A diffusion-controlled step in the catalytic cycle of nitrous oxide reductase from Wolinella succinogenes

The viscosity test was applied to the highly active nitrous oxide reductase purified from Wolinella succinogenes to determine whether the catalytic cycle may contain a diffusion-controlled step. For this test, the benzyl viologen cation (BV+)-N2O oxidoreductase reaction, which exhibits a kcatBV/KmBV approximately 2 x 10(8) M-1 s-1 at 23 degrees C and pH 7.1, was run in solutions of nominally nondenaturing viscogens that were used to increase microviscosity. The parameters kcatN2O and kcatBV were unaffected by viscosity in the range of viscogen concentrations that were not inhibitory and where the data were well behaved. KmBV, but not KmN2O, was observed to increase linearly with relative viscosity with a slope of 0.14 for all viscogens surveyed. The results, when considered in the context of a plausible kinetic model for the BV(+)-N2O oxidoreductase reaction, suggest that one of the two one-electron reactions between BV+ and enzyme is diffusion-controlled but only partially rate determining. The ratio of the second-order-rate constants for these two one-electron steps is estimated to be about 6.1, and the larger rate constant to be about 1.1 x 10(9) M-1 s-1. There would appear to be no diffusion-controlled step associated with the half-reaction which results in reduction of N2O to N2.