title: Relative timing of hydrogen and proton transfers in the reaction of flavin oxidation catalyzed by choline oxidase.
abstract: 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.
authors: Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA.
authors: Swathi
authors: S
authors: Gannavaram
authors: Giovanni
authors: G
authors: Gadda
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D003903:Deuterium; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011522:Protons; D012997:Solvents / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods; D014783:Viscosity
chemical_list: D005415:Flavins; D011522:Protons; D012997:Solvents; D006859:Hydrogen; D003903:Deuterium; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019888:choline oxidase