N2-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by hupSL) and a bidirectional enzyme (encoded by hoxEFUYH). The biosynthesis\\/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS

Daniela Ferreira; Filipe Pinto; Pedro Moradas-Ferreira; Marta V Mendes; Paula Tamagnini

2009-01-01

127

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

SciTech Connect

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

Arp, D.J.

1991-01-01

128

Characterization of Fe-hydrogenase genes diversity and hydrogen-producing population in an acidophilic sludge  

Microsoft Academic Search

According to the DNA sequences of six Fe-hydrogenase genes (FHG) of Clostridium species retrieved from the GenBank, a set of primers specific for Fe-hydrogenase genes were identified from their common conserved regions. The length of DNA fragments amplified using these two primers averaged 313bps. This primer set was then used to investigate the FHG diversity in an acidophilic rice-degrading sludge

Herbert H. P. Fang; Tong Zhang; Chenlin Li

2006-01-01

129

Phenotypic evidence that the function of the [Fe]-hydrogenase Hmd in Methanococcus maripaludis requires seven hcg (hmd co-occurring genes) but not hmdII.  

PubMed

The H2 -dependent methylene-tetrahydromethanopterin dehydrogenase (Hmd), also known as the [Fe]-hydrogenase, is found only in methanogens without cytochromes. In contrast to the binuclear metal centers of the [NiFe]- and [FeFe]-hydrogenases, the [Fe]-hydrogenase contains only a single Fe atom, which is coordinated by a novel guanylylpyridinol cofactor in the active site. The biosynthesis of the cofactor is not well understood and the responsible genes are unknown. However, seven genes (hmd co-occurring genes, hcg) encoding proteins of unknown function are always associated with the hmd gene. In the model methanogen Methanococcus maripaludis, we used a genetic background in which a deletion of hmd had a distinct growth phenotype, and made null-mutations in each hcg gene as well as in a gene encoding the Hmd paralog HmdII, which is hypothesized to function as a scaffold for cofactor synthesis. Deletions in all seven hcg genes resulted in the same growth phenotype as a deletion in hmd, suggesting they are required for Hmd function. In all cases, genetic complementation of the mutation restored the wild-type phenotype. A deletion in hmdII had no effect. PMID:23551135

Lie, Thomas J; Costa, Kyle C; Pak, Daniel; Sakesan, Varun; Leigh, John A

2013-04-18

130

Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging.  

PubMed

Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ? H(2) while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H(+)/H(2) half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum , CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules. PMID:21916466

Madden, Christopher; Vaughn, Michael D; Díez-Pérez, Ismael; Brown, Katherine A; King, Paul W; Gust, Devens; Moore, Ana L; Moore, Thomas A

2011-10-03

131

Studies on hydrogenase activity and chlorobenzene respiration in Dehalococcoides sp. strain CBDB1.  

PubMed

Hydrogen oxidation and electron transport were studied in the chlorobenzene-utilizing anaerobe Dehalococcoides sp. strain CBDB1. While Cu(2+) and Hg(2+) ions irreversibly inhibited hydrogenase activity in intact cells, Ni(2+) ions inhibited reversibly. About 80% of the initial hydrogenase activity was inactivated within 30 s when the cells were exposed to air. In contrast, hydrogenase was active at a redox potential of +10 mV when this redox potential was established anoxically with a redox indicator. Viologen dyes served both as electron acceptor for hydrogenase and electron donor for the dehalogenase. A menaquinone analogue, 2,3-dimethyl 1,4-naphthoquinone, served neither as electron acceptor for the hydrogenase nor as electron donor for the dehalogenase. In addition, the menaquinone antagonist 2-n-heptyl-4-hydroxyquinoline-N-oxide had no effect on dechlorination catalyzed by cell suspensions or isolated membranes with hydrogen as electron donor, lending further support to the notion that menaquinone is not involved in electron transport. The ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone did not inhibit dechlorination by cell suspensions, indicating that strain CBDB1 does not require reverse electron transport. The ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the dechlorination reaction with cell suspensions; however, the latter effect was partially relieved by the addition of tetrachlorosalicylanilide. 1,2,3,4-tetrachlorobenzene strongly inhibited dechlorination of other chlorobenzenes by cell suspensions with hydrogen as electron donor, but it did not interfere with either hydrogenase or dehalogenase activity. PMID:15490122

Jayachandran, Gopalakrishnan; Görisch, Helmut; Adrian, Lorenz

2004-10-15

132

The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis.  

PubMed

The activity of the bidirectional hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was found not to be regulated in parallel to respiration but to photosynthesis. A mutant with a deletion in the large hydrogenase subunit gene (hoxH), which contains the active site, was impaired in the oxidation of photosystem I (PSI) when illuminated with light, which excites either PSI alone or both photosystems. The fluorescence of photosystem II (PSII) of this mutant was higher than that of wild-type cells. The transcript level of the photosynthetic genes psbA, psaA and petB was found to be different in the hydrogenase-free mutant cells compared to wild-type cells, which indicates that the hydrogenase has an effect on the regulation of these genes. Collectively, these results suggest that the bidirectional hydrogenase functions as a valve for low-potential electrons generated during the light reaction of photosynthesis, thus preventing a slowing down of electron transport. This conclusion is supported by growth curves demonstrating that the mutant cells need more time to adapt to changing light intensities. Investigations of the wild-type and deltahoxH strains strongly suggest that Synechocystis contains only the bidirectional hydrogenase, which seems to be essentially insensitive to oxygen. PMID:10896211

Appel, J; Phunpruch, S; Steinmüller, K; Schulz, R

133

Three-dimensional carbon nanotube–polypyrrole–[NiFe] hydrogenase electrodes for the efficient electrocatalytic oxidation of H 2  

Microsoft Academic Search

Hydrogenase electrodes for hydrogen oxidation are elaborated by an innovative immobilization strategy of [NiFe] hydrogenases from Desulfovibrio fructosovorans on highly porous single-walled (SWCNT) and multi-walled (MWCNT) carbon nanotube electrodes.The bioelectrode fabrication involved the adsorption of hydrogenase and amphiphilic pyrrole monomer functionalized by a methylviologen moiety on the nanotube deposits.The electropolymerization of the adsorbed monomer then leads to the enzyme entrapment

Jessica Baur; Alan Le Goff; Sébastien Dementin; Michael Holzinger; Marc Rousset; Serge Cosnier

2011-01-01

134

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

Microsoft Academic Search

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

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

2007-01-01

135

Decolorization and degradation of textile dyes with biosulfidogenic hydrogenases.  

PubMed

Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480-620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds. PMID:17880103

Mutambanengwe, C C Z; Togo, C A; Whiteley, C G

2007-09-19

136

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

PubMed Central

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

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

2012-01-01

137

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

PubMed

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H(2) metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H(2) suggests that its role is anaplerotic. Indeed, H(2) via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for. PMID:22872868

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

2012-08-07

138

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

NASA Astrophysics Data System (ADS)

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.

McIntosh, Chelsea Lee

139

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

SciTech Connect

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

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

1991-12-01

140

Spectroscopic and Electrochemical Characterization of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F: Reversible Redox Behavior and Interactions between Electron Transfer Centers.  

PubMed

Characterizing a new hydrogenase: The newly isolated [NiFeSe] hydrogenase from Desulfovibrio vulgaris Miyazaki F displays catalytic properties distinct from other hydrogenase proteins. Here we apply site-specific spectroscopic and electrochemical techniques to characterize these unique features at the molecular level. PMID:24038675

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

2013-09-03

141

Nickel-Iron Dithiolates Related to the Deactivated [NiFe]-Hydrogenases  

PubMed Central

Described herein are preparations of synthetic models for the deactivated Ni(II)Fe(II) states of the [NiFe]-hydrogenases. Iodination of the S = ½ species [(dppe)Ni(pdt)Fe(CO)3]+ afforded the diamagnetic iodo complex [(dppe)Ni(pdt)IFe(CO)3]+. Crystallographic analysis of this species confirmed the presence of square-pyramidal Ni linked to an octahedral Fe centre. The Ni···Fe separation of 3.018 Å indicated the absence of metal-metal bonding. This complex could be reduced to give (dppe)Ni(pdt)Fe(CO)3 and, in the presence of iodide, decarbonylated to afford (dppe)Ni(pdt)FeI2. Derivatives of the type [(diphosphine)Ni(dithiolate)XFe(CO)2L]+ (X = Cl, Br, I) were prepared by halogenation of mixed-valence precursors [(diphosphine)Ni(dithiolate)Fe(CO)2L]+ (diphosphine = dppe, dcpe; L = tertiary phosphine or CO). The Fe(CO)2(PR3)-containing derivatives are more robust than the related tricarbonyl derivatives. Exploiting this greater stability, we characterised examples of chloride and bromide derivatives. Related fluorides could be prepared by F? abstraction from BF4?. Spectroscopic evidence is presented for the hydroperoxide [(diphosphine)Ni(dithiolate)(OOH)Fe(CO)2L]+, which was prepared by oxidation of a model for Ni-SU and Ni-SIr states.

Schilter, David

2012-01-01

142

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

PubMed Central

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

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

1988-01-01

143

Regulation and Genetic Organization of Hydrogenase: Final Progress Report for the Period June 1, 1985-July 31, 1988.  

National Technical Information Service (NTIS)

Hydrogenase is an enzyme which plays an important role in the anaerobic metabolism of many bacteria. The objectives of the research were to elucidate the regulation and genetic organization of hydrogenase in microorganisms. A mutation in the E. coli hydE ...

A. I. Krasna

1988-01-01

144

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

SciTech Connect

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

Arp, D.J.

1990-01-01

145

Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermpholic archaebacterium Pyrodictium brockii  

SciTech Connect

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105C. P. brokii is also a chemolithotroph, requiring H{sub 2} and CO{sub 2} for growth. The authors have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 {plus minus} 0.6 mol of Fe and 6.2 {plus minus} 1.2 mol of S per mol of hydrogenase. Growth of cells in {sup 63} NiCl{sub 2} resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H{sub 2} in the presence of reduced methyl viologen. The K{sub m} of membrane-bound hydrogenase for H{sub 2} was approximately 19 {mu}M with methylene blue as the electron acceptor, whereas the purified enzyme had a higher K{sub m} value.

Pihl, T.D. (Johns Hopkins Univ., Baltimore, MD (United States)); Maier, R.J. (Johns Hopkins Univ., Baltimore, MD (United States) Univ. of Maryland, Baltimore (United States))

1991-03-01

146

Molecular dynamics and experimental investigation of H2 and O2 diffusion in [Fe]-hydrogenase  

PubMed Central

The [Fe]-hydrogenase enzymes are highly efficient H2 catalysts found in ecologically, and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. Although these enzymes can occur in several forms, H2 catalysis takes place at a unique [FeS] prosthetic group, or H-cluster, located at the active site. Significant to the function of hydrogenases is how the surrounding protein structure facilitates substrate-product transfer, and protects the active site H-cluster from inactivation. To elucidate the role of protein structure in O2 inactivation of [Fe]-hydrogenases, experimental and theoretical investigations have been performed. Molecular dynamics was used to comparatively investigate O2 and H2 diffusion in [Fe]-hydrogenase CpI. The results are compared to initial investigations of H2 diffusion in [NiFe]-hydrogenase [1]. Our preliminary results suggest that H2 diffuses more easily and freely than O2, which is restricted to a small number of allowed pathways to and from the active site. These O2 pathways are located in the conserved active site domain, shown experimentally to have an essential role in active site protection.

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

2008-01-01

147

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

PubMed Central

A 0.6-kb fragment of DNA involved in intracellular Ni metabolism was isolated and cloned from a cosmid containing 23.2 kb of hydrogenase-related genes of Bradyrhizobium japonicum. This locus is located 8.3 kb upstream of the hydrogenase structural genes. The hydrogenase activity of a mutant with a gene-directed mutation at this locus (strain JHK7) showed dependency on nickel provided during hydrogenase derepression. The hydrogenase activity was only 20% of that in the wild-type strain, JH, at a concentration of 0.5 microM NiCl2. The hydrogenase activity in JH reached its maximum at 3 microM NiCl2, whereas the mutant (JHK7) reached wild-type levels of hydrogenase activity when derepressed in 50 microM NiCl2. Studies with the hup-lacZ transcriptional fusion plasmid pSY7 in JHK7 showed that the mutant JHK7 expressed less promoter activity under low-nickel conditions than did strain JH. The mutant accumulated less nickel during a 45-h hydrogenase derepression period than did the wild type. However, both JHK7 and the JH wild-type strain had the same short-term Ni transport rates, and the KmS for Ni of both strains were about 62 microM. When incubated under non-hydrogenase-derepression conditions, the mutant accumulated Ni at the same rate as strain JH. However, this stored source of nickel was unable to restore hydrogenase expression ability of the mutant to wild-type levels during derepression without nickel. The results indicate that the locus identified in B. japonicum is not involved in nickel-specific transport; indeed, it was not at all homologous to the "nickel transporter" hoxN gene of Alcaligenes eutrophus.(ABSTRACT TRUNCATED AT 250 WORDS) Images

Fu, C L; Maier, R J

1991-01-01

148

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

PubMed Central

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.

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

2010-01-01

149

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

PubMed Central

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

2012-01-01

150

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

PubMed

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

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

2010-07-01

151

Chemical vapor deposition growth of Fe 3O 4 thin films and Fe\\/Fe 3O 4 bi-layers for their integration in magnetic tunnel junctions  

Microsoft Academic Search

A possible route for the synthesis of Fe3O4, Fe, and Fe\\/Fe3O4 bi-layers with chemical vapor deposition by employing the same Fe3(CO)12 carbonyl precursor is presented. The comprehensive structural, chemical, and morphological investigation of the as deposited thin single films and bi-layers is performed by X-ray diffraction, X-ray reflectivity, Raman spectroscopy, and time-of-flight secondary ion mass spectrometry depth profiling. We present

S. Vangelista; R. Mantovan; S. Cocco; A. Lamperti; O. Salicio; M. Fanciulli

152

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

SciTech Connect

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

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

2008-10-22

153

[The effect of sodium salts and pH on hydrogenase activity of the haloalkaliphilic sulfate-reducing bacteria].  

PubMed

Hydrogenase is the main catabolic enzyme of hydrogen-utilizing sulfate-reducing bacteria. In haloalkaliphilic sulfate reducers, hydrogenase, particularly if it is periplasmic, functions at high concentrations of Na+ ions and low concentrations of H+ ions. The hydrogenases of the newly isolated sulfate-reducing bacteria Desulfonatronum thiodismutans, D. lacustre, and Desulfonatrovibrio hydrogenovorans exhibit different sensitivity to Na+ ions and remain active at NaCl concentrations between 0 and 4.3 M and NaHCO3 concentrations between 0 and 1.2 M. The hydrogenases of D. lacustre and D. thiodismutans remain active at pH values between 6 and 12. The optimum pH for the hydrogenase of D. thiodismutans is 9.5. The optimum pH for the cytoplasmic and periplasmic hydrogenases of D. lacustre is 10. Thus, the hydrogenases of D. thiodismutans, D. lacustre, and Dv. hydrogenovorans are tolerant to high concentrations of sodium salts and extremely tolerant to high pH values, which makes them unique objects for biochemical studies and biotechnological applications. PMID:16211848

Detkova, E N; Soboleva, G S; Pikuta, E V; Pusheva, M A

154

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

PubMed Central

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.

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

2013-01-01

155

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

PubMed Central

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.

Wang, Shuning; Huang, Haiyan; Kahnt, Jorg

2013-01-01

156

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

NASA Astrophysics Data System (ADS)

Natural single-crystal specimens of barbosalite from Brazil, with general formula FeFe23+()2( were investigated by Raman and infrared spectroscopy. The mineral occurs as secondary products in granitic pegmatites. The Raman spectrum of barbosalite is characterized by bands at 1020, 1033 and 1044 cm-1 cm-1, assigned to ?1 symmetric stretching mode of the HOPO33- and PO43- units. Raman bands at around 1067, 1083 and 1138 cm-1 are attributed to both the HOP and PO antisymmetric stretching vibrations. The set of Raman bands observed at 575, 589 and 606 cm-1 are assigned to the ?4 out of plane bending modes of the PO4 and H2PO4 units. Raman bands at 439, 461, 475 and 503 cm-1 are attributed to the ?2 PO4 and H2PO4 bending modes. Strong Raman bands observed at 312, 346 cm-1 with shoulder bands at 361, 381 and 398 cm-1 are assigned to FeO stretching vibrations. No bands which are attributable to water vibrations were found. Vibrational spectroscopy enables aspects of the molecular structure of barbosalite to be assessed.

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

2013-11-01

157

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

Microsoft Academic Search

Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichlo- roethene (TCE) to vinyl chloride and ethene using H2 as an electron donor. PCE- and TCE-reductive dehalogenase (RD) activities were mainly membrane associated, whereas only about 20% of the hydrogenase activity was membrane associated. Experiments with methyl viologen (MV) were consistent with a periplasmic location for the RDs or a

Ivonne Nijenhuis; Stephen H. Zinder

2005-01-01

158

Kinetic Mechanism Studies of the Soluble Hydrogenase from Alcaligenes Eutrophus H161.  

National Technical Information Service (NTIS)

Purified soluble hydrogenase (112:NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N2 and H2 and then adding substoichiometric quantities of NADH. 112-dependen...

R. G. Keefe M. J. Axley A. L. Harabin

1995-01-01

159

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...

160

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS  

Microsoft Academic Search

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H 2. The enzyme showed a maximal activity of 120±40 µmol H 2 oxidized · min -1 · mg -1 with methyl viologen as an electron acceptor, a maximal hydrogen

J. M. Kemner; J. G. Zeikus

1994-01-01

161

Artificial hydrogenases: assembly of an H-cluster analogue within a functionalised poly(pyrrole) matrix.  

PubMed

We show that a redox active {Fe(4)S(4)}(2+)-cubane assembly covalently bound within a cysteinyl-alkylammonium functionalised polypyrrole can be modified with a diiron dithiolate carbonyl unit to give an artificial hydrogenase H-cluster framework confined within the polymer matrix. PMID:20922263

Ibrahim, Saad; Woi, Pei Meng; Alias, Yatimah; Pickett, Christopher J

2010-10-05

162

Molecular and immunological comparison of membrane-bound, H2-oxidizing hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii.  

PubMed Central

The membrane-bound hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii were purified extensively and compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of each hydrogenase revealed two prominent protein bands, one near 60 kilodaltons and the other near 30 kilodaltons. The migration distances during nondenaturing polyacrylamide gel electrophoresis were similar for all except A. vinelandii hydrogenase, which migrated further than the other three. The amino acid composition of each hydrogenase was determined, revealing substantial similarity among these enzymes. This was confirmed by calculation of S delta Q values, which ranged from 8.0 to 26.7 S delta Q units. S delta Q is defined as sigma j(Xi,j-Xk,j)2, where i and k identify the proteins compared and Xj is the content (residues per 100) of a given amino acid of type j. The hydrogenases of this study were also compared with an enzyme-linked immunosorbent assay. Antibody raised against B. japonicum hydrogenase cross-reacted with all four hydrogenases, but to various degrees and in the order B. japonicum greater than A. latus greater than A. eutrophus greater than A. vinelandii. Antibody raised against A. eutrophus hydrogenase also cross-reacted with all four hydrogenases, following the pattern of cross-reaction A. eutrophus greater than A. latus = B. japonicum greater than A. vinelandii. Antibody raised against B. japonicum hydrogenase inhibited B. japonicum hydrogenase activity to a greater extent than the A. eutrophus and A. latus activities; no inhibition of A. vinelandii hydrogenase activity was detected. The results of these experiments indicated remarkable homology of the hydrogenases from these four microorganisms. Images

Arp, D J; McCollum, L C; Seefeldt, L C

1985-01-01

163

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

PubMed Central

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

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

2012-01-01

164

Distribution of Hydrogenase Genes in Desulfovibrio spp. and Their Use in Identification of Species from the Oil Field Environment.  

PubMed

The distribution of genes for [Fe], [NiFe], and [NiFeSe] hydrogenases was determined for 22 Desulfovibrio species. The genes for [NiFe] hydrogenase were present in all species, whereas those for the [Fe] and [NiFeSe] hydrogenases had a more limited distribution. Sulfate-reducing bacteria from 16S rRNA groups other than the genus Desulfovibrio (R. Devereux, M. Delaney, F. Widdel, and D. A. Stahl, J. Bacteriol. 171:6689-6695, 1989) did not react with the [NiFe] hydrogenase gene probe, which could be used to identify different Desulfovibrio species in oil field samples following growth on lactate-sulfate medium. PMID:16348376

Voordouw, G; Niviere, V; Ferris, F G; Fedorak, P M; Westlake, D W

1990-12-01

165

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

Microsoft Academic Search

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

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

2005-01-01

166

Role of the Hya Hydrogenase in Recycling of Anaerobically Produced H2 in Salmonella enterica Serovar Typhimurium  

Microsoft Academic Search

Double and triple uptake-type hydrogenase mutants were used to determine which hydrogenase recycles fermentatively produced hydrogen. The hyb hya and hyd hya double mutants evolved H2 at rates similar to that of the triple mutant strain, so Hya alone oxidizes the bulk of H2 produced during fermentation. When only Hya was present, no hydrogen production was observed in nutrient-limited medium.

