Peripheral Ligand Binding and Allostery in Cytochrome P450 3A4

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The mechanisms of ligand binding and allostery in the major human drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, Fluorol‐7GA (F7GA), as a model substrate. Incorporation into the enzyme of an SH‐reactive FRET donor, pyrene iodoacetamide (PIA), allowed us to use FRET from the pyrene donor to the F7GA acceptor to detect its binding to CYP3A4. Cooperativity of the interactions detected by FRET indicates that the enzyme possess at least two F7GA‐binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of the sites we studied FRET in a series of mutants bearing PIA at different positions, and measured the distances from each of the two binding sites to the donor. Our results demonstrate the presence of a high‐affinity binding site at the periphery of the enzyme. Molecular modeling and docking show that the most probable location of this site is at the distal surface of the enzyme, similar to the site of peripheral binding of progesterone in a prior X‐ray crystal structure. The binding of F7GA to this site causes a substantial spin shift and is a prerequisite for subsequent binding of F7GA in the active site. Our results demonstrate the importance of peripheral ligand binding and substrate‐induced conformational changes in substrate recognition. (Supported by GM054995).