The amino acid sequence of the copper,zinc superoxide dismutase from Saccharomyces cerevisiae has been determined by automated Edman degradation. Peptides were obtained from cyanogen bromide cleavage, Staphylococcus aureus V8 protease digestion, tryptic and chymotryptic digests of the citraconylated reduced and carboxymethylated enzyme, and by further fragmentation of selected peptides with trypsin. From the alignment of these peptides and the previously published sequence of the first 54 amino terminal residues (24) the complete sequence was deduced by direct sequence identification of all 153 amino acid residues and of all peptide overlaps. The amino acid sequence corresponds to a molecular weight of 15,950 for each of the two identical subunits in the native enzyme. The primary structure of yeast copper, zinc superoxide dismutase is 55 96 identical with the sequence of the copper, zinc enzyme from bovine erythrocytes. Importantly, all the copper and zinc ligands, six histidine residues and one aspartate residue from the bovine enzyme, are conserved in the ye,~st enzyme. The high overall sequence homology and conservation of important metal binding active site amino acid residues suggest that the three-dimensional structure and in particular the active site geometry is virtually the same for the bovine and yeast enzyme. In contrast no sequence homology is apparent by comparison with the manganese or iron class of superoxide dismutases indicating that the two classes have not evolved from a common ancestor. Superoxide dismutases (SOD) are a group of metalloenzymes, whose function is to scavenge the superoxide radical anion 02 (12) by catalyzing its dismutation to hydrogen peroxide and dioxygen:
The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding. This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure. Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r. spectrum of the enzymes. This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins.
High resolution nuclear magnetic resonance (n.m.r.) spectroscopy of the copper-zinc superoxide dismutase from Saccharomyces cerevisiae has revealed a substantial structural homology with the bovine enzyme. N.m.r. spectra of the apo enzyme and the holo-reduced and holo-oxidized enzymes are reported and assignments are made to the histidines in the active site and the single tyrosine residue. All the assignments are in agreement with the known amino-acid sequence and the geometry of the active site is virtually unchanged.Addition of halide ions to the reduced holo enzyme results in the perturbation of the chemical shift of three histidine C2 protons and the degree of perturbation is CI-~Br->I->F-for 1 M solutions of the anions. The enzyme has also been shown to retain its structure at 75~
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