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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 genomic DNA fragment from Desulfovibrio fructosovorans, which strongly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenborough, was cloned and sequenced. This fragment was found to contain four genes, named hndA, hndB, hndC, and hndD. Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taurus complex I, the 25-kDa subunit from Paracoccus denitrificans NADH dehydrogenase type I, and the N-terminal domain of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively. HndB does not show any significant homology with any known protein. HndC shows 37 and 33% identity with the C-terminal domain of HoxF and the 51-kDa subunit from B. taurus complex I, respectively, and has the requisite structural features to be able to bind one flavin mononucleotide, one NAD, and three [4Fe-4S] clusters. HndD has 40, 42, and 48% identity with hydrogenase I from Clostridium pasteurianum and HydC and HydA from D. vulgaris Hildenborough, respectively. The 4.5-kb length of the transcripts expressed in D. fructosovorans and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit. The sizes of the four polypeptides were measured by performing heterologous expression of hndABCD in E. coli, using the T7 promoter/polymerase system. The products of hndA, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectively. One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis. Immunoblotting studies carried out on cell extracts from D. fructosovorans wild-type and SM3 strains, using antibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only. In soluble extracts from D. fructosovorans wild type, a 10-fold induction of NADP reduction was observed when H(2) was present, but no H(2)-dependent NAD reduction ever occurred. This H(2)-dependent NADP reductase activity disappeared completely in extracts from SM3. These results indicate that the hnd operon actually encodes an NAdP-reducing hydrogenase in D. fructosovorans.
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PMID:Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans. 775 Dec 70

Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 100 degrees C by the fermentation of carbohydrates yielding acetate, CO2, and H2 as the primary products. If elemental sulfur (S0) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). From the cytoplasm of this organism, we have now purified an enzyme, sulfide dehydrogenase (SuDH), which catalyzes the reduction of polysulfide to H2S with NADPH as the electron donor. SuDH is a heterodimer with subunits of 52,000 and 29,000 Da. SuDH contains flavin and approximately 11 iron and 6 acid-labile sulfide atoms per mol, but no other metals were detected. Analysis of the enzyme by electron paramagnetic resonance spectroscopy indicated the presence of four iron-sulfur centers, one of which was specifically reduced by NADPH. SuDH has a half-life at 95 degrees C of about 12 h and shows a 50% increase in activity after 12 h at 82 degrees C. The pure enzyme has a specific activity of 7 mumol of H2S produced.min-1.mg of protein-1 at 80 degrees C with polysulfide (1.2 mM) and NADPH (0.4 mM) as substrates. The apparent Km values were 1.25 mM and 11 microM, respectively. NADH was not utilized as an electron donor for polysulfide reduction. P. furiosus rubredoxin (K(m) = 1.6 microM) also functioned as an electron acceptor for SuDH, and SuDH catalyzed the reduction of NADP with reduced P. furiosus ferredoxin (K(m) = 0.7 microM) as an electron donor. The multiple activities of SuDH and its proposed role in the metabolism of S(o) and polysulfide are discussed.
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PMID:Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur. 796 1

A flavodoxin was purified to homogeneity from the nitrogen-fixing heterocystous cyanobacterium Anabaena sphaerica grown under iron-limited conditions. The protein has a molecular mass of 21 kDa, and its spectral properties and amino-acid composition are very close to that of flavodoxins from other cyanobacteria. A. sphaerica flavodoxin supported the activities of A. sphaerica NADP reductase and Clostridium butyricum hydrogenase in reconstituted systems with illuminated plant chloroplasts as reductant. With the use of polyclonal anti-flavodoxin antiserum it was found that nitrogen-fixing cultures of A. sphaerica grown under iron-sufficient conditions contain low but significant amounts of flavodoxin (0.2-0.6 micrograms/mg crude extract protein) which increased dramatically (to 8-15 micrograms/mg crude extract protein) after the iron concentration in the medium was decreased to below 1 microM Fe. The flavodoxin content of both iron-limited and iron-sufficient. A. sphaerica was also shown to depend upon the growth phase of the (batch) cultures with a maximum at early exponential phase, coinciding with maximal in-vivo nitrogenase activity. These results suggest that A. sphaerica flavodoxin not only substitutes for ferredoxin under iron-limiting conditions, but also fulfills some specific role under iron-sufficient conditions.
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PMID:Purification and properties of a flavodoxin from the heterocystous cyanobacterium Anabaena sphaerica. 834 30

