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Query: EC:1.12.7.2 (hydrogenase)
 3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.
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PMID:Enzymology and bioenergetics of respiratory nitrite ammonification. 1216 29

ZORA relativistic DFT calculations are presented which aim to model the geometric and electronic structure of the active site of NiFe hydrogenases in its EPR-active oxidized states Ni-A (unready state) and Ni-B (ready state). Starting coordinates are taken from the X-ray structure of a mutant of Desulfovibrio fructosovorans hydrogenase refined at 1.81 A resolution. Nine possible candidates for Ni-A and Ni-B are analyzed in terms of their geometric and electronic structure. Comparison of calculated geometric and magnetic resonance parameters with available experimental data indicates that both oxidized states have a micro-hydroxo bridge between the two metal centers. The different electronic structures of both forms can be explained by a modification of a terminal cysteine in Ni-B, best modeled by protonation of the sulfur atom. A possible mechanism for the activation of both oxidized forms is presented.
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PMID:Density functional calculations for modeling the active site of nickel-iron hydrogenases. 2. Predictions for the unready and ready States and the corresponding activation processes. 1218 59

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic eukaryotes scattered throughout the eukaryotic tree. All of the eukaryotic enzymes characterized so far are iron-only [Fe] hydrogenases. In contrast, it has previously been suggested that hydrogenosomes of the best-studied anaerobic fungus Neocallimastix frontalis L2 contain an unrelated iron-nickel-selenium [NiFeSe] hydrogenase. We have isolated a gene from strain L2 that encodes a putative protein containing all of the characteristic features of an iron-only [Fe] hydrogenase, including the cysteine residues required for the co-ordination of the unique 'hydrogen cluster'. As is the case for experimentally verified hydrogenosomal matrix enzymes from N. frontalis, the [Fe] hydrogenase encodes a plausible amino terminal extension that resembles mitochondrial targeting signals. Phylogenetic analyses of an expanded [Fe] hydrogenase dataset reveal a complicated picture that is difficult to interpret in the light of current ideas of species relationships. Nevertheless, our analyses cannot reject the hypothesis that the novel [Fe] hydrogenase gene of Neocallimastix is specifically related to other eukaryote [Fe] hydrogenases, and thus ultimately might be traced to the same ancestral source.
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PMID:An [Fe] hydrogenase from the anaerobic hydrogenosome-containing fungus Neocallimastix frontalis L2. 1238 2

Nickel has been shown to be an essential trace element involved in the metabolism of several species of bacteria, archea, and plants. In these organisms, nickel is involved in enzymes that catalyze both non-redox (e.g., urease, glyoxalase I) and redox (e.g., hydrogenase, carbon monoxide dehydrogenase, superoxide dismutase) reactions, and proteins involved in the transport, storage, metallocenter assembly, and regulation of nickel concentration have evolved. Studies of structure/function relationships in nickel biochemistry reveal that cysteine ligands are used to stabilize the Ni(III/II) redox couple. Certain nickel compounds have also been shown to be potent human carcinogens. A likely target for carcinogenic nickel is nuclear histone proteins. Here we present X-ray absorption spectroscopic studies of a model Ni peptide designed to help characterize the structure of the nickel complexes formed with histones and place them in the context of nickel structure/function relationships, to gain insights into the molecular mechanism of nickel carcinogenesis.
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PMID:Use of XAS for the elucidation of metal structure and function: applications to nickel biochemistry, molecular toxicology, and carcinogenesis. 1242 16

NiFe-hydrogenases have an Ni-Fe site in which the iron has one CO and two CN groups as ligands. Synthesis of the CN ligands requires the activity of two hydrogenase maturation proteins: HypF and HypE. HypF is a carbamoyltransferase that transfers the carbamoyl moiety of carbamoyladenylate to the COOH-terminal cysteine of HypE and thus forms an enzyme-thiocarbamate. HypE dehydrates the S-carbamoyl moiety in an adenosine triphosphate-dependent process to yield the enzyme thiocyanate. Chemical model reactions corroborate the feasibility of this unprecedented biosynthetic route and show that thiocyanates can donate CN to iron. This finding underscores a striking parallel between biochemistry and organometallic chemistry in the formation of an iron-cyano complex.
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PMID:Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands. 1258 41

