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J. Biol. Chem., Vol. 282, Issue 37, 26865-26873, September 14, 2007

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The One-electron Autoxidation of Human Cytochrome P450 3A4^*

Ilia G. Denisov, Yelena V. Grinkova, Mark A. McLean, and Stephen G. Sligar^¶||1

From the Departments of Biochemistry and Chemistry, ^¶Center for Biophysics and Computational Biology, and ^||College of Medicine, University of Illinois, Urbana, Illinois 61801

Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450.

Received for publication, June 8, 2007 , and in revised form, July 2, 2007.

^* This work was supported by National Institutes of Health Grants GM31756 and GM33775. 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, 116 Morrill Hall, University of Illinois, 505 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-244-7395; Fax: 217-265-4073; E-mail: s-sligar{at}uiuc.edu.

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