Dept. of Environmental and Biomolecular Systems
Oregon Health & Science University
Selenocysteine-containing mutants of human copper chaperone for superoxide dismutase (hCCS) were constructed using intein-mediated peptide ligation. These mutants were studied with respect to their ability to transfer Cu to E,Zn superoxide dismutase (SOD1) and their Cu-binding and X-ray absorption spectroscopic (XAS) properties. Previous studies have shown that three functionally distinct polypeptide domains are present in CCS: the N-terminal domain 1 (D1, residues 1-85) contains the copper-binding MXCXXC motif, domain 2 (D2, residues 86-234) has sequence homology to residues associated with the native SOD1 dimer interface, and the C-terminal domain 3 (D3, residues 235-274) contains a CXC motif. Recent results suggest the formation of a D3- D3 cluster within a dimeric or tetrameric protein and suggest that this cluster may be an important element of the copper transfer machinery. D3 cysteine-to-selenocysteine mutants of wild-type and D1 mutants of hCCS were constructed to investigate the D3 copper cluster in more detail. These mutants display similar activity to wild-type protein. The structure of the Cu centers of selenocysteine-containing mutants as shown by Cu EXAFS is similar to that of wild-type protein, with clear indications of a Cu cluster. Cu and Se EXAFS of these constructs reveal a unique adamantane-like cluster formed between two molecules of CCS at the D3-D3 interface. These results confirm the existence of a D3-D3 copper cluster in hCCS and suggest that a unique copper cluster may exist in this protein.
OGI School of Science and Engineering
Barry, Amanda Nell, "Spectroscopic studies of the human copper chaperone for superoxide dismutase probing the active cluster with selenocysteine variants" (2007). Scholar Archive. 152.