Through the research outlined in this proposal we seek a precise understanding of biological substrate hydroxylation, the interaction of iron-tetraphyrroles with terminal oxidants, particularly the reductive cleavage of oxygen and nitrogen compounds, and the related concomitant electron transfer processes. The primary system in these investigations is the cytochrome P450 mixed function oxidase, which plays a central and crucial role in mammalian, plant, and microbe metabolism. Primary reactions catalyzed by P450 monoxygenases of immediate relevance include the detoxification of ingested environmental toxins and pollutants, carcinogen activation and deactivation carried out in the human liver, and steroid hormone synthesis in adrenal and other tissues. All of these monoxygenation reactions reduce atmospheric O2 producing peroxy anion and monoxygenated substrate. Central questions as to the mechanisms of these important reactions are the mode of regulation and control of biological activity, the precise chemistry of carbon substrate and oxygen activation related to the catalytic event, the mechanistic details surrounding concerted inter- and intra-protein electron transfer, and the role of multi-enzyme complexes in catalytic oxygenation and redox movement. Systems to be utilized include the hepatic microsomal P450 detoxification hydroxylase and the camphor monoxygenases analogous to the adrenal 11-beta mixed function oxidase on the aldosteronepathway. Through these efforts, the fundamental and current ideas, techniques, methods, and theories of chemistry, biology, and physics will be brought to bear in concerted fashion on some of the most important problems of modern molecular biochemistry, providing insight into the inner workings of these processes and aiding therapeutic prescription through detailed knowledge of regulation.
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