Crystal structure solution and refinement of the semisynthetic cobalt-substituted bovine erythrocyte superoxide dismutase at 2.0 Å resolution

Abstract

The semisynthetic Co-substituted bovine erythrocyte superoxide dismutase (SOD) has been crystallized in a new crystalline form and the structure determined at 2.0 Å (1 Å = 0.1 nm) resolution. The crystals belong to space group P2 12 12 1 with cell constants: a = 51.0, b = 147.6, c = 47.5 A ̊ , and contain one dimeric molecule of 32,000 M r per asymmetric unit. The structure has been solved by molecular replacement techniques using the Cu,Zn bovine enzyme as a search model, and refined by molecular dynamics with the crystallographic pseudo-energy term, followed by conventional crystallographic refinement. The R-factor for the 18,964 unique reflections in the resolution range from 10.0 to 2.0 Å is 0.176 for a model comprising 2188 protein atoms and 200 solvent molecules; the root-mean-square deviation from the ideal bond lengths is 0.010 Å, and the average atomic temperature factor is 26.5 Å 2. The dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. The subunit has as its structural scaffolding the conventional SOD-flattened antiparallel eight-stranded β-barrel, with three external loops. The co-ordination geometry of the metal center in the active site is fairly well preserved when compared with the native Cu,Zn bovine enzyme. Co 2+ is in tetrahedral co-ordination, while the Cu 2+ ligands show an uneven distortion from the square planar geometry. The least-squares superposition of the metals ligands and the catalytically important Arg141 of the native and Co-substituted enzyme yields a root-mean-square value of 0.401 Å, the largest deviation occurring at the Co 2+ ligand Asp81. An additional copper ligand, compatible with a water molecule, is observed at 2.38 Å from Cu 2+ in the active-site channel, at the supposed binding site of the O 2 − anion substrate. Several ordered water molecules have been observed on the protein surface and in the active-site channel; their structural locations coincide remarkably with those of related water molecules found in the crystal structure of the phylogenetically distant superoxide dismutase from yeast.