KINETICS OF METAL DISSOCIATION IN THE YEAST Cu2,Zn2-SUPEROXIDE DISMUTASE. APPARENT ASYMMETRY IN THE METAL BINDING SITES

The inactivation and metal depletion of the yeast Cu2,Zn2-superoxide dismutase have been examined in the presence of a number of chelating reagents. Total inactivation and metal depletion occurred only at pH < 3.5. The loss of enzyme activity exhibited biphasic kinetics and was directly related to the rate of loss of copper from the protein. Two rate constants characterized also the dissociation of zinc from the native enzyme. At low pH, in the absence of chelating reagents, there is a fifty percent reduction in dismutase activity and a concommitant decrease in the metal concentration to one equivalent each of copper and zinc per mole dimer. It is apparent that the dissociation constants for the copper-protein and zinc-protein complexes differ between the subunits of the dimeric enzyme. Comparison of the hydrodymanic and optical properties of the native and apo-SOD reveals changes in the structure and stability of the protein as a result of metal removal.