Overall structure of NiSOD. (A) The solvent-accessible surface of NiSOD viewed along the hexamer's threefold symmetry axis. The outer surface (black mesh) is sliced to allow the view to the inner solvent-filled space (in orange) and the protein backbone trace (chain A, yellow; chain B, blue; chain C, red; chain D, magenta; chain E, cyan; chain F, green; Ni ions, salmon-colored spheres). Arrows indicate the three twofold symmetry axes and the entrance to channels that render the inner space accessible to solvent molecules. (B) Ribbon representation of a NiSOD subunit. The N-terminal loop hosting the Ni ion protrudes from the body of the four-helix bundle. Residues involved in aromatic stacking are shown as a ball-and-stick representation. (C) Residues linking the active-site loop (subunit A) to neighboring chains by polar interactions. His-1 Nε takes part in a hydrogen-bonding triangle with Glu-17 and Arg-47 of subunit C. Main-chain oxygen atoms of Asp-3 and Leu-4 hydrogen bond to the side chain of Arg-39 in subunit C. The side-chain oxygen atoms of Asp-3 hydrogen bond to the side chains of Lys-52, Ser-86, and Lys-89 of subunit F.

σ_A-weighted 2 F_o – F_c electron density maps of the Ni ion environment. (A–C) Structures of subunit F captured at successively increasing x-ray doses. (A) The fifth ligand His-1 Nδ (2.5 Å distant to Ni here) is revealed at low x-ray exposure (map resolution 2.2 Å, 1.0 σ contour level). (B) After longer exposure of the same crystal as in A, the imidazolate ligation is disrupted. (C) Map at 1.6-Å resolution obtained from a different crystal as in A and B, applying a maximum total x-ray dose. The ligands show a distorted cis square-planar geometry, equaling the thiosulfate-reduced NiSOD. The average angle between planesdefined by N(His-1)-Ni-N(Cys-2) and S(Cys-2)-Ni-S(Cys-6) is 7.5°.(D) Superposition of models from A in green, B in magenta, and C in gray illustrates the His-1 imidazole rotation upon which Nδ reaches a distance of ≈2.9 Å to Val-8 O, thereby maintaining the hydrogen-bond triangle of His-1 Nε to Glu-17 Oε and Arg-47 Nη of the adjacent subunit. (E) Electron density map of thiosulfate-reduced NiSOD contoured at 1.1 σ showing the square-planar Ni(II) coordination. One thiosulfate-ion (S₂O₃^2–) per subunit is found 7–8 Å away from each metal center (subunit A). The precise bonding pattern of these ions varies among the 12 subunits in the asymmetric unit, indicating a high degree of disorder or low-binding specificity.

Active-site accessibility. (A) Active-site environment from His-1 to Tyr-9 of the high-dose structure (Fig. 2C). Nitrogen atoms are labeled with the respective residue number. For Cys-2 to Gly-7, only backbone atoms are shown and oxygen atoms are omitted for clarity. Backbone nitrogen atoms from His-1, Asp-3, Cys-6, and Gly-7 separate the Ni ion and all other ligands (Cys-2 N, Cys-2 Sγ, and Cys-6 Sγ, right to the vertical line) from the solvent-accessible pocket (hosting two water molecules, left to the line). In all but the thiosulfate-reduced NiSOD structures, a water molecule (here W 828) is found close to the vacant axial position opposite the His-1 imidazole at ≈3.5 Å from the Ni ion, 3.3 Å from His-1 N, and hydrogen bonds to Cys-6 N. High temperature factors for this solvent molecule indicate elevated mobility. (B) Surface representation of thiosulfate-reduced NiSOD at the active-site loop and the innermost end of a channel that allows thiosulfate-ions to approach the Ni sites (only selected side chains are shown). The solvent-accessible pocket close to the Ni center is marked by an arrow and exhibits a bottleneck formed by Pro-5 and Tyr-9 ≈5 Å away from the Ni ion, conferring to NiSOD a selectivity for small molecules as substrate or inhibitors. A sulfate ion (not included in the surface calculation) from the crystallization liquor is found at the pocket's entrance in all described structures.

Sequence alignment of NiSOD with putative ORFs from cyanobacteria and secondary structure prediction. Sequences are given for S. seoulensis (S. seo), S. coelicolor (S. coe), Trichodesmium erythraeum IMS101 (T. ery), Synechococcus sp. WH 8102 (Synec), Prochlorococcus marinus subsp. Pastoris (P. ma1), and Prochlorococcus marinus str. MIT 9313 (P. ma2). Identical residues are marked with asterisks and similar residues are marked with colons or periods (strongly or weakly conserved groups). Predicted α-helices are highlighted in red, predicted β-sheets in cyan, and α-helices in S. seoulensis NiSOD (based on our structures) are shown as cylinders, colored as in Fig. 1B. Ni ligands in S. seoulensis NiSOD are highlighted in yellow, and residues involved in aromatic stacking at the N terminus are highlighted in magenta.