Cytochrome P450s are responsible for metabolism of numerous foreign compounds that are ingested or absorbed in the form of drugs and environmental contaminants. It is generally believed that 50 to 200 P450 forms may be expressed in a given organism. Each P450 form can metabolize numerous structurally unrelated compounds. A single compound can also sometimes be metabolized by numerous P450 forms. Frequently, certain chemicals are converted to high energy electrophiles that escape from the P450 active site and can damage DNA. We have taken advantage of two results of P450 evolution to determine the structure-function relationship of their active sites in i) closely related subfamily members that share considerable sequence similarities but have differing catalytic activities, and ii) allelic variants with differing catalytic activities but only a few amino acid differences. We have made chimeric P450s through use of cDNA manipulation and cDNA expression to determine which regions of the P450 proteins determine substrate specificities. Further studies using site directed mutagenesis will be undertaken to determine which amino acid residues are responsible for specific catalytic activities. We are also carrying out in situ reconstitution studies to determine the requirements of P450 forms for NADPH-P450 oxidoreductase and cytochrome b5 using the vaccinia virus expression system. Hepatoma cells are coinfected with recombinant vaccinia containing P450, NADPH-P450 oxidoreductase and cytochrome b5 cDNAs. Catalytic activities are then measured using intact cells and subcellular fraction.