| dc.description.abstract | Copper is required for the activity of cytochrome c oxidase (CcO), the mitochondrial enzyme that catalyzes cellular respiration. Copper delivery to CcO is a complex process requiring a number of proteins, and loss-of-function mutations in these proteins diminish CcO activity, causing rare mitochondrial disorders, for which no effective therapy currently exists. A lack of understanding of the basic mechanisms for copper transport to the mitochondria and ultimately to CcO has been the main bottleneck in developing therapeutic strategies for these disorders. To address this gap in our knowledge, I utilized a multi-disciplinary approach involving chemical biology, structural biology, and yeast genetics to identify small molecules and genes that facilitate copper transport to the mitochondrial CcO. First, using a chemical biology approach, I determined that elesclomol (ES), an investigational anticancer drug, is a potent copper delivery agent to mitochondria. I showed that ES supplementation rescues respiratory growth of several yeast copper metabolism mutants, including cells lacking Coa6, a CcO assembly factor. ES also restored CcO levels in a series of copper-deficient mammalian cells and in a zebrafish model of copper deficiency. These findings demonstrate the applicability of ES to more complex eukaryotic cells and intact multicellular organisms.
Second, I utilized nuclear magnetic resonance (NMR) spectroscopy to uncover the structure and function of COA6, a new member of the CcO copper delivery pathway that was discovered in our laboratory. I collaborated with structural biologists to solve the solution structure of human COA6, which revealed a CHCH domain typically found in he redox-active proteins in the mitochondrial intermembrane space. Consistent with its redox role, I showed that COA6 function can be bypassed in the reducing environment. Interaction mapping and redox potential determination of COA6 and its client proteins showed that COA6 facilitates copper delivery to CcO by acting as a thiol-disulfide reductase. Finally, I utilized yeast genetics to uncover overlapping functions of COA6 and SCO2, a well-known member of the copper delivery pathway to CcO. Taken together, this dissertation describes molecular mechanisms of copper delivery to the mitochondrial CcO by both the interplay of CcO assembly factors and through pharmacological means. | |
