The cytochrome P450s metabolize a wide variety of xenobiotic and endogenous compounds. We apply biochemical, biophysical and computational approaches to examine the structure-function relationships which govern the interactions of P450s with substrates, inhibitors, and membrane components. Since these interactions modulate P450 activity, elucidation of their molecular mechanism will aid in a) clarifying the mechanism of P450 mediated drug and carcinogen metabolism, b) defining the role of individual P450s in the metabolism of endogenous and environmental chemicals and c) development of specific P450 inhibitors. We employed the flash photolysis technique to study the kinetics of CO binding to P450s in a natural biological membrane environment, as a unique probe of P450 conformation/dynamics and P450-substrate interactions. The interactions of various drugs and carcinogens with several rat and human P450s were thus assessed, and provided new insights into their modes of binding. Of particular interest is the finding that both 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 provides a basis for P450 recognition of a wide array of structurally diverse substrates. In addition, our results reveal that flavones, a class of dietary phytochemicals, differentially interact with and modulate the activity of specific P450s. We employed computer-aided homology modeling to generate a mammalian P450 model which was used to predict P450 recognition sites for NADPH cytochrome P450 reductase. Synthetic peptides corresponding to these regions on P450 2B1 were prepared and inhibited the P450-reductase interaction. In addition, the substrate binding site of this model was consistent with the known substrate specificity of this P450. This model thus successfully predicts both reductase and substrate binding domains of a mammalian P450.
