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

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• 作者：Swathi Gannavaram ; Giovanni Gadda
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：February 19, 2013
• 年：2013
• 卷：52
• 期：7
• 页码：1221-1226
• 全文大小：332K
• 年卷期：v.52,no.7(February 19, 2013)
• ISSN：1520-4995

文摘

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.