Of the known biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. Rapid progress in recent years as revealed a P450 gene superfamily with numerous members in bacteria, fungi, plants, invertebrates, and vertebrates, including the human. These enzymes are of great interest from a fundamental point of view because of their remarkable versatility and reaction mechanisms involving the generation of a powerful oxidant from molecular oxygen, and also from the viewpoint of their biomedical relevance. Because the substrates include physiologically important compounds such as steroids, bile acids, fatty acids, prostaglandins, retinoids, biogenic amines, and lipid hydroperoxides, as well as a host of """"""""environmental chemicals,"""""""" it is no exaggeration to say that improved knowledge of P450 function will contribute to progress in drug metabolism and design, as well as to better insights into chemical carcinogenesis, alcoholism, endocrine disorders, and oxidative stress. Our main objectives are: 1. To obtain detailed evidence on the important question of whether multiple oxygenating species contribute to substrate oxidation by cytochrome P450. Mutant P450s blocked in proton delivery to the active site will be examined for their rates of oxidation of various substrates as an indication of the role of peroxo-hydroperoxo- and oxenoid-iron as discrete oxidants. Attempts will be made to characterize these labile species by chemical and physical methods in order to correlate the apparent steady state level of a particular oxidant with the catalytic rates. In addition, the possibility will be examined that the levels of the oxidants are subject to regulation by effectors, including cytochrome b5 and flavonoids. 2. To continue our attempts to obtain one or more mammalian P450s in a crystalline form suitable for structure determination by x-ray diffraction. Various full-length and truncated P450s will be studied for this purpose. 3. To determine the role of phospholipids and other membrane components in influencing the formation of binary and ternary complexes of P450, NADPH-P450 reductase, and cytochrome b5, and in altering the rates and specificities of substrate oxidation in lipid bilayers. The effect of lipid composition in membrane bilayers. The effect of lipid composition in membrane bilayers will be examined with respect to single-phase fluid membranes with co-existing solid-phase obstacles to protein diffusion.
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