The cytochrome P450s oxidize a wide variety of xenobiotic and endogenous compounds. Biochemical, biophysical and computational approaches were applied to examine the structure-function relationships which govern the interactions of P450s with substrates, membrane lipids and microsomal proteins. The CO binding kinetics was used as a probe of P450 conformation and dynamics, to define the effect of various drugs and carcinogens on P450s. Of particular interest is the finding that both human P450 1A1, which metabolizes carcinogens, and P450 3A4, which metabolizes a variety of important drugs, are composed of multiple conformers with distinct substrate specificities. This finding was used to elucidate 7,8-naphthoflavone activation of P450 3A4 and inhibition of P450 1A1, and the mechanism of quinidine inhibition of P450 3A4 mediated nifedipine metabolism. In addition, CO binding kinetics was applied to examine the differential binding of erythromycin to rat P450s 3A1 and 3A2, which exhibit 89% sequence similarity. The results indicate a model of the P450 substrate binding site in which erythromycin forms a more rigid complex with P450 3A1 than P450 3A2. These results show that CO binding kinetics can distinguish among closely related P450s in the same microsomal membrane. We employed molecular modeling to generate a P450 2B1 model. P450 recognition surfaces for NADPH cytochrome P450 reductase were predicted, and the corresponding peptides were prepared and assessed for their ability to inhibit the P450-reductase interaction. The most potent peptide inhibitors were topographically derived from spatially proximate P450 sequences in the C and L-helices and the meander region. The model also suggests a membrane binding domain which consists of the amino terminal region, the pre-A helix region and the F-G loop. In addition, when several known substrates were docked into the substrate binding site, the observed substrate-P450 interactions were consistent with the known substrate specificity of P450 2B1. This model thus suggests reductase, membrane and substrate binding domains of this P450.
