C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.