This project represents a continuing effort to determine the role of protein structure in protein-protein interaction and electron transfer in the P-450cam system from Pseudomonas putida. The P-450cam system consists of three soluble proteins, cytochrome P-450 cam, putidaredoxin (Pdx), a 2Fe-2S ferredoxin, and the flavin-containing NADH-dependent putidaredoxin reductase (PdR). The cytochrome catalyzed the 5-exo-hydroxylation of camphor by molecular oxygen, a reaction which requires two electrons, which are supplied to the cytochrome sequentially by Pdx, which is also required as an effector substrate turnover. The electrons are supplied to Pdx by PdR by oxidation of PdR-bound NADH. The camphor hydroxylase system is a good model for human P-450 enzymes involved in steroid hormone biosynthesis and processing of xenobiotics and carcinogens, which can result in carcinogen activation. The P. putida system has been studied in detail because it is easy to purify, all the components are soluble in their active forms, and the genes have been cloned for all components. This project aims to refine the NMR-derived solution structure of Pdx by obtaining more NOE and dihedral angle restraints using multidimensional 13C, 15N-edited NMR methods. The current structure was determined using 1H NMR methods, from which a limited number of unambiguous NOE restraints were obtained. The paramagnetism of the 2Fe- 2S cluster is another impediment to structure refinement, and selective labeling schemes and diamagnetic metal center substitutions are proposed to overcome this problem. Several bound waters are suspected in the structure, and are expected on the surface of Pdx, and pulsed-field- gradient NMR methods will be employed to identify these. Redox-dependent structural changes have been identified in Pdx, and the dynamics of two accessible oxidation states will be examined by amide proton exchange measurements. Based on the known structures of Pdx and p-450cam, models for the diprotein complex will be proposed and tested using heteronuclear- edited NMR methods. Crystallization trials for pdx, PdR and their complexes with each other and with P-450cam are in progress for determination of crystal structures. Finally, the mechanism of metal center incorporation into Pdx will be investigated by a variety of physico-chemical methods.
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