Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

The reduction of CoM-S-S-HTP, the heterodisulfide of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP), with H2 is an energy-conserving step in methanogenic archaea. We report here that in Methanosarcina barkeri this reaction is catalyzed by a membrane-bound multienzyme complex, designated H2:heterodisulfide oxidoreductase complex, which was purified to apparent homogeneity. The preparation was found to be composed of nine polypeptides of apparent molecular masses 46 kDa, 39 kDa, 28 kDa, 25 kDa, 23 kDa, 21 kDa, 20 kDa, 16 kDa, and 15 kDa and to contain 3.2 nmol cytochrome b, 70 to 80 nmol non-heme iron and acid-labile sulfur, 5 nmol Ni, and 0.6 nmol FAD per mg protein. The 23 kDa polypeptide possessed heme-derived peroxidase activity indicating that this polypeptide is the cytochrome b. The purified H2:heterodisulfide oxidoreductase complex catalyzed the reduction of CoM-S-S-HTP with H2 at a specific activity of 6 U/mg protein (1 U = 1 mumol.min-1), the reduction of benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP HTP with reduced benzylviologen at a specific activity of 24 U/mg protein. The complex did not mediate the reduction of coenzyme F420 with H2 nor the oxidation of reduced coenzyme F420 with CoM-S-S-HTP. The reduced cytochrome b in the enzyme complex could be oxidized by CoM-S-S-HTP and re-reduced by H2. The specific rates of cytochrome oxidation and reduction were too high to be resolved under our experimental conditions. The findings suggest that the H2:heterodisulfide oxidoreductase complex is composed of a F420-non-reducing hydrogenase, a cytochrome b and heterodisulfide reductase and that cytochrome b is a redox carrier in the electron transport chain involved in CoM-S-S-HTP reduction with H2.
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PMID:Purification of a cytochrome b containing H2:heterodisulfide oxidoreductase complex from membranes of Methanosarcina barkeri. 847 25

Two genes, hynA and hynB, encode the two subunits of the periplasmic [NiFe] hydrogenase in Desulfovibrio fructosovorans. Sequencing downstream from hynB revealed a third open reading frame (hynC) that has the potential for encoding a polypeptide showing 21% identity with the HyaD, HoxM, and HupD proteins, belonging to putative operons encoding Escherichia coli hydrogenase 1, Alcaligenes eutrophus H16 membrane-bound hydrogenase, and Rhizobium leguminosarum uptake hydrogenase, respectively. Northern (RNA) blotting with a structural gene probe revealed the existence of a major transcript of 2.9 kb, which is the appropriate length to contain the two hydrogenase subunits only. In addition, two minor 4.4- and 5.8-kb transcripts that could contain hynABC and additional genes were found. The 5' end of the most abundant [NiFe] hydrogenase mRNA was found 170 bp upstream from the translational start site of hynA. The sequences at -10 and -35 relative to the transcriptional starting site showed 55% homology with the consensus sequences of the Escherichia coli sigma 70-type promoter. The cloning of that particular region as a promoter to control transcription of the lacZ gene in E. coli DH5 alpha or the hynA, hynB, and hynC genes in D. fructosovorans MR400 led to strong expression in both systems.
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PMID:Analysis of the periplasmic [NiFe] hydrogenase transcription unit from Desulfovibrio fructosovorans. 850 Oct 43

A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3'-terminal part encoding the C-terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase. The truncated gene was transferred to the in situ position on the chromosome. Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity. The activity was not restored by high nickel concentrations in the medium. The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of 63Ni2+. These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal.
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PMID:Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit. 863 25

The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism.
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PMID:An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure. 908 73

Formation of enzymatically active [NiFe] hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C-terminal peptide from the precursor, and oligomerisation of the subunits. Two amino acid replacements were introduced by site-directed mutagenesis at the C-terminal proteolytic cleavage site of HoxH, the Ni-containing subunit of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus H16. Replacement of Ala465, the first residue of the 24-amino-acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C-terminal proteolysis. This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme. In the second mutant, the C-terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon. Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni. Both mutant proteins contained only traces of H2-activating functions. H2-dependent reduction of NAD and benzylviologen, and D2/H+-exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained. These results allow the following conclusions: the C-terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C-terminal peptide is not competent for assembly.
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PMID:C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydrogenase in Alcaligenes eutrophus. 915 77

