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)

We have investigated the action of sodium nitrite and other nitrosyl complexes, such as sodium nitroprusside and Roussin's black salt, on the growth of metronidazole-sensitive and resistant strains of Trichomonas vaginalis and their hydrogenosomal enzymes. All three chemicals inhibited the growth of T. vaginalis: sodium nitrite at 8 mM, sodium nitroprusside at 1.2 mM and Roussin's black salt at 0.2 mM. Metronidazole-sensitive (KT9) and resistant (CDC85) isolates showed similar cytotoxicity against these molecules. Specific activities of pyruvate:ferredoxin oxidoreductase and hydrogenase and oxygen uptake rates were decreased in the T. vaginalis isolate treated with sodium nitrite and sodium nitroprusside. However, Roussin's black salt increased the specific activity of pyruvate:ferredoxin oxidoreductase or hydrogenase in CDC85 or KT9 cells and increased the oxygen uptake rate in the KT9 isolate.
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PMID:Cell cytotoxicity of sodium nitrite, sodium nitroprusside and Roussin's black salt against Trichomonas vaginalis. 764 39

Purified soluble hydrogenase (H2: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. H2-dependent NAD+ reduction activities > or = 110 mumol NADH formed/min/mg protein at pH 8.0 and 30 degrees C were obtained which were stable for several hours at 4 degrees C. Kinetic studies were conducted anaerobically using activated enzyme for the purpose of evaluating the potential of using hydrogenase to enhance decompression of mammals breathing H2/O2 mixtures under hyperbaric conditions (i.e., at ambient pressures greater than 1 atm). Using nonlinear curve fitting of the kinetic data, it was found that H2 and NAD+ bind hydrogenase via a ping pong bi bi mechanism with Km values (+/- SE) of 11 +/- 0.9 and 138 +/- 11 microM, respectively, at 30 degrees C and pH 8.0. Sodium ions were found to reversibly inhibit hydrogenase via a dead-end type of inhibition in which two catalytic forms of the enzyme bind Na+ with dissociation constants calculated to be 8.3 +/- 1.2 and 49.8 +/- 11.5 mM. In the absence of NaCl, maximum NAD+ reduction activity was measured at pH 8.3 at 30 and 37 degrees C. In the presence of 50 mM NaCl, inhibition was observed primarily at alkaline pH, and at assay pH values < or = 7.0, little or no difference was observed in activity in the presence or absence of 50 mM NaCl at a given temperature. Least squares analyses of the kinetic data indicated that substrate inhibition by H2 occurs at high substrate concentrations (Ki = 1.46 +/- 0.64 mM), which would become a significant influence on enzyme catalytic activity at hyperbaric levels of H2.
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PMID:Kinetic mechanism studies of the soluble hydrogenase from Alcaligenes eutrophus H16. 789 62

A novel iron-containing blue protein, named neelaredoxin, was isolated from the sulfate-reducing bacterium Desulfovibrio gigas. It is a monomeric protein with a molecular mass of 15 kDa containing two iron atoms/molecule. The N-terminal sequence of neelaredoxin has similarity to the second domain of desulfoferrodoxin, a protein purified from Desulfovibrio vulgaris Hildenborough. This finding supports the hypothesis that the gene coding for desulfoferrodoxin (rbo) might have arisen from a gene fusion [Brumlik, M. J., Leroy, G., Bruschi, M. & Voordouw, G. (1990) J. Bacteriol. 172, 7289-7292]. The visible spectrum exhibits a single band at 666 nm, responsible for the blue color of the protein, which is completely bleached upon reduction with sodium ascorbate. In the oxidized state the EPR spectrum is complex, exhibiting well-resolved features at g = 7.6, 7.0, 5.9, and 5.8 which are assigned to two high-spin (S = 5/2) mononuclear-iron (III) centers with different rhombic distortions (E/D approximately 0.05 and approximately 0.08). The two iron atoms contribute identically to the visible spectrum as judged from visible redox titrations, from which a reduction potential of +190 mV was determined for both iron sites at pH 7.5. At high pH the visible and the EPR spectra become pH-dependent with a pKa above 9: the 666-nm band shifts to 590 nm and the EPR signals are converted into a signal with gmax approximately 4.7. Neelaredoxin is readily reduced both by H2/hydrogenase/cytochrome c3 and by NADH/NADH-rubredoxin oxidoreductase.
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PMID:A blue non-heme iron protein from Desulfovibrio gigas. 800 76

Thermotoga maritima is a H2-producing, fermentative anaerobe and is one of the most thermophilic bacteria known. Its iron-only (Fe-)hydrogenase was previously shown to be a homotetramer and to contain two [4Fe-4S] and two [2Fe-2S] clusters per monomer, but the enzyme lacked the characteristic EPR signal of the oxidized H cluster, the proposed site of H2 catalysis in mesophilic Fe-hydrogenases (Juszczak, A., Aono, S. and Adams, M.W.W. (1991) J. Biol. Chem. 266, 13834-13841). The two types of cluster were shown by spectroelectrochemistry to have reduction potentials (Em) of -390 and -440 mV, respectively. We have now identified two additional redox centers in the enzyme, a [2Fe-2S] center with a higher reduction potential (Em = -365 mV) and an unusual paramagnetic species (Em > -200 mV). The higher potential [2Fe-2S] center can be reduced by sodium dithionite at pH 6.0 and exhibits an axial-type EPR signal with gz = 2.026 and gy = gx = 1.940. The two lower potential [2Fe-2S] centers are fully reduced by sodium dithionite only at pH 10.0. Both of these clusters in their reduced states exhibit rhombic-type EPR signals with gz = 2.005, gy = 1.955, and gx = 1.921. This hydrogenase is therefore thought to contain three [2Fe-2S] clusters, as well as two [4Fe-4S] clusters. In addition, a nearly isotropic EPR signal (g = 2.01) was observed when the enzyme was anaerobically oxidized by organic dyes such as thionine (E alpha = 64 mV) or 2,6-dichlorophenolindophenol (E alpha = 217 mV). This resonance was not observed at 20 K due to relaxation broadening and therefore did not arise from a conventional organic radical. The oxidized enzyme was fully active in an H2 production assay, and also reacted directly with H2. In contrast, the air-oxidized enzyme was inactive and did not exhibit the g = 2.01 EPR signal. This resonance was assigned to a novel paramagnetic species with an approximate Em value of -70 mV. It is thought to be associated with the H2 activating site of this atypical Fe-hydrogenase.
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PMID:Identification of an unusual paramagnetic species and of three [2Fe-2S] clusters in the iron-only hydrogenase from the hyperthermophilic bacterium Thermotoga maritima. 818 40

