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 superoxide dismutase (CuZnSOD). CCS proteins from different organisms share high sequence homology and consist of three distinct domains; a CuZnSOD-like central domain 2 flanked by domains 1 and 3, which contain putative metal-binding motifs. We report deduced protein sequences from tomato and Arabidopsis, the first functional homologues of CCS identified in plants. We have purified recombinant human (hCCS) and tomato (tCCS) copper chaperone proteins, as well as a truncated version of tCCS containing only domains 2 and 3. Their cobalt(2+) binding properties in the presence and absence of mercury(2+) were characterized by UV-vis and circular dichroism spectroscopies and it was shown that hCCS has the ability to bind two spectroscopically distinct cobalt ions whereas tCCS binds only one. The cobalt binding site that is common 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 conserved in other CCS sequences including hCCS. Thus, an interaction between domain 1 and domain 3 is concluded, and it may be important in the copper chaperone mechanism of these proteins.

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery.

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.