Total Reconstitution of Copper-Zinc Superoxide Dismutase

The total reconstitution of copper-zinc superoxide dismutase by stoichiometric addition of the metals to completely metal-free apoprotein is described. Reconstitution has been monitored by electron paramagnetic resonance (EPR) and absorption spectroscopy, enzymic activity, and acrylamide gel electrophoresis. Under the same conditions, it has been possible to prepare highly active enzymes with cobalt, mercury, or cadmium in place of zinc. The properties of these derivatives demonstrate that the replacement is exact, that copper has the major influence on the spectral properties of the enzyme, and that occupation of the zinc site is necessary in order to attain fully native properties. A technique is described which allows the extraction of superhyperfine patterns from the bulk of magnetic resonance spectra. Use of this technique shows that the superhyperfine patterns of superoxide dismutase from animal sources differ from those of fungal enzymes. In addition, the technique of shows that the superhyperfine patterns in the EPR spectra reconstituted Cu 2- , Cu 2 , Cd 2- , Cu 2 , Hg 2- , and Cu 2 , Zn 2 -derivatives are very similar to that in the EPR spectrum of native enzyme. Reconstitution with 63 Cu and treatment with cyanide demonstrates that three magnetically equivalent nitrogens are the source of these patterns. The results of this study are shown to be consistent with the copper binding site being a distorted square planar arrangement of ligands and neighboring the zinc-binding site.