title: Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.
abstract: 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 (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) 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 FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6β-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded.
authors.affiliation: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and the Division of Metabolism, Diabetes and Endocrinology, Departments of Internal Medicine and Pharmacology, University of Michigan, Ann Arbor, Michigan 48019.
authors.forename: Francis K
authors.initials: FK
authors.lastname: Yoshimoto
authors.affiliation: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and.
authors.forename: Eric
authors.initials: E
authors.lastname: Gonzalez
authors.affiliation: the Division of Metabolism, Diabetes and Endocrinology, Departments of Internal Medicine and Pharmacology, University of Michigan, Ann Arbor, Michigan 48019.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
authors.affiliation: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and f.guengerich@vanderbilt.edu.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D006801:Humans; D006900:Hydroxylation; D007553:Isotope Labeling; D010100:Oxygen / Q000737:chemistry*; D011374:Progesterone / Q000737:chemistry*; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry*
chemical_list: D011374:Progesterone; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; D010100:Oxygen
keywords: cytochrome P450; enzyme catalysis; enzyme mechanism; mass spectrometry (MS); nuclear magnetic resonance (NMR); oxygenase; steroid; steroidogenesis