Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination.

The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.