Relative Timing of Hydrogen and Proton Transfers in the Reaction of Flavin Oxidation Catalyzed by Choline Oxidase

The oxidation of the reduced flavin in choline oxidase was investigated with pH, solvent viscosity, and kinetic isotope effects (KIEs) in steady-state kinetics and time-resolved absorbance spectroscopy of the oxidative half-reaction in a stopped-flow spectrophotometer. Both the effects of isotopic substitution on the KIEs and the multiple KIEs suggest a mechanism for flavin oxidation in which the H atom from the reduced flavin and a H+ from the solvent or a solvent exchangeable site are transferred in the same kinetic step. Stopped-flow kinetic data demonstrate flavin oxidation without stabilization of flavin-derived species. Solvent viscosity effects establish an isomerization of the reduced enzyme. These results allow us to rule out mechanisms for flavin oxidation in which C4a-peroxy and -hydroperoxy flavin intermediates accumulate to detectable levels in the reaction of flavin oxidation catalyzed by choline oxidase. A mechanism of Flavin oxidation that directly results in the formation of oxidized flavin and hydrogen peroxide without stabilization of reaction intermediates is consistent with the data presented.