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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 hydrogenase activity of the intact cells of a thermophilic hydrogen-oxidizing bacterium Pseudomonas thermophila K-2 was determined using methylene blue; it was several times higher than the rate of hydrogen uptake in the presence of oxygene and carbon dioxide. The activity of membrane-associated hydrogenase was assayed with the aid of phenazine methosulphate and 2,6-dichlorphenolindophenol as a cascade electron carrier. The enzyme is sufficiently stable in the air. The stability increases in the atmosphere of hydrogen. The membrane-bound enzyme was activated by Mn2+ ions. The pH-optimum of the enzyme activity in 0.1 M Tris-HCI buffer was 8,5-9,0. Natural electron acceptors tested, such as NAD, FMN, riboflavin, and cytochrome c, had no effect on the reaction rate. The enzyme is relatively thermostable: its activity was halved after heating at 78 degrees C for 10 min or at 80 degrees C for 8 min. Energy of activation was calculated. It was 14.5 kcal-mol-1 within the range of 23-40 degrees C and 10.3 kcal-mol-1 within the range of 40-60 degrees C.
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PMID:[Hydrogenase activity of the thermophilic hydrogen-oxidizing bacterium Pseudomonas thermophila]. 2 May 54

Extracts of aerobically, CO-autotrophically grown cells of Pseudomonas carboxydovorans were shown to catalyze the oxidation of CO to CO(2) in the presence of methylene blue, pyocyanine, thionine, phenazine methosulfate, or toluylene blue under strictly anaerobic conditions. Viologen dyes and NAD(P)(+) were ineffective as electron acceptors. The same extracts catalyzed the oxidation of formate and of hydrogen gas; the spectrum of electron acceptors was identical for the three substrates, CO, formate, and H(2). The CO- and the formate-oxidizing activities were found to be soluble enzymes, whereas hydrogenase was membrane bound exclusively. The rates of oxidation of CO, formate, and H(2) were measured spectrophotometrically following the reduction of methylene blue. The rate of carbon monoxide oxidation followed simple Michaelis-Menten kinetics; the apparent K(m) for CO was 45 muM. The reaction rate was maximal at pH 7.0, and the temperature dependence followed the Arrhenius equation with an activation energy (DeltaH(0)) of 35.9 kJ/mol (8.6 kcal/mol). Neither free formate nor hydrogen gas is an intermediate of the CO oxidation reaction. This conclusion is based on the differential sensitivity of the activities of formate dehydrogenase, hydrogenase, and CO dehydrogenase to heat, hypophosphite, chlorate, cyanide, azide, and fluoride as well as on the failure to trap free formate or hydrogen gas in coupled optical assays. These results support the following equation for CO oxidation in P. carboxydovorans: CO + H(2)O --> CO(2) + 2 H(+) + 2e(-) The CO-oxidizing activity of P. carboxydovorans differed from that of Clostridium pasteurianum by not reducing viologen dyes and by a pH optimum curve that did not show an inflection point.
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PMID:Oxidation of carbon monoxide in cell extracts of Pseudomonas carboxydovorans. 3 64

Spheroplasts that were osmotically stable in 0.2M Tris-HCl--0.02M EDTA were prepared from the autotrophically grown cells of Pseudomonas thermophila K-2. The spheroplasts possessed 90--95% of the hydrogenase activity of the whole cells. The half-life time of hydrogenase in the spheroplasts at 80 degrees C was 8.5 min. A spectrophotometric technique was developed for determining the membrane-bound hydrogenase in the presence of sulfhydryl compounds with methylene blue as electron acceptor. The maximal specific activity of hydrogenase in extracts prepared in the anaerobic conditions in the presence of dithiothreitol and Mg2+ and Mn2+ ions was 10 +/- 3 units per 1 mg of protein, which closely corresponded with the activity of hydrogenase in the whole cells. Almost all activity of hydrogenase assayed with methylene blue was localized in the membrane fraction. The activity of soluble NAD-specific hydrogenase was not detected. Large particles located in 60-70% sucrose had the highest hydrogenase activity upon fractionation in a continuous sucrose concentration gradient. The second, lower peak of the hydrogenase activity was detected in fractions of 40--50% sucrose. As was found by electron microscopy, the size of membrane vesicles with the hydrogenase activity varied within the range of 68--156 nm. The membrane preparations possessed the activity of NADH-dehydrogenase, NADH-oxidase and succinate dehydrogenase as well.
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PMID:[Localization of hydrogenase in the cells of the thermophilic hydrogen bacterium, Pseudomonas thermophila]. 21 85

