Metal-deficient copper-zinc superoxide dismutases

Abstract

The simultaneous addition of copper and zinc ions directly to bovine apo-superoxide dismutase at pH 5.0 leads to the formation of electrophoretically distinct metal-deficient enzymes in addition to the native enzyme. The EDTA-insensitive metal ion incorporation and the enzymatic activity has been studied at various ratios of added metal ions. The simultaneous addition of metal ions was carried out with either equimolar copper and zinc or with zinc maintained at 1.1 per active site. The addition of equimolar copper and zinc results in the incorporation of the metal ions in a one-to-one ratio; however, at low ratios of metal ions per active site not all of the metal ions added are incorporated and the yield of reconstituted metal-deficient enzyme and native enzyme is low. On the other hand, the maintenance of zinc at 1.1 per active site results not only in the incorporation of zinc at about one per active site but also in the incorporation of all the added copper, a result not seen in the addition of equimolar metal ions except at ratios of greater than 0.5 per active site. Furthermore, there is an increase in the formation of a metal-deficient species as well as native enzyme at the low ratios of copper. The formation of electrophoretically distinct metal-deficient dismutases in the reconstitution of apodismutase by direct addition of metal ions to nondenatured enzyme may account for previous observations of lower yields of native proteins using these types of procedures. The nature and enzymatic activity of the metal-deficient proteins is discussed.