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)

A new FMN-containing flavoprotein isolated from Desulfovibrio gigas provided maximum coupling efficiency for the reduction of bisulfite from molecular H2. This protein, which is distinct from flavodoxin and for which the name flavoredoxin is proposed, is required for reconstitution of an electron transfer chain between hydrogenase and bisulfite reductase. A Ca(2+)-binding protein functions as a modulator in the presence of Ca2+ in the process. The finding of a membrane-bound cytochrome c with a molecular weight of 104,000 Da that is also active in this electron transfer chain provides an explanation for the energetic linkage between periplasmic and cytoplasmic proteins in this sulfate-reducing bacterium.
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PMID:Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas. 838 52

Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H2 oxidized min-1 (mg protein)-1 with benzyl viologen as electron acceptor and an apparent Km value for H2 of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 +/- 1.7 mol Fe, 11.4 +/- 1.4 mol S2-, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)+. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H2-dependent cytochrome b reduction at a rate of 0.18 nmol min-1 (mg protein)-1.
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PMID:Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport. 859 1

The catalytic and spectroscopic properties of the reversible hydrogenase from the cyanobacterium Anabaena variabilis have been examined. The hydrogenase required reductive activation in order to elicit hydrogen-oxidation activity. Carbon monoxide was a weak (Ki=35 microM), reversible and competitive inhibitor. A flavin with the chromatographic properties of FMN, and nickel were detected in the purified enzyme. A. variabilis hydrogenase exhibited electron paramagnetic resonance (EPR) spectra in its hydrogen-reduced state, indicative of [2Fe-2S] and [4Fe-4S] clusters. Although no EPR signals due to nickel were detected, the results are consistent with the enzyme being a flavin-containing hydrogenase of the nickel-iron type.
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PMID:Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres. 861 97

The specific activity of purified soluble hydrogenase of Alcaligenes eutrophus H16 was found to vary with enzyme concentration. Specific activity as a function of concentration of purified enzyme could be fit to an equation describing the dissociation of a compound into two components. An association constant, kappa(a), was determined in this way to be 39.4 +/- 8.7 micrograms protein/ml. The concentration of the enzyme affected its kinetic parameters: a tenfold decrease in enzyme concentration caused by a reduction of the V(max) and Kappa(m) (NAD) values to 45% and 58%, respectively, of the values for undiluted (0.64 mg/ml) enzyme. Diaphorase (NAD-dependent reduction of benzyl viologen) specific activity of the hydrogenase was unaffected by dilution. The extent of dilution-induced activity loss was dependent on pH, with greater activity loss observed at higher pH values. The substrate NAD prevented loss of specific activity due to dilution, while the product NADH did not. Specific activity loss due to dilution as reversed with the addition of the cofactor FMN. Dilution of the hydrogenase caused an increase in the enzyme's specific flavin fluorescence. These results suggest that dilution of the soluble hydrogenase of Alcaligenes eutrophus causes dissociation of the cofactor FMN, and this activity loss should be taken into account as an important factor governing hydrogenase activity and kinetic properties.
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PMID:FMN cofactor dissociation from the soluble hydrogenase of Alcaligenes eutrophus H16. 872 22

A monomeric flavoprotein (18.8 kDa) was isolated from the soluble cell fraction of Wolinella succinogenes and was identified as a flavodoxin based on its N-terminal sequence, FMN content, and redox properties. The midpoint potentials of the flavodoxin (Fld) at pH 7. 5 were measured as -95 mV (Fldox/Flds) and -450 mV (Flds/Fldred) relative to the standard hydrogen electrode. The cellular flavodoxin content [0.3 micromol (g protein)-1] was the same in bacteria grown with fumarate or with polysulfide as the terminal acceptor of electron transport. The flavodoxin did not accept electrons from hydrogenase or formate dehydrogenase, the donor enzymes of electron transport to fumarate or polysulfide. Pyruvate:flavodoxin oxidoreductase activity [180 U (g cellular protein)-1] was detected in the soluble cell fraction of W. succinogenes grown with fumarate or polysulfide. The enzyme was equally active with Fldox or Flds at high concentrations. The Km for Flds (80 microM) was larger than that for Fldox and for the ferredoxin isolated from W. succinogenes (15 microM). We conclude that flavodoxin serves anabolic rather than catabolic functions in W. succinogenes.
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PMID:Flavodoxin from Wolinella succinogenes. 877 74

Biochemical decompression has been proposed as a method for reducing the amount of time required for deep-sea divers to return to the surface. Divers breathing H2/O2 mixtures would be presented with hydrogenase enzyme, and decompression would be accelerated by means of the enzymic removal of excess H2 from the tissues. We have studied FAD as a hydrogenase electron acceptor that is capable of transferring electrons derived from H2 oxidation directly to O2. Kinetic activity constants for the soluble hydrogenase from the bacterium Alcaligenes eutrophus H16 were determined with FAD, FMN and riboflavin as electron acceptors, and these values were compared with those obtained with the physiological electron acceptor NAD+. The Michaelis constants (K(m)) were similar for FAD, FMN and NAD. However, the maximal catalytic-centre activity (Kcat) was much lower for the flavins, and the catalytic efficiency (Kcat/K(m)) with FAD was 1/20th the value for NAD+. After enzyme-catalysed FAD reduction to FADH2, the FAD could be regenerated by addition of O2 and reduced again by the enzyme in the presence of H2. Thus FAD served as a regenerable electron shuttle between H2 and O2. H2O2, a by-product of FADH2 oxidation by O2, inhibited the enzyme. Much greater inhibition was observed with the reduced form of the enzyme. Active hydrogenase was efficiently encapsulated into human and pig red blood cells. Hydrogen consumption was seen with lysed carrier cells, but was demonstrated with unlysed carrier cells only when FAD was co-encapsulated along with enzyme. These results demonstrate that red blood cells encapsulating hydrogenase and FAD act as a system for continuous H2 consumption in a mammalian tissue without addition of exogenous factors, and such cells may provide a biotherapeutic method for reducing the risk and treatment of decompression sickness.
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PMID:Hydrogenase encapsulation into red blood cells and regeneration of electron acceptor. 886 3

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 microM WO42-. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (Km < 20 microM) and aromatic aldehydes (Km < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD+ or NADP+. 185WO42- was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (>/= 98%). Anionic-exchange chromatography demonstrated coelution of 185W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity.
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PMID:Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes. 938 39

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.
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PMID:Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I). 1100 44

Hydrogenases have clear evolutionary links to the much more complex NADH-ubiquinone oxidoreductases (Complex I). Certain membrane-bound [NiFe]-hydrogenases presumably pump protons. From a detailed comparison of hydrogenases and Complex I, it is concluded here that the TYKY subunit in these enzymes is a special 2[4Fe-4S] ferredoxin, which functions as the electrical driving unit for a proton pump. The comparison further revealed that the flavodoxin fold from [NiFe]-hydrogenases is presumably conserved in the PSST subunit of Complex I. It is proposed that bovine Complex I and the soluble NAD(+)-reducing hydrogenase from Ralstonia eutropha each contain a second FMN group.
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PMID:Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH--ubiquinone oxidoreductase (Complex I). 1108 55

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.
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PMID:Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae. 1110 87


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