Thermotoga maritima grows optimally at 80 degrees C by fermenting carbohydrates to organic acids, CO(2), and H(2). The production of H(2) is catalyzed by a cytoplasmic, heterotrimeric (alphabetagamma) Fe-hydrogenase. This is encoded by three genes, hydC (gamma), hydB (beta) and hydA (alpha), organized within a single operon that contains five additional open reading frames (ORFs). The recombinant form of the first ORF of the operon, TM1420, was produced in Escherichia coli. It has a molecular mass of 8537+/-3 Da as determined by mass spectrometry, in agreement with the predicted amino acid sequence. Purified TM1420 is red in color, has a basic p I (8.8), and contains 1.9 Fe atoms/mol that are present as a single [2Fe-2S] cluster, as determined by UV-visible absorption and EPR spectroscopy. The protein contains five cysteine residues, but their arrangement is characteristic of a subunit or domain rather than of a ferredoxin-type protein. The reduction potential of the [2Fe-2S] cluster (-233 mV at pH 6.5 and 25 degrees C) is pH independent but decreases linearly with temperature to -296 mV (-1.15 mV/ degrees C) at 80 degrees C. TM1420 is not reduced, in vitro, by the Fe-hydrogenase nor by a pyruvate ferredoxin oxidoreductase. The protein was unstable at 70 degrees C under anaerobic conditions with a half-life of approximately 30 min. The basic nature of TM1420, its instability at the growth temperature of T. maritima, and the unusual spacing of its cysteine residues suggest that this protein does not function as a ferredoxin-type electron carrier for the Fe-hydrogenase. Instead, TM1420 is more likely part of a thermostable multi-protein complex that is involved in metal cluster assembly of the hydrogenase holoenzyme.
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PMID:Characterization of a [2Fe-2S] protein encoded in the iron-hydrogenase operon of Thermotoga maritima. 1260 55

F(420)-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis is a [NiFe] hydrogenase composed of the three subunits MvhA, MvhG, and MvhD. Subunits MvhA and MvhG form the basic hydrogenase module conserved in all [NiFe] hydrogenases, whereas the 17-kDa MvhD subunit is unique to Mvh. The function of this extra subunit is completely unknown. In this work, the physiological function of this hydrogenase, and in particular the role of the MvhD subunit, is addressed. In cells of Mt. marburgensis from Ni(2+)-limited chemostat cultures the amount of Mvh decreased about 70-fold. However, the amounts of mvh transcripts did not decrease in these cells as shown by competitive RT-PCR, arguing against a regulation at the level of transcription. In cells grown in the presence of non-limiting amounts of Ni(2+), Mvh was found in two chromatographically distinct forms-a free form and in a complex with heterodisulfide reductase. In cells from Ni(2+)-limited chemostat cultures, Mvh was only found in a complex with heterodisulfide reductase. The EPR spectrum of the purified enzyme reduced with sodium dithionite was dominated by a signal with g(zyx)=2.006, 1.936 and 1.912. The signal could be observed at temperatures up to 80 K without broadening, indicative of a [2Fe-2S] cluster. Subunit MvhD contains five cysteine residues that are conserved in MvhD homologues of other organisms. Four of these conserved cysteine residues can be assumed to coordinate the [2Fe-2S] cluster that was detected by EPR spectroscopy. The MvhG subunit contains 12 cysteine residues, which are known to ligate three [4Fe-4S] clusters. Data base searches revealed that in some organisms, including the Methanosarcina species and Archaeoglobus fulgidus, a homologue of mvhD is fused to the 3' end of an hdrA homologue, which encodes a subunit of heterodisulfide reductase. These data allow the conclusion that the only function of Mvh is to provide reducing equivalents for heterodisulfide reductase and that the MvhD subunit is an electron transfer protein that forms the contact site to heterodisulfide reductase.
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PMID:Physiological role of the F420-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis. 1285 8

The number of accessible SH-groups was determined in membrane vesicles prepared from Escherichia coli growing in fermentation conditions at slightly alkaline pH on glucose with or without added formate. Addition of ATP or formate to the vesicles caused a approximately 1.4-fold increase in the number of accessible SH-groups. The increase was inhibited by treatment with N-ethylmaleimide or the presence of the F(0)F(1)-ATPase inhibitors N,N(')-dicyclohexylcarbodiimide or sodium azide. The increase in accessible SH-groups was also absent in strains with the ATP synthase operon deleted or with the single F(0) domain cysteine Cysb21 changed to Ala. Using hyc and hyf mutants, it was shown that the increase was also largely dependent on hydrogenase 4 or hydrogenase 3, main components of formate hydrogen lyase, when bacteria were grown in the absence or presence of added formate. These results suggest a relationship between the F(0)F(1)-ATP synthase and hydrogenase 4 or hydrogenase 3 under fermentation conditions.
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PMID:The number of accessible SH-groups in Escherichia coli membrane vesicles is increased by ATP or by formate. 1291

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.
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PMID:Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy. 1457 Apr 80

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions led to the modification of additional His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the activity of the hydrogenase. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.
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PMID:Chemical modification of catalytically essential functional groups of NAD-dependent hydrogenase from Ralstonia eutropha H16. 1460 42


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