Inhibition of membrane‐bound electron transport of the methanogenic archaeon Methanosarcina mazei Gö1 by diphenyleneiodonium

Abstract

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...

Membrane-Bound Electron Transport in Methanosaeta thermophila

“…thermophila DSM6194 as described previously (17) with cell disruption by French pressure treatment (1,000 lb/in 2 ). Enzyme assays were carried out at 55°C (optimal growth temperature) (3,17). Benzyl viologen-dependent heterodisulfide reductase activity was high, with 878 Ϯ 90 mU mg Ϫ1 membrane protein (Table 1), and was comparable to activities found in Methanosarcina species (3) (Fig.…”

Extracellular Iron Reduction Is Mediated in Part by Neutral Red and Hydrogenase inEscherichia coli

“…For example, methanophenazine is of similar structure to NR and occurs in electron transport chains of Methanosarcina mazei (1,9). Methanophenazine has been proposed as an electron mediator in a symbiotic relationship in consortia of methanogens and sulfate-reducing bacteria (30).…”

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…It was initially thought that this activity was mediated by a single hypothetical enzyme complex, the F 420 H 2 :heterodisulfide oxidoreductase (303). However, it is now appreciated that this system is in fact formed from two respiratory complexes (304-306), the F 420 H 2 -dependent methanophenazine reductase (Fpo) (162) and the methanophenazine-dependent heterodisulfide reductase (Hdr) (307), which are linked by the redox-active membrane-diffusible cofactor methanophenazine (305,(308)(309)(310) (Fig. 10).…”