Mitochondrial cytochrome c oxidase catalyzes the four-electron reduction of oxygen to water. The functional form of the protein contains two copper ions and two heme irons. Two of these metal centers, cytochrome a3 and Cua3, constitute the oxygen reduction site. The other two metal centers, cytochrome a and Cua, are involved in electron transfer from cytochrome c to the oxygen reduction site. It is the structure and function of these four metal centers in cytochrome c oxidase which is the subject of this thesis.
The metal centers constituting the oxygen reduction site of cytochrome c oxidase have traditionally been difficult to study because their coupled nature renders them EPR silent. It is shown that nitric oxide can uncouple these metal centers, and under appropriate conditions renders both metal centers observable by EPR. One of the nitric oxide complexes is an NO-bridge complex, demonstrating that the oxygen reduction site is between the two metal centers. Furthermore, this NO-bound complex allows us to calculate a distance between the metal centers of about 5 Å. Finally, with 15N-his isotopically labeled yeast oxidase it is shown that the fifth endogenous ligand to cytochrome a3 is a histidine.
The copper metal center involved in electron transfer, Cua, is shown to have a cysteine and a histidine as ligands. The substitution of 12CD2-cysteine into yeast oxidase does not perturb the eight line hyperfine pattern first seen on the Cua EPR signal at 3 GHz. This demonstrates that the S = 1/2 center interacting with Cua is probably cytochrome a at a distance of 10-13 Å. These studies represent the first time that ligands to any of the metal centers in cytochrome c oxidase have been unequivocally elucidated.
