Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport ofMethanosarcina mazeiGö1

Abstract

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.

Involvement of Ech hydrogenase in energy conservation of Methanosarcina mazei

“…The F 420 nonreducing hydrogenase oxidizes H 2 on the outside of the cytoplasmic membrane [7], thereby releasing two protons. The electrons and two H + from the cytoplasm are used for the reduction of methanophenazine, which is a membrane-integral electron carrier in Methanosarcina species [17]. Reduced methanophenazine transfers electrons to heterodisulfide reductase (Fig.…”

Re‐evaluation of the function of the F420 dehydrogenase in electron transport of Methanosarcina mazei

“…Purification of Fd from Clostridium pasteurianum was performed as outlined by Mortenson [32] with replacement of the last two steps (crystallization and dialysis) by ultrafiltration. Heterodisulfide was synthesized as specified previously [33], and cofactor F 420 was purified and reduced as described by Abken et al [34]. Cell lysate was obtained by anaerobically harvesting trimethylaminegrown cells and resuspending in Buffer A (40 mm K 2 HPO 4 ⁄ KH 2 PO 4 , pH 7.0, 5 mm dithioerythritol, 1 lgAEmL )1 of resazurin) containing desoxyribonuclease to yield a final protein content of about 5 mgAEmL )1 .…”

Exocellular electron transfer in anaerobic microbial communities

“…Some natural redox mediators have a midpoint redox potential that is sufficiently low (< −100 mV) to be relevant for methanogenic environments, e.g. riboflavin and phenazines (Abken et al, 1998;Murakami et al, 2001). In some methanogenic environments, proton-reducing acetogenic bacteria and methanogenic archaea have direct physical contact and a direct transfer of electrons without the involvement of hydrogen or formate cannot be excluded.…”