Andrea L. Zbell; Robert J. Maier

2009-01-01

167

Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.  

PubMed

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

Sun, Junsong; Hopkins, Robert C; Jenney, Francis E; McTernan, Patrick M; Adams, Michael W W

2010-05-06

168

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

PubMed Central

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

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

2010-01-01

169

Monitoring dark hydrogen fermentation performance of indigenous Clostridium butyricum by hydrogenase gene expression using RT-PCR and qPCR  

Microsoft Academic Search

Hydrogenase is the key enzyme responsible for H2 production in dark fermentation. Therefore, the expression of hydrogenase gene may be a good indicator for the performance of a dark H2 fermentation culture. In this study, we investigated the correlation between expression of the functional gene (hydA encoding for hydrogenase in Clostridium butyricum) and bioH2 production activity during batch growth of

Mei-Yun Wang; Yu-Li Tsai; Betty H. Olson; Jo-Shu Chang

2008-01-01

170

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.  

PubMed Central

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-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 last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.

Fu, C; Javedan, S; Moshiri, F; Maier, R J

1994-01-01

171

Functionally relevant interplay between the Fe(4)S(4) cluster and CN(-) ligands in the active site of [FeFe]-hydrogenases.  

PubMed

[FeFe]-hydrogenases are highly efficient H(2)-evolving metalloenzymes that include cyanides and carbonyls in the active site. The latter is an Fe(6)S(6) cluster (the so-called H-cluster) that can be subdivided into a binuclear portion carrying the CO and CN(-) groups and a tetranuclear subcluster. The fundamental role of cyanide ligands in increasing the basicity of the H-cluster has been highlighted previously. Here a more subtle but crucial role played by the two CN(-) ligands in the active site of [FeFe]-hydrogenases is disclosed. In fact, QM/MM calculations on all-atom models of the enzyme from Desulfovibrio desulfuricans show that the cyanide groups fine-tune the electronic and redox properties of the active site, affecting both the protonation regiochemistry and electron transfer between the two subclusters of the H-cluster. Despite the crucial role of cyanides in the protein active site, the currently available bioinspired electrocatalysts generally lack CN(-) groups in order to avoid competition between the latter and the catalytic metal centers for proton binding. In this respect, we show that a targeted inclusion of phosphine ligands in hexanuclear biomimetic clusters may restore the electronic and redox features of the wild-type H-cluster. PMID:20302340

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

2010-04-14

172

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

PubMed

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

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

2009-12-01

173

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

NASA Astrophysics Data System (ADS)

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

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

1996-03-01

174

Expression and Regulation of a Silent Operon, hyf, Coding for Hydrogenase 4 Isoenzyme in Escherichia coli†  

PubMed Central

On the basis of hyf-lacZ fusion studies, the hyf operon of Escherichia coli, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain. However, mutant FhlA proteins (constitutive activators of the hyc-encoded hydrogenase 3 isoenzyme) activated hyf-lacZ. HyfR, an FhlA homolog encoded by the hyfR gene present at the end of the hyf operon, also activated transcription of hyf-lacZ but did so only when hyfR was expressed from a heterologous promoter. The HYD4 isoenzyme did not substitute for HYD3 in H2 production. Optimum expression of hyf-lacZ required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator.

Self, William T.; Hasona, Adnan; Shanmugam, K. T.

2004-01-01

175

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

SciTech Connect

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.

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

1989-02-01

176

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

PubMed Central

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

Teixeira, Vitor H.; Baptista, Antonio M.; Soares, Claudio M.

2006-01-01

177

Putative signal peptide on the small subunit of the periplasmic hydrogenase from Desulfovibrio vulgaris.  

PubMed Central

We sequenced the NH2 terminus of the large and small subunits of the periplasmic hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) and found that the small subunit lacks a region of 34 NH4-terminal amino acids coded by the gene for the small subunit (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). We suggest that this region constitutes a signal peptide based on comparison with known procaryotic signal peptides. Images

Prickril, B C; Czechowski, M H; Przybyla, A E; Peck, H D; LeGall, J

1986-01-01

178

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

Microsoft Academic Search

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

A. J. Pierik

1993-01-01

179

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

PubMed Central

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.

Kow, Y W; Burris, R H

1984-01-01

180

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

PubMed

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

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

2009-12-22

181

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

PubMed Central

Nitrite, NO, CO, and C2H2 inhibited O2-dependent H2 uptake (H3H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N2O or NO3-. The apparent Ki values for inhibition of O2-dependent H2 uptake were 20 microM for NO2-, 0.4 microM for NO, 28 microM for CO, and 88 microM for C2H2. These inhibitors also affected methylene blue-dependent H2 uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H2 uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N2. The C2H2 inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO2- inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO2- on H2-dependent respiration. These results suggest that the low hydrogenase activities observed in NO3(-)-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO2- and NO produced by NO3- reduction.

Tibelius, K H; Knowles, R

1984-01-01

182

The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation.  

PubMed Central

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

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

1994-01-01

183

[NiFe] Hydrogenase from Alteromonas macleodii with Unusual Stability in the Presence of Oxygen and High Temperature ? †  

PubMed Central

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 H2 evolution and H2 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 O2 was evaluated by the hydrogen-deuterium (H-D) exchange assay. In 1% O2, 20.4% of its H-D exchange activity was retained. The great stability of HynSL makes it a potential candidate for biotechnological applications.

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

2011-01-01

184

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

PubMed Central

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

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

2012-01-01

185

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

PubMed

Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO(2) to formylmethanofuran. The best candidates for F(420)-dependent N(5),N(10)-methyl-H(4) MPT and N(5),N(10),-methylene-H(4)MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases. PMID:20884694

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

2010-09-30

186

A second soluble Hox-type NiFe enzyme completes the hydrogenase set in Thiocapsa roseopersicina BBS.  

PubMed

Three functional NiFe hydrogenases were previously characterized in Thiocapsa roseopersicina BBS: two of them are attached to the periplasmic membrane (HynSL and HupSL), and one is localized in the cytoplasm (HoxEFUYH). The ongoing genome sequencing project revealed the presence of genes coding for another soluble Hox-type hydrogenase enzyme (hox2FUYH). Hox2 is a heterotetrameric enzyme; no indication for an additional subunit was found. Detailed comparative in vivo and in vitro activity and expression analyses of HoxEFUYH (Hox1) and the newly discovered Hox2 enzyme were performed. Functional differences between the two soluble NiFe hydrogenases were disclosed. Hox1 seems to be connected to both sulfur metabolism and dark/photofermentative processes. The bidirectional Hox2 hydrogenase was shown to be metabolically active under specific conditions: it can evolve hydrogen in the presence of glucose at low sodium thiosulfate concentration. However, under nitrogen-fixing conditions, it can oxidize H(2) but less than the other hydrogenases in the cell. PMID:20543059

Maróti, Judit; Farkas, Attila; Nagy, Ildikó K; Maróti, Gergely; Kondorosi, Eva; Rákhely, Gábor; Kovács, Kornél L

2010-06-11

187

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

PubMed Central

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

Lim, Soo T.

1978-01-01

188

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

PubMed

We studied the electrocatalytic activity of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaH2ase) immobilized on single-wall carbon nanotube (SWNT) networks. SWNT networks were prepared on carbon cloth by ultrasonic spraying of suspensions with predetermined ratios of metallic and semiconducting nanotubes. Current densities for both proton reduction and hydrogen oxidation electrocatalytic activities were at least 1 order of magnitude higher when hydrogenase was immobilized onto SWNT networks with high metallic tube (m-SWNT) content in comparison to hydrogenase supported on networks with low metallic tube content or when SWNTs were absent. We conclude that the increase in electrocatalytic activities in the presence of SWNTs was mainly due to the m-SWNT fraction and can be attributed to (i) substantial increases in the active electrode surface area, and (ii) improved electronic coupling between CaH2ase redox-active sites and the electrode surface. PMID:21384925

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

2011-03-08

189

Hydrogen production by termite gut protists: characterization of iron hydrogenases of Parabasalian symbionts of the termite Coptotermes formosanus.  

PubMed

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H(2)) that is emitted by the termites; however, little is known about the physiology and biochemistry of H(2) production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H(2) 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 H(2) evolution rather than H(2) uptake. H(2) evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H(2) evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H(2) uptake activity of the endosymbiotic bacteria compensated for the strong H(2) 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 H(2) production in termite guts has been largely underestimated. PMID:17766465

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

2007-08-31

190

A QM/MM investigation of the activation and catalytic mechanism of Fe-only hydrogenases.  

PubMed

Fe-only hydrogenases are enzymes that catalyze dihydrogen production or oxidation, due to the presence of an unusual Fe(6)S(6) cluster (the so-called H-cluster) in their active site, which is composed of a Fe(2)S(2) subsite, directly involved in catalysis, and a classical Fe(4)S(4) cubane cluster. Here, we present a hybrid quantum mechanical and molecular mechanical (QM/MM) investigation of the Fe-only hydrogenase from Desulfovibrio desulfuricans, in order to unravel key issues regarding the activation of the enzyme from its completely oxidized inactive state (Hoxinact) and the influence of the protein environment on the structural and catalytic properties of the H-cluster. Our results show that the Fe(2)S(2) subcluster in the Fe(II)Fe(II) redox state - which is experimentally observed for the completely oxidized form of the enzyme - binds a water molecule to one of its metal centers. The computed QM/MM energy values for water binding to the diferrous subsite are in fact over 70 kJ mol(-1); however, the affinity toward water decreases by 1 order of magnitude after a one-electron reduction of H(ox)(inact), thus leading to the release of coordinated water from the H-cluster. The investigation of a catalytic cycle of the Fe-only hydrogenase that implies formation of a terminal hydride ion and a di(thiomethyl)amine (DTMA) molecule acting as an acid/base catalyst indicates that all steps have reasonable reaction energies and that the influence of the protein on the thermodynamic profile of H(2) production catalysis is not negligible. QM/MM results show that the interactions between the Fe(2)S(2) subsite and the protein environment could give place to structural rearrangements of the H-cluster functional for catalysis, provided that the bidentate ligand that bridges the iron atoms in the binuclear subsite is actually a DTMA residue. PMID:17602468

Greco, Claudio; Bruschi, Maurizio; De Gioia, Luca; Ryde, Ulf

2007-06-29

191

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

PubMed

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

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

2012-01-05

192

Heterologous expression of a hydrogenase gene in Enterobacter aerogenes to enhance hydrogen gas production  

Microsoft Academic Search

The hydrogenase gene from Enterobacter cloacae (IIT-BT 08) was amplified and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-4T-2-Cat\\/hydA).\\u000a The recombinant plasmid was transformed into a hydrogen-producing strain of Enterobacter aerogenes (ATCC13408). SDS–PAGE and western blot analysis confirmed the successful expression of the GST-tagged hydA protein. Anaerobic\\u000a fermentation for the production of hydrogen from glucose was

Jin-Fang Zhao; Wen-Lu Song; Jun Cheng; Chuan-Xi Zhang

2010-01-01

193

Construction of a chassis for hydrogen production: physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking a functional bidirectional hydrogenase.  

PubMed

Cyanobacteria are photosynthetic prokaryotes that are promising 'low-cost' microbial cell factories due to their simple nutritional requirements and metabolic plasticity, and the availability of tools for their genetic manipulation. The unicellular non-nitrogen-fixing Synechocystis sp. PCC 6803 is the best studied cyanobacterial strain and its genome was the first to be sequenced. The vast amount of physiological and molecular data available, together with a relatively small genome, makes Synechocystis suitable for computational metabolic modelling and to be used as a photoautotrophic chassis in synthetic biology applications. To prepare it for the introduction of a synthetic hydrogen producing device, a Synechocystis sp. PCC 6803 deletion mutant lacking an active bidirectional hydrogenase (?hoxYH) was produced and characterized at different levels: physiological, proteomic and transcriptional. The results showed that, under conditions favouring hydrogenase activity, 17 of the 210 identified proteins had significant differential fold changes in comparisons of the mutant with the wild-type. Most of these proteins are related to the redox and energy state of the cell. Transcriptional studies revealed that only six genes encoding those proteins exhibited significant differences in transcript levels. Moreover, the mutant exhibits similar growth behaviour compared with the wild-type, reflecting Synechocystis plasticity and metabolic adaptability. Overall, this study reveals that the Synechocystis ?hoxYH mutant is robust and can be used as a photoautotrophic chassis for the integration of synthetic constructs, i.e. molecular constructs assembled from well characterized biological and/or synthetic parts (e.g. promoters, regulators, coding regions, terminators) designed for a specific purpose. PMID:22096147

Pinto, Filipe; van Elburg, Karin A; Pacheco, Catarina C; Lopo, Miguel; Noirel, Josselin; Montagud, Arnau; Urchueguía, Javier F; Wright, Phillip C; Tamagnini, Paula

2011-11-17

194

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

PubMed Central

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

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

2012-01-01

195

Spectroelectrochemical study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F in solution and immobilized on biocompatible gold surfaces.  

PubMed

The catalytic cycle of the anaerobic [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) both in solution and immobilized on an Au electrode was studied by IR spectroscopic and electrochemical methods. IR spectroelectrochemistry in solution at different pH values allows the identification of the various redox-states of the active site and the determination of the midpoint potentials, as well as their acid-base equilibria. The spectroscopic characterization was based on the unique marker bands of the CN and CO stretching modes of the Ni-Fe center and served as reference for the surface-enhanced IR absorption (SEIRA) study of the immobilized enzyme. Using structural models of hydrogenases from DvMF and Desulfovibrio gigas , dipole moment calculations were carried out to guide the immobilization strategy. In view of the high dipole moment of about 1100 D pointing through the negatively charged area surrounding the distal [FeS] cluster, the Au electrode was coated by a self-assembled monolayer of amino-terminated mercaptanes which, due to the positively charged head groups, permit a durable electrostatic binding of the protein. SEIRA spectroscopy revealed a structurally and functionally intact active site as demonstrated by the reversible activation and inactivation under hydrogen and argon, respectively. Cyclic voltammetry on the immobilized enzyme demonstrate a reversible anaerobic inactivation upon changing the applied potential. The "switch" potential (E(switch)) associated with the reductive reactivation was determined to be -33 mV (vs normal hydrogen electrode). However, the catalytic current decreased on the time scale of hours during continuous cycling. SEIRA experiments demonstrate that the loss of catalytic activity is not due to protein desorption but is rather related to a slow degradation of the active site, possibly initiated by the attack of reactive species electrochemically generated from residual traces of oxygen in solution. PMID:19845323

Millo, Diego; Pandelia, Maria-Eirini; Utesch, Tillmann; Wisitruangsakul, Nattawadee; Mroginski, Maria A; Lubitz, Wolfgang; Hildebrandt, Peter; Zebger, Ingo

2009-11-19

196

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

PubMed Central

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

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

1988-01-01

197

Purification and characterization of putative alkaline [Ni-Fe] hydrogenase from unicellular marine green alga, Tetraselmis kochinensis NCIM 1605.  

PubMed

Hydrogenase enzyme from the unicellular marine green alga Tetraselmis kochinensis NCIM 1605 was purified 467 fold to homogeneity. The molecular weight was estimated to be approximately 89kDa by SDS-PAGE. This enzyme consists of two subunits with molecular masses of approximately 70 and approximately 19kDa. The hydrogenase was found to contain 10g atoms of Fe and 1g of atom of Ni per mole of protein. The specific activity of hydrogen evolution was 50micromol H(2)/mg/h of enzyme using reduced methyl viologen as an electron donor. This hydrogenase enzyme has pI value approximately 9.6 representing its alkaline nature. The absorption spectrum of the hydrogenase enzyme showed an absorption peak at 425nm indicating that the enzyme had iron-sulfur clusters. The total of 16 cysteine residues were found per mole of enzyme under the denaturing condition and 20 cysteine residues in reduced denatured enzyme indicating that it has two disulfide bridges. PMID:17321121

Bhosale, S H; Pant, A; Khan, M I

2007-02-23

198

GOLLUM [FeFe]-hydrogenase-like proteins are essential for plant development in normoxic conditions and modulate energy metabolism.  

PubMed

[FeFe]-hydrogenase-like genes encode [Fe4 S4 ]-containing proteins that are ubiquitous in eukaryotic cells. In humans, iron-only hydrogenase-like protein 1 (IOP1) represses hypoxia inducible factor-1? subunit (HIF1-?) at normal atmospheric partial O2 pressure (normoxia, 21?kPa O2 ). In yeasts, the nar1 mutant cannot grow at 21?kPa O2 , but can develop at a lower O2 pressure (2?kPa O2 ). We show here that plant [FeFe]-hydrogenase-like GOLLUM genes are essential for plant development and cell cycle progression. The mutant phenotypes of these plants are seen in normoxic conditions, but not under conditions of mild hypoxia (5?kPa O2 ). Transcriptomic and metabolomic experiments showed that the mutation enhances the expression of some hypoxia-induced genes under normal atmospheric O2 conditions and changes the cellular content of metabolites related to energy metabolism. In conclusion, [FeFe]-hydrogenase-like proteins play a central role in eukaryotes including the adaptation of plants to the ambient O2 partial pressure. PMID:23639116

Mondy, Samuel; Lenglet, Aurore; Cosson, Viviane; Pelletier, Sandra; Pateyron, Stéphanie; Gilard, Françoise; Scholte, Marije; Brocard, Lysiane; Couzigou, Jean-Malo; Tcherkez, Guillaume; Péan, Michel; Ratet, Pascal

2013-05-01

199

Expression of Shewanella oneidensis MR-1 [FeFe]-hydrogenase genes in Anabaena sp. strain PCC 7120.  

PubMed

H(2) generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H(2) from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O(2) sensitive. Certain filamentous cyanobacteria protect nitrogenase from O(2) by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P(hetN). Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O(2). PMID:23023750

Gärtner, Katrin; Lechno-Yossef, Sigal; Cornish, Adam J; Wolk, C Peter; Hegg, Eric L

2012-09-28

200

Attenuated total reflectance infrared spectroelectrochemistry at a carbon particle electrode; unmediated redox control of a [NiFe]-hydrogenase solution.  

PubMed

We report a versatile infrared spectroscopic method for studying redox chemistry of metalloproteins, and demonstrate for the first time electrochemically-induced changes to the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. A carbon particle network working electrode allows control over a wide potential window without the need for solution mediators. PMID:23552374

Healy, Adam J; Ash, Philip A; Lenz, Oliver; Vincent, Kylie A

2013-05-21

201

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803*  

PubMed Central

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S]+ and at least one [2Fe2S]+ cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.

Germer, Frauke; Zebger, Ingo; Saggu, Miguel; Lendzian, Friedhelm; Schulz, Rudiger; Appel, Jens

2009-01-01

202

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

PubMed

Complex enzymes containing Fe-S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear. Here we show the structure of HydA(DeltaEFG) (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe-4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes. PMID:20418861

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

2010-04-25

203

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

PubMed

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

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

2011-10-07

204

Connecting [NiFe]- and [FeFe]-hydrogenases: mixed-valence nickel-iron dithiolates with rotated structures.  

PubMed

New mixed-valence iron-nickel dithiolates are described that exhibit structures similar to those of mixed-valence diiron dithiolates. The interaction of tricarbonyl salt [(dppe)Ni(pdt)Fe(CO)(3)]BF(4) ([1]BF(4), where dppe = Ph(2)PCH(2)CH(2)PPh(2) and pdt(2-) = -SCH(2)CH(2)CH(2)S-) with P-donor ligands (L) afforded the substituted derivatives [(dppe)Ni(pdt)Fe(CO)(2)L]BF(4) incorporating L = PHCy(2) ([1a]BF(4)), PPh(NEt(2))(2) ([1b]BF(4)), P(NMe(2))(3) ([1c]BF(4)), P(i-Pr)(3) ([1d]BF(4)), and PCy(3) ([1e]BF(4)). The related precursor [(dcpe)Ni(pdt)Fe(CO)(3)]BF(4) ([2]BF(4), where dcpe = Cy(2)PCH(2)CH(2)PCy(2)) gave the more electron-rich family of compounds [(dcpe)Ni(pdt)Fe(CO)(2)L]BF(4) for L = PPh(2)(2-pyridyl) ([2a]BF(4)), PPh(3) ([2b]BF(4)), and PCy(3) ([2c]BF(4)). For bulky and strongly basic monophosphorus ligands, the salts feature distorted coordination geometries at iron: crystallographic analyses of [1e]BF(4) and [2c]BF(4) showed that they adopt "rotated" Fe(I) centers, in which PCy(3) occupies a basal site and one CO ligand partially bridges the Ni and Fe centers. Like the undistorted mixed-valence derivatives, members of the new class of complexes are described as Ni(II)Fe(I) (S = ½) systems according to electron paramagnetic resonance spectroscopy, although with attenuated (31)P hyperfine interactions. Density functional theory calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the spin for [1e](+) is mostly localized in a Fe(I)-centered d(z(2)) orbital, orthogonal to the Fe-P bond. The PCy(3) complexes, rare examples of species featuring "rotated" Fe centers, both structurally and spectroscopically incorporate features from homobimetallic mixed-valence diiron dithiolates. Also, when the NiS(2)Fe core of the [NiFe]-hydrogenase active site is reproduced, the "hybrid models" incorporate key features of the two major classes of hydrogenase. Furthermore, cyclic voltammetry experiments suggest that the highly basic phosphine ligands enable a second oxidation corresponding to the couple [(dxpe)Ni(pdt)Fe(CO)(2)L](+/2+). The resulting unsaturated 32e(-) dications represent the closest approach to modeling the highly electrophilic Ni-SI(a) state. In the case of L = PPh(2) (2-pyridyl), chelation of this ligand accompanies the second oxidation. PMID:22838645

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

2012-07-27

205

Konetsu kosansei suiso saikin kara seiseishita hidorogenase no denkyoku shokubai to shite no tekisei. (Characterization of the hydrogenase from thermoacidophilic aerobic hydrogen-oxidizing bacterium, hydrogenobacter acidophilus).  

