A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F420H2 dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2 was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. TheKm values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.

The proton translocating electron transport systems (F 420 H 2 :heterodisulfide oxidoreductase and H 2 :heterodisulfide oxidoreductase) of Methanosarcina mazei Go È1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC 50 values of 20 nmol DPI´mg ±1 protein and 45 nmol DPI´mg ±1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2-hydroxyphenazine-dependent reactions as catalyzed by F 420 -nonreducing hydrogenase, F 420 H 2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H 2 -dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane-bound cytochromes indicated that DPI inhibited CoB-S-S-CoM-dependent oxidation of reduced cytochromes and H 2 -dependent cytochrome reduction. Membrane-bound and purified F 420 H 2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2-hydroxyphenazine were used as electron acceptors. Detailed examination of 2-hydroxy-phenazine-dependent F 420 H 2 -oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the K m value for 2-hydroxyphenazine, which increased from 35 mm to 100 mm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron transport systems take place. Thus, electron transfer from reduced 2-hydroxyphenazine to cytochrome b 2 as part of the heterodisulfide reductase and from H 2 to cytochrome b 1 as subunit of the membranebound hydrogenase is affected in the presence of DPI. In case of the F 420 H 2 dehydrogenase electron transport from FAD or from FeS centers to 2-hydroxyphenazine is inhibited.Keywords: cytochrome, diphenyleneiodonium; heterodisulfide reductase; methanogenic Archaea; Methanosarcina; NADH dehydrogenase; phenazine.The process of methanogenesis as performed by methanogenic archaea is coupled to energy conservation by electron transport phosphorylation [1]. In Methanosarcina mazei Go È1 two membrane-bound electron transport systems have been discovered. The F 420 H 2 :heterodisulfide oxidoreductase is involved in methanol degradation and consists of F 420 H 2 dehydrogenase [2,3] and heterodisulfide reductase [4]. Electron transfer between the enzymes is probably mediated by methanophenazine, a membrane integral electron carrier which was recently isolated from Ms. mazei Go È1 [5]. The F 420 H 2 dehydrogenase catalyzes the oxidation of F 420 H 2 , which is formed during oxidation of one of four methanol molecules. The remaining methyl moities are transferred to 2-mercaptoethanesulfonate (CoM-SH). In the final step of methanogenesis the methyl-S-CoM reductase catalyzes t...

The F420H2 dehydrogenase is part of the energy‐conserving F420H2:heterodisulfide oxidoreductase system in Methanosarcina mazei Gö1. The enzyme was purified 75‐fold to apparent homogeneity from washed membranes. The molecular mass as determined by both native gel electrophoresis and gel filtration was 115 000. The purified enzyme was composed of five different polypeptides with molecular masses of 40, 37, 22, 20, and 16 kDa and contained 7 mol S2− and 7 mol Fe. The specific activity was 17 U mg protein−1 (apparent Vmax) using F420H2 as electron donor (Km= 7 μM) and methylviologen and metronidazole as electron acceptors at pH 8.5 at a temperature of 39°C. The enzyme also catalyzed the reduction of 2,3‐dimethyl‐1,4‐naphthoquinone, 2‐methyl‐1,4‐naphthoquinone and tetramethyl‐p‐benzoquinone. The protein did not exhibit hydrogenase activity since F420 was not reduced by hydrogen and H2 production from F420H2 was not observed. In contrast to the F420H2 dehydrogenase from Archaeoglobus fulgidus, the enzyme from Methanosarcina mazei Gö1 was similar to the corresponding protein of Methanolobus tindarius with respect to molecular mass and subunit composition. However, the proteins of the methanogenic organisms are different in cofactor content, since evidence is presented that the enzyme from Methanosarcina mazei Gö1 contains FAD.

The F420H2 dehydrogenase is part of the energy‐conserving F420H2:heterodisulfide oxidoreductase system in Methanosarcina mazei Gö1. The enzyme was purified 75‐fold to apparent homogeneity from washed membranes. The molecular mass as determined by both native gel electrophoresis and gel filtration was 115 000. The purified enzyme was composed of five different polypeptides with molecular masses of 40, 37, 22, 20, and 16 kDa and contained 7 mol S2− and 7 mol Fe. The specific activity was 17 U mg protein−1 (apparent Vmax) using F420H2 as electron donor (Km= 7 μM) and methylviologen and metronidazole as electron acceptors at pH 8.5 at a temperature of 39°C. The enzyme also catalyzed the reduction of 2,3‐dimethyl‐1,4‐naphthoquinone, 2‐methyl‐1,4‐naphthoquinone and tetramethyl‐p‐benzoquinone. The protein did not exhibit hydrogenase activity since F420 was not reduced by hydrogen and H2 production from F420H2 was not observed. In contrast to the F420H2 dehydrogenase from Archaeoglobus fulgidus, the enzyme from Methanosarcina mazei Gö1 was similar to the corresponding protein of Methanolobus tindarius with respect to molecular mass and subunit composition. However, the proteins of the methanogenic organisms are different in cofactor content, since evidence is presented that the enzyme from Methanosarcina mazei Gö1 contains FAD.