2-hydroxyphenazine and diphenyleneiodonium

Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.

Topics: Carbon Monoxide; Electron Spin Resonance Spectroscopy; Electron Transport; Flow Injection Analysis; Iron-Sulfur Proteins; Mersalyl; Mesna; Methanosarcina; Onium Compounds; Oxidoreductases; Phenazines; Phosphothreonine

The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H2:heterodisulfide oxidoreductase) of Methanosarcina mazei Gö1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 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 F420-nonreducing hydrogenase, F420H2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H2-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 H2-dependent cytochrome reduction. Membrane-bound and purified F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2-hydroxyphenazine were used as electron acceptors. Detailed examination of 2-hydroxy-phenazine-dependent F420H2-oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the Km value for 2-hydroxyphenazine, which increased from 35 microm to 100 microm 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 b2 as part of the heterodisulfide reductase and from H2 to cytochrome b1 as subunit of the membrane-bound hydrogenase is affected in the presence of DPI. In case of the F420H2 dehydrogenase electron transport from FAD or from FeS centers to 2-hydroxyphenazine is inhibited.

Topics: Bacterial Proteins; Binding, Competitive; Cytochrome b Group; Electron Transport; Enzyme Inhibitors; Ferritins; Kinetics; L-Lactate Dehydrogenase; L-Lactate Dehydrogenase (Cytochrome); Membrane Proteins; Methanosarcina; Models, Biological; Onium Compounds; Oxidoreductases; Phenazines