Fluorescent Probes for Cytochrome P450 Structural Characterization and Inhibitor Screening

Abstract

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (K_d = 0.83 μM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (K_d ≈ 0.02 μM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 Å resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B‘, F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.