Studies on the reconstitution of bovine erythrocyte superoxide dismutase: III. Evidence for a strong interdependence between Cu 2+ and Zn 2+ binding in the expression of the spectroscopic properties of the native protein and for a close proximity of the Zn 2+ and Cu 2+ sites

Abstract

1. 1. Conditions are described for reconstitution of apo superoxide dismutase to native protein in high yield with no side products. 2. 2. 67 Zn (I = 5 2 ) has been substituted in place of normal zinc with no observable broadening of the EPR spectrum. 3. 3. Excess cyanide added to the 63Cu-substituted protein yields a low-field hyperfine line having a superhyperfine pattern clearly arising from an interaction with three nitrogen nuclei. 4. 4. Cu 2+ may bind so as to form a Zn 2+-binding site and Zn 2+ may bind forming a native Cu 2+-binding site. 5. 5. When present in excess, Cu 2+ bind in close proximity to one another, and an EPR spectrum characteristic of a magnetic dipolar coupling between at least two Cu 2+ has been observed.