Selenocysteine-containing mutants of the human copper chaperone for superoxide dismutase (hCCS) were constructed using intein-mediated peptide ligation to investigate copper transfer and binding. These mutants retain wild-type activity and display similar Cu-binding properties as shown by X-ray absorption spectroscopy. Cu and Se EXAFS analyses reveal a unique adamantane-like copper cluster formed at the D3–D3 interface between CCS molecules. These results confirm the presence of a D3-mediated copper cluster and support its functional role in copper transfer to SOD1.
