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)

The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.
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PMID:Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro. 199 34

Formate hydrogenlyase activity in a cell extract of Methanobacterium formicicum was abolished by removal of coenzyme F420; addition of purified coenzyme F420 restored activity. Formate hydrogenlyase activity was reconstituted with three purified components from M. formicicum: coenzyme F420-reducing hydrogenase, coenzyme F420-reducing formate dehydrogenase, and coenzyme F420. The reconstituted system required added flavin adenine dinucleotide (FAD) for maximal activity. Without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F420-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of formate dehydrogenase or coenzyme F420-reducing hydrogenase from the cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. The formate hydrogenlyase activity was reversible, since both the cell extract and the reconstituted system produced formate from H2 plus CO2 and HCO3-.
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PMID:Reconstitution and properties of a coenzyme F420-mediated formate hydrogenlyase system in Methanobacterium formicicum. 266 36

The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any amino acid) was conserved in the N-termini of the alpha subunits of the F420-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F420-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F420. Maximal coenzyme F420-reducing activity was obtained at 55 degrees C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H2 production at a rate threefold lower than that for H2 uptake and reduced coenzyme F420, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited the coenzyme F420-dependent but not the methyl viologen-dependent activity of the purified enzyme.
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PMID:Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum. 273 24

Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum delta H have been determined by electron microscopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, indicating a holoenzyme molecular weight of 8.0 X 10(5). Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis.
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PMID:Electron microscopy of nickel-containing methanogenic enzymes: methyl reductase and F420-reducing hydrogenase. 380 76

Hydrophobic interaction chromatography of coenzyme F420-reducing hydrogenase purified from Methanobacterium formicicum depleted protein-bound FAD and eliminated the ability to reduce coenzyme F420. Preincubation of the FAD-depleted hydrogenase with FAD restored 85% of the coenzyme F420-reducing activity. FMN did not replace FAD. A Kd of 12 microM was estimated for FAD. Analysis of the reactivated hydrogenase following molecular sieve column chromatography showed that FAD was bound to protein. The results indicate that protein-bound FAD is reversibly removed from the coenzyme F420-reducing hydrogenase and that this flavin is required for the reduction of coenzyme F420.
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PMID:FAD requirement for the reduction of coenzyme F420 by hydrogenase from Methanobacterium formicicum. 637 61

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.
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PMID:Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg. 800 93

The membrane-associated coenzyme F420-reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95-fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F420-reducing activity (82.8 mumol.min-1.mg protein-1 versus 11.5 mumol.min-1.mg protein-1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79-88]. The predominant coenzyme F420-reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non-identical subunits with apparent molecular masses of 48 (alpha), 33 (beta), and 30 kDa (gamma), apparently in a stoichiometry of alpha 2 beta 2 gamma 1. This minimal coenzyme F420-reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198-kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28-32 mol non-heme iron, and 34 mol acid-labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N-termini of the putative nickel-binding subunits of most [NiFe]- and [NiFeSe]hydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F420-reducing activity was obtained with reductively reactivated enzyme at 55 degrees C in the pH range 6.5-7.25. The Km values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3 microM and 4 microM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5 x 10(7) M-1.s-1 with coenzyme F420 and 6.9 x 10(7) M-1.s-1 with methylviologen.
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PMID:Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro. 852 35

The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically purified to a specific hydrogen-uptake activity of 350 U/mg protein as determined with the natural electron acceptor. The concentrated enzyme was used for EPR-spectroscopic investigations. As isolated, the enzyme showed an EPR spectrum with g(xyz) values of 2.21, 2.15 and 2.01. Illumination of such samples at low temperatures led to an EPR spectrum with g(xyz) values of 2.05, 2.11 and 2.29. These spectra are typical for [NiFe]hydrogenases in the active state. Spectra of samples enriched in 77Se showed a hyperfine interaction between the unpaired spin of the nickel ion and the nuclear spin of one 77Se atom before and after illumination. A 90 degree flip of the electronic z-axis is proposed to explain the hyperfine interaction in both states. This has been demonstrated previously only for the F420-non-reducing hydrogenase from M. voltae, where the selenium atom is present as a selenocysteine residue on an unusually small separate subunit [Sorgenfrei, O., Klein, A. & Albracht, S. P. J. (1993) FEBS Lett. 332, 291-297]. The results demonstrate that the three-dimensional structures of the active sites in the selenium-containing F420-reducing and F420-non-reducing hydrogenases from M. voltae are highly similar and hence are not influenced by the unusual subunit structure of the latter enzyme. Oxidized samples containing either natural selenium or 77Se were prepared from the F420-reducing and the selenium-containing F420-non-reducing hydrogenase. Both enzymes exhibited EPR spectra typical for [NiFe]hydrogenases in the inactive 'ready' state. In contrast to the reduced form, no splitting of the nickel-derived signal due to the nuclear spin of 77Se was observed in the oxidized state, indicating that the electronic z-axis is perpendicular to the Ni-Se direction.
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PMID:Changes in the electronic structure around Ni in oxidized and reduced selenium-containing hydrogenases from Methanococcus voltae. 926 13

LANGUAGE="EN">Summary In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F420 (F420) with H2 are present: (1) the F420-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N5, N10-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H2-forming methylenetetrahydromethanopterin dehydrogenase and F420-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F420-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H2-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F420-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F420 with H2 is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system.
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PMID:Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2 947 54

Methanogenic archaea are known to contain two types of [NiFe] hydrogenases designated F420-reducing hydrogenase and F420-non-reducing hydrogenase. We report here that they additionally contain Escherichia coli hydrogenase-3-type [NiFe] hydrogenases. The evidence is based on biochemical studies and analysis of the subunit primary structure of this hydrogenase (designated Ech) purified from membranes of acetate-grown cells of Methanosarcina barkeri. The subunits EchE and EchC of the EchABCDEF complex showed 34% and 45% sequence identity to the nickel-containing large subunit HycE and to the iron-sulfur cluster containing small subunit HycG, respectively, of the hydrogenase in the formate hydrogen lyase complex from E. coli. Analysis of the totally sequenced genomes of Methanococcus jannaschii and Methanobacterium thermoautotrophicum strain deltaH revealed that these organisms contain similar open reading frames, indicating the presence of an E. coli hydrogenase-3-type hydrogenase also in these methanogenic archaea.
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PMID:An Escherichia coli hydrogenase-3-type hydrogenase in methanogenic archaea. 954 62


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