title: Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate.
abstract: The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1. The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol, was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios of rearranged and unrearranged products to be 120, 13 and 1ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9×10(10)s(-1), 7.2×10(10)s(-1) and 1.0×10(12)s(-1). For the model iron porphyrin, FeTMPyP, a radical lifetime of 81ps and a rebound rate of 1.2×10(10)s(-1) were determined. These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron porphyrin suggest that for these systems a considerable amount of the intermediate carbon radical is trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon radical in [Fe(IV)-OH ()R] can compete with cage collapse to form a C-O bond. The results could be significant with regard to our understanding of iron-catalyzed C-H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
authors.forename: Harriet L R
authors.initials: HL
authors.lastname: Cooper
authors.forename: John T
authors.initials: JT
authors.lastname: Groves
mesh_terms: D000818:Animals; D003521:Cyclopropanes / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D008665:Metalloporphyrins / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D000439:Pentanols / Q000378:metabolism; D010422:Pentanones / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
chemical_list: D003521:Cyclopropanes; D008665:Metalloporphyrins; D000439:Pentanols; D010422:Pentanones; C417967:1,1,2,2-tetramethylcyclopropane; D019392:Cytochrome P-450 CYP2E1; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C080344:diacetone alcohol; D010100:Oxygen