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)

Cadmium and lead metals deposited on CdS particles are shown to act as substrates--electron donors for enzymes, hydrogenase from Thiocapsa roseopersicina (HG), NAD-dependent hydrogenase from Alcaligenes eutrophus (NLH), and ferredoxin:NADP oxidoreductase (FNR) from Chlorella in the formation of hydrogen, NADH and NADPH, respectively. Adsorption of the enzyme on the surface of the metallized CdS particle is required for enzymatic oxidation of metal. The maximum rates for the formation of hydrogen and NADH catalyzed by hydrogenase and NAD-dependent hydrogenase with metals as electron donors are comparable with the rates obtained for these enzymes using soluble substrates. Kinetic analysis of the enzymatic oxidation of cadmium metal has revealed that the rate decreases mainly due to the formation of a solid product, which is supposed to be Cd(OH)2. The deceleration of lead oxidation catalyzed by hydrogenase proceeds at the expense of the inhibitory effect of the formed Pb2+. The enzymatic oxidation of electrochemically prepared cadmium metal is also shown. Based on these results, a new mechanism of action of the enzymes involved in anaerobic biocorrosion is proposed. By this mechanism, the enzyme accelerates the process of metal dissolution through a mediatorless catalysis of the reduction of the enzyme substrate.
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PMID:Enzymatic oxidation of cadmium and lead metals photodeposited on cadmium sulfide. 1120 26

The reduction potentials of the metalloproteins pyruvate ferredoxin oxidoreductase (POR), ferredoxin, and hydrogenase isolated from hyperthermophilic Thermococcus celer (Topt = 88 degrees C) were determined as a function of temperature from 10 to 85 degrees C. Square-wave voltammetry experiments were carried out on 15 microL samples directly at an unmodified "edge-polished" pyrolytic graphite electrode using MgCl2 as an electrode promoter. POR exhibited two voltammetric waves with peaks at -280 and -403 mV at room temperature, indicating multiple redox centers, and a single wave at -420 mV at 85 degrees C. These waves displayed different temperature-dependent peak positions and peak heights, indicating that these redox centers have different thermodynamic and kinetic properties. Ferredoxin displayed a single linear temperature-dependent voltammetric wave at -280 mV at room temperature and -327 mV at 85 degrees C. Hydrogenase displayed a single biphasic temperature-dependent voltammetric wave at -197 mV at room temperature and -211 mV at 85 degrees C. Thermodynamic parameters associated with electron transfer, namely standard enthalpies and entropies for the redox centers in the various proteins, are reported.
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PMID:Direct electrochemical characterization of hyperthermophilic Thermococcus celer metalloenzymes involved in hydrogen production from pyruvate. 1131 58

Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolated gamma-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was -356 mV at pH 7.0 and 25 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7. The oxidized, recombinant gamma-subunit was stable at 80 degrees C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively.
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PMID:Heterologous expression and properties of the gamma-subunit of the Fe-only hydrogenase from Thermotoga maritima. 1133 85

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO2. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.
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PMID:Energy yield of respiration on chloroaromatic compounds in Desulfitobacterium dehalogenans. 1152 91

The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H(2):benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H(2)-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H(2)-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO(4) and 0.5 mM NiCl(2). Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.
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PMID:Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha. 1180 65

A new tetraheme cytochrome c3 was isolated from the membranes of Desulfovibrio vulgaris Hildenborough (DvH). This cytochrome has a molecular mass of 13.4 kDa and a pI of 5.5 and contains four heme c groups with apparent reduction potentials of -170 mV, -235 mV, -260 mV and -325 mV at pH 7.6. The complete sequence of the new cytochrome, retrieved from the preliminary data of the DvH genome, shows that this cytochrome is homologous to the "acidic" cytochrome c3 from Desulfovibrio africanus (Da). A model for the structure of the DvH cytochrome was built based on the structure of the Da cytochrome. Both cytochromes share structural features that distinguish them from other cytochrome c3 proteins, such as a solvent-exposed heme 1 surrounded by an acidic surface area, and a heme 4 which lacks most of the surface lysine patch proposed to be the site of hydrogenase interaction in other cytochrome c3 proteins. Furthermore, in contrast to previously discovered cytochrome c3 proteins, the genes coding for these two cytochromes are adjacent to genes coding for two membrane-associated FeS proteins, which indicates that they may be part of membrane-bound oxidoreductase complexes. Altogether these observations suggest that the DvH and Da cytochromes are a new type of cytochrome c3 proteins (Type II: TpII-c3) with different redox partners and physiological function than the other cytochrome c3 proteins (Type I: TpI-c3). The DvH TpII-c3 is reduced at considerable rates by the two membrane-bound [NiFe] and [NiFeSe] hydrogenases, but catalytic amounts of TpI-c3 increase these rates two- and fourfold, respectively. With the periplasmic [Fe] hydrogenase TpII-c3 is reduced much slower than TpI-c3, and no catalytic effect of TpI-c3 is observed.
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PMID:A membrane-bound cytochrome c3: a type II cytochrome c3 from Desulfovibrio vulgaris Hildenborough. 1194 78

Development of resistance against metronidazole and mechanisms responsible for this process were studied in a sexually transmitted pathogen of humans, Trichomonas vaginalis. Monitoring of changes in metabolism and protein expression that accompanied increasing resistance of strains derived from a common drug-susceptible parent (TV 10-02) showed the multistep character of the process. The aerobic type of resistance known to occur in isolates from patients non-responsive to treatment appeared at the earliest stage, followed by development of the anaerobic type of resistance which was accompanied by gradual loss of hydrogenosomal proteins associated with drug-activating pathways [pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin]. Unexpectedly, the loss of PFOR did not result in acquisition of full anaerobic resistance, thus indicating an alternative source of electrons required for the drug activation. These data suggest involvement of the oxidative decarboxylation of malate in hydrogenosomes, catalysed by NAD(+)-dependent malic enzyme and subsequent transfer of reduced equivalents to the drug via NADH:ferredoxin oxidoreductase and ferredoxin. Accordingly, all components of this pathway were eliminated before the resistance was fully developed. Resistant Trichomonas vaginalis compensated the impaired function of hydrogenosomes by enhanced conversion of pyruvate to lactate in the cytosol. Further analysis of the two key enzymes involved in metronidazole activation by Northern blotting and assay for nascent mRNA showed that the insufficient expression of the PFOR protein results from decreased gene transcription, while down-regulation of malic enzyme is controlled at the mRNA level.
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PMID:Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalis. 1217 40

Nicotinamide adenine dinucleotide-reduced form (NADH):quinone oxidoreductase (respiratory Complex I), F420H2 oxidoreductase and complex, membrane-bound NiFe-hydrogenase contain protein subunits homologous to a certain type of bona fide antiporters. In Complex I, these polypeptides (NuoL/ND5, NuoM/ND4, NuoN/ND2) are most likely core components of the proton pumping mechanism, and it is thus important to learn more about their structure and function. In this work, we have determined the transmembrane topology of one such polypeptide, and built a 2D structural model of the protein valid for all the homologous polypeptides. The experimentally determined transmembrane topology was different from that predicted by majority vote hydrophobicity analyses of members of the superfamily. A detailed phylogenetic analysis of a large set of primary sequences shed light on the functional relatedness of these polypeptides.
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PMID:Transmembrane topology of the NuoL, M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters. 1246 Jun 69

Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.
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PMID:Mitochondria and hydrogenosomes are two forms of the same fundamental organelle. 1259 27

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


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