Structural analyses of various Cu 2+, Zn 2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy

Abstract

The thermal denaturation profile of the Cu 2+, Zn 2+ metalloenzyme, bovine Superoxide dismutase, consists of two primary components, the major component denatures irreversibly at T m = 104 °C with a total enthalpy (Δ H cal) of 7.30 cal/g. Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers T m to 96 °C and Δ H cal to 6.96 cal/ g. The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 °C with an enthalpy only one-half that of the holo-form. The reduced thermal stability, which indicates a greater ability to change conformation, may explain the previously observed much greater membrane binding of the apo-enzyme. Reconstitution with Zn 2+, Cu 2+, or Zn 2+ and Cu 2+ raises T m to 80, 89, or 102 °C, respectively, with corresponding increases in the enthalpy. Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation. The effect of Cu 2+ alone is greater than that of Zn 2+, although both are needed for full stability. Raman spectroscopy indicates little difference in secondary structure between the apo- and holo-forms, implying that the increased stability due to metal binding is not caused by an extreme structural reorganization. The value of T m of canine and yeast superoxide dismutase is also lowered by reduction of Cu(II). The reduced form of the yeast enzyme denatures irreversibly, as do all forms of the bovine and canine enzymes, but the oxidized form is unique in that it denatures reversibly. Thus, the copper ion must be oxidized for renaturation and appears to act as a nucleation site.