Kinetic Solvent Isotope Effect in Human P450 CYP17A1-Mediated Androgen Formation: Evidence for a Reactive Peroxoanion Intermediate

Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydro­epi­andro­sterone, preg­nenol­one is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17–C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C–C “lyase” activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1. Our experiments revealed the expected small positive (∼1.3) isotope effect for the hydroxylase chemistry. However, a surprising result was the large inverse isotope effect (∼0.39) observed for the C–C bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion.