The focus of this project is the characterization of structure-function relationships the cytochromes P-450. A cross-linking study of intact rat liver microsomal membranes revealed that P-450c, which metabolizes polycyclic hydrocarbons, is specifically associated with both P-450 reductase as well as P450 2a, which metabolizes testosterone. These results support the membrane cluster model in which P-450s and P-450 reductase both exist as stable complexes rather that as monomers. Such specific P-450 interactions may influence the secondary metabolism of P-450 substrates. The active site structure of P-450c was examined in binding studies using the substrate benzopyrene (BP). BP fluorescence was quenched upon binding to either microsomal or purified P-450c. The fluorescence of the BP-P-450c complex was used to probe the interaction between BP substrate and active site heme. Addition of P-450 reductase promoted a closer association of heme and BP, which demonstrates that the reductase does not function solely as an electron carrier. Flash photolysis experiments of CO recombination with the P-450 heme yielded parallel kinetic data on the effect of BP on active site dynamics. A three dimensional model of mammalian P-450 is being developed using both theoretical and experimental approaches. The latter includes identification of exposed surface regions on P-450s by protease digestion experiments, and the use of antibodies to synthetic P-450 peptides to identify functionally significant sequences.
