The focus of this project is the characterization of structure-function relationships for the cytochromes P450. A cross-linking study of rat liver microsomes revealed that P450c is specifically associated with both P450 2a and P450 reductase. Benzo[a]pyrene (BP) metabolism data indicate that P450 2a further metabolizes one of the phenol metabolites generated by P450c. These results support the membrane cluster model in which P450s and P450 reductase exist as stable complexes rather than as monomers. The binding of BP to purified P450c was examined by fluorescence energy transfer and polarization techniques. The results showed that the role of reductase extends beyond that of an electron donor since it also enhances binding of BP to P450c. Monoclonal antibody (MAb) 1-7-1 inhibited reductase-mediated changes, which suggests binding near the reductase receptor region of P450c. A thermodynamic study of the interaction of benzphetamine with microsomal P450b revealed that MAb 4-7-1 alters the P450 spin equilibrium of substrate-bound but not substrate-free P450b, and that a membrane phase transition near 200C alters the binding of benzphetamine to P450b. A three dimensional model of mammalian P450 is being developed using both theoretical and experimental approaches. The latter includes identification of exposed surface regions on P450s by protease digestion experiments, cross-linking experiments to identify spatially proximate regions within the P450 primary sequence, and the use of antibodies to synthetic P450 peptides to identify functionally significant sequences. A membrane topography study involving microsomal P450c, P450b, and P450j revealed a proteolytically sensitive region common to all three P450s. This region has been identi- fied and corresponds to a predicted turn in a relatively exposed region of the P450 surface. This finding is consistent with the concept of a common tertiary structure for different classes of mammalian P450s. Experiments are underway to evaluate whether the structural integrity of this region is essential for substrate binding and activity.
