| Cytochromes P450 are a superfamily of heme-thiolate enzymes that play a crucial role in the oxidation of both endogenous and exogenous compounds in biological systems. Cytochrome P450cam (CYP101) catalyzes the 5- exo hydroxylation of camphor in the first step of camphor catabolism by the soil bacterium Pseudomonas putida. Comparison of RDC-derived structures determined in the presence and absence of substrate show structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or heme iron spin state.;15N T1 and T2 relaxation and H-D exchange experiments were performed to investigate the dynamics of substrate-free CYP101 relative to camphor-bound CYP101 over a range of time scales. Backbone and side chain fluctuations occur on the picosecond to nanosecond timescale, and these are typically studied by measuring heteronuclear T1 and T2 relaxation rates, and H-D exchange reflects dynamics on the time scale of seconds to hours. The general trend of T1 and T 2 increasing upon substrate removal implies more freedom of motion overall. The ratio of 15N T1/T2 also provides important information about dynamics on the millisecond timescale, and in substrate-free CYP101, many of the residues most affected seem to be in the area believed to be the substrate access/egress route. Overall, H-D exchange follows the same trends and is faster in substrate-free CYP101 than camphor-bound. Some notable areas of faster exchange in the substrate-free enzyme are in the F-G loop, beta1 sheet, B' helix, and B-B' loop, which are all believed to be involved in substrate access to the active site.;Despite most P450 reactions being oxidation reactions, there is still a considerable diversity of reduction reactions catalyzed by P450s, most of which are observed more readily under hypobaric or anaerobic conditions, in which O2 is not available to act as the ultimate electron acceptor. Very few studies of CYP101 reducing its substrates have been reported, but it has been shown to be able to reduce carbon-halogen bonds under anaerobic conditions. Chapter 3 examines the reduction of nitroacetophenones by CYP101, which has not been reported. While the reduction of nitro compounds by P450s is known, this marks the first example of such a reaction catalyzed by CYP101. When dithionite is used as the reducing agent, the P450 is not required for reduction, and all isomers of nitroacetophenone are reduced to aminoacetophenone. It is still unclear whether or not the P450 is required for the reduction to occur when NADH is used as a reducing agent. It seems to be necessary for at least the reduction of m-nitroacetophenone, but that may not be the case for the ortho and para isomers. |