National Technical Information Service (NTIS)

Hydrogenate is an enzyme of bacteria, which catalyzes oxidation and reduction of molecular hydrogen. The enzyme has been expected as an alternative catalyst for expensive platinum. However, hydrogenase is very sensitive to oxygen and high temperature, so ...

K. Sasaki S. Nakasono

1995-01-01

206

Electronic structure of a binuclear nickel complex of relevance to [NiFe] hydrogenase.  

PubMed

The binuclear complex [Ni(2)(L)(MeCN)(2)](3+) (L(2-) = compartmental macrocycle incorporating imine N and thiolate S donors) has a Ni(III) center bridged via two thiolate S-donors to a diamagnetic Ni(II) center. The ground-state has dominant 3d(z)(1)(2) character similar to that observed for [NiFe] hydrogenases in which Ni(III) is bridged via two thiolate donors to a diamagnetic center (Fe(II)). The system has been studied by X-ray crystallography and pulse EPR, ESEEM, and ENDOR spectroscopy in order to determine the extent of spin-delocalization onto the macrocycle L(2-). The hyperfine coupling constants of six nitrogen atoms have been identified and divided into three sets of two equivalent nitrogens. The most strongly coupled nitrogen atoms (a(iso) approximately 53 MHz) stem from axially bound solvent acetonitrile molecules. The two macrocycle nitrogens on the Ni(III) side have a coupling of a(iso) approximately 11 MHz, and those on the Ni(II) side have a coupling of a(iso) approximately 1-2 MHz. Density functional theory (DFT) calculations confirm this assignment, while comparison of the calculated and experimental (14)N hyperfine coupling constants yields a complete picture of the electron-spin density distribution. In total, 91% spin density is found at the Ni(III) of which 72% is in the 3d(z)(2) orbital and 16% in the 3d(xy) orbital. The Ni(II) contains -3.5% spin density, and 7.5% spin density is found at the axial MeCN ligands. In analogy to hydrogenases, it becomes apparent that binding of a substrate to Ni at the axial positions causes a redistribution of the electron charge and spin density, and this redistribution polarizes the chemical bonds of the axial ligand. For [NiFe] hydrogenases this implies that the H(2) bond becomes polarized upon binding of the substrate, which may facilitate its heterolytic splitting. PMID:18998627

van Gastel, Maurice; Shaw, Jennifer L; Blake, Alexander J; Flores, Marco; Schröder, Martin; McMaster, Jonathan; Lubitz, Wolfgang

2008-12-15

207

Metabolic engineering for solvent productivity by downregulation of the hydrogenase gene cluster hupCBA in Clostridium saccharoperbutylacetonicum strain N1-4  

Microsoft Academic Search

The selective production of acetone and butanol is highly desirable from the viewpoint of biofuel production. We have manipulated\\u000a the activity level of a hydrogenase for this purpose because hydrogen and solvent production are closely correlated with each\\u000a other. First, we cloned the hydrogenase gene cluster from Clostridium saccharoperbutylacetonicum strain N1-4 and downregulated its expression using an antisense RNA strategy.

Shun-ichi Nakayama; Tomoyuki Kosaka; Hanako Hirakawa; Kentaro Matsuura; Sadazo Yoshino; Kensuke Furukawa

2008-01-01

208

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

PubMed

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

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

2013-09-02

209

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

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

Changlin Fu; Robert J. Maier

1992-01-01

210

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

Microsoft Academic Search

Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H2 and is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). To elucidate the physiological role(s) of Ech a mutant lacking this enzyme was constructed. The mutant was unable to grow on methanol\\/H2\\/CO2,

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

2002-01-01

211

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

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.

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

1983-01-01

212

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

PubMed

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

La Favre, J S; Focht, D D

1983-08-01

213

H?-driven cofactor regeneration with NAD(P)?-reducing hydrogenases.  

PubMed

A large number of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amounts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H?-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H? as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H?-driven cofactor-recycling systems and examples of their integration with enzyme reactions are summarized in this article. The O?-tolerant NAD?-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for technical applications. PMID:23497170

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

2013-04-17

214

Spin dynamics and criteria for onset of exchange bias in superspin glass Fe/?-Fe2O3 core-shell nanoparticles  

NASA Astrophysics Data System (ADS)

A detailed study is presented on Fe/?-Fe2O3 core-shell structured nanoparticles (mean size ˜10 nm) to understand the spin dynamics of the core and shell independently and their role in triggering exchange bias (EB) phenomena. The particle dynamics critically slow down at Tg ˜ 68 K, below which they exhibit memory effect in field-cooled and zero-field-cooled protocols associated with a superspin glass state. The field dependence of mean blocking temperature fits the de Almeida-Thouless line and shows two different linear responses in the low and high field regimes corresponding to the core and shell, respectively. We show that the energy barrier distribution estimated from the temperature decay of isothermal remanent magnetization shows two maxima that mark the freezing temperatures of the core (Tf-cr ˜ 48 K) and shell (Tf-sh ˜ 21 K). Last, hysteresis measurements after field cooling reveal strong EB indicated by a loop shift associated with unidirectional anisotropy. The onset of EB is at 35 K when the ferromagnetic core is frozen and the moments in the ferrimagnetic shell begin to block, resulting in enhanced exchange coupling.

Chandra, Sayan; Khurshid, H.; Li, Wanfeng; Hadjipanayis, G. C.; Phan, M. H.; Srikanth, H.

2012-07-01

215

Magnetic and transport properties of nanocomposite Fe/Fe3-?O4 and Fe3-?O4 films prepared by plasma-enhanced chemical vapour deposition  

NASA Astrophysics Data System (ADS)

Reflective, pinhole-free Fe/Fe3O4 and Fe3-?O4/Fe2O3 nanocomposite films were obtained by reacting iron pentacarbonyl, Fe(CO)5, in an inductively-coupled radio frequency glow discharge reactor. The conductivity of the Fex/(Fe3O4)1-x (x > 7%) composite films exhibits metallic characteristics and the conductivity decreases as the ?-Fe content decreases. The metal-to-insulator transition temperature of the Fex/(Fe3O4)1-x (x ~0.07) films shifts to a higher temperature as compared with Fe3-?O4 due to the increased conductivity from ?-Fe. The magnetization versus temperature (M-T) curves show the transition temperature ranging from 95 to 136 K, corresponding to the Verwey temperature, which is dependent on the film composition. The Fe3-?O4 film exhibits a negative magnetoresistance of about 4% and 8% at room temperature and 80 K, respectively.

Yang, J. B.; Malik, S. K.; Zhou, X. D.; Kim, M. S.; Yelon, W. B.; James, W. J.; Anderson, H. U.

2005-04-01

216

Genetic Diversity and Expression of the (NiFe) Hydrogenase Large-Subunit Gene of Desulfovibrio spp. in Environmental Samples  

Microsoft Academic Search

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

CATHRIN WAWER; MIKE S. M. JETTEN; GERARD MUYZER

1997-01-01

217

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

PubMed Central

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

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

1985-01-01

218

Mechanistic insight into the blocking of CO diffusion in [NiFe]-hydrogenase mutants through multiscale simulation  

PubMed Central

[NiFe]-hydrogenases are fascinating biological catalysts with potential application in biofuel cells. However, a severe problem in practical application is the strong sensitivity of hydrogenase to gaseous inhibitor molecules such as CO and O2. Recently, a number of successful protein engineering studies have been reported that aimed at lowering the access of diatomic inhibitors to the active site pocket, but the molecular mechanism conferring increased resistance remained unclear. Here we use a multiscale simulation approach combining molecular dynamics with a master equation formalism to explain the steady drop in CO diffusion rate observed for the mutants V74M L122A, V74M L122M, and V74M of Desulfovibrio fructosovorans [NiFe]-hydrogenase. We find that diffusion in these variants is controlled by two gates, one between residues 74 and 476 and the other between residues 74 and 122. The existence of two control points in different locations explains why the reduction in the experimental diffusion rate does not simply correlate with the width of the main gas channel. We also find that in the more effective mutation (V74M) CO molecules are still able to reach the active site through transitions that are gated by the microsecond dihedral motions of the side chain of R476 and the thermal fluctuations of the width of the gas channel defined by M74 and L122. Reflecting on the molecular information gained from simulation, we discuss future mutation experiments that could further lower the diffusion rates of small ligands inhibiting [NiFe]-hydrogenase.

Wang, Po-hung; Blumberger, Jochen

2012-01-01

219

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

Microsoft Academic Search

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 hydro- genase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p

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

2004-01-01

220

Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein nifA.  

PubMed

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupSp). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential -173 to -88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule. PMID:9177161

Brito, B; Martínez, M; Fernández, D; Rey, L; Cabrera, E; Palacios, J M; Imperial, J; Ruiz-Argüeso, T

1997-06-10

221

Biochemical Analysis of the Interactions between the Proteins Involved in the [FeFe]-Hydrogenase Maturation Process*  

PubMed Central

[FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.

Vallese, Francesca; Berto, Paola; Ruzzene, Maria; Cendron, Laura; Sarno, Stefania; De Rosa, Edith; Giacometti, Giorgio M.; Costantini, Paola

2012-01-01

222

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

PubMed

Sensitive electrochemical sensing for the DNA damage in situ based on a cathodic process of Fe@Fe(2)O(3) core-shell nanonecklace and Au nanoparticles was performed by a novel biosensor, which was constructed via a glassy carbon electrode (GCE) modified with a multilayer film comprising of separate layers of poly(dimethyldiallylammonium chloride) (PDDA), the mixture of Fe@Fe(2)O(3) core-shell nanonecklace and Au nanoparticles, PDDA and double strand DNA (ds-DNA). Iron ions and H(2)O(2) (Fenton reagents) were generated continuously at a constant rate by the cathodic process. The two Fenton reagents reacted further to generate hydroxyl radicals in situ, which attacked ds-DNA in the film and caused severe damage of ds-DNA molecules. These courses of DNA damage were just like those happened in organism. It could be used to mimic metal toxicity pathways in vivo. Fe@Fe(2)O(3) core-shell nanonecklace and Au nanoparticles played considerable synergistic effects for DNA damage. Differential pulse voltammetry and cyclic voltammetry were applied to monitor the DNA damage. Different from the previously reported metal-mediated DNA damage sensor, the process of the ds-DNA damage was not achieved in the solution of metal ions and H(2)O(2), but merely in buffer solution, and the instable enzymes were not used in the whole course. The biosensor possesses the potential as a screening tool for rapid assessment of the genotoxicity of existing and new chemicals. PMID:19734034

Wang, Xueliang; Yang, Tao; Jiao, Kui

2009-04-24

223

A new cumulene diiron complex related to the active site of Fe-only hydrogenases and its phosphine substituted derivatives: synthesis, electrochemistry and structural characterization.  

PubMed

A new cumulene diiron complex related to the Fe-only hydrogenase active site [(?-SCH(2)C(S)CCH(2))Fe(2)(CO)(6)] (1) was obtained by treatment of (?-LiS)(2)Fe(2)(CO)(6) with excess 1,4-dichloro-2-butyne. By controllable CO displacement of 1 with PPh(3) and bis(diphenylphosphino)methane (dppm), mono- and di-substituted complexes, namely [(?-SCH(2)C(S)CCH(2))Fe(2)(CO)(5)L] (2: L=PPh(3); 3: L=dppm) and [(?-SCH(2)C(S)CCH(2))Fe(2)(CO)(4)L(2)] (4: L=PPh(3); 5: L=dppm) could be prepared in moderate yields. Treatment of 1 with bis(diphenylphosphino)ethane (dppe) afforded a double butterfly complex [(?-SCH(2)C(S)CCH(2))Fe(2)(CO)(5)](2)(?-dppe) (7). With dppm in refluxing toluene, a dppm-bridged complex [(?-SCH(2)C(S)CCH(2))Fe(2)(CO)(4)(?-dppm)] (6) was obtained. These model complexes were characterized by IR, (1)H, (31)P NMR spectra and the molecular structures of 1, 2 and 5-7 were determined by single crystal X-ray analyses. The electrochemistry of 1-3 was studied and the electrocatalytic property of 1 was investigated for proton reduction in the presence of HOAc. PMID:21704584

Wen, Na; Xu, Fenfen; Feng, Yanan; Du, Shaowu

2011-06-06

224

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

PubMed

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

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

2013-02-27

225

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

PubMed Central

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

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

1989-01-01

226

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

SciTech Connect

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

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

1989-03-01

227

A dithiolate-bridged (CN)2(CO)Fe-Ni complex reproducing the IR bands of [NiFe] hydrogenase.  

PubMed

A dithiolate-bridged dinuclear Fe-Ni complex, which has the desired fac-(CN)(2)(CO) ligand set at iron, has been synthesized. Its CN/CO bands in the IR spectrum reproduce those of the Ni-A, Ni-B, and Ni-SU states, which indicate that these octahedral Fe(II) centers have similar electronic properties. This result verifies the assignment of a (CN)(2)(CO)Fe(II) moiety in the active site of [NiFe] hydrogenase. PMID:19222192

Tanino, Soichiro; Li, Zilong; Ohki, Yasuhiro; Tatsumi, Kazuyuki

2009-03-16

228

FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791.  

PubMed Central

Rhizobium leguminosarum bv. viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr. This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome. Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-like proteins. Individual R. leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix+ phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules. In contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities. Unlike the wild-type strain and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis. In the search for symbiotic genes that could be controlled by FnrN, a fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain UPM791 and partially sequenced. The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the fixN promoter region. Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain. A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant. The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype. These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum bv. viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene.

Gutierrez, D; Hernando, Y; Palacios, J M; Imperial, J; Ruiz-Argueso, T

1997-01-01

229

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

PubMed Central

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

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

2011-01-01

230

Proton management as a design principle for hydrogenase-inspired catalysts  

SciTech Connect

The properties of the hydrogenase-inspired [Ni(PNP)2]2+ (PNP ¼ Et2PCH2NMeCH2PEt2) catalyst for homogeneous hydrogen oxidation in acetonitrile solution are explored from a theoretical perspective for hydrogen production. The defining characteristic of this catalyst is the presence of pendent bases in the second coordination sphere that function as proton relays between the solution and the metal center. DFT calculations of the possible intermediates along proposed catalytic pathways are carried out and used to construct coupled Pourbaix diagrams of the redox processes and free-energy profiles along the reaction pathways. Analysis of the coupled Pourbaix diagrams reveals insights into the intermediate species and the mechanisms favored at different pH values of the solution. Consideration of the acid-base behavior of the metal hydride and H2 adduct species imposes additional constraints on the reaction mechanism, which can involve intramolecular as well as intermolecular proton-coupled electron-transfer steps. The efficacy of the catalyst is shown to depend critically on the pKa values of these potential intermediates, as they control both the species in solution at a given pH and the freeenergy profile of reaction pathways. Optimal relationships among these pKa values can be identified, and it is demonstrated that ‘‘proton management’’, i.e., the manipulation of these pKa values (e.g., through choice of metal or substituents on ligands), can serve as a design principle for improved catalytic behavior. This material is based upon work supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.

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

2011-06-01

231

Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation.  

PubMed

Proton motive force (?p) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical-the membrane potential (??) and chemical-the pH transmembrane gradient (?pH), were established and the effects of external pH (pHex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pHex at pH 7.5 and 6.5, respectively; and it was higher than pHex at pH 5.5. At high pHex, the increase of ?? (-130 mV) was only partially compensated by a reversed ?pH, resulting in a low ?p. At low pHex ?? and consequently ?p were decreased. The generation of ?p during glycerol fermentation was compared with glucose fermentation, and the difference in ?p might be due to distinguished mechanisms for H(+) transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F0F1-ATPase. H(+) efflux was determined to depend on pHex; overall and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibitory H(+) efflux was maximal at pH 6.5. Moreover, ?pH was changed at pH 6.5 and ?p was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, ?p generation by E. coli during glycerol fermentation is different than that during glucose fermentation. ?p is dependent on pHex, and a role of Hyd enzymes in its generation is suggested. PMID:23271421

Trchounian, Karen; Blbulyan, Syuzanna; Trchounian, Armen

2012-12-28

232

Proton Management as a Design Principle for Hydrogenase-inspired Catalysts  

SciTech Connect

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

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

2011-08-01

233

Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.  

PubMed Central

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown. Images FIG. 2 FIG. 3

Menon, N K; Robbins, J; Peck, H D; Chatelus, C Y; Choi, E S; Przybyla, A E

1990-01-01

234

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

PubMed Central

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.

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

2004-01-01

235

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

PubMed Central

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

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

1983-01-01

236

Radioassay for hydrogenase activity in viable cells and documentation of aerobic hydrogen-consuming bacteria living in extreme environments  

SciTech Connect

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

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

1983-05-01

237

Disruption of the Operon Encoding Ehb Hydrogenase Limits AnabolicCO2 Assimilation in the Archaeon Methanococcus maripaludis  

SciTech Connect

Methanococcus maripaludis is a mesophilic archaeon thatreduces CO2 to methane with H2 or formate as an energy source. Itcontains two membrane-bound energy-conserving hydrogenases, Eha and Ehb.To determine therole of Ehb, a deletion in the ehb operon wasconstructed to yield the mutant, strain S40. Growth of S40 was severelyimpaired in minimal medium. Both acetate and yeast extract were necessaryto restore growth to nearly wild-type levels, suggesting that Ehb wasinvolved in multiple steps in carbon assimilation. However, nodifferences in the total hydrogenase specific activities were foundbetween the wild type and mutant in either cell extracts ormembrane-purified fractions. Methanogenesis by resting cells withpyruvate as the electron donor was also reduced by 30 percent in S40,suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetylcoenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specificactivities in the mutant, and genes encoding these enzymes, as well asAMP-forming acetyl-CoA synthetase, were expressed at increased levels.These observations support a role for Ehb in anabolic CO2 assimilation inmethanococci.

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

2006-02-01

238

Sequence analysis and interposon mutagenesis of the hupT gene, which encodes a sensor protein involved in repression of hydrogenase synthesis in Rhodobacter capsulatus.  

PubMed

The hupT gene, which represses hydrogenase gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus, has been identified and sequenced. The nucleotide sequence of hupT and of the contiguous downstream open reading frame, hupU, is reported. The HupT protein of 456 amino acids (48,414 Da) has sequence similarity with the FixL, DctB, NtrB, and ArcB proteins and is predicted to be a soluble sensor kinase. Insertional inactivation of the hupT gene led to deregulation of transcriptional control, so that the hydrogenase structural operon hupSLC became overexpressed in cells grown anaerobically or aerobically. The HupT- mutants were complemented in trans by a plasmid containing an intact copy of the hupT gene. The hupU open reading frame, capable of encoding a protein of 84,879 Da, shared identity with [NiFe]hydrogenase subunits; the strongest similarity was observed with the periplasmic hydrogenase of Desulfovibrio baculatus. PMID:8226687

Elsen, S; Richaud, P; Colbeau, A; Vignais, P M

1993-11-01

239

Theoretical Spectroscopy of the Ni(II) Intermediate States in the Catalytic Cycle and the Activation of [NiFe] Hydrogenases.  

