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The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P21212 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a noncrystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded β-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 β-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c β-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 β-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers. The β-barrel of the bovine enzyme is closed at opposite ends by apolar interactions involving Leu104 and Leu36; the latter is substituted by Ser36 in the yeast enzyme. Three concerted substitutions Ala87 → Thr, His41 → Arg and Glu119 → Ala allow for the formation of a “plug” for the β-barrel by means of a hydrogen bonding scheme. |
