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Cytochromes are found in all living beings and play an essential role in life processes. The two proteins studied in this thesis, cytochrome P450 carries out enzymatic reactions, metabolizes drugs, and synthesizes steroids while cytochrome c553 is a protein involved in electron transfer and photosynthesis. Despite the diverse functions cytochromes have, they all have the same core, a heme porphyrin that makes this class of proteins very interesting and worth studying. The cryogenic crystal structure of cytochrome P450 BM-3, a bacterial model enzyme for mammalian P450s, shows the substrate N-palmitoylglycine (NPG) too far away from the heme to be the catalytically relevant binding mode. Previous evidence suggests that the substrate moves as a function of temperature and molecular dynamics simulations have shown that at room temperature the ligand is positioned correctly for chemistry. However, how the substrate moves and the correct position of the substrate is not known. In order to gain more insight on the position of the substrate, we conducted solid-state nuclear magnetic resonance (SSNMR) experiments, enzymatic assays, and solved a new crystal structure. Enzymatic assays determined there is a kinetic preference for room temperature and a large difference in Km and Kd, indicates multiple conformations of the ligand, some of which are unproductive. SSNMR experiments also provided evidence for multiple conformations since changes in the chemical shift of NPG showed the terminal region of the ligand is moving as a function of temperature and the solvent exposed region of the ligand has dynamics between multiple conformations. In addition, our new crystal structure shows the first structural evidence of the solvent exposed end of NPG interacting with Arg47, which is involved in substrate binding shown by our R47E mutation. However, in our cryogenic crystal structure the ligand is still in a conformation not amenable for catalysis. Therefore, we also laid the foundation to obtain structural information on the enzyme at room temperature by assigning the ligand, identifying important alanine marker peaks, and determining the best method is selectively labeled samples with TEDOR experiments. In addition, cytochrome c553 was used as a model to evaluate the role of the thioether linkages in c-type cytochromes. Since c-type and b-type cytochromes are the most abundant and c-type cytochromes are biosynthesized from apo-protein and b-type heme, the fundamental question arises as to why Nature has made the covalent attachment in c-type cytochromes. We performed thermodynamic analyses and structural studies of a c-to-b conversion in a mutated cytochrome c, in which the thioether bonds cannot form, to determine the covalent attachment is necessary for structure and function of c-type cytochromes. |