PubMed

[NiFe] hydrogenases catalyze the reversible oxidation of dihydrogen. The corresponding catalytic cycle involves a formidable number of redox states of the Ni-Fe active site; these can be distinguished experimentally by the IR stretching frequencies of their CN and CO ligands coordinated to iron. These spectroscopic fingerprints serve as sensitive probes for the intrinsic electronic structure of the metal core and, indirectly, for the structural composition of the active site. In this study, density functional theory (DFT) was used to calculate vibrational frequencies, by focusing on the EPR-silent intermediate states that contain divalent metal centers. By using the well-characterized Ni-C and Ni-B states as references, we identified candidates for the Ni-SIr , Ni-SIa , and Ni-R states by matching the predicted relative frequency shifts with experimental results. The Ni-SIr and Ni-SIa states feature a water molecule loosely bound to nickel and a formally vacant bridge. Both states are connected to each other through protonation equilibria; that is, in the Ni-SIa state one of the terminal thiolates is protonated, whereas in Ni-SIr this thiolate is unprotonated. For the reduced Ni-R state two feasible models emerged: in one, H2 coordinates side-on to nickel, and the second features a hydride bridge and a protonated thiolate. The Ni-SU state remains elusive as no unequivocal correspondence between the experimental data and calculated frequencies of the models was found, thus indicating that a larger structural rearrangement might occur upon reduction from Ni-A to Ni-SU and that the bridging ligand might dissociate. PMID:23703916

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

2013-05-22

240

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

PubMed Central

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

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

2012-01-01

241

Mixed-Valence Nickel-Iron Dithiolate Models of the [NiFe]-Hydrogenase Active Site  

PubMed Central

A series of mixed-valence iron-nickel dithiolates is described. Oxidation of (diphosphine)Ni(dithiolate)Fe(CO)3 complexes 1, 2, and 3 with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)Ni(pdt)Fe(CO)3]+ ([1]+), [(dppe)Ni(edt)Fe(CO)3]+ ([2]+) and [(dcpe)Ni(pdt)Fe(CO)3]+ ([3]+), respectively, where dppe = Ph2PCH2CH2PPh2, dcpe = Cy2PCH2CH2PCy2, pdtH2 = HSCH2CH2CH2SH and edtH2 = HSCH2CH2SH. The cation [2]+ proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as Cs-symmetric species. Crystallographic characterization of [3]BF4 shows that Ni is square planar. Interaction of [1]BF4 with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)Ni(pdt)Fe(CO)2L]BF4 for L = P(OPh)3 ([4a]BF4), P(p-C6H4Cl)3 ([4b]BF4), PPh2(2-py) ([4c]BF4), PPh2(OEt) ([4d]BF4), PPh3 ([4e]BF4), PPh2(o-C6H4OMe) ([4f]BF4), PPh2(o-C6H4OCH2OMe) ([4g]BF4), P(p-tol)3 ([4h]BF4), P(p-C6H4OMe)3 ([4i]BF4), PMePh2 ([4j]BF4). EPR analysis indicates that ethanedithiolate [2]+ exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of 1 and oxidized S = ½ [4e]BF4 are both consistent with a low-spin Fe(i) state. The hyperfine coupling tensor of [4e]BF4 has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in a Fe(i)-centered d(z2) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [1]+ with CN- and compact basic phosphines results in complex reactions. With dppe, [1]+ undergoes quasi-disproportionation to give 1 and the diamagnetic complex [(dppe)Ni(pdt)Fe(CO)2(dppe)]2+ ([5]2+), which features square-planar Ni linked to an octahedral Fe center.

Schilter, David; Nilges, Mark J.; Chakrabarti, Mrinmoy; Lindahl, Paul A.; Rauchfuss, Thomas B.; Stein, Matthias

2012-01-01

242

Iron Acyl Thiolato Carbonyls: Structural Models for the Active Site of the [Fe]-Hydrogenase (Hmd)  

PubMed Central

Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls via the reaction of phosphine thioesters and sources of Fe(0). The reaction generates both Fe(SPh)(Ph2PC6H4CO)(CO)3 (1) and the diferrous diacyl Fe2(SPh)2(CO)3(Ph2PC6H4CO)2, which carbonylates to give 1. For the extremely bulky arylthioester Ph2PC6H4C(O)SC6H4-2,6-(2,4,6-trimethylphenyl)2, oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained. Complex 1 reacts with cyanide to give Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2] (Et4N[2]). 13C and 31P NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2]? in CH2Cl2 solution very closely matches that for HmdCN. XANES and EXAFS measurements also indicate close structural and electronic similarity of Et4N[2] to the active site of wild-type Hmd. Complex 1 also stereospecifically forms a derivative with TsCH2NC, but the adduct is more labile than Et4N[2]. Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd.

Royer, Aaron M.; Salomone-Stagni, Marco

2012-01-01

243

Mixed-valence nickel-iron dithiolate models of the [NiFe]-hydrogenase active site.  

PubMed

A series of mixed-valence nickel-iron dithiolates is described. Oxidation of (diphosphine)Ni(dithiolate)Fe(CO)(3) complexes 1, 2, and 3 with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)Ni(pdt)Fe(CO)(3)](+) ([1](+)), [(dppe)Ni(edt)Fe(CO)(3)](+) ([2](+)) and [(dcpe)Ni(pdt)Fe(CO)(3)](+) ([3](+)), respectively, where dppe = Ph(2)PCH(2)CH(2)PPh(2), dcpe = Cy(2)PCH(2)CH(2)PCy(2), (Cy = cyclohexyl), pdtH(2) = HSCH(2)CH(2)CH(2)SH, and edtH(2) = HSCH(2)CH(2)SH. The cation [2](+) proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as C(s)-symmetric species. Crystallographic characterization of [3]BF(4) shows that Ni is square planar. Interaction of [1]BF(4) with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)Ni(pdt)Fe(CO)(2)L]BF(4) for L = P(OPh)(3) ([4a]BF(4)), P(p-C(6)H(4)Cl)(3) ([4b]BF(4)), PPh(2)(2-py) ([4c]BF(4)), PPh(2)(OEt) ([4d]BF(4)), PPh(3) ([4e]BF(4)), PPh(2)(o-C(6)H(4)OMe) ([4f]BF(4)), PPh(2)(o-C(6)H(4)OCH(2)OMe) ([4g]BF(4)), P(p-tol)(3) ([4h]BF(4)), P(p-C(6)H(4)OMe)(3) ([4i]BF(4)), and PMePh(2) ([4j]BF(4)). EPR analysis indicates that ethanedithiolate [2](+) exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of 1 and oxidized S = 1/2 [4e]BF(4) are both consistent with a low-spin Fe(I) state. The hyperfine coupling tensor of [4e]BF(4) has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in an Fe(I)-centered d(z(2)) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [1](+) with CN(-) and compact basic phosphines results in complex reactions. With dppe, [1](+) undergoes quasi-disproportionation to give 1 and the diamagnetic complex [(dppe)Ni(pdt)Fe(CO)(2)(dppe)](2+) ([5](2+)), which features square-planar Ni linked to an octahedral Fe center. PMID:22304696

Schilter, David; Nilges, Mark J; Chakrabarti, Mrinmoy; Lindahl, Paul A; Rauchfuss, Thomas B; Stein, Matthias

2012-02-03

244

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

SciTech Connect

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

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

2011-08-02

245

Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae  

PubMed Central

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (?hupG, ?hupH, ?hupI, and ?hupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The ?hupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the ?hupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from ?hupH, ?hupI, and ?hupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the ?hupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

Manyani, Hamid; Rey, Luis; Palacios, Jose M.; Imperial, Juan; Ruiz-Argueso, Tomas

2005-01-01

246

Multiscale simulation reveals multiple pathways for H2 and O2 transport in a [NiFe]-hydrogenase.  

PubMed

Hydrogenases are enzymes that catalyze the reversible conversion of hydrogen molecules to protons and electrons. The mechanism by which the gas molecules reach the active site is important for understanding the function of the enzyme and may play a role in the selectivity for hydrogen over inhibitor molecules. Here, we develop a general multiscale molecular simulation approach for the calculation of diffusion rates and determination of pathways by which substrate or inhibitor gases can reach the protein active site. Combining kinetic data from both equilibrium simulations and enhanced sampling, we construct a master equation describing the movement of gas molecules within the enzyme. We find that the time-dependent gas population of the active site can be fit to the same phenomenological rate law used to interpret experiments, with corresponding diffusion rates in very good agreement with experimental data. However, in contrast to the conventional picture, in which the gases follow a well-defined hydrophobic tunnel, we find that there is a diverse network of accessible pathways by which the gas molecules can reach the active site. The previously identified tunnel accounts for only about 60% of the total flux. Our results suggest that the dramatic decrease in the diffusion rate for mutations involving the residue Val74 could be in part due to the narrowing of the passage Val74-Arg476, immediately adjacent to the binding site, explaining why mutations of Leu122 had only a negligible effect in experiment. Our method is not specific to the [NiFe]-hydrogenase and should be generally applicable to the transport of small molecules in proteins. PMID:21341658

Wang, Po-hung; Best, Robert B; Blumberger, Jochen

2011-02-22

247

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

PubMed Central

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.

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

2012-01-01

248

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

PubMed Central

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

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

249

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

PubMed Central

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

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

2004-01-01

250

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

PubMed

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

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

2012-03-08

251

Electronic structure of the unique [4Fe-3S] cluster in O2-tolerant hydrogenases characterized by 57Fe M?ssbauer and EPR spectroscopy  

PubMed Central

Iron–sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H2 catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O2-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent 57Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron–sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron–sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher.

Pandelia, Maria-Eirini; Bykov, Dmytro; Izsak, Robert; Infossi, Pascale; Giudici-Orticoni, Marie-Therese; Bill, Eckhard; Neese, Frank; Lubitz, Wolfgang

2013-01-01

252

Electronic structure of the unique [4Fe-3S] cluster in O2-tolerant hydrogenases characterized by 57Fe Mossbauer and EPR spectroscopy.  

PubMed

Iron-sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H(2) catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O(2)-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent (57)Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron-sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron-sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher. PMID:23267108

Pandelia, Maria-Eirini; Bykov, Dmytro; Izsak, Robert; Infossi, Pascale; Giudici-Orticoni, Marie-Thérèse; Bill, Eckhard; Neese, Frank; Lubitz, Wolfgang

2012-12-24

253

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

Microsoft Academic Search

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order

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

2004-01-01

254

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

Microsoft Academic Search

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

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

2005-01-01

255

Investigation of the FeFe-hydrogenase gene diversity combined with phylogenetic microbial community analysis of an anaerobic domestic sewage sludge.  

PubMed

Biological hydrogen production through the anaerobic digestion is an environmental friendly alternative for satisfying future hydrogen demands. Microorganisms residing into waste water treatment plants are far from being exhaustively characterized and surveys on hydrogen production through FeFe-hydrogenase in such ecosystems are scarce. This study combined the analysis of 16S rRNA and [FeFe]-hydrogenase (hydA) genes with statistical tools to estimate richness and diversity of the microbial community of a domestic sewage treatment plant at the phylogenetic and functional levels. Archaeal groups were represented by 69 % of sequences assigned to Methanosarcinales and the remaining belonged to Methanomicrobiales. Within the bacterial library, 136 operational taxonomic units (OTUs) were distributed into 9 phyla, being 86 OTUs related to uncultivated bacteria. From these, 25 OTUs represented potential novel taxa within Synergistetes. Proteobacteria was the most predominant (36 % of the OTUs) and diversified phylogenetic group in the bacterial library, most of them assigned to the class Betaproteobacteria. Twenty-two putative hydA sequences were recovered into four distinct clusters and most of them were more closely related to each other than with sequences retrieved from databases, indicating they are hitherto undetected [Fe-Fe]-hydrogenase gene sequences. The richness estimates revealed that the number of sampled sequences was enough for full coverage of the archaeal diversity but not sufficient to cover both bacterial and hydA gene diversities. The results confirmed a great richness and diversity of bacterial and hydA sequences retrieved from the sewage sludge sample, suggesting such environment as a potential reservoir of new hydrogenase genes for biotechnological exploration. PMID:23632909

Tomazetto, Geizecler; Oliveira, Valéria M

2013-04-30

256

New Nitrosyl Derivatives of Diiron Dithiolates Related to the Active Site of the [FeFe]-Hydrogenases  

PubMed Central

Nitrosyl derivatives of diiron dithiolato carbonyls have been prepared starting from the versatile precursor Fe2(S2CnH2n)(dppv)(CO)4 (dppv = cis-1,2-bis(diphenylphosphinoethylene). These studies expand the range of substituted diiron(I) dithiolato carbonyl complexes. From [Fe2(S2C2H4)(CO)3(dppv)(NO)]BF4 ([1(CO)3]BF4), the following compounds were prepared: [1(CO)2(PMe3)]BF4, [1(CO)(dppv)]BF4, NEt4[1(CO)(CN)2], and 1(CO)(CN)(PMe3). Some if not all of these substitution reactions occur via the addition of two equiv of the nucleophile followed by dissociation of one nucleophile and decarbonylation. Such a double adduct was characterized crystallographically in the case of [Fe2(S2C2H4)(CO)3(dppv)(NO)(PMe3)2]BF4. 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)(NO)(CO) derivative. Compounds [1(CO)3]BF4, [1(CO)2(PMe3)]BF4, and [1(CO)(dppv)]BF4 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.

Olsen, Matthew T.; Justice, Aaron K.; Gloaguen, Frederic; Wilson, Scott R.

2008-01-01

257

Secondary coordination sphere interactions within the biomimetic iron azadithiolate complexes related to Fe-only hydrogenase: dynamic measure of electron density about the Fe sites.  

PubMed

A series of iron azadithiolate complexes possessing an intramolecular secondary coordination sphere interaction and an ability to reduce HOAc at the potential near the first electron-transfer process are reported. A unique structural feature in which the aza nitrogen has its lone pair point toward the apical carbonyl carbon is observed in [Fe(2)(mu-S(CH(2))(2)NR(CH(2))(2)S)(CO)(6-x)L(x)](2) (R = (n)Pr, x = 0, 1a; R = (i)Pr, x = 0, 1b; R = (n)Pr, L = PPh(3), x = 1, 2; R = (n)Pr, L = P(n)Bu(3), x = 1, 3) as biomimetic models of the active site of Fe-only hydrogenase. The presence of this weak N...C(CO(ap)) interaction provides electronic perturbation at the Fe center. The distance of the N...C(CO(ap)) contact is 3.497 A in 1a. It increases by 0.455 A in 2 when electronic density of the Fe site is slightly enriched by a weak sigma-donating ligand, PPh(3). A longer distance (4.040 A) is observed for the P(n)Bu(3) derivative, 3. This N...C(CO(ap)) distance is thus a dynamic measure of electronic nature of the Fe(2) core. Variation of electronic richness within the Fe(2) moiety among the complexes reflects on their electrochemical response. Reduction of 2 is recorded at the potential of -2.17 V, which is 270 mV more negative than that of 1. Complex 3 requires additional 150 mV for the same reduction. Such cathodic shift results from CO substitution by phosphines. Electrocatalytic hydrogen production from HOAc by both kinds of complexes (all-CO and phosphine-substituted species) requires the potential close to that for reduction of the parent molecules in the absence of acids. The catalytic mechanism of 1a is proposed to involve proton uptake at the Fe(0)Fe(I) redox level instead of the Fe(0)Fe(0) level. This result is the first observation among the all-CO complexes with respect to electrocatalysis of HOAc. PMID:20557034

Liu, Yu-Chiao; Tu, Ling-Kuang; Yen, Tao-Hung; Lee, Gene-Hsiang; Yang, Shu-Ting; Chiang, Ming-Hsi

2010-07-19

258

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

SciTech Connect

Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H2 as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0- Angstroms resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a Kd (dissociation constant) of {approx}400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]2 heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.

Rangarajan,E.; Asinas, A.; Proteau, A.; Munger, C.; Baardsnes, j.; Iannuzzi, P.; Matte, A.; Cygler, m.

2008-01-01

259

Hydrogen-oxidizing hydrogenases 1 and 2 of Escherichia coli regulate the onset of hydrogen evolution and ATPase activity, respectively, during glucose fermentation at alkaline pH.  

PubMed

Simultaneous measurement of redox potential (Eh ) and determination of H2 evolution kinetics using a pair of titanium silicate and platinum redox electrodes in fermenting cultures of Escherichia coli wild type and different mutants lacking hydrogenases 1 (Hyd-1) or 2 (Hyd-2) revealed that Hyd-1 controls the onset of H2 evolution at slightly alkaline pH (pH 7.5) and under oxidizing Eh . In addition, Hyd-2 influences the N,N'-dicyclohexylcarbodiimide-inhibited ATPase activity in fermenting cells and thus regulates the proton F0 F1 -ATPase at the alkaline pH but under reducing Eh . PMID:24111652

Poladyan, Anna; Trchounian, Karen; Sawers, R Gary; Trchounian, Armen

2013-10-10

260

Analysis of a pentacoordinate iron dicarbonyl as synthetic analogue of the Hmd or mono-iron hydrogenase active site.  

PubMed

Pentacoordinate iron dicarbonyls, (NS)Fe(CO)(2)P (NS=2-amidothiophenylate, P=PCy(3) (4), PPh(3), (5), and P(OEt)(3) (6)) were prepared as potential biomimetics of the active site of the mono-iron hydrogenase, [Fe]-H(2)ase. Full characterization including X-ray diffraction, density functional theory (DFT) computations, and Mössbauer studies for complexes 5 and 6 find that, despite similar infrared v(CO) pattern and absorption frequencies as the active site of the [Fe]-H(2)ase, the geometrical distortions towards trigonal bipyramidal, the negative isomer shift parameters, and the differences in CO-uptake reactivity are due to the "non-innocence" of the NS ligand. Ligand-based protonation with a strong acid, HBF(4).Et(2)O, interrupted the extensive pi-delocalization over Fe and NS ligand of complex 4 and switched on CO uptake (1 bar) to form a CO adduct, mer-[(H-NS)Fe(CO)(3)(PCy(3))](+) or 4(CO)-H(+). The extrinsic CO is reversibly removed on deprotonation with Et(3)N to regenerate complex 4. In a (13)CO atmosphere, concomitant CO uptake by 4-H(+) and exchange with intrinsic CO groups provide a facile route to (13)C-labeled 4(CO)-H(+) and, upon deprotonation, (13)C-labeled complex 4. DFT calculations show substantial Fe character in the LUMO of 4-H(+) typical of the d(6) Fe(II) in a regular square-pyramidal geometry. Thus, the Lewis acidity of 4-H(+) makes it amenable for CO binding, whereas the dianionic NS ligand renders the iron center of 4 insufficiently electrophilic and largely of Fe(I) character. PMID:20119989

Liu, Tianbiao; Li, Bin; Popescu, Codrina V; Bilko, Andrey; Pérez, Lisa M; Hall, Michael B; Darensbourg, Marcetta Y

2010-03-01

261

Stabilization role of a phenothiazine derivative on the electrocatalytic oxidation of hydrogen via Aquifex aeolicus hydrogenase at graphite membrane electrodes.  

PubMed

The [NiFe] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic Aquifex aeolicus bacterium (Aa Hase) presents oxygen, carbon monoxide, and temperature resistances. Since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. Efficient immobilization of the enzyme onto electrodes is however a mandatory step. To gain further insight into the parameters governing the interfacial electron process, cyclic voltammetry was performed combining the use of a phenothiazine dye with a membrane electrode design where the enzyme is entrapped in a thin layer. In the absence of the phenothiazine dye, direct electron transfer (DET) for H(2) oxidation is observed due to Aa Hase adsorbed onto the PG electrode. An unexpected loss of the catalytic current with time is however observed. The effect of toluidine blue O (TBO) on the catalytic process is first studied with TBO in solution. In addition to the expected mediated electron transfer process (MET), TBO is demonstrated to reconnect directly some Aa Hase molecules possibly released from the electrode but still entrapped in the thin layer. On adsorbed TBO the two same processes occur demonstrating the ability of the TBO film to connect Aa Hase via a DET process. Loss of activity is however observed due to the poor stability of adsorbed TBO at high temperatures. Aa Hase immobilization is then studied on electropolymerized TBO (pTBO). The effect of film thickness, temperature, presence of inhibitors and pH is evaluated. Given a film thickness less than 20 nm, H(2) oxidation proceeds via a mixed DET/MET process through the pTBO film. A high and very stable H(2) oxidation activity is reached, showing the potential applicability of the bioelectrode for biotechnologies. Finally, the multifunctional roles of TBO-based matrix are underlined, including redox mediator, Aa Hase anchor, but also buffering and ROS scavenger capabilities to drive pH local changes and avoid oxidative damage. PMID:21043442

Ciaccafava, Alexandre; Infossi, Pascale; Giudici-Orticoni, Marie-Thérèse; Lojou, Elisabeth

2010-11-02

262

Characterization of Photochemical Processes for H2 Production by CdS Nanorod-[FeFe] Hydrogenase Complexes  

SciTech Connect

We have developed complexes of CdS nanorods capped with 3-mercaptopropionic acid (MPA) and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) that photocatalyze reduction of H{sup +} to H{sub 2} at a CaI turnover frequency of 380-900 s{sup -1} and photon conversion efficiencies of up to 20% under illumination at 405 nm. In this paper, we focus on the compositional and mechanistic aspects of CdS:CaI complexes that control the photochemical conversion of solar energy into H{sub 2}. Self-assembly of CdS with CaI was driven by electrostatics, demonstrated as the inhibition of ferredoxin-mediated H{sub 2} evolution by CaI. Production of H{sub 2} by CdS:CaI was observed only under illumination and only in the presence of a sacrificial donor. We explored the effects of the CdS:CaI molar ratio, sacrificial donor concentration, and light intensity on photocatalytic H{sub 2} production, which were interpreted on the basis of contributions to electron transfer, hole transfer, or rate of photon absorption, respectively. Each parameter was found to have pronounced effects on the CdS:CaI photocatalytic activity. Specifically, we found that under 405 nm light at an intensity equivalent to total AM 1.5 solar flux, H{sub 2} production was limited by the rate of photon absorption ({approx}1 ms{sup -1}) and not by the turnover of CaI. Complexes were capable of H{sub 2} production for up to 4 h with a total turnover number of 106 before photocatalytic activity was lost. This loss correlated with inactivation of CaI, resulting from the photo-oxidation of the CdS capping ligand MPA.

Brown, K. A.; Wilker, M. B.; Boehm, M.; Dukovic, G.; King, P. W.

