Biophysical characterization of Cox17, the metallochaperone to the mitochondria

Cox17 is involved in copper delivery to the cytochrome oxidase complex. Cox17 binds Cu(I) ions via cysteinyl thiolate ligands. Cysteines 23, 24 and 26 of Cox17 are required to produced a functional cytochrome oxidase complex in vivo. These residues appear to be important for Cox17 copper binding, since a double mutant lacking cysteines 23 and 24 fails to bind copper. This double mutant localizes to the intermembrane space, indicating that copper binding does not signal mitochondrial Cox 17 uptake. The lack of function of the CYS57Tyr Cox17 variant appears to be due to a nonconservative change since a CYS57Ser Cox17 variant is functional. Cox17 binds three Cu(I) ions per monomer in a solvent shielded, polynuclear cluster. The Cox17 Cu(I)-thiolate bonds are thermodynamically stable, but kinetically labile. Therefore, Cox17 has the ability to prevent copper from reacting nonspecifically during copper transport through the mitochondria, yet still retains the ability to transfer the copper when Cox17 contacts its cellular target. At physiologically important concentrations, Cox17 exists in a dimer/tetramer equilibrium with a kd of 20 µM ± 10 µM. Cox17 variants with changes to essential cysteinyl residues fail to tetramerize under physiologically relevant concentrations. These mutants exist in a monomer/dimer equilibrium. The concentration of Cox17 in the cytoplasm appear to be close to .4 µm, whereas the Cox17 concentration of Cox17 in the intermembrane space is probably greater than 60 µM. Therefore, Cox17 is largely dimeric in the cytoplasm and tetrameric in the intermembrane space. Oligomerization may be important in copper delivery to cytochrome oxidase.