Through the research outlined in this competitive renewal, we seek a precise understanding of the mechanisms of biological oxidations through knowledge of the detailed chemistry and physical operation of oxygenase and oxidase catalysis. Proposed for investigation are the heme protein reductases, termed cytochromes P450, which play central and crucial roles in mammalian metabolism and human health. Understanding the functioning of the cytochrome P450 systems is critical to defining the control points of drug metabolism, pro- carcinogen processing, and the regulation and method for hormonal control of development and gene regulation. Our approach is problem rather than technique or system focused. We choose amongst the 800+ known P450 sequences those cytochrome systems where there is substantial structural and functional background information. One such system is P450cam, from Pseudomonas putida, which catalyzes the regiospecific hydroxylation of camphor and with which we have been working since the inception of this grant. Other structurally defined P450 systems to be employed include P450(BM3) and P450eryF for which we have the genetic basis in hand to utilize the powerful tools of recombinant DNA technology to express a variety of variant proteins. We have focused our next funding period on four highly specific aims. First, to obtain further high resolution structural information from xray crystallography and physical - chemical definition of the intermediate states of iron and oxygen involved in the P450 catalytic cycle. Second, to further define and elucidate the mechanisms of acid base catalysis and proton delivery that are responsible for the unique chemistry displayed by the P450 cytochromes. Third, to understand the controlling features of the branch points in the catalytic cycle where the path is open to productive commitments for catalysis as well as various abortive autoxidative processes that leak reducing equivalents into the cell and form potentially toxic reduced dioxygen species. Finally, we seek to understand the role of amino acid residues proximal to the heme plane in control of redox movement in protein-protein complexes and the chemical structure/activities of intermediate states. Through these interdisciplinary efforts we hope 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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