Metal ion reconstitution studies of yeast copper-zinc superoxide dismutase: the "phantom" subunit and the possible role of Lys7p

Using a corrected molar extinction coefficient for yeast apo copper-zinc su peroxide dismutase (CuZnSOD), we have confirmed that the metal binding prop erties of this protein in vitro differ greatly from those of the bovine and human CuZnSOD enzymes. Thus yeast apo CuZnSOD was found to bind only one C o2+ per protein dimer under the conditions in which the bovine and human Cu ZnSOD apoenzymes readily bind two per dimer. The spectroscopic properties c haracteristic of the two Cu2+ plus two Co2+ per dimer or four Cu2+ per dime r metal-substituted bovine apo CuZnSOD derivatives were obtained for the ye ast apoprotein but by the addition of only half of the appropriate metals, i.e., one Cu2+ plus one Co2+ per dimer or two Cu2+ per dimer. This half-met allated yeast CuZnSOD has been characterized by UV-visible and EPR spectros copy as well as by native polyacrylamide gel electrophoresis. We conclude t hat yeast apo CuZnSOD, unlike the bovine and human apoproteins, cannot be r econstituted fully with metal ions under the same conditions. Instead, only one subunit of the homodimer, the "normal" subunit, can be remetalled in a fashion reminiscent of the well-characterized bovine protein. The other "p hantom" subunit is not competent to bind metals in this fashion. Furthermor e, we have shown that CuZnSOD protein isolated from Saccharomyces cerevisia e that lacks the gene coding for the copper chaperone, Lys7p, contains only one metal ion, Zn2+, per protein dimer. The possibility that yeast CuZnSOD can exist in multiple conformational states may represent an increased pro pensity of the yeast protein to undergo changes that can occur in all CuZnS ODs, and may have implications for amyotrophic lateral sclerosis.