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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...

Mutants of Azotobacter vinelandii negative to N, N, N', N'-tetramethyl-p-phenylenediamine[Ph(NMe2)2] oxidase were obtained after mutagenesis by screening colonies with the 'oxidase reagent' Ph(NMe2)z. These mutants appear to be the first respiratory mutants reported for Azotobacter. Characterization of one of these mutants (AV-11) revealed similar growth rates under N2-fixing conditions, comparable P/O ratios (about 0.6, NADH) and H/O quotients (about 4), and similar respiration rates as the parent strain AV-OP. No oxygen consumption or net synthesis of ATP could be demonstrated with phosphorylating membranes of mutant AV-11 using reduced Ph(NMe2)z as substrate. The oxidase-negative properties of mutant AV-11 appear to be associated with an inability of the terminal oxidases cytochromes o and al to reoxidize cytochromes c4 + c5 in membrane particles. Cytochrome c4 + c5 of mutant AV-11 could not be reoxidized by normal procedures (bubbling with oxygen or with 0.05 mM ferricyanide). These could only be reoxidized by excess ferricyanide (10-20 mM). Oxidized cytochromes c4 + c5 of mutant AV-11 are readily reduced by reduced Ph(NMe2)Z and studies on partially purified cytochromes c4 and c5 showed no unusual properties.A comparison of the respiratory kinetics for membrane particles of strains AV-11 and AV-OP showed no differences in the oxidation of NADH or malate via cytochrome oxidase d [ V = 3.2 pmol oxygen consumed x min-' x mg protein-'; K , ( 0 2 ) = 18 pM]. The respiratory kinetics exhibited for oxidation of reduced Ph(NMe& via the oxidases cytochrome o and a1 could only be determined for strain AV-OP (V = 0.7 pmol 0 2 x min-' x mg protein-'; K, = 3.1 pM). The very high V value observed for oxidation of cytochrome d (strains AV-11 and AV-OP) suggests that this oxidase is capable of handling the electron flow generated by the very active dehydrogenases. Since the respiratory chain of mutant AV-11 appears to be blocked between cytochromes c4 + c5 and the oxidases cytochromes o and al, we suggest that for the Ph(NMe2)2-oxidase-negative mutants, cytochrome d is the only functional oxidase.The respiratory chain of Azotobacter vinelandii has been extensively studied (for a partial review, see [l]) as has its role in protection of the oxygen-sensitive nitrogenase complex [2 -41. Azotobacter membranes contain a rich variety of redox carriers, cytochromes, and dehydrogenases, arranged to support two or possibly three sites of respiration-driven proton translocation [5 -91. The cytochrome chain appears to be branched at cytochrome bl [7] and is terminated by three oxidases [5,7,10,11]. Inhibitor studies [5,7,11, 121 as well as studies by photochemical action spectral methods [8,10] suggest that the major flow of elecAbbreviation. Ph(NMe*)z, N,N, N', N'-Tetramethyl-p-phenylenediamine, also known as TMPD.trons from the dehydrogenases are oxidized via cytochrome d. Cytochrome oxidases o and a1 terminate the minor branch and appear to be associated with cytochromes c4 + c5 [5,8,11,13]. The minor pathway contains a phosphor...

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