2012-03-28

263

Crystallization and preliminary X-ray crystallographic studies of the [NiFe] hydrogenase maturation proteins HypC and HypD.  

PubMed

HypC and HypD proteins are required for the insertion of the Fe atom with diatomic ligands into the large subunit of [NiFe] hydrogenases, an important step in the maturation process of this type of hydrogenase. The crystallization and preliminary crystallographic analysis of HypC and HypD from Thermococcus kodakaraensis KOD1 are reported. Crystals of HypC grew in two different forms. Monoclinic crystals of HypC in space group C2 with unit-cell parameters a = 78.2, b = 59.1, c = 54.0 A, beta = 109.0 degrees were obtained using PEG 4000 and ammonium sulfate or sodium bromide as precipitants. They diffracted X-rays to 1.8 A resolution and were suitable for structure determination. Crystals of HypD were also obtained in two different forms. The monoclinic crystals obtained using PEG 4000 and magnesium chloride diffracted X-rays to beyond 2.1 A resolution, despite growing as clusters. They belong to space group P2(1), with unit-cell parameters a = 42.3, b = 118.4, c = 81.2 A, beta = 100.9 degrees , and are suitable for data collection. PMID:17554182

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

2007-05-31

264

Evidence for acyl-iron ligation in the active site of [Fe]-hydrogenase provided by mass spectrometry and infrared spectroscopy.  

PubMed

[Fe]-hydrogenase catalyzes the reversible heterolytic cleavage of H(2) and stereo-specific hydride transfer to the substrate methenyltetrahydromethanopterin in methanogenic archaea. This enzyme contains a unique iron guanylylpyridinol (FeGP) cofactor as a prosthetic group. It has recently been proposed-on the basis of crystal structural analyses of the [Fe]-hydrogenase holoenzyme-that the FeGP cofactor contains an acyl-iron ligation, the first one reported in a biological system. We report here that the cofactor can be reversibly extracted with acids; its exact mass has been determined by electrospray ionization Fourier transform ion cyclotron resonance mass-spectrometry. The measured mass of the intact cofactor and its gas-phase fragments are consistent with the proposed structure. The mass of the light decomposition products of the cofactor support the presence of acyl-iron ligation. Attenuated total reflection infrared spectroscopy of the FeGP cofactor revealed a band near wave number 1700 cm(-1), which was assigned to the C=O (double bond) stretching mode of the acyl-iron ligand. PMID:22080303

Shima, Seigo; Schick, Michael; Kahnt, Jörg; Ataka, Kenichi; Steinbach, Klaus; Linne, Uwe

2011-11-14

265

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

PubMed

The reaction of NiBr(2)(EtOH)(4) with a 1:2-3 mixture of FeBr(2)(CO)(4) and Na(SPh) generated a linear trinuclear Fe-Ni-Fe cluster (CO)(3)Fe(mu-SPh)(3)Ni(mu-SPh)(3)Fe(CO)(3), 1, whereas the analogous reaction system FeBr(2)(CO)(4)/Na(S(t)Bu)/NiBr(2)(EtOH)(4) (1:2-3:1) gave rise to a linear tetranuclear Fe-Ni-Ni-Fe cluster [(CO)(3)Fe(mu-S(t)Bu)(3)Ni(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 complexes, the structures of which mimic [NiFe] hydrogenase active sites. The reactions of 2 with SC(NMe(2))(2) (tmtu), Na{S(CH(2))(2)SMe} and ortho-NaS(C(6)H(4))SR (R = Me, (t)Bu) led to isolation of (CO)(3)Fe(mu-S(t)Bu)(3)NiBr(tmtu), 3, (CO)(3)Fe(S(t)Bu)(mu-S(t)Bu)(2)Ni{S(CH(2))(2)SMe}, 4, and (CO)(3)Fe(S(t)Bu)(mu-S(t)Bu)(2)Ni{S(C(6)H(4))SR}, 5a (R = Me) and 5b (R = (t)Bu), respectively. On the other hand, treatment of 2 with 2-methylthio-phenolate (ortho-O(C(6)H(4))SMe) in methanol resulted in (CO)(3)Fe(mu-S(t)Bu)(3)Ni(MeOH){O(C(6)H(4))SMe}, 6a. The methanol molecule bound to Ni is labile and is readily released under reduced pressure to afford (CO)(3)Fe(S(t)Bu)(mu-S(t)Bu)(2)Ni{O(C(6)H(4))SMe}, 6b, and the coordination geometry of nickel changes from octahedral to square planar. Likewise, the reaction of 2 with NaOAc in methanol followed by crystallization from THF gave (CO)(3)Fe(mu-S(t)Bu)(3)Ni(THF)(OAc), 7. The dinuclear complexes, 3-7, are thermally unstable, and a key to their successful isolation is to carry out the reactions and manipulations at -40 degrees C. PMID:18511566

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

2008-05-29

266

Density functional theory calculations on the mononuclear non-heme iron active site of Hmd hydrogenase: role of the internal ligands in tuning external ligand binding and driving H2 heterolysis.  

PubMed

DFT calculations on active-site models of the non-heme Fe site of Hmd hydrogenase are reported. Binding of several biologically relevant ligands (e.g., CN(-), CO, H(-), H(2), and O(2)) to the active site of Hmd was investigated using a method that reproduced the geometric and vibrational properties of the resting site. The results indicate that this neutral ferrous active site has higher affinity toward anionic ligands (e.g., H(-) and CN(-)) than ?-acidic ligands (e.g., CO and O(2)). Natural population analysis and molecular orbital analysis revealed that this is due to extensive delocalization of electron density into the low-lying unoccupied orbitals of the CO, acyl, and pyridinol ligands present in the active site. In addition to normal d-? back-bonding, metal 3d orbital-mediated charge transfer from occupied ligand orbitals to the unoccupied orbitals of the internal ligands was observed. This charge transfer leads to systematic variations in the experimentally observed C-O stretching frequencies. Protonation of the thiolate ligand present in the active site significantly enhances these anion ligand binding affinities. In fact, the calculated vibrational frequencies indicate that CN(-) binding is possibly associated with protonation of the thiolate ligand. The high affinity for binding of the anionic H(-) ligand (where 81% of the electron density of H(-) is delocalized into the active site) is calculated to play a dominating role in the H-H bond heterolysis step during catalysis. The binding energies of these ligands relative to the substrate, H(2), highlight the importance of a proposed structural reorganization during catalysis. PMID:20831194

Dey, Abhishek

2010-10-01

267

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

SciTech Connect

Partition coefficients (D) for metal/sulfide liquid, troilite/sulfide liquid, and schreibersite/sulfide liquid have been experimentally determined for Ag, Au, Mo, Ni, Pb, Pd, and Tl. With these partition coefficients, it should be possible to better understand the [sup 107]Pd-[sup 107]Ag and [sup 205]Tl-[sup 205]Pb systems of iron meteorites. In general, the schreibersite/metal and troilite/metal partition coefficients for compatible' elements are quite similar to those inferred from natural assemblages. In contrast, these same partition coefficients for incompatible' elements often do not even qualitatively agree with analyses of iron meteorites. The authors suggest that analyses of incompatible elements in iron meteorites may sometimes be dominated by minor/trace phases. Identification of such phases may allow refinement of the initial isotopic composition of Pb in the early solar system. If the trace impurity model is correct, it may also be possible to reconcile iron meteorite cooling rates inferred from silver isotopic systematics with those deduced from metallography. 36 refs., 2 figs., 6 tabs.

Jones, J.H. (NASA Johnson Space Center, Houston, TX (United States)); Hart, S.R. (Woods Hole Oceanographic Institution, MA (United States)); Benjamin, T.M. (Los Alamos National Lab., NM (United States))

1993-01-01

268

Active-Site Models for the Nickel-Iron Hydrogenases: Effects of Ligands on Reactivity and Catalytic Properties  

PubMed Central

Described are new derivatives of the type [HNiFe(SR)2(diphosphine)(CO)3]+, which feature a Ni(diphosphine) group linked to a Fe(CO)3 group via two bridging thiolate ligands. Previous work had described [HNiFe(pdt)(dppe)(CO)3]+ ([1H]+) and its activity as a catalyst for the reduction of protons. Work described in this paper focused on the effects of the diphosphine attached to nickel as well as the dithiolate bridge, 1,3-propanedithiolate (pdt) vs 1,2-ethanedithiolate (edt). A new synthetic route to these Ni-Fe dithiolates is described, involving reaction of Ni(SR)2(diphosphine) with FeI2(CO)4 followed by in situ reduction with cobaltocene. Evidence is presented that this route proceeds via metastable ?-iodo derivatives. Attempted isolation of such species led to the crystallization of NiFe(Me2pdt)(dppe)I2, which features tetrahedral Fe(II) and square planar Ni(II) centers (Me2pdt = 2,2-dimethylpropanedithiol). The new tricarbonyls prepared in this work are NiFe(pdt)(dcpe)(CO)3 (2, dcpe = 1,2-bis(dicyclohexylphosphino)ethane), NiFe(edt)(dppe)(CO)3 (3), and NiFe(edt)(dcpe)(CO)3 (4). Attempted preparation of a phenylthiolate-bridged complex via the FeI2(CO)4 + Ni(SPh)2(dppe) route gave the tetrametallic species [(CO)2Fe(SPh)2Ni(CO)]2(?-dppe)2. Crystallographic analysis of the edt-dcpe compund [2H]BF4 and the edt-dppe compound [3H]BF4 verified their close resemblance. Each features pseudo-octahedral Fe and square pyramidal Ni centers. Starting from [4H]BF4 we prepared the PPh3 derivative [HNiFe(edt)(dppe)(PPh3)(CO)2]BF4 ([5H]BF4), which was obtained as a ~2:1 mixture of unsymmetrical and symmetrical isomers. Acid-base measurements indicate that changing from Ni(dppe) to Ni(dcpe) decreases the acidity of the cationic hydride complexes by 2.5 pKaMeCN units, from ~11 to ~13.5 (previous work showed that substitution at Fe leads to more dramatic effects). The redox potentials are more strongly affected by the change from dppe to dcpe, for example the [2]0/+ couple occurs at E1/2 = ?820 for [2]0/+ vs ?574 mV (vs Fc+/0) for [1]0/+. Changes in the dithiolate do not affect the acidity or the reduction potentials of the hydrides. The acid-independent rate of reduction of CH2ClCO2H by [2H]+ is ca. 50 s?1 (25 °C), twice that of [1H]+. The edt-dppe complex [2H]+ proved to be the most active catalyst, with an acid-independent rate of 300 s?1.

Carroll, Maria E.; Barton, Bryan E.; Gray, Danielle L.; Mack, Amanda E.; Rauchfuss, Thomas B.

2011-01-01

269

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

PubMed

The direct conversion of sunlight into biofuels is an intriguing alternative to a continued reliance on fossil fuels. Natural photosynthesis has long been investigated both as a potential solution, and as a model for utilizing solar energy to drive a water-to-fuel cycle. The molecules and organizational structure provide a template to inspire the design of efficient molecular systems for photocatalysis. A clear design strategy is the coordination of molecular interactions that match kinetic rates and energetic levels to control the direction and flow of energy from light harvesting to catalysis. Energy transduction and electron-transfer reactions occur through interfaces formed between complexes of donor-acceptor molecules. Although the structures of several of the key biological complexes have been solved, detailed descriptions of many electron-transfer complexes are lacking, which presents a challenge to designing and engineering biomolecular systems for solar conversion. Alternatively, it is possible to couple the catalytic power of biological enzymes to light harvesting by semiconductor nanomaterials. In these molecules, surface chemistry and structure can be designed using ligands. The passivation effect of the ligand can also dramatically affect the photophysical properties of the semiconductor, and energetics of external charge-transfer. The length, degree of bond saturation (aromaticity), and solvent exposed functional groups of ligands can be manipulated to further tune the interface to control molecular assembly, and complex stability in photocatalytic hybrids. The results of this research show how ligand selection is critical to designing molecular interfaces that promote efficient self-assembly, charge-transfer and photocatalysis. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. PMID:23541891

King, Paul W

2013-03-27

270

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

PubMed Central

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.

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

271

Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stable-isotope labeling.  

PubMed

[Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized. PMID:22260087

Schick, Michael; Xie, Xiulan; Ataka, Kenichi; Kahnt, Jörg; Linne, Uwe; Shima, Seigo

2012-02-06

272

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

PubMed

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

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

2013-03-01

273

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

PubMed Central

Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)?1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg?1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg?1. Owing to the combinatorial power exhibited by the presented cloning platform, the system might represent an important step towards new routes in synthetic biology.

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

2013-01-01

274

ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O2 Regulation of Gene Expression in Escherichia coli  

PubMed Central

Transcription of the Escherichia coli hydrogenase-1 operon (hyaABCDEF) is increased by the transcription factors ArcA and AppY under anaerobic growth conditions. However, IscR, which represses transcription of the hyaA promoter (PhyaA) under aerobic conditions, was not known to repress transcription of this promoter under anaerobic conditions. Here, we report that ArcA and AppY increase PhyaA expression under anaerobic conditions by antagonizing IscR binding at PhyaA, since IscR repression is observed when either ArcA or AppY is eliminated. The ability of ArcA and AppY to act as antirepressors of IscR repression of PhyaA depended on IscR levels, suggesting that IscR competes with ArcA and/or AppY for binding. In support of this competition model, electrophoretic mobility shift assays and DNase I footprinting showed that the ArcA and IscR binding sites overlap and that binding of ArcA and IscR is mutually exclusive. Unexpectedly, IscR with a C92A mutation (IscR-C92A), which mimics the clusterless form of the protein that is present predominantly under aerobic conditions, was a better repressor under anaerobic conditions of both PhyaA and a constitutive promoter containing the IscR binding site from PhyaA than wild-type IscR, which is predominantly in the [2Fe-2S] form under anaerobic conditions. This observation could not be explained by differences in DNA binding affinities or IscR levels, so we conclude that [2Fe-2S]-IscR is a weaker repressor of PhyaA than clusterless IscR. In sum, a combination of ArcA and AppY antirepression of IscR function, lower levels of IscR, and weak repression by [2Fe-2S]-IscR leads to increased PhyaA expression under anaerobic conditions.

Nesbit, A. D.; Fleischhacker, A. S.; Teter, S. J.

2012-01-01

275

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

NASA Astrophysics Data System (ADS)

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.

Best, Stephen P.; Cheah, Mun Hon

2010-02-01

276

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

SciTech Connect

A series of dipeptide nickel complexes with the general formula, [Ni(PPh2NNNA-amino acid/ester2)2](BF4)2, have been synthesized and characterized (P2N2= 1,5-diaza-3,7-diphosphacyclooctane, amino acid/esters = glutamic acid, alanine, lysine, and aspartic acid). Each of these complexes is an efficient electrocatalyst for H2 production. The contribution of the outer-coordination sphere, specifically the impact of sterics, the ability to protonate and the pKa of amino acid side chain on the hydrogen production activity of these complexes, was investigated. The rates of all of the catalysts ranged over an order of magnitude. The amino acid containing complexes display 2-3 times higher rates of hydrogen production than the corresponding ester complexes, suggesting the significance of protonated species (side chains/backbone of amino acids) in the outer-coordination sphere. The largest had the fastest rates suggesting that catalytic activity is not hindered by sterics. However, the shapes of catalytic waves are indicative of hindered electron transfer and may suggest a competing mechanism for catalysis than that observed for the unsubstituted parent complex. These studies demonstrate the significant contribution that the outer-coordination sphere can have in tuning the catalytic activity of small molecule hydrogenase mimics.

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

2012-06-18

277

Escherichia coli multiple [Ni-Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs.  

PubMed

Escherichia coli evolves H2 via multiple [Ni-Fe]-hydrogenases (Hyd). This activity under hyper- and hypo-osmotic stress was investigated with mutants lacking different Hyd enzymes during glycerol fermentation. Inhibitory effects of hypo-stress on H2 production was stronger at pH 6.5 in wild type and mutants except fhlA, which encodes a transcriptional activator for Hyd-3, compared with the effects of N,N'-dicyclohexylcarbodiimide. These results indicate that Hyd-3 and Hyd-4 are osmosensitive at pH 7.5. Hyd-4 and FhlA are implicated in osmotic stress response at pH 6.5. Hyd-1 and FhlA might be osmosensitive at pH 5.5. Thus, osmosensitivity of Hyd enzymes is a novel property that depends on pH. This is significant for mechanisms of cell osmoregulation and H2 production biotechnology when glycerol is used as a fermentation substrate. PMID:24060380

Trchounian, Karen; Trchounian, Armen

2013-09-20

278

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

PubMed

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

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

2005-02-01

279

Three diiron complexes bearing an aromatic ring as mimics of the diiron subunit of [FeFe]-hydrogenase: synthesis, electron transfer and coupled chemical reactions.  

PubMed

Three diiron complexes (1-Ph, 2-OH, and 3-OCOFc) as mimics of the diiron subunit of [FeFe]-hydrogenase were electrochemically investigated in 0.1 mol L(-1) [NBut(4)]BF(4)-acetonitrile (MeCN) under CO and Ar atmosphere. Complex 3-OCOFc was prepared from the reaction of complex 2-OH with ferrocenylacyl chloride (FcOCCl). The complex was fully characterised using a variety of spectroscopic techniques. Its structure was established using X-ray single crystal diffraction analysis. In addition to the well-established ECE (E for electrochemical and C for chemical) mechanism, it was revealed that a further reversible reduction at a potential more negative by ca. 600 mV was observed under CO atmosphere. It was further proposed based on the analysis of electrochemical and infrared spectroscopic data that the second redox was due to a two-electron process of supposedly a tetrairon cluster. This product was formed in situ from the reaction between the dianion generated from the ECE process and its parent complex (1-Ph, 2-OH, and 3-OCOFc) and is supposedly of a core "Fe(4)(II)". This reaction occurred only when CO was presented. Under Ar atmosphere, bulk electrolysis led to fully-reduced products, that is, with the iron at the oxidation state of zero, but complex was an exception. An overall mechanism to describe the electron transfer and coupled chemical reactions under CO atmosphere was proposed. PMID:22864673

Zhao, Jia; Wei, Zhenhong; Zeng, Xianghua; Liu, Xiaoming

2012-08-28

280

Binding of Iron(III) to Organic Soils: Exafs Spectroscopy And Chemical Equilibrium Modeling  

SciTech Connect

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.

Gustafsson, J.P.; Persson, I.; Kleja, D.B.; Schaik, J.W.J.van

2007-07-09

281

Computer Modeling in Biotechnology  

NASA Astrophysics Data System (ADS)

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.

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

282

Bis{?-[4-(1,3-benzothia-zol-2-yl)phen-yl]methane-thiol-ato-?4 S,S?:S,S?}bis-[tricarbonyl-iron(I)](Fe--Fe)  

PubMed Central

The title compound, [Fe2(C14H10NS2)2(CO)6], was synthesized as a structural and biochemical model for the active site of [FeFe]-hydrogenase. The bond lengths (Fe—Fe, Fe—S and Fe—C) and angles (C—Fe—Fe and Fe—S—Fe) are within expected ranges. The S?S distance [2.9069?(12)?Å] and the dihedral angle between two Fe—S—Fe planes [78.5?(3)°] of the butterfly-shaped Fe2S2 core are enlarged compared with related bridged dithiol­ate diiron analogues. The calculated 4-benzothia­zolebenzyl best planes are almost parallel [dihedral angle = 3.7?(7)°].

Gao, Shang; Jiang, Da-yong; Liang, Qing-cheng; Duan, Qian

2012-01-01

283

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

PubMed Central

The photoreaction of Fe(CO)5 and cyanide salts in MeCN solution affords the dianion [Fe(CN)2(CO)3]2?, conveniently isolated as [K(18-crown-6)]2[Fe(CN)2(CO)3]. Solutions of [Fe(CN)2(CO)3]2? oxidize irreversibly at ?600 mV (vs. Ag/AgCl) to give primarily [Fe(CN)3(CO)3]?. Protonation of the dianion affords the hydride [K(18-crown-6)][HFe(CN)2(CO)3] with a pKa ? 17 (MeCN). The ferrous hydride exhibits enhanced electrophilicity vs. its dianionic precursor, which resists substitution. Treatment of [K(18-crown-6)][Fe(CN)2(CO)3] with tertiary phosphines and phosphites gives isomeric mixtures of [HFe(CN)2(CO)2L]? (L = P(OPh)3 and PPh3). Carbonyl substitution on [1H(CO)2]? by P(OPh)3 is first-order in both the phosphite and iron (k = 0.18 M?1 s?1 at 22 °C) with ?H‡ = 51.6 kJ mol?1 and ?S‡ = ?83.0 J K?1 mol?1. These ligands are displaced under an atmosphere of CO. With cis-Ph2PCH=CHPPh2 (dppv), we obtained the monocarbonyl, [HFe(CN)2(CO)(dppv)]?, a highly basic hydride (pKa > 23.3) that rearranges in solution to a single isomer. Treatment of [K(18-crown-6)][HFe(CN)2(CO)3] with Et4NCN resulted in rapid proton transfer to give [Fe(CN)2(CO)3]2? and HCN. The tricyano hydride [HFe(CN)3(CO)2]2? is prepared by the reaction of [HFe(CN)2(CO)2(PPh3)]? and [K(18-crown-6)]CN. Similar to the phosphine and phosphite derivatives, [HFe(CN)3(CO)2]2? exists as a mixture of all three possible isomers. Protonation of the hydrides [HFe(CN)2(CO)(dppv)]? and [HFe(CN)3(CO)2]? in acetonitrile solutions releases H2 and gives the corresponding acetonitrile complexes [K(18-crown-6)][Fe(CN)3(NCMe)(CO)2] and Fe(CN)2(NCMe)(CO)(dppv). Alkylation of [K(18-crown-6)]2[Fe(CN)2(CO)3] with MeOTf gives the thermally-unstable [MeFe(CN)2(CO)3]?, which was characterized spectroscopically at ?40 °C. Reaction of dppv with [MeFe(CN)2(CO)3]? gives the acetyl complex, [Fe(CN)2(COMe)(CO)(dppv)]?. Whereas [Fe(CN)2(CO)3]2? undergoes protonation and methylation at Fe, acid chlorides give the iron(0) N-acylisocyanides [Fe(CN)(CO)3(CNCOR)]? (R = Ph, CH3). The solid state structures of [K(18-crown-6)][HFe(CN)2(CO)(dppv)], Fe(CN)2(NCMe)(CO)(dppv), and [K(18-crown-6)]2[HFe(CN)3(CO)2] were confirmed crystallographically. In all three cases, the cyanide ligands are cis to the hydride or acetonitrile ligands.

