Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

Abstract

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45 % yield and a final specific activity of 270 pmol H2 evolved min-' (mg protein)-'. The hydrogenase has a high molecular mass of z 800 kDa and subunits with molecular masses of z 60 kDa. The enzyme is stable to heating at 65 "C and to exposure to air at 4°C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8 -10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit.The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24,2.20 and = 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g= 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center I1 has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only obser\'able at temperatures higher than 20 K. In the native preparation, at high temperatures ( T > 35 K) or in partially red wed samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80 "/, Me2S0 presents an axial type of spectrum only detectable below 30 K.The methanogenic bacteria are strict anaerobic microorganisms that synthesize methane from a limited number of substrates (e. g. formate, acetate, methylamines, methanol, CO or C 0 2 and H2). They belong to a bacterial group designated as Archaebacteria which are only distantly related in the evolutionary scale to eukaryotes and the strictly anaerobic bacteria such as Clostridia and sulfate-reducing organisms [ 11. Supporting this clasification, they contain unusual cofactors not found in other organisms such as: coenzyme M [2], F420 [3], F342 [4], and F,,, [5,6,52] which may be isolated in a protein-bound form. In contrast, the amino acid sequence of Methanosarcina barkeri ferredoxin is quite similar to the clostridial type [7], but the evolutionary significance of this observation is not understood.Methanogens are basically chemoautotrophs in their metabolism and molecular hydrogen is often used as a subst...

Nickel hydrogenases: in search of the active site

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Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg

“…Two types of hydrogenase have been detected in numerous methanogenic bacteria, including M. thermoautotrophicum (3,7,9,10,13,21,25,27,29,36,37,48), i.e., an F420 (8-hydroxy-5-deazaflavin)-reducing hydrogenase and a non-F420-reducing hydrogenase, both of which can be assayed via the reduction of viologen dyes with H2 (16). The F420-reducing hydrogenase also reduces the physiological two-electron acceptor F420.…”

Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum

“…The other (MV-hydrogenase) reduces MV but not F420. F420-hydrogenases from several H2-oxidizing methanogens have been purified and characterized (11,14,27,37,41). All of these enzymes form large aggregates with molecular weights of 720,000 to 1,300,000 and contain nickel and iron-sulfur centers.…”