Spectroscopic and computational investigation of three Cys-to-Ser mutants of nickel superoxide dismutase: insight into the roles played by the Cys2 and Cys6 active-site residues

Nickel-dependent superoxide dismutase (Ni-SOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O(2)(·−)). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel–thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O(2)(·−), demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, 2010). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys → Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni(2+) ion in each mutant adopts a high-spin (S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin (S = 0) configuration observed for the four-coordinate Ni(2+) center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys → Ser mutant possesses an essentially six-coordinate Ni(2+) center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys → Ser NiSOD mutants are explored

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

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