Whaley, C. Matthew; Wilson, Scott R.

2009-01-01

284

Coordination chemistry of [HFe(CN)(2)(CO)(3)](-) and its derivatives: toward a model for the iron subsite of the [NiFe]-hydrogenases.  

PubMed

The photoreaction of Fe(CO)(5) and cyanide salts in MeCN solution affords the dianion [Fe(CN)(2)(CO)(3)](2-), conveniently isolated as [K(18-crown-6)](2)[Fe(CN)(2)(CO)(3)]. Solutions of [Fe(CN)(2)(CO)(3)](2-) oxidize irreversibly at -600 mV (vs Ag/AgCl) to give primarily [Fe(CN)(3)(CO)(3)](-). Protonation of the dianion affords the hydride [K(18-crown-6)][HFe(CN)(2)(CO)(3)] with a pK(a) approximately 17 (MeCN). The ferrous hydride exhibits enhanced electrophilicity vs its dianionic precursor, which resists substitution. Treatment of [K(18-crown-6)][Fe(CN)(2)(CO)(3)] with tertiary phosphines and phosphites gives isomeric mixtures of [HFe(CN)(2)(CO)(2)L](-) (L = P(OPh)(3) and PPh(3)). Carbonyl substitution on [1H(CO)(2)](-) by P(OPh)(3) is first-order in both the phosphite and iron (k = 0.18 M(-1) s(-1) at 22 degrees C) with DeltaH(double dagger) = 51.6 kJ mol(-1) and DeltaS(double dagger) = -83.0 J K(-1) mol(-1). These ligands are displaced under an atmosphere of CO. With cis-Ph(2)PCH=CHPPh(2) (dppv), we obtained the monocarbonyl, [HFe(CN)(2)(CO)(dppv)](-), a highly basic hydride (pK(a) > 23.3) that rearranges in solution to a single isomer. Treatment of [K(18-crown-6)][HFe(CN)(2)(CO)(3)] with Et(4)NCN resulted in rapid deprotonation to give [Fe(CN)(2)(CO)(3)](2-) and HCN. The tricyano hydride [HFe(CN)(3)(CO)(2)](2-) is prepared by the reaction of [HFe(CN)(2)(CO)(2)(PPh(3))](-) and [K(18-crown-6)]CN. Similar to the phosphine and phosphite derivatives, [HFe(CN)(3)(CO)(2)](2-) exists as a mixture of all three possible isomers. Protonation of the hydrides [HFe(CN)(2)(CO)(dppv)](-) and [HFe(CN)(3)(CO)(2)](-) in acetonitrile solutions releases H(2) and gives the corresponding acetonitrile complexes [K(18-crown-6)][Fe(CN)(3)(NCMe)(CO)(2)] and Fe(CN)(2)(NCMe)(CO)(dppv). Alkylation of [K(18-crown-6)](2)[Fe(CN)(2)(CO)(3)] with MeOTf gives the thermally unstable [MeFe(CN)(2)(CO)(3)](-), which was characterized spectroscopically at -40 degrees C. Reaction of dppv with [MeFe(CN)(2)(CO)(3)](-) gives the acetyl complex, [Fe(CN)(2)(COMe)(CO)(dppv)](-). Whereas [Fe(CN)(2)(CO)(3)](2-) undergoes protonation and methylation at Fe, acid chlorides give the iron(0) N-acylisocyanides [Fe(CN)(CO)(3)(CNCOR)](-) (R = Ph, CH(3)). The solid state structures of [K(18-crown-6)][HFe(CN)(2)(CO)(dppv)], Fe(CN)(2)(NCMe)(CO)(dppv), and [K(18-crown-6)](2)[HFe(CN)(3)(CO)(2)] were confirmed crystallographically. In all three cases, the cyanide ligands are cis to the hydride or acetonitrile ligands. PMID:19374433

Whaley, C Matthew; Rauchfuss, Thomas B; Wilson, Scott R

2009-05-18

285

First-principles thermodynamic modeling of lanthanum chromate perovskites  

NASA Astrophysics Data System (ADS)

Tendencies toward local atomic ordering in (A,A')(B,B')O3-? mixed composition perovskites are modeled to explore their influence on thermodynamic, transport, and electronic properties. In particular, dopants and defects within lanthanum chromate perovskites are studied under various simulated redox environments. (La1-x,Srx)(Cr1-y,Fey)O3-? (LSCF) and (La1-x,Srx)(Cr1-y,Ruy)O3-? (LSCR) are modeled using a cluster expansion statistical thermodynamics method built upon a density functional theory database of structural energies. The cluster expansions are utilized in lattice Monte Carlo simulations to compute the ordering of Sr and Fe(Ru) dopant and oxygen vacancies (Vac). Reduction processes are modeled via the introduction of oxygen vacancies, effectively forcing excess electronic charge onto remaining atoms. LSCR shows increasingly extended Ru-Vac associates and short-range Ru-Ru and Ru-Vac interactions upon reduction; LSCF shows long-range Fe-Fe and Fe-Vac interaction ordering, inhibiting mobility. First principles density functional calculations suggest that Ru-Vac associates significantly decrease the activation energy of Ru-Cr swaps in reduced LSCR. These results are discussed in view of experimentally observed extrusion of metallic Ru from LSCR nanoparticles under reducing conditions at elevated temperature.

Dalach, P.; Ellis, D. E.; van de Walle, A.

2012-01-01

286

Hydrogen Production by Photosynthesis and Hydrogenase Activity.  

National Technical Information Service (NTIS)

This report presents evidence for the biophotolysis of water to hydrogen and oxygen by systems: (1) Spinach chloroplasts forming reduced triphosphopyridine (TPNH) nucleotide photosynthetically and hydrogen formation from it as catalyzed by a bacterial hyd...

L. O. Kamptiz

1973-01-01

287

Isomerization of the hydride complexes [HFe2(SR)2(PR3)x(CO)6–x]+ (x = 2, 3, 4) relevant to the active site models for the [FeFe]-hydrogenases†  

PubMed Central

The stepwise formation of bridging (?-) hydrides of diiron dithiolates is discussed with attention on the pathway for protonation and subsequent isomerizations. Our evidence is consistent with protonations occurring at a single Fe center, followed by isomerization to a series of ?-hydrides. Protonation of Fe2(edt)(CO)4(dppv) (1) gave a single ?-hydride with dppv spanning apical and basal sites, which isomerized at higher temperatures to place the dppv into a dibasal position. Protonation of Fe2(pdt)(CO)4(dppv) (2) followed an isomerization pathway similar to that for [1H]+, except that a pair of isomeric terminal hydrides were observed initially, resulting from protonation at the Fe(CO)3 or Fe(CO)(dppv) site. The first observable product from low temperature protonation of the tris-phosphine Fe2(edt)(CO)3(PMe3)(dppv) (3) was a single ?-hydride wherein PMe3 is apical and the dppv ligand spans apical and basal sites. Upon warming, this isomer converted fully but in a stepwise manner to a mixture of three other isomeric hydrides. Protonation of Fe2(pdt)(CO)3(PMe3)(dppv) (4) proceeded similarly to the edt analogue 3, however a terminal hydride was observed, albeit only briefly and at very low temperatures (?90 °C). Low-temperature protonation of the bis-chelates Fe2(xdt)(CO)2(dppv)2 produced exclusively the terminal hydrides [HFe2(xdt)(?-CO)(CO)(dppv)2]+ (xdt = edt and pdt), which subsequently isomerized to a pair of ?-hydrides. At room temperature these (dppv)2 derivatives convert to an equilibrium of two isomers, one C2-symmetric and the other Cs-symmetric. The stability of the terminal hydrides correlates with the (C2-isomer)/(Cs-isomer) equilibrium ratio, which reflects the size of the dithiolate. The isomerization was found to be unaffected by the presence of excess acid, by solvent polarity, and the presence of D2O. This isomerization mechanism is proposed to be intramolecular, involving a 120° rotation of the HFeL3 subunit to an unobserved terminal basal hydride as the rate-determining step. The observed stability of the hydrides was supported by DFT calculations, which also highlight the instability of the basal terminal hydrides. Isomerization of the ?-hydride isomers occurs on alternating FeL3 via 120° rotations without generating D2O-exchangeable intermediates.

Barton, Bryan E.; Zampella, Giuseppe; Justice, Aaron K.; Wilson, Scott R.

2012-01-01

288

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

SciTech Connect

Abstract Advances in DNA microarray and proteomics technologies have enabled high-throughput measurement of mRNA expression and protein abundance. Parallel profiling of mRNA and protein on a global scale and integrative analysis of these two data types could provide additional insight into the metabolic mechanisms underlying complex biological systems. However, because protein abundance and mRNA expression are affected by many cellular and physical processes, there have been conflicting results on the correlation of these two measurements. In addition, as current proteomic methods can detect only a small fraction of proteins present in cells, no correlation study of these two data types has been done thus far at the whole-genome level. In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), Zero-inflated Poisson models were used to define the correlation pattern of mRNA and protein abundance. The models assumed that there is a probability mass at zero representing some of the undetected proteins because of technical limitations. The models thus use abundance measurements of transcripts and proteins experimentally detected as input to generate predictions of protein abundances as output for all genes in the genome. We demonstrated the statistical models by comparatively analyzing D. vulgaris grown on lactate-based versus formate-based media. The increased expressions of Ech hydrogenase and alcohol dehydrogenase (Adh)-periplasmic Fe-only hydrogenase (Hyd) pathway for ATP synthesis were predicted for D. vulgaris grown on formate.

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

2006-05-04

289

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

PubMed

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

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

2012-05-01

290

Construction of Martian Interior Model  

NASA Astrophysics Data System (ADS)

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).

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

2005-09-01

291

Synthesis and Mössbauer characterization of octahedral iron(II) carbonyl complexes FeI2(CO)3L and FeI2(CO)2L2: developing models of the [Fe]-H(2)ase active site.  

PubMed

A series of mono- and disubstituted complexes, FeI(2)(CO)(x)L(4-x), x = 2 or 3, is conveniently accessed from simple mixing of N-heterocyclic carbenes, phosphines, and aromatic amines with FeI(2)(CO)(4), first reported by Hieber in 1928. The highly light sensitive complexes yield to crystallization and X-ray diffraction studies for six complexes showing them to be rudimentary structural models of the monoiron hydrogenase, [Fe]-H(2)ase or Hmd, active site in native (Fe(II)(CO)(2)) or CO-inhibited (Fe(II)(CO)(3)) states. Diatomic ligand (nu(CO)) vibrational and Mossbauer spectroscopies are related to those reported for the Hmd active site. The importance of a serial approach for relating such parameters in model compounds to low spin Fe(II) in the diverse ligation of enzyme active sites is stressed. PMID:19860458

Li, Bin; Liu, Tianbiao; Popescu, Codrina V; Bilko, Andrey; Darensbourg, Marcetta Y

2009-12-01

292

Modeling  

SciTech Connect

Slurry flows occur in many circumstances, including chemical manufacturing processes, pipeline transfer of coal, sand, and minerals; mud flows; and disposal of dredged materials. In this section we discuss slurry flow applications related to radioactive waste management. The Hanford tank waste solids and interstitial liquids will be mixed to form a slurry so it can be pumped out for retrieval and treatment. The waste is very complex chemically and physically. The ARIEL code is used to model the chemical interactions and fluid dynamics of the waste.

Loth, E.; Tryggvason, G.; Tsuji, Y.; Elghobashi, S. E.; Crowe, Clayton T.; Berlemont, A.; Reeks, M.; Simonin, O.; Frank, Th; Onishi, Yasuo; Van Wachem, B.

2005-09-01

293

?4-Orthothio-carbonato-tetra-kis-[tri-carbonyl-iron(I)](2 Fe--Fe)  

PubMed Central

The fused bis-butterfly-shaped title compound, [Fe4(CS4)(CO)12], possesses an orthothio­carbonate (CS4 4?) ligand that acts as a bridge between two Fe2(CO)6 units. A short intra­molecular S?S contact [2.6984?(8) and 2.6977?(8)?Å] occurs in each S2Fe2(CO)6 fragment.

Shi, Yao-Cheng; Cheng, Huan-Ren; Yuan, Li-Min; Li, Qian-Kun

2011-01-01

294

?(4)-Orthothio-carbonato-tetra-kis-[tri-carbonyl-iron(I)](2 Fe-Fe).  

PubMed

The fused bis-butterfly-shaped title compound, [Fe(4)(CS(4))(CO)(12)], possesses an orthothio-carbonate (CS(4) (4-)) ligand that acts as a bridge between two Fe(2)(CO)(6) units. A short intra-molecular S?S contact [2.6984?(8) and 2.6977?(8)?Å] occurs in each S(2)Fe(2)(CO)(6) fragment. PMID:22219779

Shi, Yao-Cheng; Cheng, Huan-Ren; Yuan, Li-Min; Li, Qian-Kun

2011-10-12

295

Hard magnetic nanocrystalline alloys of Fe–Fe 2O 3 system  

Microsoft Academic Search

The structure and magnetic properties of Fe2O3 and Fe powders as well as their mixtures, subjected to treatment in a high-energy ball mill, were studied. It was shown that the phase composition of all the starting materials (except that of the Fe powder) was changed in the milling process. The nanocrystalline composite alloys, containing FeO and ?-Fe with an average

A. S. Lileev; Yu. D. Yagodkin; E. N. Grishina; E. S. Khanenya; V. S. Nefedov; O. I. Popova

2005-01-01

296

Percolation of Fe-FeS Melts in Partially Molten Peridotite  

Microsoft Academic Search

In order to establish a percolation threshold of FeS melts in peridotite matrix, two types of experiments have been done on partially molten garnet peridotite. Peridotite powders with 100-200 mu and 20-30 mu grain size were mixed with 5-15 vol% Fe70S30 composition. The first type of experiments has been done in the centrifuging piston cylinder at ETH, Zürich. The mixed

N. Bagdassarov; G. Golabek; G. Solferino; M. W. Schmidt

2006-01-01

297

Percolation of Fe-FeS Melts in Partially Molten Peridotite  

NASA Astrophysics Data System (ADS)

In order to establish a percolation threshold of FeS melts in peridotite matrix, two types of experiments have been done on partially molten garnet peridotite. Peridotite powders with 100-200 ? and 20-30 ? grain size were mixed with 5-15 vol% Fe70S30 composition. The first type of experiments has been done in the centrifuging piston cylinder at ETH, Zürich. The mixed powders were sealed in graphite capsules, D=2.6 mm and H=3 mm, and annealed at 1 GPa and 1150-1260° C in a piston-cylinder over 70 h. Then, the capsules have been rotated at 500 g during 2-10 h at 1 GPa. The distribution of Fe70S30 and partial melts has been quantified on polished sections. In the second type of experiments, the mixtures of peridotite and Fe70S30 were annealed at 1 GPa from 650 to 1300° C over 24-70 h in a piston- cylinder. The centrifuge experiments revealed a negligible percolation of Fe70S30 melts through the partially molten peridotite matrix. Only at 1260° C (18 % melting) at starting 5 vol% of Fe70S30 the vertical gradient is 1 %/mm, and in samples with starting 15 % Fe70S30 the vertical separation achieved 2 %/mm after 10 h of centrifuging. The progressive melting contributes only to the increase of Fe70S30 droplet size, in agreement with Yoshino & Watson (2005). In conductivity experiments, during the 1st heating cycle, the initially high conductivity of peridotite+Fe70S30 drops for 1-1.5 orders of magnitude within 1h after reaching of Fe70S30 melting. At T<1000° C the activation energy of conductivity is about that of a pure peridotite sample, 2.2 eV. Above the melting point of Fe70S30, the activation energy increases to 2.35 eV, and, then, drops to 0.6-0.7 eV, when the peridotite melting is started. In contradiction to Yoshino et al. (2004), the conductivity measurements show that 5-15 vol% Fe70S30 melts never built an interconnected pattern.

Bagdassarov, N.; Golabek, G.; Solferino, G.; Schmidt, M. W.

2006-12-01

298

Interface controlled electrical and magnetic properties in Fe-Fe3O4-silica gel nanocomposites  

Microsoft Academic Search

Iron nanoparticles with a shell of Fe3O4 phase with a total diameter of 5.3 nm have been grown within a silica gel matrix in the percolative configuration by suitable reduction followed by oxidation treatments. dc electrical resistivity measurements were carried out in the temperature range 80-300 K. The resistivity of the nanocomposites was found to be about 7 orders of

D. Das; S. Roy; J. W. Chen; D. Chakravorty

2002-01-01

299

Interface controlled electrical and magnetic properties in Fe-Fe3O4-silica gel nanocomposites  

NASA Astrophysics Data System (ADS)

Iron nanoparticles with a shell of Fe3O4 phase with a total diameter of 5.3 nm have been grown within a silica gel matrix in the percolative configuration by suitable reduction followed by oxidation treatments. dc electrical resistivity measurements were carried out in the temperature range 80-300 K. The resistivity of the nanocomposites was found to be about 7 orders of magnitude lower than that of the reference gel. The electrical conduction has been explained on the basis of a small polaron hopping mechanism. The activation energy in the case of the composites was calculated from experimental data to be about one-fifth that for the reference sample. An interfacial amorphous phase is believed to cause such reduction in resistivity. The effective dielectric constant of this phase was estimated to be about four times that of the reference glass. Magnetization measurements on these specimens were carried out in the temperature range 5-300 K both in zero field cooled and field cooled states. A peak in the magnetization at ~120 K was ascribed to an order-disorder (Verwey) transition. Another peak at ~55 K was explained as arising due to a spin glass like disorder at the interface between the ferromagnetic iron ores and the ferrimagnetic Fe3O4 shell. A loop shift was observed as a result of the spin freezing below this temperature.

Das, D.; Roy, S.; Chen, J. W.; Chakravorty, D.

2002-04-01

300

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

PubMed

We present a genome-scale metabolic model for the archaeal methanogen Methanosarcina barkeri. We characterize the metabolic network and compare it to reconstructions from the prokaryotic, eukaryotic and archaeal domains. Using the model in conjunction with constraint-based methods, we simulate the metabolic fluxes and resulting phenotypes induced by different environmental and genetic conditions. This represents the first large-scale simulation of either a methanogen or an archaeal species. Model predictions are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for wild type and mutants of the methanogenic pathway have a high level of agreement with experimental findings. We further examine the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction, and determine a stoichiometry for the nitrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation and further uncover the metabolic characteristics of methanogenesis. PMID:16738551

Feist, Adam M; Scholten, Johannes C M; Palsson, Bernhard Ø; Brockman, Fred J; Ideker, Trey

2006-01-31

301

Biochemical and Physiological Characterization of the Pyruvate Formate-Lyase Pfl1 of Chlamydomonas reinhardtii, a Typically Bacterial Enzyme in a Eukaryotic Alga?  

PubMed Central

The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.

Hemschemeier, Anja; Jacobs, Jessica; Happe, Thomas

2008-01-01

302

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

SciTech Connect

We present a genome-scale metabolic reconstruction for the archaeal methanogen Methanosarcina barkeri. This reconstruction represents the first large-scale, predictive model of a methanogen and an archael species. We characterize this reconstruction and compare it to those from the prokaryotic, eukaryotic, and archael domains. We further apply constraint-based methods to stimulate the metabolic fluxes and resulting phenotypes under different environmental and genetic conditions. These results are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for mutants of the methanogenic pathway were found to have a high level of agreement with experimental findings. The active reactions and pathways under selected growth conditions are presented and characterized. We also examined the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an in silico analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation, and further uncover the metabolic characteristics of methanogenesis.

Feist, Adam; Scholten, Johannes C.; Palsson, Bernard O.; Brockman, Fred J.; Ideker, Trey

2006-01-31

303

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

SciTech Connect

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

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

2011-05-01

304

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

NASA Astrophysics Data System (ADS)

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.

