Regulation of carcinogen activation is affected by interaction of cytochrome P450 and cytochrome P450 reductase, as well as by other factors. We propose to define the factors which govern the interaction of reductase and P450 and thereby influence carcinogen activation. The factors include the structural domains of P450 and reductase, i.e. membrane binding domains, substrate binding domains, and the binding domains which interact through complementary charge pairing. We will define these domains through a series of chemical modifications of the proteins and a series of site directed mutagenesis studies. In each aspect, the aim of the study is to specify which amino acid residues in a particular domain govern the interaction of that domain with substrate, membrane, or pair protein and how these residues/domains act in concert to regulate carcinogen activation. In pursuit of these goals, we will define the amino acid residues of P450s responsible for charge pairing with critical carboxyl groups of the reductase. We will define the amino acid residues involved in substrate binding to P450 using a photoaffinity substrate analogue and site directed mutagenesis. We will define the NADPH binding region and the flavin binding regions of P450 reductase. These parameters will be evaluated for their role in overall control of substrate metabolism. For example, we will determine how substrate binding to P450 affects the interaction of P450 with reductase.
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