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J. Biol. Chem., Vol. 283, Issue 11, 7293-7308, March 14, 2008

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Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

HIGHLY COOPERATIVE PYRENE HYDROXYLATION AND MULTIPHASIC KINETICS OF LIGAND BINDING^*

Christal D. Sohl¹, Emre M. Isin¹², Robert L. Eoff, Glenn A. Marsch, Donald F. Stec^¶, and F. Peter Guengerich³

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, the Department of Physics, Grove City College, Grove City, Pennsylvania 16127-2104, and the ^¶Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of -naphthoflavone (NF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for NF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (NF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-NF complex show active site space for only one NF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.

Received for publication, November 29, 2007 , and in revised form, January 9, 2008.

^* This work was supported in part by United States Public Health Service Grants R37 CA090426 (to F. P. G.), T32 ES007028 (to C. D. S. and F. P. G.), F32 CA119776 (to R. L. E.), and P30 ES000267 (to F. P. G.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S10.

¹ Both authors contributed equally to this work.

² Present address: Biotransformation Section, Dept. of Discovery DMPK and Bioanalytical Chemistry, AstraZenecaR&DMölndal, SE-431 83 Mölndal, Sweden.

³ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 2200 Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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