Through the research outlined in this proposal we seek a precise understanding of the mechanisms of biological recognition, the collective interactions of organized protein systems, and the detailed chemistry and physical operation of oxygenase and oxidase catalysis. Focus will be made 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, as 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 that we proposed to investigate include: the mode of the regulation and control of biological activity, the precise chemistry involved in the activation of oxygen and substrate and the identity of metal-oxygen-carbon intermediates in the catalytic event, the detailed physical description of inter- and intra-protein electron and proton transfer, and the role of multi-enzyme complexes and conformer equilibria in catalytic oxygenation and redox movement. A particular goal is the delineation of the collective properties of organized assemblies of biological macromolecules and their role in determining function. The metalloprotein systems to be utilized in these investigations have known solution and/or crystal structures, or will be defined through support from this grant. All are expressible either through cloned or totally synthetic genes at gram levels in common microbial hosts. Specific metalloproteins to be investigated include two P-450 monoxygenases and their associated electron transfer partners which are found in the soil Pseudomonads, human hemoglobins, cytochromes b5 and c, and the four-helical bundle cytochrome b562. Detailed knowledge is sought with regard to the molecular mechanisms of protein-protein and protein-small molecule recognition, the means by which cooperative subunits communicate, the role of protein dynamics in the control of biological redox events, and the questions of self-recognition involved in protein folding and subunit assembly. 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 modem 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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