Structural analyses of various Cu2+, Zn2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy.
The thermal denaturation profile of the Cu2+, Zn2+ metalloenzyme, bovine superoxide dismutase, consists of two primary components, the major component denatures irreversibly at Tm = 104 degrees C with a total enthalpy (delta Hcal) of 7.30 cal/g. Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers Tm to 96 degrees C and delta Hcal to 6.96 cal/g. The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 degrees 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 Zn2+, Cu2+, or Zn2+ and Cu2+ raises Tm to 80, 89, or 102 degrees C, respectively, with corresponding increases in the enthalpy. Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation. The effect of Cu2+ alone is greater than that of Zn2+, 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 Tm 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.
D000818:Animals; D002151:Calorimetry; D002417:Cattle; D003300:Copper; D004285:Dogs; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D013059:Spectrum Analysis, Raman; D013482:Superoxide Dismutase; D013816:Thermodynamics; D015032:Zinc
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc