Structure-Function Relationship of Nickel-containing Superoxide Dismutase from Streptomyces seoulensis

Su peroxide dismutases (SOD) are enzymes that play an important role in the defence against oxidative stress and are found ubiquitously in oxygen metabolising organisms. The substrate of SOD is the superoxide radical anion which is responsible both for direct damage of biological molecules and for generating other reactive oxygen species. Superoxide is generated by a one electron transfer to dioxygen and its concentration is kept in low limits by SODs. SODs are generally classified according to the metal species which acts as redox-active centre to catalyse the overall dismutation reaction 2O2- + 2H+ → O2 + H2O2. Until recently, three different metal species have been found; copper- and zinc-containing SOD, with copper as the catalytically active metal, manganese-containing SOD, and iron-containing SOD. More recently a new superoxide dismutase containing nickel (NiSOD) was purified from a variety of Streptomyces species and characterized biochemically as well as spectroscopically. NiSOD was suggested to represent a novel class of superoxide dismutases on its own since no sequence homology is found to enzymes of the Cu,ZnSOD class or the MnSOD /FeSOD class. The present PhD thesis has the aim to provide structmal information which is needed to understand the function of NiSOD in detail. NiSOD from the aerobic soil bacterium Streptomyces seoulensis was crystallized in two crystal forms. The phase problem was solved by the multiplewavelength anomalous dispersion (MAD) method exploiting anomalous scattering at the K-absorption edge of nickel. The three-dimensional molecular structures of resting, chemically and X-ray reduced enzyme were refined to a resolution of up to 1.6 A and are analysed. NiSOD is found to be distinct from SODs of both other classes regarding oligomeric state, subunit structure and active site. It is a homohexamer of four-helix-bundle subunits where each subunit hosts an active site in an N-terminal loop. In the resting enzyme, the Ni(III) site is five-coordinate in a square pyramidal geometry by the amino-terminal and imidawle nitrogen No of His 1, the amide nitrogen and thiolate sulfur of Cys 2, and the thiolate sulfur of Cys 6. The reduced enzyme state was obtained either by thiosulfate soaking of crystals or by X-ray induced reduction in the course of exposure to high X-ray doses. Loss of the axial ligand His 1 Nc5 is observed upon reduction to Ni(II). Neither the nickel ion nor its ligands except His 1 N appear to be solvent accessible through a small pocket in the enzyme's surface. This pocket shows a narrow bottleneck at its entrance and may account for substrate specificity. Longrange electrostatic attraction of superoxide anions to this pocket is not of importance for NiSOD's activity in contrast to other SOD classes.