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J Biol Chem, Vol. 273, Issue 27, 17036-17049, July 3, 1998

Identification of the Binding Site on Cytochrome P450 2B4 for Cytochrome b₅ and Cytochrome P450 Reductase

Angela Bridges, Larry Gruenke^¶, Yan-Tyng Chang, Ilya A. Vakser^**, Gilda Loew, and Lucy Waskell^¶

From the ^¶ Department of Anesthesia and the Liver Center, University of California, San Francisco, California 94143, the  Department of Anesthesia, Veterans Affairs Medical Center, San Francisco, California 94121, the  Molecular Research Institute, Palo Alto, California 94304, and the ^** Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b₅. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b₅ and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b₅. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b₅ and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.

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