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)

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.
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PMID:An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase. 1 76

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

The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized. Bacteroids were prepared, then broken by sonication. The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography. The specific activity has been increased 196-fold to 19.6 units/mg protein. The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air. The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM. Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c. Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced. The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal. H2 evolution by the enzyme has been demonstrated. The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.
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PMID:Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules. 4 Jun 1

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.
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PMID:Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16. 18 26

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.
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PMID:Physicochemical properties of flavodoxin from Desulfovibrio vulgaris. 23 84

A purified preparation of hydrogenase from D. gigas was inactive toward ferredoxin, flavodoxin or rubredoxin in the absence of cytochrome c3 (M.W. 13,000), in an atmosphere of hydrogen, although direct reduction of benzyl viologen or FMN was possible. The hydrogen evolution reaction from dithionite was possible with methyl viologen. The same reaction also occured with cytochrome c3 (M.W. 13,000) or cytochrome c3 (M.W. 26,000). Addition of either ferredoxin or flavodoxin did not accelerate the reaction.
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PMID:Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors. 66 85

The isolation method and some peoperties of purple sulphur bacteria (Thiocapsa roseopersicina strain BBS) hydrogenase are described Hydrogenase molecular weight is found to be 66000; it contains 3.7 moles of S2- and 3.9 moles of Fe2+ per one mole of the enzyme;pI=4.2. The enzyme absorption spectrum has the maximum at 400-412 nm which is characteristic of proteins containing non-haem iron. Hydrogenase is suggested to consist pf 4 subunits of two types: with molar weight 27000 and 6000. Unlike other hydrogenases, this enzyme is rather resistant to O2 and is more thermostable: the inactivation of the enzyme was observed at the temperature above 80 degrees C; Hydrogenase preparation catalyses D2-H2O exchange reaction, H2 evolution from the reduced methyl viologene (MV) and H2 absorption in the presense of MV or benzylviologene but not in the presense of NAD(P), FAD, FMN, azocarmine, methylene blue and ferricyanide.
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PMID:[Purification and properties of hydrogenase from phototrophic bacterium Thiocapsa roseopersicina]. 102 87

The primary structures of the nuclear-encoded 51 kDa and 78 kDa subunits of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria were determined by sequencing cDNA and the N-terminus of the mature proteins. Both subunits are related to the soluble NAD-reducing hydrogenase of the bacterium Alcaligenes eutrophus. Sequence comparison between these subunits, the corresponding subunits of the bovine complex I and the bacterial NAD-reducing hydrogenase further confirms the binding sites of NAD(H), FMN and three iron-sulfur clusters.
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PMID:Primary structures of two subunits of NADH: ubiquinone reductase from Neurospora crassa concerned with NADH-oxidation. Relationship to a soluble NAD-reducing hydrogenase of Alcaligenes eutrophus. 183 16

Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.
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PMID:Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase. 190 Jan 94

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with [32P]NADH [Yagi, T., & Dinh, T.M. (1990) Biochemistry 29, 5515-5520]. Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.
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PMID:The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structure. 190 52


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