The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis

We have investigated factors that influence the properties of the zinc bind ing site in yeast copper-zinc superoxide dismutase (CuZnSOD). The propertie s of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, be low this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity f rom strong to weak, (2) the expulsion of the metal bound at this site, or ( 3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserve d among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupanc y and redox status of the adjacent copper binding site. Furthermore, we hav e investigated the effect of single site mutations on the pH- and redox-sen sitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However , they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc bindi ng site seen in the wild-type is conserved in the mutants, albeit with an a ltered pK(a). The mutants H71C and D83A did not bind Co2+ in a wildtype-lik e fashion under any of the conditions tested. This study reveals that the z inc binding site is exquisitely sensitive to changes in the protein environ ment. Since three of the mutant yeast proteins investigated here contain mu tations analogous to those that cause ALS (amyotrophic lateral sclerosis) i n humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.