A novel vanadium bromoperoxidase: Investigation of enzyme kinetics and reaction mechanism

The enzyme kinetics and reaction mechanism of vanadium bromoperoxidase (V-BrPO) isolated from the marine alga Ascophyllum nodosum (i.e., the first naturally occurring vanadium-containing metalloenzyme) were investigated.The steady state reaction kinetics of dioxygen formation were analyzed as a function of (HV-BrPO was inhibited by fluoride and azide. Fluoride is a competitive inhibitor with respect to hydrogen peroxide as monitored by MCD bromination and bromide-assisted disproportionation of hydrogen peroxide. Azide inhibits V-BrPO by mechanism-based inactivation which irreversibly inactivates the enzyme.The electronic state of the dioxygen produced was shown to be singlet oxygen as determined by chemiluminescence measurements. Spectral analysis of the chemiluminescence (i.e., emission maximum at 1268 nm), the effect of deuterated solvent on the emission intensity, and the effect of singlet oxygen quenchers confirmed the production of V-BrPO catalyzes the halogenation of suitable organic substrates (e.g., monochlorodimedone (MCD)), by catalyzing the oxidation of bromide by hydrogen peroxide. In the absence of an organic substrate, V-BrPO catalyzes the halide-assisted disproportionation of hydrogen peroxide forming dioxygen, determined by oxygen-probe techniques. V-BrPO did not exhibit direct catalase activity but required bromide or iodide in addition to hydrogen peroxide to effect dioxygen formation. The rate of dioxygen formation in the absence of MCD (0.1 M Br...