Cytochrome P450 and P450 11A1 retain Compound I (FeO) chemistry with electrophilic substrates poised for Compound 0 (FeO) reactions.

The catalytic cycle of cytochrome P450 (P450) enzymes involves ferric peroxide anion (FeO, Compound 0) and perferryl oxygen (FeO, Compound I) intermediates. Compound I is generally viewed as responsible for most P450-catalyzed oxidations, but Compound 0 has been implicated in the oxidation of some carbonyl compounds, particularly deformylation reactions. We considered the hypothesis that Compound 0 could also attack other electrophilic carbon atoms and accordingly positioned keto groups at preferred hydroxylation sites of substrates for two P450s with well-defined catalytic reactions, bacterial P450 (102A1), and human P450 11A1. The predicted products of Compound I and Compound 0 reactions were analyzed. With the normally preferred ω-1 site blocked, P450 oxidized 12-oxotridecanoic acid (12-oxo C13:0) only at the ω-2 position (yielding 11-hydroxy,12-oxotridecanoic acid), indicative of a Compound I oxidation. P450 11A1 is highly selective for catalyzing the 22R-hydroxylation of cholesterol (and some other sterols) in the first step of its overall side-chain cleavage reaction. With 22-oxocholesterol as the substrate, P450 11A1 (slowly) generated only 23-hydroxy,22-oxocholesterol, indicative of Compound I oxidation. Neither P450 generated the products expected from nucleophilic Compound 0 reactions. We conclude that the strategic placement of electrophilic oxo substituents at sites of substrate hydroxylation failed to divert the oxidation mechanism to a Compound 0 pathway with either enzyme. Instead, the Compound I mechanism-blocked at the preferred reaction site-was redirected to neighboring carbons, suggesting that the basis for Compound 0-mediated reactions lies in chemical properties of the enzyme rather than those of the substrate.