Original Paper

JBIC Journal of Biological Inorganic Chemistry

, Volume 15, Issue 5, pp 777-793

First online:

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. JohnsonAffiliated withDepartment of Chemistry, University of Wisconsin-Madison
  • , Kelly C. RyanAffiliated withDepartment of Chemistry, University of Massachusetts at Amherst
  • , Michael J. MaroneyAffiliated withDepartment of Chemistry, University of Massachusetts at Amherst
  • , Thomas C. BrunoldAffiliated withDepartment of Chemistry, University of Wisconsin-Madison Email author 

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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