From enrichment cultures four carbon monoxide utilizing bacteria were isolated; strain OM5 isolated from waste water was studied in detail. The cells are Gram-negative, slightly curved rods, motile by a single subpolarly inserted flagellum. The colonies are smooth, translucent and not slimy. The cells are able to grow autotrophically in mineral medium under an atmosphere of 40% CO, 5% O2 and 55% N2 at a doubling time of 20h (30 degrees C) or of 85% H2, 5% O2 and 10% CO2 at a doubling time of 7h. Heterotrophic growth occurred on organic acids such as acetate(td = 8h), pyruvate(td = 8h), lactate, crotonate, malate, succinate (td = 8h), formate (td = 35h) and glyoxylate as substrates. The enzyme system for carbon monoxide utilization is formed only during growth on CO; hydrogenase is present in cells grown on CO or on H2 + CO2 as well as grown on pyruvate. The rate of oxygen reduction by intact CO-grown cells is 3.7-fold higher in the presence of hydrogen than in the presence of carbon monoxide. During growth the stoichiometry of gas uptake was 6.1 CO + 2.8 O2 + H2O leads to CH2O +5.1 CO2. For the new isolate the name Pseudomonas carboxydovorans (Kistner) comb. nov. has been proposed.
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PMID:Reisolation of the carbon monoxide utilizing hydrogen bacterium Pseudomonas carboxydovorans (Kistner) comb. nov. 69 1

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction. While the second enzyme was found to be particulate and unable to react with NAD. All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gently methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis. Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 micronmoles H2 oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme for A. eutrophus H 16 which was shown to be more stable under aerobic conditions.
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PMID:Localization and stability of hydrogenases from aerobic hydrogen bacteria. 87 Dec 26

The cells of Pseudomonas methylica, strain 2, cultivated in a medium containing methanol, displayed the activity of hydrogenase in the exchange reaction (D2--H2O) and in the absorption of H2 in the presence of methylviologen, azocarmine, methylene blue, and ferricyanide. The rate of H2 utilization was highest in the presence of methylviologen. Cell extracts absorb H2 in the presence of methylviologen, NAD, and NADP, but much faster in the presence of flavin mononucleotide. The bulk of the hydrogenase remains, during centrifugation of the initial cell extract (3,000 g), in the soluble fraction (144,000 g). The absorption of oxygen by the cell suspensions and the incorporation of 14C of formiate into the cells are stimulated by H2. The cells, however, cannot grow in the autotrophic conditions at the account of molecular hydrogen and CO2.
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PMID:[Hydrogenase activity of the methylotroph, Pseudomonas methylica]. 120 95

Denitrification and methylotrophy in Paracoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced from P. denitrificans. A number of sequences are available for enzymes from Escherichia coli, Pseudomonas stutzeri and Pseudomonas aeruginosa. It is concluded that pathway specific c-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification. In methanol oxidation at least 20 genes are involved. In this case too pathway specific c-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholde-hydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. The moxFJGI operon determines the structural components of methanol dehydrogenase and the associated c-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The moxY protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy. The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA of Thiosphaera pantotropha is identical to that of P. denitrificans. Still these bacteria show a number of differences. T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the beta-and gamma-subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification in T. pantotropha, which belongs to the alpha-subdivision of purple non-sulfur bacteria is a remarkable property. Furthermore T. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochrome cd1 type as in P. denitrificans. Also the cytochrome b of the Qbc complex of T. pantotropha is highly similar to its counterpart in P. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the alpha-group of purple non sulphur bacteria is reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes. 157 65

The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.
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PMID:Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16. 218 45

Pleiotropic mutants of Alcaligenes eutrophus with the phenotype Hno- have been characterized previously. They are deficient in several diverse metabolic activities, including hydrogen oxidation, nitrate and urea assimilation, denitrification, and various substrate transport systems. Phenotypically similar mutants were identified among hydrogenase-deficient strains of Pseudomonas facilis. The Tn5-labeled hno gene was cloned from a genomic DNA library of A. eutrophus and used to identify the corresponding unimpaired wild-type DNA sequence. The recombinant plasmid pCH148 contained an insert of 12.3 kilobase pairs and was shown to restore the Hno+ phenotype to mutants of A. eutrophus and P. facilis. A cosmid isolated from a DNA library of P. facilis also exhibited intergeneric Hno-complementing activity. The cloned hno loci from both organisms showed DNA homology by Southern blot hybridization. A subclone of pCH148 which contained a 6.5-kilobase-pair insert was constructed. The resulting hybrid, pCH170, not only was able to complement Hno- mutants but also relieved glutamine auxotrophy in NtrA- mutants of enteric bacteria. This suggests that the hno gene product from A. eutrophus is functionally similar to the NtrA protein, which has been identified as a novel sigma factor (sigma 54) of RNA polymerase.
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PMID:An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation. 253 72

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.
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PMID:Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803. 253 87


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