Membrane-associated cytochrome P450s (P450s) work as members of a large enzymatic complex (the "P450 monooxygenase system") that catalyze the oxidation of a variety of exogenous and endogenous compounds, particularly the detoxification or bioactivation of thousands of pharmaceutical compounds. Allelic variants of P450s are also implicated in the development of certain cancers and are considered important biomarkers for cancer susceptibility. Catalytic activities of cytP450 are modulated by the microsomal membrane-bound cytochrome b5 (cytb5). The influence of cytb5 on cytP450 activity has been shown to depend on the cytP450 isozyme and the substrate involved. Remarkably, cytb5 enhances some catalytic reactions of cytP450 but does not affect or even inhibits others. The main objective of the proposed research is to obtain an in-depth understanding of the molecular basis of the effects of cytb5 on cytP450 activity by determining the structural interactions between the membrane-bound full-length rabbit cytP4502B4 and cytb5. Structure of the membrane-bound ~70-kDa cytP450-cytb5 complex will provide insights into the mechanism by which cytb5 regulates the enzymatic function of cytP450. We have already determined the first full-length membrane- bound rabbit cytb5 structure. We propose to use a combination of solution and solid-state (both static and magic angle spinning) NMR techniques to obtain structural and dynamic interactions from the rabbit cytP450- cytb5 complex embedded in model membranes such as bicelles, lipid vesicles and nanodiscs. Mutational and functional studies will also be carried out to identify the residues in the interacting interface of the cytP450- cytb5 complex. The effect of selected substrates on the cytP450-cytb5 complex will be investigated using NMR and functional measurements. While our preliminary results demonstrate that NMR measurements on the cytP450-cytb5 complex embedded in bicelles are feasible, we also plan to use nanodisc as it would enable the investigation of the roles of membrane components on the structure and dynamics of the cytP450-cytb5 complex by varying the membrane composition. After completing our investigation on rabbit cytP4502B4, we will use other cytP450s (such as cytP4503A4, cytP4502D6 and cytP45017A1) to compare their structures and membrane orientations and to further understand the role of cytb5 on the function of cytP450.