Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of CuA in human cytochrome oxidase.

Maturation of cytochrome oxidases is a complex process requiring assembly of several subunits and adequate uptake of the metal cofactors. Two orthologous Sco proteins (Sco1 and Sco2) are essential for the correct assembly of the dicopper CuA site in the human oxidase, but their function is not fully understood. Here, we report an in vitro biochemical study that shows that Sco1 is a metallochaperone that selectively transfers Cu(I) ions based on loop recognition, whereas Sco2 is a copper-dependent thiol reductase of the cysteine ligands in the oxidase. Copper binding to Sco2 is essential to elicit its redox function and as a guardian of the reduced state of its own cysteine residues in the oxidizing environment of the mitochondrial intermembrane space (IMS). These results provide a detailed molecular mechanism for CuA assembly, suggesting that copper and redox homeostasis are intimately linked in the mitochondrion.