Inhibition of membrane-bound electron transport of the methanogenic archaeon Methanosarcina mazei Go1 by diphenyleneiodonium

The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H-2:heterodisulfide oxidoreductase) of Methanosarcina m azei Go1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 values of 20 nmol DPI.mg(-1) protein and 45 nmol DPI.mg(-1) protein, r espectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2 hydroxyphena zine-dependent reactions as catalyzed by F-420-nonreducing hydrogenase, F42 0H2 dehydrogenase and heterodisulfide reductase were inhibited. Interesting ly, the H-2-dependent methylviologen reduction and the heterodisulfide redu ction by reduced methylviologen as catalyzed by the hydrogenase and the het erodisulfide 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 cyt ochromes and H-2-dependent cytochrome reduction. Membrane-bound and purifie d F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylv iologen + metronidazole or 2-hydroxyphenazine were used as electron accepte rs. Detailed examination of 2-hydroxy-phenazine-dependent F420H2-oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by th e K-m value for 2-hydroxyphenazine, which increased from 35 mu M to 100 mu M in the presence of DPI. As DPI and phenazines are structurally similar wi th respect to their planar configuration we assume that the inhibitor is ab le 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 bl as part of the heterodisulfide reductase and from H2 to cytochrome bl as subunit of the membrane-bound hy drogenase is affected in the presence of DPI. In case of the F420H2 dehydro genase electron transport from FAD or from FeS centers to 2-hydroxyphenazin e is inhibited.