Mechanism of 17α20-Lyase and New Hydroxylation Reactions ofHuman Cytochrome P450 17A1

摘要

Cytochrome P450 (P450) reactions can involve C–C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2⁻) versus perferryl (FeO³⁺, compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one ¹⁸O atom (from ¹⁸O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127–132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2–12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO³⁺ species) but not by H2O2. We propose three mechanisms that can involve the FeO³⁺ entity and that explain the ¹⁸O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found toperform 16-hydroxylation reactions on its 17α-hydroxylated products toyield 16,17α-dihydroxypregnenolone and progesterone, suggesting thepresence of an active perferryloxo active species of P450 17A1 when its lyasesubstrate is bound. The 6β-hydroxylation of16α,17α-dihydroxyprogesterone and the oxidation of both16α,17α-dihydroxyprogesterone and16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were alsoobserved. We provide evidence for the contribution of a compound I mechanism,although contribution of a ferric peroxide pathway in the 17α,20-lyasereaction cannot be excluded.