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 c2 mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c2 accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c2), Michaelis constants, KM,DMS and KM,cyt of 53 and 21 microM, 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 c2 immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible FeIII/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The FeIII/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c2 is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c2 effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant K'M,DMS of 13(+/-1) microM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c2 as the electron acceptor was found to be approximately 8.3.