| Description |
Cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain, requires three copper ions for function. The copper ions need to be ferried from the cytoplasm to the mitochondrial inter-membrane (IM) space, and subsequently inserted into COX subunits 1 and 2. Cox17 is proposed to be the copper chaperone that transports the copper ions to the mitochondria. Consistent with this role, Cox17 is present in both the cytosol and mitochondrial IM space, and binds Cu(I) within a polycopper cluster. Sco1, a mitochondrial inner membrane protein, is proposed to accept the ions from Cox17, and donate them to Cox2. Sco1 was first implicated in this pathway by the observation that the respiration-defective phenotype of ?cox17 cells could be suppressed by high copy SCO1. The aim of this thesis is to further characterize Cox17 and Sco1, which would lead to better understanding of the process of copper metallation of COX. In this study mutational analysis on yeast Cox17 was used to identify three of seven cysteinyl residues as Cu(I)-binding ligands. These residues, which are present in a conserved CxCC motif, are also critical for Cox17 function. Mitochondrial import of Cox17 is independent of Cu(I) binding. Mutation of a cysteinyl residue located near the C-terminal end of the protein results in reduced levels of Cox17 inside the mitochondria, suggesting that this portion of the protein may contain a mitochondrial targeting sequence or protein docking site. The work described in this thesis shows for the first time that purified Sco1 binds one Cu(I) ion per monomer. The Cu(I) ion in Sco1 is ligated via two cysteines present in a conserved CxxxC motif, and a conserved histidine. As with Cox17, all three residues in Sco1 involved in Cu(I) ligation are also essential for function. Size-exclusion chromatography of solubilized mitochondrial and cytoplasmic lysates shows that Sco1 forms oligomers in vivo. Oligomerization is not dependent on Sco1 function. Lack of either Cox17 or Cox2 also does not affect Sco1 oligomerization. |