The genes encoding the basic and acidic tetraheme cytochromes c3 from Desulfovibrio africanus have been sequenced. The corresponding amino acid sequences of the basic and acidic cytochromes c3 indicate that the mature proteins consist of a single polypeptide chain of 117 and 103 residues, respectively. Their molecular masses, 15102 and 13742 Da, respectively, determined by mass spectrometry, are in perfect agreement with those calculated from their amino acid sequences. Both D. africanus cytochromes c3 are synthesized as precursor proteins with signal peptides of 23 and 24 residues for the basic and acidic cytochromes, respectively. These cytochromes c3 exhibit the main structural features of the cytochrome c3 family and contain the 16 strictly conserved cysteine + histidine residues directly involved in the heme binding sites. The D. africanus acidic cytochrome c3 differs from all the other homologous cytochromes by its low content of basic residues and its distribution of charged residues in the amino acid sequence. The presence of four hemes per molecule was confirmed by EPR spectroscopy in both cytochromes c3. The g-value analysis suggests that in both cytochromes, the angle between imidazole planes of the axial histidine ligands is close to 90 degrees for one heme and much lower for the three others. Moreover, an unusually high exchange interaction (approximately 10[-2] cm[-1]) was evidenced between the highest potential heme (-90 mV) and one of the low potential hemes in the basic cytochrome c3. The reactivity of D. africanus cytochromes c3 with heterologous [NiFe] and [Fe] hydrogenases was investigated. Only the basic one interacts with the two types of hydrogenase to achieve efficient electron transfer, whereas the acidic cytochrome c3 exchanges electrons specifically with the basic cytochrome c3. The difference in the specificity of the two D. africanus cytochromes c3 has been correlated with their highly different content of basic and acidic residues.
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PMID:Further characterization of the two tetraheme cytochromes c3 from Desulfovibiro africanus: nucleotide sequences, EPR spectroscopy and biological activity. 939 24

The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most of hoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of hoxF and hoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH. Deletion of hoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.
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PMID:Subforms and in vitro reconstitution of the NAD-reducing hydrogenase of Alcaligenes eutrophus. 949 38

H2 evolution by direct electron transfer from the dithionite-reduced photosystem I (PSI) complex to both hydrogenase I and hydrogenase II from Clostridium pasteurianum was observed. Evidence indicates that the electron carriers on PSI that transfer electrons to hydrogenase in this system are the FA/FB iron-sulfur clusters on the PsaC polypeptide, the terminal bound electron acceptors in PSI. Light-dependent H2 evolution was also observed, using high potential electron donors to PSI, from a combination of hydrogenase I and either solubilized purified PSI or thylakoids. Mediators capable of transferring electrons from the PSI complex to hydrogenase were not necessary for H2 evolution, indicating again that the mechanism of H2 evolution is direct electron transfer from PSI to hydrogenase, and that this can occur with light-reduced as well as chemically reduced PSI, and with PSI in thylakoids as well as the solubilized complex. Light-dependent H2 evolution was also observed from a mixture of thylakoids and the oxygen-resistant hydrogenase of Rhodococcus sp. MR11. These results suggest that direct electron transfer from PSI to hydrogenase could be engineered to occur in vivo in a photosynthetic organism to create an organism that would efficiently produce H2 from H2O.
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PMID:Hydrogen evolution by direct electron transfer from photosystem I to hydrogenases. 953 56

[NiFe]-hydrogenases generally carry the bimetallic Ni-Fe reaction center on their largest subunit. The [NiFeSe]-hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu-hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild-type enzyme. M. voltae carries the genetic information for four different [NiFe]-hydrogenases. In addition to the Vhu-hydrogenase, a second selenium-containing enzyme, Fru, has been purified. Two selenium-free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium-containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc andfrc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.
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PMID:Fusion of two subunits does not impair the function of a [NiFeSe]-hydrogenase in the archaeon Methanococcus voltae. 976 Jan 86

The primary structure of Clostridium pasteurianum hydrogenase I appears to be composed of modules suggesting that the various iron-sulfur clusters present in this enzyme might be segregated in structurally distinct domains. On the basis of this observation, a gene fragment encoding the 76 N-terminal residues of this enzyme has been expressed in Escherichia coli. The polypeptide thus produced contains a [2Fe-2S]n+ cluster of which the oxidized level (n = 2) has been monitored by UV-visible absorption, circular dichroism, and resonance Raman spectroscopy. This cluster can be reduced by dithionite or electrochemically to the n = 1 level which has been investigated by EPR and by low-temperature magnetic circular dichroism. The redox potential of the +2 to +1 transition is -400 mV (vs the normal hydrogen electrode). The spectroscopic and redox results indicate a [2Fe-2S]2+/+ chromophore coordinated by four cysteine ligands in a protein fold similar to that found in plant- and mammalian-type ferredoxins. Among the five cysteines present in the N-terminal hydrogenase fragment, four (in positions 34, 46, 49, and 62) are conserved in other sequences and are therefore the most likely ligands of the [2Fe-2S] site. The fifth cysteine, in position 39, can be dismissed on the grounds that the Cys39Ala mutation does not alter any of the properties of the iron-sulfur cluster. The spectroscopic signatures of this chromophore are practically identical with some of those reported for full-size hydrogenase. This confirms that C. pasteurianum hydrogenase I contains a [2Fe-2S] cluster and indicates that the polypeptide fold around the metal site of the N-terminal fragment is very similar, if not identical, to that occurring in the full-size protein. The N-terminal sequence of this hydrogenase is homologous to sequences of a number of proteins or protein domains, including a subunit of NADH-ubiquinone oxidoreductase of respiratory chains. From that, it can be anticipated that the structural domain isolated and described here is a building block of electron transfer complexes involved in various bioenergetic processes.
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PMID:Heterologous biosynthesis and characterization of the [2Fe-2S]-containing N-terminal domain of Clostridium pasteurianum hydrogenase. 984 4


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