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

305

[?-3-(Methyl-sulfan-yl)benzene-1,2-di-thiol-ato-1:2?4 S,S?:S,S?]bis-[tri-carbonyl-iron(I)  

PubMed Central

The title compound, [Fe2(C7H6S3)(CO)6], was prepared as a biomimic for the active site of [FeFe]-hydrogenases. The central Fe2S2 core is in a butterfly conformation and each FeI atom has a pseudo-square-pyramidal coordination by three O atoms and two S atoms. The Fe—Fe distance is 2.471?(2)?Å and the dihedral angle between the two Fe—S—Fe planes is 78.96?(7)°. The least-squares plane through the –S(C7H6S)S– bridge nearly bis­ects the mol­ecular structure: except for the two Fe(CO)3 units, all atoms are in this plane with an average deviation from the plane of 0.028?(3)?Å. In the crystal, the mol­ecules are linked into chains along [001] by C—H??(arene) inter­actions.

Yang, Yong; Wang, Ning; Chen, Lin

2013-01-01

306

Biomimetic chemistry: Merging the old with the new  

NASA Astrophysics Data System (ADS)

The classic organometallic compound ferrocene has been combined with a unique diiron unit in the latest synthetic analogue of an enzyme active site, achieving the three functionalities needed for a working model of diiron hydrogenase, itself of ancient origin.

Darensbourg, Marcetta Y.; Bethel, Ryan D.

2012-01-01

307

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

NASA Astrophysics Data System (ADS)

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.

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

2006-07-01

308

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

SciTech Connect

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.

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

2011-08-12

309

Regioselectivity of H Cluster Oxidation  

PubMed Central

The H2-evolving potential of [FeFe] hydrogenases is severely limited by the oxygen sensitivity of this class of enzymes. Recent experimental studies on hydrogenase from C. reinhardtii point to O2-induced structural changes in the [Fe4S4] subsite of the H cluster. Here, we investigate the mechanistic basis of this observation by means of density functional theory. Unexpectedly, we find that the isolated H cluster shows a pathological catalytic activity for the formation of reactive oxygen species such as O2– and HO2–. After protonation of O2–, an OOH radical may coordinate to the Fe atoms of the cubane, whereas H2O2 specifically reacts with the S atoms of the cubane-coordinating cysteine residues. Both pathways are accompanied by significant structural distortions that compromise cluster integrity and thus catalytic activity. These results explain the experimental observation that O2-induced inhibition is accompanied by distortions of the [Fe4S4] moiety and account for the irreversibility of this process.

2011-01-01

310

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

PubMed Central

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.

Xue, Genqiang; Fiedler, Adam T.; Martinho, Marlene; Munck, Eckard; Que, Lawrence

2008-01-01

311

Metabolic network reconstruction and genome-scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052  

PubMed Central

Background Solventogenic clostridia offer a sustainable alternative to petroleum-based production of butanol--an important chemical feedstock and potential fuel additive or replacement. C. beijerinckii is an attractive microorganism for strain design to improve butanol production because it (i) naturally produces the highest recorded butanol concentrations as a byproduct of fermentation; and (ii) can co-ferment pentose and hexose sugars (the primary products from lignocellulosic hydrolysis). Interrogating C. beijerinckii metabolism from a systems viewpoint using constraint-based modeling allows for simulation of the global effect of genetic modifications. Results We present the first genome-scale metabolic model (iCM925) for C. beijerinckii, containing 925 genes, 938 reactions, and 881 metabolites. To build the model we employed a semi-automated procedure that integrated genome annotation information from KEGG, BioCyc, and The SEED, and utilized computational algorithms with manual curation to improve model completeness. Interestingly, we found only a 34% overlap in reactions collected from the three databases--highlighting the importance of evaluating the predictive accuracy of the resulting genome-scale model. To validate iCM925, we conducted fermentation experiments using the NCIMB 8052 strain, and evaluated the ability of the model to simulate measured substrate uptake and product production rates. Experimentally observed fermentation profiles were found to lie within the solution space of the model; however, under an optimal growth objective, additional constraints were needed to reproduce the observed profiles--suggesting the existence of selective pressures other than optimal growth. Notably, a significantly enriched fraction of actively utilized reactions in simulations--constrained to reflect experimental rates--originated from the set of reactions that overlapped between all three databases (P = 3.52 × 10-9, Fisher's exact test). Inhibition of the hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed. Conclusions Microbial production of butanol by C. beijerinckii offers a promising, sustainable, method for generation of this important chemical and potential biofuel. iCM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production.

2011-01-01

312

Metabolic Deficiences Revealed in the Biotechnologically Important Model Bacterium Escherichia coli BL21(DE3)  

Microsoft Academic Search

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all

Constanze Pinske; Markus Bönn; Sara Krüger; Ute Lindenstrauß; R. Gary Sawers

2011-01-01

313

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

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

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

2009-10-14

314

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 Central

In order to model the syn disposition of histidine residues in carboxylate-bridged non-heme diiron enzymes, we prepared a new dinucleating ligand, H2BPG2DEV, that provides this geometric feature. The ligand incorporates biologically relevant carboxylate functionalities, which have not been explored as extensively as nitrogen-only analogs. Three novel oxo-bridged diiron(III) complexes [Fe2(?-O)(H2O)2-(BPG2DEV)](ClO4)2 (6), [Fe2(?-O)(?-O CAriPrO)(BPG2DEV)](ClO4) (7), and [Fe2(?-O)(?-CO3)(BPG2DEV)] (8) were prepared. Single crystal X-ray structural characterization confirms that two pyridines 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 CO2 in organic solvents. A common maroon-colored intermediate (?max = 490 nm; ? = 1500 M?1 cm?1) forms in reactions of 6, 7, or 8 with H2O2 and NEt3 in CH3CN/H2O solutions. Mass spectrometric analyses of this species, formed using 18O-labeled H2O2, indicate the presence of a peroxide ligand bound to the oxo-bridged diiron(III) center. The Mössbauer spectrum at 90 K of the EPR-silent intermediate exhibits a quadrupole doublet with ?. = 0.58 mm/s and ?EQ = 0.58 mm/s. The isomer shift is typical for a peroxodiiron(III) species, but the quadrupole splitting parameter is unusually small compared to related complexes. These Mössbauer parameters are comparable to those observed for a peroxo intermediate formed in the reaction of reduced toluene/o-xylene monooxygenase hydroxylase (ToMOH) 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 reformed in significant quantities after a second addition of H2O2, as observed spectroscopically and by mass spectrometry.

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

2009-01-01

315

Undeca-carbonyl-?2-methane-thiol-ato-?2-[(pyridin-2-yl)methane-thiol-ato]-?4-sulfido-tetra-iron(II)(2 Fe--Fe)  

PubMed Central

The title compound, [Fe4(C6H6NS)(CH3S)S(CO)11], com­prises two butterfly-shaped sub-cluster cores, Fe2S2N and Fe2S2, joined together by a spiro-type ?4-S atom. The (pyridin-2-yl)methane­thiol­ate ligand is attached to the Fe2(CO)5 unit in a ?-?N:?2 S mode, and the methane­thiol­ate ligand is coordinated to the Fe2(CO)6 unit in a ?-?2 S fashion.

Shi, Yao-Cheng; Lai, Liang; Shen, Wen-Bin; Yuan, Li-Min

2011-01-01

316

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

Microsoft Academic Search

Partition coefficients (D) for metal\\/sulfide liquid, troilite\\/sulfide liquid, and schreibersite\\/sulfide liquid have been experimentally determined for Ag, Au, Mo, Ni, Pb, Pd, and Tl. With these partition coefficients, it should be possible to better understand the [sup 107]Pd-[sup 107]Ag and [sup 205]Tl-[sup 205]Pb systems of iron meteorites. In general, the schreibersite\\/metal and troilite\\/metal partition coefficients for compatible' elements are quite

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

1993-01-01

317

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

NASA Astrophysics Data System (ADS)

Partitioning coefficients for metal/sulfide liquid, troilite/sulfide liquid, and schreibersite/sulfide liquid were determined for Ag, Au, Mo, Ni, Pd, and Tl (using EMPA and proton-induced X-ray microprobe and ion microprobe analyses) in order to understand the chronometer systems of iron meteorites. In general, the obtained schreibersite/metal and troilite/metal partition coefficients for 'compatible' elements were quite similar to those inferred from natural assemblages, reinforcing an earlier made conclusion that there is a class of elements for which experimental troilite/metal and schreibersite/metal partition coefficients approximate those inferred from natural samples. The consistency between experimental and natural assemblages, however, was not observed for Ag, Pb, and Tl, indicating that the abundances of these elements determined in 'metal' and 'troilite' separates from iron meteorites are influenced by trace minerals that concentrate incompatible elements.

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

1993-01-01

318

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

Microsoft Academic Search

BackgroundSurface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism

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

2010-01-01

319

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

PubMed

The generation of H2 from protons and electrons by complexes of cobalt has an extensive history. During the past decade, interest in this subject has increased as a result of developments in hydrogen generation that are driven electrochemically or photochemically. This article reviews the subject of hydrogen generation using Co complexes as catalysts and discusses the mechanistic implications of the systems studied for making H2. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. PMID:23689026

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

2013-05-17

320

Comparison of Extracellular Polysaccharide Composition, Rhizobitoxine Production, and Hydrogenase Phenotype among Various Strains of Bradyrhizobium japonicum  

Microsoft Academic Search

A survey of 51 strains of Bradyrhizobium japonicum was performed with respect to the com- position of extracellular polysaccharide (EPS), the production of rhizobitoxine and the hydro- genase phenotype. A good correlation was found among these three different characteristics. Thirty-six strains producing an EPS composed of glucose, mannose, galactose, 4-O-methyl galac- tose and galacturonic acid did not synthesize rhizobitoxine, whereas

Kiwamu Minamisawa

321

Hydrogenase Activity of Soybean Nodules Doubly Infected with Bradyrhizobium japonicum and B. elkanii  

Microsoft Academic Search

Rhizobitoxine (2-amino-4-(2-amino-3-hydropropoxy)-trans-but-3-erioic acid) is a phytotoxin produced by some strains of Bradyrhizobium species. Rhizobitoxine-producing strains often induce chlorosis in new leaves of soybean as a result of the synthesis of the toxin in nodules (Owens and Wright 1964; Owens et al. 1972). Some of the B. japonicum bacteroids possessing the hydrogen uptake (Hup) system are capable of ATP production by

Kiwamu Minamisawa; Kuniko Ebihara

1996-01-01

322

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

Microsoft Academic Search

Interconversion of water and hydrogen in unitized regenerative fuel cells is a promising energy storage framework for smoothing out the temporal fluctuations of solar and wind power. However, replacement of presently available platinum catalysts by lower-cost and more abundant materials is a requisite for this technology to become economically viable. Here, we show that the covalent attachment of a nickel

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

2009-01-01

323

Local structures of Sr2FeMnO5+y (y=0, 0.5) and Sr2Fe1.5Cr0.5O5 from reverse Monte Carlo modeling of pair distribution function data and implications for magnetic order  

NASA Astrophysics Data System (ADS)

The local structures of the oxygen deficient perovskites Sr2FeMnO5, Sr2FeMnO5.5, and Sr2Fe1.5Cr0.5O5 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 Sr2FeMnO5+y compounds it is found that there is no short range ordering of the Fe and Mn cations. For Sr2Fe1.5Cr0.5O5 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 Sr2FeMnO5 it is found that the Fe3+ cations are most often in 4-fold coordination but there is also a large amount of Fe3+ in 5-fold coordination and a small amount in 6-fold coordination. The Mn3+ is split between 5-fold and 6-fold coordination. The Mn?O bond length distributions indicate that the Mn3+O6 octahedra and Mn3+O5 square pyramids are locally Jahn-Teller distorted. In Sr2FeMnO5.5 the Fe3+ is almost entirely 5 coordinate while the Mn4+ is almost entirely 6 coordinate. The Cr3+ in Sr2Fe1.5Cr0.5O5 is almost entirely 6-fold coordinated, giving the Fe3+ an average coordination number of 4.67. In Sr2FeMnO5 and Sr2Fe1.5Cr0.5O5 the Fe3+ and Sr2+ cations undergo local displacements which are driven by the oxygen vacancies, while the Mn3+ and Cr3+ cations remain near their positions in the average structures. In Sr2FeMnO5.5 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.

King, Graham; Ramezanipour, Farshid; Llobet, Anna; Greedan, John E.

2013-02-01

324

Structured Modeling and Model Management  

Microsoft Academic Search

\\u000a We discuss Geoffrion’s contribution to model management and the practice of modeling through his structured modeling formalism.\\u000a We review the trajectory of structured model management research, enumerating the contributions and limitations of both structured\\u000a modeling and model management in general. We summarize by suggesting how Geoffrion’s work could be leveraged to contribute\\u000a to a next generation of model management.

Daniel Dolk

325

Fair Model  

NSDL National Science Digital Library

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

Blecha, Betty

326

Flexibility of syntrophic enzyme systems in desulfovibrio species ensures their adaptation capability to environmental changes.  

PubMed

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

Meyer, Birte; Kuehl, Jennifer V; Deutschbauer, Adam M; Arkin, Adam P; Stahl, David A

2013-08-23

327

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

PubMed

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

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

2011-04-19

328

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

PubMed Central

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

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

2011-01-01

329

Toilet Model  

NSDL National Science Digital Library

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

Rathjen, Don

2005-01-01

330

Understanding Models  

NSDL National Science Digital Library

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

Ireton, Shirley W.; Gilbert, Steven W.

2003-01-01

331

Supermatrix models  

SciTech Connect

Radom matrix models based on an integral over supermatrices are proposed as a natural extension of bosonic matrix models. The subtle nature of superspace integration allows these models to have very different properties from the analogous bosonic models. Two choices of integration slice are investigated. One leads to a perturbative structure which is reminiscent of, and perhaps identical to, the usual Hermitian matrix models. Another leads to an eigenvalue reduction which can be described by a two component plasma in one dimension. A stationary point of the model is described.

Yost, S.A.

1991-05-01

332

ENTRAINMENT MODELS  

EPA Science Inventory

This presentation presented information on entrainment models. Entrainment models use entrainment hypotheses to express the continuity equation. The advantage is that plume boundaries are known. A major disadvantage is that the problems that can be solved are rather simple. The ...

333

Higgs models  

NASA Astrophysics Data System (ADS)

This lecture presented at the Baikal summer school on physics of elementary particles and astrophysics in 2011 is devoted to the Higgs mechanism of the electroweak symmetry breaking within the Standard Model and in some models beyond it.

Ivanov, I. P.

2012-12-01

334

Landscape Models  

NSDL National Science Digital Library

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

Marchetti, David

335

Model Fundamentals  

NSDL National Science Digital Library

The module provides background information for the Characteristics of Operational NWP Models module (also in the NWP PDS), which contains current information about the characteristics and architecture of commonly used operational models, their operationally significant strengths and weaknesses, and model assessment tools. The subject matter expert for this module is Dr. Ralph Petersen of the National Centers for Environmental Prediction, Environmental Modeling Center (NCEP/EMC).

Spangler, Tim

1999-12-10

336

Model Completeness  

Microsoft Academic Search

\\u000a We already defined model completeness in Chapter 1: a theory T is called model complete if every embedding between models of T is elementary. We dealt with this notion also in Chapter 2, where we considered its connection with quantifier elimination\\u000a and completeness. But now we wish to examine model completeness in a closer and more direct way, to discuss

Annalisa Marcja; Carlo Toffalori

337

Better Models  

NSDL National Science Digital Library

At the end of the first chapter, you realized, hopefully, that the model of an atom that we have so far (Rutherford's model of a concentrated positive nucleus with negative charges around it) doesn't go very far in helping us explain observations. So, why not try to make that model better? This chapter shows you how it's done.

Robertson, William C.

2007-01-01

338

Thinkable models  

Microsoft Academic Search

A primary objective of technical education should be the development of thinkable models in the minds of students. Thinkable models are representations of things and processes simple enough that people can use them in thought experiments. The organization of cognitive structures for technical domains can be imagined to be a network of appropriately connected thinkable models. Artificial intelligence (AI), as

Robert W. Lawler

1996-01-01

339

Energizer Model  

NSDL National Science Digital Library

The EJS Energizer model explores the relationship between kinetic, potential, and total energy. Users create a potential energy curve and observe the resulting motion. The Energizer model was created using the Easy Java Simulations (EJS) modeling tool. It is distributed as a ready-to-run (compiled) Java archive. Double clicking the jar file will run the program if Java is installed.

Gallis, Michael R.

2008-10-27

340

Hydrological models are mediating models  

NASA Astrophysics Data System (ADS)

Despite the increasing role of models in hydrological research and decision-making processes, only few accounts of the nature and function of models exist in hydrology. Earlier considerations have traditionally been conducted while making a clear distinction between physically-based and conceptual models. A new philosophical account, primarily based on the fields of physics and economics, transcends classes of models and scientific disciplines by considering models as "mediators" between theory and observations. The core of this approach lies in identifying models as (1) being only partially dependent on theory and observations, (2) integrating non-deductive elements in their construction, and (3) carrying the role of instruments of scientific enquiry about both theory and the world. The applicability of this approach to hydrology is evaluated in the present article. Three widely used hydrological models, each showing a different degree of apparent physicality, are confronted to the main characteristics of the "mediating models" concept. We argue that irrespective of their kind, hydrological models depend on both theory and observations, rather than merely on one of these two domains. Their construction is additionally involving a large number of miscellaneous, external ingredients, such as past experiences, model objectives, knowledge and preferences of the modeller, as well as hardware and software resources. We show that hydrological models convey the role of instruments in scientific practice by mediating between theory and the world. It results from these considerations that the traditional distinction between physically-based and conceptual models is necessarily too simplistic and refers at best to the stage at which theory and observations are steering model construction. The large variety of ingredients involved in model construction would deserve closer attention, for being rarely explicitly presented in peer-reviewed literature. We believe that devoting more importance to identifying and communicating on the many factors involved in model development might increase transparency of model building.

Babel, L. V.; Karssenberg, D.

2013-08-01

341

Station Models  

NSDL National Science Digital Library

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

Ertl, Mr.

2007-11-03

342

Analytical models  

NASA Astrophysics Data System (ADS)

A system-level design and analysis model was developed. This model was conceived to have several key elements: a solar pond thermodynamic performance model, a power generation subsystem model, and an economic analysis element. The basic approach was to create these elements or modules and refine them on an individual basis yet retain the capability to easily couple them into a full system design model. This building block approach allows for maximum flexibility and substitution of refined descriptions as the technology develops. A general overview of interconnecting these subsystem models is presented. The primary program control element will perform the administrative functions of data input, data output, information storage and transfer, and sequential calling of the subsystem models. From the point of view of the requirements of a system design model, a power conversion subsystem model was developed. The goal of the effort was a preliminary subsystem model compatible with the solar pond subsystem model so that a first order system simulation analysis could be performed.

1984-09-01

343

ICRF modelling  

SciTech Connect

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

Phillips, C.K.

1985-12-01

344

Computer Models  

NSDL National Science Digital Library

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

Nielsen-Gammon, John

1996-01-01

345

Modeling patterns for task models  

Microsoft Academic Search

Models allow us to describe complex systems at different abstract and conceptual levels, hence amplify our analytical and problem solving capabilities, However, a lot of human effort and experience is needed to build correct models, and to translate them to concrete artifacts: in our case a usable user interface. This paper introduces the concept of task and pattern models to

Ashraf Gaffar; Daniel Sinnig; Ahmed Seffah; Peter Forbrig

2004-01-01

346

Functions and Models: Mathematical Models  

NSDL National Science Digital Library

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

Freeze, Michael

2003-01-22

347

Ventilation Model  

SciTech Connect

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.

H. Yang

1999-11-04

348

Gimbal Model  

NSDL National Science Digital Library

The Gimbal Model illustrates the pitch, roll and yaw of a 3D object. Independent axis mode allows each axes to be rotated without affecting the others axes. If this mode is not selected, the model can be used to explore the phenomena of gimbal lock. This model tests the Java 3D implementation of the EJS 3D library. If the Java 3D option is selected, a airplane VMRL file (wrl) is rendered inside the gimbals. A warning message will appear if the Java 3D library is not available. The Gimbal Model was developed using the Easy Java Simulations (EJS) modeling tool. It is distributed as a ready-to-run (compiled) Java archive. Double clicking the ejs_ntnu_Gimbal.jar file will run the program if Java is installed. You can modify this simulation if you have EJS installed by right-clicking within the map and selecting "Open Ejs Model" from the pop-up menu item.

Hwang, Fu-Kwun; Wee, Loo K.

