Abstract
Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized benzphetamine to norbenzphetamine in high yield in bulk studies. In direct kinetic studies of benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (K(bind)) for each. The first-order oxidation rate constants (k(ox)) were comparable for Compounds I of CYP2B4, the E301Q mutant, and the T302A mutant, whereas the k(ox) for Compound I of the F429H mutant was reduced by a factor of 2. CYP119 Compound I, studied for comparison purposes, reacted with benzphetamine with a binding constant that was nearly an order of magnitude smaller than that of CYP2B4 but a rate constant that was similar. Substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions, and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.