Abstract
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.
Highlights
Mechanism of H2O2 Elimination in Pyranose 2-oxidase fore, the unique property of P2O2 in that the enzyme reacts with oxygen and stabilizes the C4a-hydroperoxyflavin intermediate makes the pyranose 2-oxidase (P2O) system an ideal model for investigating solvent kinetic isotope effects (SKIE) on the H2O2 elimination step in a flavoprotein oxidase and for understanding the general mechanism underlying H2O2 elimination from enzyme-bound C4a-hydroperoxyflavins
The results indicated that for P2O from T. multicolor, an SKIE (D2Ok2) of 2.8 Ϯ 0.2 was found for the H2O2 elimination step and that the N5 proton of reduced flavin is the proton-in-flight that causes this SKIE
The currently accepted mechanism of the reaction between reduced flavin and oxygen (Fig. 1) predicts that the first step of the reaction involves a one-electron transfer to form a radical pair of flavin semiquinone and superoxide radical, which rapidly collapses to form C4a-hydroperoxyflavin [1, 2, 15,16,17,18]
Summary
P2O, pyranose 2-oxidase; FMN, flavin mononucleotide; KIE, kinetic isotope effect; SKIE, solvent kinetic isotope effect(s); ABTS, 2,2Ј-azino-bis[3]-ethybenzenethiazoline-6-sulfonic acid diammonium salt; D/H, D2O/H2O; N5-D, the deuterium incorporated flavin N5; N5-H, the protium incorporated flavin N5. Eliminates H2O2 (path 1 in Fig. 1) [12,13,14]. The formation of C4a-hydroperoxy-FAD in P2O is known to require optimized interactions, especially around the FAD N5/C4a locus because mutations of Thr169 to Ser/Ala/Asn/Gly resulted in the abolishment of the intermediate formation [31]. We elucidated the mechanism underlying the elimination of H2O2 from C4a-hydroperoxy-FAD in P2O using the results of kinetic isotope effects and transient kinetics. The data clearly show that the transfer of the flavin N5 proton to the peroxide leaving group is the key step controlling the process of H2O2 elimination from C4a-hydroperoxyflavin
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