Structural Studies on Nitric Oxide Synthase

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This proposal centers on structure-function relationships in heme proteins with a special emphasis on the heme thiolate enzymes cytochrome P450 and nitric oxide synthase (NOS). P450s play critical roles in drug detoxification, steroid biosynthesis, and in the oxidative assimilation of various natural organic compounds by microorganisms. With P450s our efforts currently are focused on the interaction between P450s and their redox partners. There are only 3 crystal structures of a P450-partner complex and one of these, the complex formed between P450cam and its redox partner (a ferredoxin called Pdx), shows that Pdx induces a large structural change in P450cam that we hypothesize is required for proton coupled electron transfer. P450cam is quite specific for Pdx and no other ferredoxin or related protein can support P450cam catalysis. The goal now is to ask whether or not this property is a more general feature of P450s and if not, why not. What is the biological advantage for such a high level of control? To probe these questions we plan to study in depth other P450 redox partners to better understand how redox partner binding effects the proton coupled electron transfer reaction. Our studies on P450s also extend to mammalian P450s and especially P4503A4, the most abundant and important human P450 for drug metabolism. Our goal here is to develop a pharmacophore using novel inhibitors that probe the dynamics and adaptability of the P4503A4 active. This will provide important information on drug-drug interactions and allosteric mechanisms in P4503A4. With NOS, our efforts center on structure-based inhibitor/drug design. The overproduction of NO is well known to be associated with a number of pathological conditions and we currently are focusing on neurodegenerative diseases, melanoma, and pathogenic bacteria. In each of these 3 cases we know that inhibiting NO production by blocking NOS has potential therapeutic benefits. We now are using a broad range of approaches toward developing selective NOS inhibitors for each of the 3 potential targets. One final area is the structural biology of heme transport in bacterial pathogens. Certain bacterial pathogens must acquire host iron by taking up free heme followed by heme degradation and release of iron. This requires a complex transport system involving several proteins many of which are membrane bound. The goal here is to work out the structural biology of heme transport and especially to better understand the many protein-protein interactions required for successful delivery of heme from hemoglobin to the bacterial cytoplasm.