H2 oxidation in Azotobacter vinelandii is catalyzed by a membrane-bound, alpha beta dimeric [NiFe] hydrogenase. Maturation of the enzyme involves cleavage of a putative N-terminal signal sequence in the beta subunit and removal of 15 amino acids from the C terminus of the alpha subunit. Cells limited for nickel exhibited low hydrogenase activities and contained an apparently large form of the alpha subunit. Addition of nickel to such cells increased hydrogenase activities fivefold over 2 h. The increase in the first hour did not require transcription and translation and correlated with processing of the large form of the alpha subunit (pre-alpha) to the small form (alpha) resembling the alpha subunit from the purified enzyme. In vivo, pre-alpha appeared soluble whereas the majority of alpha was membrane bound. Processing of pre-alpha to alpha was reproduced in vitro in membrane-depleted extracts of nickel-limited cells. Processing specifically required the addition of Ni2+, whereas Co2+, Cu2+, Ca2+, Fe2+, Mn2+, and Zn2+ were ineffective. However, Zn2+, Co2+, and Cu2+ inhibited nickel-dependent processing. Mg-ATP and Mg-GTP stimulated processing, whereas anaerobic conditions and/or the addition of dithiothreitol and sodium dithionite was unnecessary. Processing was not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, E64, and pepstatin.
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PMID:In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii. 828 21

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.
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PMID:Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri. 832 4

Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1. EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals. At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared. The detection of a 4Fe-4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear. The 4Fe-4S complex intensity was slightly increased. The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.
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PMID:Electron paramagnetic resonance (EPR) studies on hydrogenase-1 (HYD1) purified from a mutant strain (AP6) of Escherichia coli enhanced in HYD1. 885 27

Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO2, acetate, butyrate, and H2. The molecular hydrogen (approximately 10 kPa; E' = -385 mV) stems from NADH generated in the 3-hydroxybutyryl-CoA dehydrogenase reaction (E degrees ' = -240 mV) of the hydroxyglutarate pathway. In contrast to growing cells, which require at least 5 mM Na+, a Na+-dependence of the H2-formation was observed with washed cells. Whereas the optimal glutamate fermentation rate was achieved already at 1 mM Na+, H2 formation commenced only at > 10 mM Na+ and reached maximum rates at 100 mM Na+. The acetate/butyrate ratio thereby increased from 2.0 at 1 mM Na+ to 3.0 at 100 mM Na+. A hydrogenase and an NADH dehydrogenase, both of which were detected in membrane fractions, are components of a model in which electrons, generated by NADH oxidation inside of the cytoplasmic membrane, reduce protons outside of the cytoplasmic membrane. The entire process can be driven by decarboxylation of glutaconyl-CoA, which consumes the protons released by NADH oxidation inside the cell. Hydrogen production commences exactly at those Na+ concentrations at which the electrogenic H+/Na+-antiporter glutaconyl-CoA decarboxylase is converted into a Na+/Na+ exchanger.
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PMID:Sodium ion-dependent hydrogen production in Acidaminococcus fermentans. 892 82

An indium tin oxide (ITO) electrode was chemically modified by one layer of viologen (VIO) derivative, which possessed a persistent and reproducible electrochemical response. A monolayer of a thermal stable hydrogenase from Thiocapsa roseopersicina was stabilized on a synthesized poly-L-lysine subphase surface and transferred onto the electrode for fabrication of an ITO-VIO-hydrogenase heterogeneous system. Electrochemical properties of both the ITO-VIO monolayer and the heterogeneous ITO-VIO-hydrogenase system have been investigated. Hydrogen evolution could be measured by potentiostating the VIO-hydrogenase-covered ITO electrode to "electroplate" [(VIO+)n]surf, and a large increase in hydrogen evolution was observed when using an electrolyte solution containing sodium dithionite. We discuss the possible electron transfer process.
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PMID:Fabrication of an electrode-viologen-hydrogenase heterogeneous system and the electrochemical hydrogen evolution. 1084 7

Carbon monoxide binding and inhibition have been investigated by electron paramagnetic resonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. Simulation of the EPR spectrum of the as-isolated state indicates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clusters. Addition of carbon monoxide to CpI in the presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristic axial EPR signal [g(eff(1)), g(eff(2)), and g(eff(3)) = 2.0725, 2.0061, and 2.0061, respectively] was observed. Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resulted in samples that exhibited the native oxidized EPR signature that could be converted to the reduced form upon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopically defined states of CpI and those observed structurally by X-ray crystallography, we have examined the CpI crystals using EPR spectroscopy. EPR spectra of the crystals in the CO-bound state exhibit the previously described axial signal associated with CO binding. The results indicate that the addition of carbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized by loss of enzyme activity and the distinctive axial EPR signal.
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PMID:Reversible carbon monoxide binding and inhibition at the active site of the Fe-only hydrogenase. 1085 94


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