2011-12-30

349

Iron-sulfur stoichiometry and structure of iron-sulfur clusters in three-iron proteins: Evidence for [3Fe-4S] clusters  

PubMed Central

Beef heart aconitase contains 3Fe clusters in its inactive and 4Fe clusters in its active form. The fully active form can be restored from the inactive one by insertion of Fe2+, whereas S2- is not required. Chemical analyses for iron and labile sulfide yield Fe/S2- ratios of 0.66-0.74 for the inactive and 0.90-1.03 for the active form. Sulfane sulfur (S0) was not detected. We propose on the basis of these data that the inactive form may arise from the active one by loss of one iron only per cluster with the sulfur remaining as S2- in a [3Fe-4S] structure. Measurements by extended x-ray absorption fine structure (EXAFS) spectroscopy on the 3Fe form of aconitase yield a Fe··S distance of 2.24 Å and a Fe··Fe distance of 2.71 Å. This Fe··Fe distance is in agreement with that obtained by EXAFS on ferredoxin II of Desulfovibrio gigas, another 3Fe protein, but disagrees with Fe··Fe distances observed for the 3Fe cluster of Azotobacter vinelandii ferredoxin I by x-ray diffraction—namely, 4.1 Å. We suggest that this difference may be due to the presence of a [3Fe-3S] structure in the Azotobacter ferredoxin I crystals vs. a [3Fe-4S] structure in liquid or frozen solutions of aconitase. The [3Fe-3S] cluster has been shown to have a relatively flat twist-boat structure, whereas a [3Fe-4S] cluster could be expected to essentially maintain the compact structure of the [4Fe-4S] cluster. This would explain the differences in Fe··Fe distances. Two possible structural models for a [3Fe-4S] cluster are discussed.

Beinert, Helmut; Emptage, Mark H.; Dreyer, Jean-Luc; Scott, Robert A.; Hahn, James E.; Hodgson, Keith O.; Thomson, Andrew J.

1983-01-01

350

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

NASA Astrophysics Data System (ADS)

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

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

2002-06-01

351

Atmospheric Modeling  

Microsoft Academic Search

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

Christian Seigneur; Robin Dennis

352

SCARP Model  

NSDL National Science Digital Library

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

Locke, Bill

353

Floodplain Modeling  

NSDL National Science Digital Library

Students explore the impact of changing river volumes and different floodplain terrain in experimental trials with table top-sized riverbed models. The models are made using modeling clay in an aluminum baking pans placed on a slight incline. Water added âupstreamâ at different flow rates and to different riverbed configurations simulates different potential flood conditions. Students study flood dynamics as they modify the riverbed with blockages or levees to simulate real-world scenarios.

Integrated Teaching And Learning Program

354

Model Cheking  

Microsoft Academic Search

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

Edmund M. Clarke

1997-01-01

355

Neurotransmission Model  

NSDL National Science Digital Library

This lesson allows students to apply engineering principles in the science classroom. Students learn how neurons convey information through designing and building a physical model of neurotransmission.

Dr. Janet M Dubinsky (University of Minnesota Neuroscience)

2011-07-20

356

PREDICTIVE MODELS  

SciTech Connect

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.

Ray, R.M. (DOE Bartlesville Energy Technology Center, Bartlesville, OK (United States))

1988-10-01

357

Model Volcanoes  

NSDL National Science Digital Library

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

358

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

ERIC Educational Resources Information Center

|A project which exposes students to biologically important transition-metal chemistry is illustrated by taking an example of the iron-carbonyl compound, [mu]-(1,3-Propanedithiolaro)-hexa-carbonyldiiron as a structural model for an iron-only hydro-genase. The project provides the students with experience of Schlenk line techniques, purification,…

Works, Carmen F.

2007-01-01

359

Computational Models  

Cancer.gov

The models work by investigating relationships between the pathways that control a cell's response to inflammation, growth factors, DNA damage, and other events. The goal is to create a dynamic model of the biological processes related to cancer initiation, progression, and metastasis.

360

Probability Models  

NSDL National Science Digital Library

This site, presented by the Department of Statistics at Yale University, gives an explanation, a definition and an example of probability models. Topics include components of probability models and the basic rules of probability. Overall, this is a great resource for any mathematics classroom studying statistics.

Lacey, Michelle

2008-12-26

361

Modeling Daisyworld  

NSDL National Science Digital Library

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

Bice, David

362

Budget Model.  

ERIC Educational Resources Information Center

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

Washington State Board for Community Coll. Education, Olympia.

363

Daisyworld Model  

NSDL National Science Digital Library

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

Lovelock, James; Watson, Andrew; Bice, Dave; Dept. Of Geosciences, Penn S.

364

Partition models  

Microsoft Academic Search

Product partition models assume that observations in different components of a random partition of the data are independent given the partition. If the probability distribution of random partitions is in a certain product form prior to making the observations, it is also in product form given the observations. The product model thus provides a convenient machinery for allowing the data

J. A. Hartigan

1990-01-01

365

Thinkable Models.  

ERIC Educational Resources Information Center

Argues that the organization of cognitive structures for technical domains can be visualized as a network of connected thinkable models. Describes a taxonomy of models that has been developed and discusses the issue of how representations relate to human modes of perception and action. Contains 25 references. (DDR)

Lawler, Robert W.

1996-01-01

366

Molecular Modeling  

Microsoft Academic Search

Molecular modeling has trickled down from the realm of pharmaceutical and research laboratories into the realm of undergraduate chemistry instruction. It has opened avenues for the visualization of chemical concepts that previously were difficult or impossible to convey. I am sure that many of you have developed exercises using the various molecular modeling tools. It is the desire of this

Jon L. Holmes

1999-01-01

367

PREDICTIVE MODELS  

Microsoft Academic Search

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)

1988-01-01

368

PREDICTIVE MODELS  

Microsoft Academic Search

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

1986-01-01

369

Supernova models  

Microsoft Academic Search

Recent progress in understanding the observed properties of type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the Ni-56 produced therein is reviewed. The expected nucleosynthesis and gamma-line spectra for this model of type I explosions and a model for type II explosions are presented. Finally, a qualitatively new approach

S. E. Woosley; T. A. Weaver

1981-01-01

370

Model Execution  

SciTech Connect

A computer software-based model is typically designed to produce a trace of system evolution over time. The actual process of computing the model state and producing the state values as the simulation time is advanced is called model execution. Models could be designed with a specific execution technique in mind, or could be generally amenable to multiple different execution techniques. Two popular methods that are used to execute models are: time-stepped method and discrete-event method. Each of these methods could in turn be executed either sequentially (on a single processor), or in parallel (using multiple processors concurrently). In this chapter, we describe the time-stepped and discrete event execution methods and outline some of the common approaches to their sequential and parallel execution. Execution concepts common to the methods are described followed by implementation details of the methods.

Perumalla, Kalyan S [ORNL

2007-01-01

371

Nucleon models  

SciTech Connect

Theoretical models for the structure of the nucleons and the hyperons are constructed for the purpose of obtaining an understanding of the dynamics that gives rise to their complex spectra. The most modest goal is to explain the ground state energies and the corresponding static observables - magnetic moments and rms radii. More ambitious models attempt in addition to explain the dynamic observables as, e.g., the electromagnetic form factors, which requires that not only overall features are understood, but that at least some parts of the short-range dynamics are understood as well. The rich structure of the baryon spectrum, with 5 known successive flavor generations [the nucleons (up and down quarks), the strange, the charmed and the bottom hyperons] makes the construction of a realistic nucleon model a complex task. Naturally, if it were possible, one would like to take the Lagrangian density of quantum chromodynamics as the starting point. This is formed of quark and gluon field operators. While the mass scale of the baryons is 1 GeV, that of the light u and d quarks and the gluons is essentially 0 (m{sub u}{congruent} 5 MeV, m{sub d} {congruent} 8 MeV, m{sub gluon} = 0). That baryons with masses of the order of 1 GeV or more can be formed of such light constituents is a consequence of the very strong gluon exchange interaction at low energies. The topics covered in this report are: Chiral Symmetry; The Chiral Bag Model; Chiral Meson Theory; The Skyrmion; Currents and Form Factors; The Skyrme Model and the Chiral Bag Model; Extensions of the Model; Explicit Vector Meson Fields; The Hyperons; The Hyperon Spectrum; The Hyperon Magnetic Moments; Exotics; The Limits of the Bound State Model; and The End of the Nucleon Models.

Riska, D.O. [Univ. of Helsinki (Finland)

1996-12-31

372

Virtual models  

US Patent & Trademark Office Database

A method of generating a virtual model of an object, comprising the step of constructing a fused model to represent the variation of the shape of the object in a plurality of configurations. In the method, position variation vectors may be calculated which describe the variation of a plurality of points in the fused model from their mean positions over all the configurations of the object; the position variation vectors may be assigned a weighting factor which represents a measure of their probabilistic confidence; a weighted covariance matrix may be constructed to represent the variations; the weighted covariance matrix may be operated on to obtain shape variation vectors representing the weighted direction and magnitude of variations; such that the fused model may describe how the shape of the object varies as a function of the shape variation vectors scaled by a shape parameter.

Fisher, III; Robert Burns (Scotland, GB); Faber; Petko (Leonberg, DE); Lukins; Timothy Campbell (Scotland, GB)

2008-10-21

373

Programming models  

SciTech Connect

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

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

2011-01-14

374

Model Lungs.  

ERIC Educational Resources Information Center

A cheap and simple model that can be made and used by pupils to study the human breathing mechanism is presented. A list of needed materials, procedures for construction, possible refinements, and method of use are included. (KR)

Taylor, Emma

1991-01-01

375

Supernova models  

SciTech Connect

Recent progress in understanding the observed properties of type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the Ni-56 produced therein is reviewed. The expected nucleosynthesis and gamma-line spectra for this model of type I explosions and a model for type II explosions are presented. Finally, a qualitatively new approach to the problem of massive star death and type II supernovae based upon a combination of rotation and thermonuclear burning is discussed. While the theoretical results of existing models are predicated upon the assumption of a successful core bounce calculation and the neglect of such two-dimensional effects as rotation and magnetic fields the new model suggests an entirely different scenario in which a considerable portion of the energy carried by an equatorially ejected blob is deposited in the red giant envelope overlying the mantle of the star.

Woosley, S.E. (California, University, Santa Cruz; California, University, Livermore, CA); Weaver, T.A. (California, University, Livermore, CA)

1981-12-29

376

Energy Models  

EPA Science Inventory

Energy models characterize the energy system, its evolution, and its interactions with the broader economy. The energy system consists of primary resources, including both fossil fuels and renewables; power plants, refineries, and other technologies to process and convert these r...

377

Device modeling  

NASA Astrophysics Data System (ADS)

A summary report is given of the activities of the device modeling workshop which was held as a part of the Space Photovoltaic Research and Technology Conference at the Lewis Research Center, October 7 to 9, 1986. The purpose of this workshop was to access the status of solar cell device modeling to see if it is meeting present and future needs of the photovoltaic community.

Schwartz, Richard

1987-06-01

378

PREDICTIVE MODELS  

SciTech Connect

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 the reservoir to wash out the oil; 2) carbon dioxide miscible flooding, where carbon dioxide mixes with the lighter hydrocarbons making the oil easier to displace; 3) in-situ combustion, which uses the heat from burning some of the underground oil to thin the product; 4) polymer flooding, where thick, cohesive material is pumped into a reservoir to push the oil through the underground rock; and 5) steamflood, where pressurized steam is injected underground to thin the oil. 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.

Ray, R.M. (DOE Bartlesville Energy Technology Technology Center, Bartlesville, OK (United States))

1986-12-01

379

Supernova models  

Microsoft Academic Search

Recent progress in understanding the observed properties of Type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the ⁵⁶Ni produced therein is reviewed. Within the context of this model for Type I explosions and the 1978 model for Type II explosions, the expected nucleosynthesis and gamma-line spectra from both kinds

S. E. Woosley; T. A. Weaver

1980-01-01

380

Modeling life.  

PubMed

We seek to construct physical and mathematical models of life. Such models allow us to test our understanding of how living systems function and how they respond to human imposed stimuli. One system is a genomically and chemically complete model of a minimal cell. This cell is a hypothetical bacterium with the fewest number of genes possible. Such a minimal cell provides a platform to ask about the essential features of a living cell and forms a platform to investigate "synthetic biology." A second system is "Body-on-a-Chip" which is a microfabricated microfluidic system with cells or tissue constructs representing various organs in the body. It can be constructed from human or animal cells and used in drug discovery development. That model is a physical representation of a physiologically based pharmacokinetic model. Both the computer and the physical models provide insight into the underlying biology and provide new tools to make use of that understanding to provide benefits to society. PMID:22527010

Shuler, Michael L

2012-04-17

381

Do stroke models model stroke?  

PubMed Central

Stroke is one of the leading causes of death worldwide and the biggest reason for long-term disability. Basic research has formed the modern understanding of stroke pathophysiology, and has revealed important molecular, cellular and systemic mechanisms. However, despite decades of research, most translational stroke trials that aim to introduce basic research findings into clinical treatment strategies – most notably in the field of neuroprotection – have failed. Among other obstacles, poor methodological and statistical standards, negative publication bias, and incomplete preclinical testing have been proposed as ‘translational roadblocks’. In this article, we introduce the models commonly used in preclinical stroke research, discuss some of the causes of failed translational success and review potential remedies. We further introduce the concept of modeling ‘care’ of stroke patients, because current preclinical research models the disorder but does not model care or state-of-the-art clinical testing. Stringent statistical methods and controlled preclinical trials have been suggested to counteract weaknesses in preclinical research. We conclude that preclinical stroke research requires (1) appropriate modeling of the disorder, (2) appropriate modeling of the care of stroke patients and (3) an approach to preclinical testing that is similar to clinical testing, including Phase 3 randomized controlled preclinical trials as necessary additional steps before new therapies enter clinical testing.

Mergenthaler, Philipp; Meisel, Andreas

2012-01-01

382

Gnomon Model  

NSDL National Science Digital Library

The EJS Gnomon model simulates the shadow cast by a gnomon (the part of a sundial that casts the shadow) over the course of a day for any day of the year and any latitude on Earth. The program gives you the option to use mean Sun (which moves relative to the stars at a constant rate throughout the year) or true Sun (which varies its apparent speed relative to the background stars). The default is to use true Sun. The program also shows the observer's horizon plane on the spherical Earth, as well as the ecliptic and the apparent path of Sun. The Earth View can be set to let Earth rotate or remain fixed EJS Gnomon model was created using the Easy Java Simulations (Ejs) modeling tool. It is distributed as a ready-to-run (compiled) Java archive. Double clicking the ejs_astronomy_Gnomon.jar file will run the program if Java is installed. Ejs is a part of the Open Source Physics Project and is designed to make it easier to access, modify, and generate computer models. Additional Ejs models for astronomy are available. They can be found by searching ComPADRE for Open Source Physics, OSP, or Ejs.

Timberlake, Todd

2009-08-19

383

Mechanistic models  

SciTech Connect

Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) interaction'' of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

Curtis, S.B.

1990-09-01

384

Mechanistic models  

SciTech Connect

Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) ``interaction`` of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

Curtis, S.B.

1990-09-01

385

Micrometer Model  

NSDL National Science Digital Library

The Micrometer Model shows the principle of operation and the physical parts of a real micrometer. Micrometers use a screw to amplify distances that are too small to measure directly into large rotations of the screw that are big enough to read from a scale. 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).

Hwang, Fu-Kwun

2009-09-11

386

Supernova models  

SciTech Connect

Recent progress in understanding the observed properties of Type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the /sup 56/Ni produced therein is reviewed. Within the context of this model for Type I explosions and the 1978 model for Type II explosions, the expected nucleosynthesis and gamma-line spectra from both kinds of supernovae are presented. Finally, a qualitatively new approach to the problem of massive star death and Type II supernovae based upon a combination of rotation and thermonuclear burning is discussed.

Woosley, S.E.; Weaver, T.A.

1980-01-01

387

Transducer Models  

NASA Astrophysics Data System (ADS)

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).

Backman, Juha Reinhold

388

Daisyworld Model  

NSDL National Science Digital Library

The Daisyworld model created by Andrew Watson and James Lovelock (1983, Tellus, v. 35B, p. 284-289) is a wonderful example of a self-regulating system incorporating positive and negative feedbacks. The model consists of a planet on which black and white daisies are growing. The growth of these daisies is governed by a parabolic shaped growth function regulated by planetary temperature and is set to zero for temperatures less than 5 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.

Menking, Kirsten

389

Climate Models  

NSDL National Science Digital Library

Climate models are tools that scientists have developed to help predict the future climate of our planet based on different scenarios of human impacts to the atmosphere. The last ice age occurred because of a drop of only a few degrees in global temperatures, so even small temperature increases are a concern.

Kqed

2012-03-27

390

Model Behavior  

ERIC Educational Resources Information Center

There are dozens of books and hundreds of resources that address the issue of character development in students: how to raise them to be good people, how to teach them to be good citizens, how to help them to make good decisions. Little is written, however, about the character development of principals and school leaders, whose behavior is a model…

Holloway, John

2006-01-01

391

OSPREY Model.  

National Technical Information Service (NTIS)

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 oper...

V. J. Rutledge

2013-01-01

392

Modeling Muscles  

ERIC Educational Resources Information Center

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

Goodwyn, Lauren; Salm, Sarah

2007-01-01

393

Modeling Convection  

ERIC Educational Resources Information Center

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

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

2004-01-01

394

Modeling Muscles  

ERIC Educational Resources Information Center

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

Goodwyn, Lauren; Salm, Sarah

2007-01-01

395

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...

396

Multivariate modeling  

NASA Astrophysics Data System (ADS)

The special session entitled Multivariate Modeling of Hydrologic and Other Geophysical Time Series was held during the AGU Fall Meeting in San Francisco on Thursday, December 8, 1983, and was sponsored by the Surface Runoff Committee of the Hydrology Section of AGU. The session brought together about 100 participants from different disciplines, including hydrologists, oceanographers, meteorologists, and statisticians, to discuss the state of the art and new developments of stochastic description and modeling of multiple time series of hydrologic and geophysical phenomena.The papers and discussion generated during the session covered a wide variety of hydrologic variables such as streamflow, precipitation, specific conductance, groundwater and water use, meteorologic variables such as air temperature, wind and pressure, and oceanographic variables such as ocean temperature and velocity. Among the topics discussed were: modeling that is oriented to data generation of multivariate processes, basic data analysis and description of statistical characteristics in time and space; modeling specifically oriented to forecasting the processes involved; transfer of hydrologic a nd geophysical information; and detection of changes in hydrologic information.

Salas, Jose D.

397

Supernova Models.  

National Technical Information Service (NTIS)

Recent progress in understanding the observed properties of Type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the exp 56 Ni produced therein is reviewed. Within the context of this model for T...

S. E. Woosley T. A. Weaver

1980-01-01

398

Supermarket Model  

Microsoft Academic Search

The flow through this supermarket is shown in Fig. 21.1. This model is adapted after an example in [1]. We first describe the crisp system. Customers arrive at the store according to the exponential distribution which will generate times between arrivals. They first go to the Carts area to pick up a shopping cart. We assume that every customer gets

James J. Buckley

399

Criticality Model  

SciTech Connect

The ''Disposal Criticality Analysis Methodology Topical Report'' (YMP 2003) presents the methodology for evaluating potential criticality situations in the monitored geologic repository. As stated in the referenced Topical Report, the detailed methodology for performing the disposal criticality analyses will be documented in model reports. Many of the models developed in support of the Topical Report differ from the definition of models as given in the Office of Civilian Radioactive Waste Management procedure AP-SIII.10Q, ''Models'', in that they are procedural, rather than mathematical. These model reports document the detailed methodology necessary to implement the approach presented in the Disposal Criticality Analysis Methodology Topical Report and provide calculations utilizing the methodology. Thus, the governing procedure for this type of report is AP-3.12Q, ''Design Calculations and Analyses''. The ''Criticality Model'' is of this latter type, providing a process evaluating the criticality potential of in-package and external configurations. The purpose of this analysis is to layout the process for calculating the criticality potential for various in-package and external configurations and to calculate lower-bound tolerance limit (LBTL) values and determine range of applicability (ROA) parameters. The LBTL calculations and the ROA determinations are performed using selected benchmark experiments that are applicable to various waste forms and various in-package and external configurations. The waste forms considered in this calculation are pressurized water reactor (PWR), boiling water reactor (BWR), Fast Flux Test Facility (FFTF), Training Research Isotope General Atomic (TRIGA), Enrico Fermi, Shippingport pressurized water reactor, Shippingport light water breeder reactor (LWBR), N-Reactor, Melt and Dilute, and Fort Saint Vrain Reactor spent nuclear fuel (SNF). The scope of this analysis is to document the criticality computational method. The criticality computational method will be used for evaluating the criticality potential of configurations of fissionable materials (in-package and external to the waste package) within the repository at Yucca Mountain, Nevada for all waste packages/waste forms. The criticality computational method is also applicable to preclosure configurations. The criticality computational method is a component of the methodology presented in ''Disposal Criticality Analysis Methodology Topical Report'' (YMP 2003). How the criticality computational method fits in the overall disposal criticality analysis methodology is illustrated in Figure 1 (YMP 2003, Figure 3). This calculation will not provide direct input to the total system performance assessment for license application. It is to be used as necessary to determine the criticality potential of configuration classes as determined by the configuration probability analysis of the configuration generator model (BSC 2003a).

A. Alsaed

2004-09-14

400

Uncertainty Modeling via Frequency Domain Model Validation.  

National Technical Information Service (NTIS)

The majority of literature on robust control assumes that a design model is available and that the uncertainty model bounds the actual variations about the nominal model. However, methods for generating accurate design models have not received as much att...

M. R. Waszak D. Andrisani

1999-01-01

401

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...

402

The Model  

Microsoft Academic Search

\\u000a The general model we propose could be applied to a wide range of specific situations, for the control of credit risk or of\\u000a any s