Three-Dimensional Structure of Cu,Zn-Superoxide Dismutase from Spinach at 2.0 Å Resolution1

Abstract

The three-dimensional structure of Cu,Zn-superoxide dismutase from spinach leaves has been determined by X-ray crystal structure analysis. The atomic coordinates were refined at 2.0 Å resolution using the Hendrickson and Konnert program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallo-graphic symmetry. The crystallographic residual error for the refined model was 24.9%, with a root mean square deviation of 0.03 Ų from the ideal bond length and an average atomic temperature factor of 9.6 Ų. A dimeric molecule of the enzyme is comprised of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit of 154 amino acid residues is composed primarily of eight anti-parallel β-strands that form a flattened cylinder, plus three external loops. The main-chain hydrogen bonds primarily link the β-strands. The overall structure of this enzyme is quite similar to that of the bovine dismutase except for some parts. The single disulfide bridge (Cys57-Cysl46) and the salt bridge (Arg79-Aspl01) may stabilize the loop regions of the structure. The Cu²⁺ and Zn²⁺ ions in the active site lie 6.1 Å apart at the bottom of the long channel. The Cu²⁺ ligands (ND1 of His-46, and NE2 of His-48, -63, and -120) show an uneven tetrahedral distortion from a square plane. The Zn²⁺ ligands (ND1 of His-63, -71, and -80 and OD1 of Asp-83) show an almost tetrahedral geometry. The imidazole ring of His-63 forms a bridge between the Cu²⁺ and Zn²⁺ ions. The side chains and main chains of the metal-liganding residues are stabilized in their orientation by a complex network of hydrogen bonds.