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Kinetic Isotope Effects in Hydroxylation Reactions Effected by Cytochrome P450 Compounds I Implicate Multiple Electrophilic Oxidants for P450-Catalyzed Oxidations^†

Xin Sheng^‡, Haoming Zhang^§, Paul F. Hollenberg^§ and Martin Newcomb*^‡

Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, and Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109

Biochemistry, 2009, 48 (7), pp 1620–1627

DOI: 10.1021/bi802279d

Publication Date (Web): January 30, 2009

†

This work was supported by grants from the National Institutes of Health (GM48722 to M.N. and CA16954 to P.F.H.).

, * To whom correspondence should be addressed. Telephone: 312-413-2106. Fax: 312-996-0431. E-mail: men@uic.edu., ‡

University of Illinois at Chicago.

, §

University of Michigan.

Abstract

Kinetic isotope effects were measured for oxidations of (S,S)-2-(p-trifluoromethylphenyl)cyclopropylmethane containing zero, two, and three deuterium atoms on the methyl group by Compounds I from the cytochrome P450 enzymes CYP119 and CYP2B4 at 22 °C. The oxidations displayed saturation kinetics, which permitted solution of both binding constants (K_bind) and first-order oxidation rate constants (k_ox) for both enzymes with the three substrates. The binding constant for CYP2B4 Compound I was about 1 order of magnitude greater than that for CYP119 Compound I, but the oxidation rate constants were similar for the two. In oxidations of 1-d₀, k_ox = 10.4 s⁻¹ for CYP119 Compound I, and k_ox = 12.4 s⁻¹ for CYP2B4 Compound I. Primary kinetic isotope effects (P) and secondary kinetic isotope effects (S) were obtained from the results with the three isotopomers. The primary KIEs were large, P = 9.8 and P = 8.9 for CYP119 and CYP2B4 Compounds I, respectively, and the secondary KIEs were small and normal, S = 1.07 and S = 1.05, respectively. Large intermolecular KIEs for 1-d₀ and 1-d₃ of k_H/k_D = 11.2 and 9.8 found for the two Compounds I contrast with small intermolecular KIEs obtained previously for the same substrate in P450-catalyzed oxidations; these differences suggest that a second electrophilic oxidant, presumably iron-complexed hydrogen peroxide, is important in cytochrome P450 oxidations under turnover conditions.