Detection of a C4a-Hydroperoxyflavin Intermediate in the Reaction of a Flavoprotein Oxidase.

This work describes for the first time the identification of a reaction intermediate, C4a- hydroperoxyflavin, during the oxidative half-reaction of a flavoprotein oxidase, pyranose 2-oxidase (P2O) from Trametes multicolor, by using rapid kinetics. The reduced P2O reacted with oxygen with a forward rate constant of 5.8 × 104 M-1 s-1 and a reverse rate constant of 2 s-1, resulting in the formation of a C4a-hydroperoxyflavin intermediate which decayed with a rate constant of 18 s-1. The absorption spectrum of the intermediate resembled the spectra of flavin-dependent monooxygenases. A hydrophobic cavity formed at the re side of the flavin ring in the closed state structure of P2O may help in stabilizing the intermediate. [ABSTRACT FROM AUTHOR]