REDOX CHEMISTRY OF SUPEROXIDE-DISMUTASE - CYCLIC VOLTAMMETRY OF WILD-TYPE ENZYMES AND MUTANTS ON FUNCTIONALLY RELEVANT RESIDUES

The reduction potential of human cuprozinc superoxide dismutase and of several of its functionally relevant mutants have been measured through cyclic voltammetry. The reduction potential of the bovine enzyme has been also measured and compared with literature values. The human enzyme has a slightly higher redox potential than the bovine isoenzyme (E-degrees = 0.36 +/- 0.01 and 0.32 +/- 0.01 V vs NHE, at pH 7.4, respectively). The redox properties of the bovine copper-cobalt derivative are very similar to those of the native protein. The pH dependence of the E-degrees value in the wild-type enzyme and its pH independence in the Asn- 1 24 mutant, which has an empty zinc binding site over the entire pH range, is ascribed to the uptake of a proton by His-63 upon reduction. A pK(a) value of 10.8 for this group is obtained from H-1 NMR titrations. It is proposed that also in the zinc-deprived derivative the copper-His-63 bond is broken upon reduction. Sizably negative reduction potentials were estimated for CN- and N3--inhibited enzymes. The values are below the reduction potential of dioxygen to superoxide.