Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate

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 1 Ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9 x 10(10) s(-1), 7.2 x 10(10) s(-1) and 1.0 x 10(12) s(-1). For the model iron porphyrin, FeTMPyP, a radical lifetime of 81 Ps and a rebound rate of 1.2 x 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-center dot] 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. (C) 2010 Elsevier Inc. All rights reserved.