The reductase component (MMOR) of the soluble methane monooxygenase isolated from Methylosinus trichosporium OB3b catalyzes transfer of 2e- from NADH to the hydroxylase component (MMOH) where oxygen activation and substrate oxidation occur. It is shown here that MMOR can also exert regulatory effects on catalysis by binding to MMOH or to the binary complex of MMOH and component B (MMOB), another regulatory protein. MMOR alters the oxidation reduction potentials of the dinuclear iron cluster at the active site of MMOH. Although little change is observed in the potential for the first electron transfer to the cluster (E1(°')) = 76 mV), the E2(°')) potential value for the second electron transfer is increased from 21 to 125 mV. This shift provides a larger driving force for electron transfer from MMOR and favors transfer of two rather than one electron as required by catalysis. Similar positive shifts in potential are observed even in the presence of MMOB which has been shown to cause a 132 mV negative shift in the midpoint potential of MMOH in the absence of MMOR. MMOR is also shown to decrease the rate of reaction between the fully reduced MMOH-MMOB and O2 approximately 20-fold at 4 °C. However, the time course of the key catalytic cycle intermediate that can react with substates, compound Q, is unaffected. This implies a compensating faster decay of one or more of the intermediates that occur between diferrous MMOH and compound Q in the reaction cycle, thereby limiting potential nonproductive autodecay of these intermediates. Accordingly, an increase in single turnover product yield is observed in the presence of MMOR. Interestingly, MMOR can cause the redox potential increases, changes in rates, and the increase in product yield when present at only 10% of the concentration of MMOH active sites. Substrate binding is shown to induce negligible changes in the redox potentials. Two alternative regulatory schemes are presented based on (i) thermodynamic coupling of component binding and redox changes or (ii) dynamic interconversion of two states of MMOH promoted by MMOR.