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)

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.
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PMID:Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b. 608 43

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.
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PMID:Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis. 621 54

A routine procedure for the growth and harvesting of large (600 1) batches of Chromatium vinosum and the isolation of hydrogenase (hydrogen: (acceptor) oxidoreductase, EC 1.12.-.-) are described. The enzyme is pure according to polyacrylamide gel electrophoresis, has a molecular weight of 61,000-63,000 and consists of a single polypeptide chain. The enzyme is stable in air but not active. Activity is obtained only after complete removal of oxygen. EPR spectroscopy at 9 GHz shows a signal indicative for a [4Fe-4S]3+(3+,2+) cluster and in addition a rather complex signal of unknown origin. This additional signal completely disappears upon removal of oxygen, by incubation with 2-mercaptoethanol or by reduction with ferrocytochrome c. No EPR signals are detected in the enzyme reduced with H2 or dithionite. The intensity of the EPR signal of the [4Fe-4S] cluster corresponds to one-quarter of the enzyme concentration, both in the untreated as well as in the He- or N2-activated enzyme. If the enzyme is activated under He and then brought in contact with air the signal increases 4-fold and represents about one free spin/enzyme molecule. When measured at 35 GHz the line shape and peak positions of the additional signal change, indicating that the signal is not originating from a simple S = 1/2 system. None of the inhibitors of the hydrogenase activity has any effect on the shape or intensity o the EPR signal fo the Fe-S cluster, 2H2O also has no effect. All EPR signals disappear after reduction with NADH or ascorbate in the presence of phenazine methosulphate. It is suggested that the Fe-S cluster is not the primary site of interaction of H2 with the enzyme.
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PMID:Purification and some properties of the soluble hydrogenase from Chromatium vinosum. 626 Jan 99

The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.
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PMID:Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16. 627 8

Extraction with n-heptane abolished over 95% of the NADH oxidase and the hydrogenase activity in membrane preparations from Azotobacter vinelandii. Incorporation of ubiquinone-8 or plastoquinone restored each reaction to about 55% of its original activity.
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PMID:Ubiquinone in hydrogen metabolism by Azotobacter vinelandii. 651 22

Electron transport from H2, NADPH, NADH and succinate to O2 or ferricytochrome c in respiratory particles isolated from Anacystis nidulans in which hydrogenase had been induced was abolished after extraction of the membranes with n-pentane; oxidation of ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine remained unaffected. Incorporation of authentic ubiquinone-10, plastoquinone-9, menaquinone-7 and phylloquinone (in order of increasing efficiency) restored the electron-transport reactions. ATP-dependent reversed electron flow from NNN'N'-tetramethyl-p-phenylenediamine to NADP+ or, via the membrane-bound hydrogenase, to H+ was likewise abolished by pentane extraction and restored by incorporation of phylloquinone. Participation of the incorporated quinones in the respiratory electron-transport reactions of reconstituted particles was confirmed by measuring the degree of steady-state reduction of the quinones. Isolation and identification of the quinones present in native Anacystis membranes yielded mainly plastoquinone-9 and phylloquinone; neither menaquinone nor alpha-tocopherolquinone could be detected. Together with the results from reconstitution experiments this suggests that phylloquinone might function as the main respiratory quinone in Anacystis nidulans.
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PMID:Restoration of respiratory electron-transport reactions in quinone-depleted particle preparations from Anacystis nidulans. 676 34

Cells, as well as crude extracts of Clostridium kluyveri or Clostridium spec. La 1, catalyze the hydrogenation of (E)- or (Z)-2-butenol to n-butanol. No single enzyme could be detected which directly accomplishes this reaction. It turned out that the reduction occurs as follows: 2-butenol leads to 2-butenal leads to n-butanal leads to n-butanol. The first step is catalyzed by the NAD-dependent alcohol dehydrogenase in C. kluyveri, the second by the recently detected enoate reductase which reduces not only nonactivated alpha, beta-unsaturated acylates but also alpha, beta-unsaturated aldehydes in a NADH-dependent reaction and the third step is again catalyzed by alcohol dehydrogenase. In Clostridium La 1 the alcohol dehydrogenase is NADP-dependent. The rate of the reduction of 2-butenol to n-butanol depends not only on the enzymes, but also on the ratio NAD(P)/NAD(P)H. In the presence of methylviologen cation radical which is formed by the reduction of methylviologen by the system H2/hydrogenase, the ratio NAD(P)/NAD(P)H is too small for the dehydrogenation of 2-butenol to 2-butenal. This explains the antagonistic effect of methylviologen in the hydrogenation of allyl alcohols and 2-enoates by both Clostridium species. Furthermore, the mechanism explains the finding that from a preparative point of view ethanol is a better electron donor than hydrogen for the stereospecific reduction of allyl alcohols.
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PMID:The reduction of allyl alcohols by Clostridium species is catalyzed by the combined action of alcohol dehydrogenase and enoate reductase. 702 92

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) which has been previously employed as an inhibitor for electron transport particles, NADH dehydrogenase, and other flavoproteins is reducible under physiological conditions. Soluble hydrogenase from Alcaligenes eutrophus H 16, several flavoproteins, and electron transport particles from baker's yeast and from beef heart were found to catalyse NADH oxidation with 9 micrometers to 2mM rhein as the electron acceptor. Dithionite or enzymatically reduced rhein (lambda max = 408 nm) is immediately reoxidized to rhein lambda max = 437 nm) by oxygen. Cyclovoltagrams reveal the midpoint redox potentials --0.240 V, -0.270 V, -0.280 V, -0.335 V at pH 6.0, 7.0, 7.7, 9.2, respectively. Due to its redox behaviour, caution should be exercised using rhein as a flavin-site-directed inhibitor for biological electron transfer systems.
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PMID:Rhein as an electron acceptor for various flavoproteins and for electron transport particles. 704 89

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.
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PMID:The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. 730 57

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


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