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J. Biol. Chem., Vol. 280, Issue 24, 22697-22705, June 17, 2005

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Calibration of the Channel That Determines the -Hydroxylation Regiospecificity of Cytochrome P4504A1

CATALYTIC OXIDATION OF 12-HALODODECANOIC ACIDS^*

Xiang He, Max J. Cryle, James J. Ortiz De Voss, and Paul R. de Montellano¶

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 and the Department of Chemistry, University of Queensland, St. Lucia, Brisbane, Australia 4072

The fatty acid -hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in H₂¹⁸₂O show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O₂. The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an -1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the -aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[²H]₂-12-chlorododecanoic acid causes a 2–3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl » I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 Å but smaller than 4.30 Å.

Received for publication, March 9, 2005 , and in revised form, April 7, 2005.

^* This work was supported by Grant GM25515 from the National Institutes of Health (to P. R. O. M.), a University of Queensland Francine Kroesen Travel Award (to M. J. C.), and Grant DP0210635 from the Australian Research Council (to J. J. D. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: University of California, Genentech Hall N572D, 600 16th St., San Francisco, CA 94143-2280. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail: ortiz{at}cgl.ucsf.edu.

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