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QM/MM Study of the Active Species of the Human Cytochrome P450 3A4, and the Influence Thereof of the Multiple Substrate Binding

Supporting Info

Dan Fishelovitch,^† Carina Hazan,^‡ Hajime Hirao,^‡ Haim J. Wolfson,^§ Ruth Nussinov,^† and Sason Shaik*^‡

Department of Human Genetics, Sackler Institute of Molecular Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel, Institute of Chemistry and the Lise-Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel, School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel, and SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, NCIFrederick, Building 469, Room 151, Frederick, Maryland 21702

J. Phys. Chem. B, 2007, 111 (49), pp 13822–13832

DOI: 10.1021/jp076401j

Publication Date (Web): November 17, 2007

†

Sackler Faculty of Medicine, Tel Aviv University.

‡

The Hebrew University of Jerusalem.

Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University.

NCIFrederick.

To whom correspondence should be addressed. Telephone: (972)-2-6585909. Fax: (972)-2-6584680. E-mail: sason@yfaat.ch.huji.ac.il.

Abstract

Cytochrome P450 3A4 is involved in the metabolism of 50% of all swallowed drugs. The enzyme functions by means of a high-valent iron−oxo species, called compound I (Cpd I), which is formed after entrance of the substrate to the active site. We explored the features of Cpd I using hybrid quantum mechanical/molecular mechanical calculations on various models that are either substrate-free or containing one and two molecules of diazepam as a substrate. Mössbauer parameters of Cpd I were computed. Our major finding shows that without the substrate, Cpd I tends to elongate its Fe−S bond, localize the radical on the sulfur, and form hydrogen bonds with A305 and T309, which may hypothetically lead to Cpd I consumption by H-abstraction. However, the positioning of diazepam close to Cpd I, as enforced by the effector molecule, was found to strengthen the NH···S interactions of the conserved I443 and G444 residues with the proximal cysteinate ligand. These interactions are known to stabilize the Fe−S bond, and as such, the presence of the substrate leads to a shorter Fe−S bond and it prevents the localization of the radical on the sulfur. This diazepam−Cpd I stabilization was manifested in the 1W0E conformer. The effector substrate did not influence Cpd I directly but rather by positioning the active substrate close to Cpd I, thus displacing the hydrogen bonds with A305 and T309, and thereby giving preference to substrate oxidation. It is hypothesized that these effects on Cpd I, promoted by the restrained substrate, may be behind the special metabolic behavior observed in cases of multiple substrate binding (also called cooperative binding). This restraint constitutes a mechanism whereby substrates stabilize Cpd I sufficiently long to affect monooxygenation by P450s at the expense of Cpd I destruction by the protein residues.

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