| Copper, zinc superoxide dismutase (SOD1) is a vital cytoplasmic enzyme that catalyzes the disproportionation of superoxide anion (O2 −) to H2O2 and O2. The copper cofactor within the SOD1 active site is essential for the protein's activity. Recent studies have demonstrated that SOD1 receives copper specifically from a previously unknown protein, the copper chaperone for SOD1 (CCS).; CCS is a multidomain protein that has a high degree of homology with both SOD1 and a newly characterized family of proteins, the metallochaperones. In agreement with in vivo experiments using S. cerevisiae, domain I of hCCS is shown here to bind Cu(I), domain II allows for homodimerization of the chaperone in solution and domain III is essential for activity.; The mechanism of protein-protein interaction between yeast CCS(yCCS) and yeast SOD1(ySOD1) was delineated using mutant forms of SOD1. This mutant ySOD1 allowed for trapping and isolation of the heterodimer complex with both apo yCCS and Cu yCCS. The effect of copper in stabilizing the heterodimer complex was also determined by fluorescence spectroscopy. Copper was shown to stabilize the heterodimer interaction by an order of magnitude and gave the first evidence of the role of the cargo metal ion in the metallochaperone:target enzyme interaction.; A role for oxygen in copper transfer was explored by developing aerobic and anaerobic assays. The results show that O2 is essential for CCS activity and proper folding/activation of nascent SOD1 in vitro and in vivo. Investigation of this system under anoxic and normoxic conditions has yielded a putative mechanism including: (1) delivery of copper to the CCS/SOD1 interface, (2) oxidation of Cu(I) to Cu(II), (3) oxidation of domain III Cys residues to disulfide bond, (4) disulfide isomerization producing an oxidized internal disulfide in SOD1. The novel sulfhydryl oxidase/disulfide isomerase function of CCS was thus established.; Finally, mutants of SOD1 (A4V, G85R and G93A) associated with the lethal neurodegenerative disease FALS were studied in vitro. Marked differences in their affinities for CCS and their homodimer subunits were found, indicating a possible mechanism for aberrant association of the disease-causing proteins. |