Three different types of tungsten-containing enzyme have been previously purified from Pyrococcus furiosus (optimum growth temperature, 100 degrees C): aldehyde ferredoxin oxidoreductase (AOR), formaldehyde ferredoxin oxidoreductase (FOR), and glyceraldehyde-3-phosphate oxidoreductase (GAPOR). In this study, the organism was grown in media containing added molybdenum (but not tungsten or vanadium) or added vanadium (but not molybdenum or tungsten). In both cell types, there were no dramatic changes compared with cells grown with tungsten, in the specific activities of hydrogenase, ferredoxin:NADP oxidoreductase, or the 2-keto acid ferredoxin oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Compared with tungsten-grown cells, the specific activities of AOR, FOR, and GAPOR were 40, 74, and 1%, respectively, in molybdenum-grown cells, and 7, 0, and 0%, respectively, in vanadium-grown cells. AOR purified from vanadium-grown cells lacked detectable vanadium, and its tungsten content and specific activity were both ca. 10% of the values for AOR purified from tungsten-grown cells. AOR and FOR purified from molybdenum-grown cells contained no detectable molybdenum, and their tungsten contents and specific activities were > 70% of the values for the enzymes purified from tungsten-grown cells. These results indicate that P. furiosus uses exclusively tungsten to synthesize the catalytically active forms of AOR, FOR, and GAPOR, and active molybdenum- or vanadium-containing isoenzymes are not expressed when the cells are grown in the presence of these other metals.
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PMID:Molybdenum and vanadium do not replace tungsten in the catalytically active forms of the three tungstoenzymes in the hyperthermophilic archaeon Pyrococcus furiosus. 855 Apr 11

The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.
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PMID:Oxidative modification of nicotinamide nucleotide transhydrogenase in submitochondrial particles: effect of endogenous ubiquinol. 895 Oct 41

The sequence of a NAD(P)-reducing hydrogenase operon of Synechocystis sp. PCC 6803 containing genes for a small and a large hydrogenase subunit and six additional ORFs was determined. Until now only 11 of the 14 polypeptides of the NADH-dehydrogenase of E. coli were found in Synechocystis. By sequence homologies we suggest that the missing subunits of the peripheral part of the dehydrogenase, containing most of the FeS-clusters, are encoded by three ORFs of this operon. This hypothesis is discussed in relation to the NAD(P)-reducing hydrogenase of Synechocystis.
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PMID:Sequence analysis of an operon of a NAD(P)-reducing nickel hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803 gives additional evidence for direct coupling of the enzyme to NAD(P)H-dehydrogenase (complex I). 898 Jun 40

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H2-dependent NADP reduction was completely abolished. The double-mutated DM4 (DeltahynABC, DeltahndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25-70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate.
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PMID:Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans. 900 Mar 40

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

Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.
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PMID:Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli. 948 16

The NADP-reducing hydrogenase of Desulfovibrio fructosovorans represents a novel class of [Fe] hydrogenases which is encoded by the well-characterized hndABCD operon containing the genes hndA, hndB, hndC, and hndD. Expression of this operon, monitored by measuring the NADP-reducing activity, was found to be maximum during the exponential phase of growth on fructose and then decreased when the concentration of the carbon and energy source became limiting. The optimum pH for the H2-driven NADP reduction was 8, and the apparent K(m) and Vmax were determined to be 0.09 mM and 13 x 10(-3) u/mg, respectively. Heterologous expression of the hnd genes in Escherichia coli was carried out to raise antisera against the different subunits of the NADP-reducing hydrogenase. The antisera were used to detect the four subunits in cell extract of D. fructosovorans after separation by SDS- and native PAGE. The four subunits of the NADP-reducing hydrogenase were demonstrated to be associated in a complex which exhibited H2-driven methyl viologen reduction. Furthermore, on native gel, a form lacking HndD, with no hydrogen-dependent methyl viologen reductase activity was also shown to be present in D. fructosovorans.
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PMID:The NADP-reducing hydrogenase of Desulfovibrio fructosovorans: evidence for a native complex with hydrogen-dependent methyl-viologen-reducing activity. 970 71


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