Cytochrome P450 is a highly interesting biological catalyst from the viewpoint of fundamental enzymology and also because of its biomedical relevance. These enzymes are remarkably versatile in the number of substrates attacked, types of reactions catalyzed, multiplicity of oxygen donors, and variety of catalytic and regulatory mechanisms. In the past twenty-five years, increasingly rapid advances in our knowledge of the biochemistry and molecular biology of P450 have revealed a gene superfamily that is widespread among microorganisms, plant, and animals, with much structural homology evident among the isoforms in mammalian species, including the human. From the viewpoint of health relevance, it is clear that better knowledge of P450 function is essential for a sophisticated understanding of environmental toxicology, chemical carcinogenesis, drug design and metabolism, alcoholism, and disorders of steroid metabolism. Furthermore, the role of these cytochromes in the generation and utilization of oxygen radicals is relevant to a host of disorders thought to be associated with """"""""oxidative stress"""""""". Our goals are as follows: 1. To characterize by rapid scanning stopped flow spectrophotometry and other methods the proposed """"""""activated"""""""" oxygen and peroxide species involved in cytochrome P450-catalyzed monooxygenation reactions, including (a) the peroxide species formed by the addition of one electron to the ferrous-O2 complex; (b) the blue- shifted intermediate G formed from iodosobenzene and ferric P450 and thought to be FeV=O; (c) the proposed alkoxy or acyloxy radicals formed in the homolytic oxygen-oxygen bond cleavage of cumyl hydroperoxide, perbenzoic acid, and other peroxy compounds; and (d) the active resonance forms in the 2-electron reduction and cleavage of O2, presumably FeV=O or one or more of its mammalian microsomal P450s by removal of the highly hydrophobic NH2-terminal signal peptide or other hydrophobic segments or by the introduction of positively charged residues in these regions by site-directed mutagenesis, with the goal of generating forms that are monomeric, stable, catalytically active, and amenable to crystallization. The modified proteins will also be used in studying the complexities of membrane binding. 3. To continue to identify and purify new isozymes of P450 and to examine their catalytic specificity toward a variety of substrates, with particular emphasis on endogenous substrates, including steroids, eicosanoids, and retinoids.
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