Cobalt(2+) binding to human and tomato copper chaperone for superoxide dismutase: Implications for the metal ion transfer mechanism

The copper chaperone for superoxide dismutase (CCS) gene encodes a protein that is believed to deliver copper ions specifically to copper-zinc superox ide dismutase (CuZnSOD), CCS proteins from different organisms share high s equence homology and consist of three distinct domains; a CuZnSOD-like cent ral domain 2 flanked by domains 1 and 3, which contain putative metal-bindi ng motifs. We report deduced protein sequences from tomato and Arabidopsis, the first functional homologues of CCS identified in plants. We have purif ied recombinant human (hCCS) and tomato (tCCS) copper chaperone proteins, a s well as a truncated version of tCCS containing only domains 2 and 3. Thei r cobalt(2+) binding properties in the presence and absence of mercury(2+) were characterized by UV-vis and circular dichroism spectroscopies and it w as shown that hCCS has the ability to bind two spectroscopically distinct c obalt ions whereas tCCS binds only one. The cobalt binding site that is com mon to both hCCS and tCCS displayed spectroscopic characteristics of cobalt (2+) bound to four or three cysteine ligands. There are only four cysteine residues in tCCS, two in domain 1 and two in domain 3; all four are conserv ed in other CCS sequences including hCCS. Thus, an interaction between doma in 1 and domain 3 is concluded, and it may be important in the copper chape rone mechanism of these proteins.