Through the research outlined in this proposal we seek a precise understanding of the mechanisms of biological oxidation, the structure of collective assemblies of organized protein and nucleic acid systems, and the detailed chemistry and physical operation of oxygenase and oxidase catalysis. A major focus will be on metalloprotein systems, including the cytochrome P-450 dependent mixed function oxidases which play central and crucial roles in mammalian, plant, insect, viral, and microbial metabolism. Biotransformations catalyzed by the P-450 monoxygenases of primary medical relevance include the detoxification of ingested toxins and pollutants, carcinogen activation and deactivation as carried out in the human liver and lung, and steroid hormone biosynthesis in the adrenals, testes, and ovaries. All P-450 catalyzed monoxygenase reactions reduce atmospheric dioxygen producing water and a monoxygenated substrate. Central questions relating to the mechanisms of these important biological oxidations include the precise chemistry involved in activation of oxygen and substrate and the identity of heretofore hypothetical metal-oxygen- carbon intermediates in the catalytic event, the detailed physical description of inter- and intra-protein electron and proton transfer, and the structure of multi-enzyme membrane complexes involved in catalytic oxygenation and redox movement. A second important goal is the development and execution of methodologies for the determination of biological structures in the 5 nm - 500 nm size range, and determination of information defining the collective properties of organized assemblies of biological macromolecules and their role in determining function. We term this size regime from 5 nm to 500 nm the 'mesoscale' domain. The actual structure of mammalian P-450's, their topological isozyme dependent distribution in membranes, the complexes of membrane bound redox components - all lie in the mesoscale region. The quantitation of forces between ordered metalloprotein structures and the direct visualization of protein dynamics involved in the control of substrate binding and biological redox events, can most directly be answered by directly attacking the problems of mesoscale structure determination. Additionally, some of the most interesting processes in biology utilize complexes of protein, nucleic acid, lipid, and sugar that occupy this mesoscale domain. Providing the important structural and functional information of specific aggregates of nucleic acid and protein which make up ribosomes, spliceosomes, and transcription complexes is an important goal of this grant. Through these efforts, fundamental ideas and techniques will be advanced, including aspects of molecular visualization, novel aspects of chemistry, physics, biology, and physiology in a truly interdisciplinary fashion in an effort to shed light on some of the most important problems of modern molecular biochemistry, providing insight into the inner workings of these processes and aiding therapeutic prescription and understanding of disease states through detailed mechanistic knowledge.
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