A mechanistic and electrochemical study of the interaction between dimethyl sulfide dehydrogenase and its electron transfer partner cytochrome c 2

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron–sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c ₂ mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c ₂ accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c ₂), Michaelis constants, K _M,DMS and K _M,cyt of 53 and 21 μM, respectively, were determined at pH 8. Further kinetic analysis revealed a “ping-pong” enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c ₂ immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible Fe^III/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The Fe^III/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c ₂ is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c ₂ effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant of 13(±1) μM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c ₂ as the electron acceptor was found to be approximately 8.3.