Cytochrome P450 monooxygenases are critical in many physiological processes, including steroid biosynthesis, drug metabolism and activation, and xenobiotic degradation. Structural and dynamic information concerning these enzymes is critical for understanding their activity (typically hdryoxylation of unactivated carbons), and will play a vital role in predicting the behavior of inhibitors and substrates as a part of drug design. Despite the large numbers of P450 enzymes found in living organisms, relatively few have been crystallized, and methodology for rapid characterization of active sites of P450s and their interactions with substrates and inhibitors is lacking. This proposal describes the first application of high-resolution multidimensional nuclear magnetic resonance (NMR) methods to P450 enzymes. Extensive sequential 1H, 15N and 13C resonance assignments have been made in cytochrome P450cam (CYP101). Residues in the active site of CYP101 can be rapidly distinguished by comparison of paramagnetic and diamagnetic forms of the enzyme. Active site resonances are observed only in the diamagnetic form. Assignments have been used to monitor the interactions of CP101 with its physiological redox partner and effector, putidaredoxin (Pdx). Based on these observations, a new model for effector activity of Pdx has been proposed. ? ? During the next period of this project, the sequential assignments of CYP101 will be completed in multiple forms of the enzyme, local dynamics will be characterized as a function of oxidation state, substrate binding and effector binding in order to shed further light on the mechanism of """"""""uncoupling"""""""", in which reducing equivalents are lost as superoxide, peroxide and water without turnover of substrate. Methodology for applying NMR to the characterization of active sites of other P450 enzymes will be tested, with CYP102 (cytochrome P450 BM-3) and CYP3A4, an important human P450, as test cases. Methodology developed during the current period for NMR structural characterization of metalloproteins will be further refined. This includes two-dimensional NMR methods that can be used near paramagnetic centers in proteins, and extraction of structural information from magnetic anisotropy data such as residual dipolar couplings and dipolar shifts. ? ?
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