Xenobiotic-metabolizing enzymes in the liver play key roles in the detoxification of lipophilic drugs, pollutants and toxins, and the bioactivation of procarcinogens to carcinogens. We propose to study the structures and active-site relationships of several cytochromes P450 isolated from rat liver microsomes, and the cytochrome P450 reductase isolated from pig and rabbit liver microsomes. The cytochrome P450 isozymes which form the basis of this study are the two induced by 3-methylcholanthrene, P450c and P450d, both of which are immunologically related. The structural studies include the determination of their primary sequences and the characterization of their shared antigenic determinants. Active site studies will be performed by labeling the enzymes with bromoacetoxy derivatives of substrates, and in the case of P450c, labeling the isozyme with the specific inhibitor acetonitrophenacyl bromide. The active site studies on the cytochrome P450 reductase include the affinity labeling of the FMN, FAD, and NADPH binding sites with the appropriate arylazido photoactive derivatives. We will study the interaction of cytochrome P450 with its reductase using antibody and peptide inhibitor probes directed at their postulated binding sites. Preliminary results suggests that these approaches will form a basis for an understanding of the structure-function relationships in these important enzyme systems.
