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

  • Olivia E. Johnson
  • Kelly C. Ryan
  • Michael J. Maroney
  • Thomas C. Brunold
Original Paper


Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O2 ·−). 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 O2 ·−, 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 Ni2+ 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 Ni2+ 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 Ni2+ 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-dependent superoxide dismutase Redox-active nickel enzymes Magnetic circular dichroism Density functional theory 



Becke’s three-parameter hybrid functional for exchange combined with the Lee–Yang–Par correlation functional


Circular dichroism

Cys → Ser

Cysteine to serine


Density functional theory


Electron paramagnetic resonance


Extended X-ray absorption fine structure


Highest occupied molecular orbital


Intermediate neglect of differential overlap/spectroscopic parameterization with configuration interaction


Ligand field


Magnetic circular dichroism


Molecular orbital


Nickel-dependent superoxide dismutase


Oxidized nickel-dependent superoxide dismutase


Reduced nickel-dependent superoxide dismutase


Superoxide dismutase


Time-dependent density functional theory





This work was supported by the National Institutes of Health (Grant GM 64631 to T.C.B.), the University of Wisconsin Chemical Biology Interface Training Grant from the National Institutes of Health (Grant T32 GM008505 to O.E.J.), and the National Science Foundation (Grant CHE-0809188 to M.J.M).

Supplementary material

775_2010_641_MOESM1_ESM.pdf (398 kb)
Supplementary material 1 (PDF 398 kb)


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Copyright information

© SBIC 2010

Authors and Affiliations

  • Olivia E. Johnson
    • 1
  • Kelly C. Ryan
    • 2
  • Michael J. Maroney
    • 2
  • Thomas C. Brunold
    • 1
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of ChemistryUniversity of Massachusetts at AmherstAmherstUSA

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