The role of CuA in the cytochrome c oxidase proton pump

Cytochrome c oxidase plays a central role in energy transduction in most aerobic organisms. It catalyzes the transfer of electrons from ferrocytochrome c on the cytosolic side of the inner mitochondrial membrane, to dioxygen. The protons consumed during this reaction are derived exclusively from the matrix space, resulting in a charge separation that contributes to the transmembrane electrochemical gradient. In addition to the dioxygen reduction activity, cytochrome oxidase is also a proton pump that can pump up to four protons from the matrix side of the inner membrane to the cytosolic side for every molecule of dioxygen reduced. This thesis investigated the role of the structure and function of CuA in the proton pumping function of the enzyme. The structure of the CuA site was studied using Extended X-ray Absorption Fine Structure (EXAFS). Using the p-(hydroxymercuri)benzoate (pHMB)-modified enzyme and a CuA-depleted form of the enzyme, we assigned the ligand structures for CuA and CuB in the resting form of the enzyme. The best fit model for the coordination environment at CuA was found to be 2 (N,O) ligands at 1.99A (presumably from histidine) and 2 (S,CI) ligands at 2.3A (presumably from cysteine). The EXAFS curve fitting techniques were further refined to investigate the copper sites in both the resting and fully reduced forms of the enzymes. These results indicated that the resting form of the enzyme contains a "long" 2.6A Cu-(S,Cl) in addition to the 2.3A Cu-(s,Cl) interactions previously reported. Using the curve-fitting results from the CuA-modified and CuA-depleted enzymes, we were able to assign this interaction to one of the two Cu-(S,CI) interactions at the CuA site. The curve-fitting results for the reduced enzyme showed no "long" interaction and indicated an average of one less sulfur per two coppers, suggesting that a a ligand rearrangement occurs upon reduction of CuA. The role of CuA in proton pumping was assessed by reconstituting the pHMB-modified enzyme into artificial phospholipid vesicles, and measuring its proton pumping activity. We found that this form of the enzyme, which contains a perturbed CuA site, exhibits a rapid proton leak. This leak is not associated with pHMB modification of the protein surface sulfhydryl groups, but appears to be specifically correlated with the modification of the CuA site. We also developed a method for specifically perturbing the CuA site using gentle heating at 43°C. It was found that heat treatment causes a specific modification of the CuA site. We showed that the products of this reaction are native CuA, a type 2 Cu, and novel "blue copper" species. Furthermore, reduction of the enzyme as well as ligand binding to the binuclear center were found to protect the CuA site from heat-induced modifications. When the heat-treated enzyme was reconstituted into vesicles, it displayed proton pumping behavior similar to the pHMB-modified enzyme, again pointing to a role for CuA in the proton pumping machinery of the enzyme. These results also suggest that the CuA site is different in the oxidized and reduced forms of the enzyme, and that a strong allosteric interaction exists between the CuA site and the binuclear center. Based on indications that there is a strong interaction between the binuclear center and CuA, we probed the protein matrix for a conformational change that could be caused by ligand binding to the binuclear center. Using the flow-flash technique, we identified a transient conformational change that appears to be associated specifically with dioxygen binding and reduction. This conformational change occurs rapidly enough to be involved in the turnover of the enzyme, and can be influenced by the redox state of cytochrome a and CuA. We conclude this thesis with a model for the turnover cycle of the enzyme, which utilizes CuA as the site of redox linkage, and a conformational switch which engages the pumping cycle