Nickel superoxide dismutase: structural and functional roles of Cys2 and Cys6

Abstract

Nickel superoxide dismutase (NiSOD) is unique among the family of SOD enzymes in that it coordinates cysteine residues (Cys2 and Cys6) to the redox-active metal center and exhibits a hexameric quaternary structure. To assess the role of the Cys residues with respect to the activity of NiSOD, mutations of Cys2 and Cys6 to serine (C2S-, C6S-, and C2S/C6S-NiSOD) were carried out. The resulting mutants do not catalyze the disproportionation of superoxide, but retain the hexameric structure found for wild-type (WT) NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic (XAS) studies of the Cys mutants reveal that the nickel active-site structure for each mutant resembles that of C2S/C6S-NiSOD and demonstrate that mutation of either Cys2 or Cys6 inhibits coordination of the remaining Cys residue. Mutation of one or both Cys residue(s) in NiSOD induces the conversion of the low-spin Ni(II) site in the native enzyme to a high-spin Ni(II) center in the mutants. This result that indicates that coordination of both Cys residues is required to generate the native low-spin configurations and maintain catalytic activity. Analysis of the quaternary structure of the cysteine mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography reveal that the cysteine ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.

Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and Its H-Bonding Network

“…Such high-spin Ni(II) centers have also been observed in Cys-to-Ser mutants of NiSOD, which are catalytically inactive. (14, 27) The formation of a high-spin six-coordinate complex for the disordered “Ni-hook” domain may also sterically prevent folding into the “Ni-hook” structure that features a His1 imidazole-Glu17 H-bond, a possible explanation for why exogenous imidazole does not complement the loss of the His1 imidazole ligand.…”

“…Qualitative analyses of the Ni K-edge EXAFS spectra from a number of NiSOD samples have shown that binding the His1 side chain results in two resolved features in the Fourier-transform (FT)-EXAFS spectrum that arise from the first-coordination sphere ligands, whereas in the absence of the apical ligand, only one broad feature is observed. (14) This presumably arises from the presence of a unique longer Ni-N vector in samples with an apical imidazole ligand. This observation is true of oxidized vs. reduced WT-NiSOD, and for H1A-NiSOD, which has only one broad feature in the FT-EXAFS spectrum.…”

“…(12-14) Single colonies were grown overnight at 37 °C with shaking in 10 mL Luria-Bertani broth, supplemented with chloramphenicol (cam) and kanamycin (kan) for selection. These cultures were added to 1L pre-warmed fresh media and grown to an OD 600 of 0.6 and then induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3-5 hours.…”

“…(13, 14, 19) Nickel K -edge XAS data were collected on beamline X3B (and X9B) at the National Synchrotron Light Source (Brookhaven National Laboratory). Samples of frozen protein solutions (1-3 mM, based on nickel content, in 20 mM Tris•HCl, pH 8.0) were placed in polycarbonate holders inserted into aluminum blocks and held near 50 K using a He displex cryostat.…”

“…(13, 14) Superoxide radicals were generated upon pulse radiolysis of an aqueous, air/O 2 saturated solution containing 10 mM phosphate, 30 mM formate, and 5 μM ethylenediaminetetraacetic acid (EDTA). Catalytic rate constants were obtained by monitoring the disappearance of O 2 •− at 260 nm in the presence of micromolar concentrations of mutant NiSOD.…”