Studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides have yielded new insight into its catalytic mechanism. A series of reductive titrations, performed over the pH range 6-10, reveal that the absorption spectrum of reduced enzyme is highly sensitive to pH. The reaction of reduced enzyme with dimethyl sulfoxide is found to be clearly biphasic throughout the pH range 6-8 with a fast, initial substrate-binding phase and substrate-concentration independent catalytic phase. The intermediate formed at the completion of the fast phase has the characteristic absorption spectrum of the established dimethyl sulfoxide-bound species. Quantitative reductive and oxidative titrations of the enzyme demonstrate that the molybdenum center takes up only two reducing equivalents, implying that the two pyranopterin equivalents of the molybdenum center are not formally redox active. Finally, the visible spectrum associated with the catalytically relevant "high-g split" Mo(V) species has been determined. Spectral deconvolution and EPR quantitation of enzyme-monitored turnover experiments with trimethylamine N-oxide as substrate reveal that no substrate-bound intermediate accumulates and that Mo(V) content remains near unity for the duration of the reaction. Similar experiments with dimethyl sulfoxide show that significant quantities of both the Mo(V) species and the dimethyl sulfoxide-bound complex accumulate during the course of reaction. Accumulation of the substrate-bound complex in the steady-state with dimethyl sulfoxide arises from partial reversal of the physiological reaction in which the accumulating product, dimethyl sulfide, reacts with oxidized enzyme to yield the substrate-bound intermediate, a process that significantly slows turnover.