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

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


Nickel superoxide dismutase (NiSOD) is unique among the family of superoxide dismutase enzymes in that it coordinates Cys 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 Ser (C2S-NiSOD, C6S-NiSOD, 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 NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic studies of the Cys mutants revealed 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 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 Cys mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography revealed that the Cys ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.


Superoxide dismutase X-ray absorption spectroscopy Differential scanning calorimetry 



Copper- and zinc-containing superoxide dismutase


Density functional theory


Differential scanning calorimetry


Electron paramagnetic resonance


Electrospray ionization mass spectrometry


Extended X-ray absorption fine structure


Iron-containing superoxide dismutase


Ligation-independent cloning


Manganese-containing superoxide dismutase


Normal hydrogen electrode


Nickel nitrilotriacetic acid


Nickel-containing superoxide dismutase


Polymerase chain reaction


Superoxide dismutase




X-ray absorption near-edge spectroscopy


X-ray absorption spectroscopy



This work was supported by grants from the National Science Foundation (CHE-0809188 to M.J.M) and the National Institutes of Health (GM 64631 to T.C.B.) and by a National Institutes of Health Chemistry–Biology Interface Training Grant (T32 GM008505 to O.E.J.). The US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, supported XAS data collection at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The National Institutes of Health supports beamline X3B (formerly X9B) at NSLS. Pulse radiolysis studies were carried out at the Center for Radiation Chemical Research, which is funded under contract DE-AC02-98CH10886 with the US Department of Energy. The authors also acknowledge Peter A. Bryngelson for contributing information regarding wild-type NiSOD and for help in mutagenesis, and Robert W. Herbst for assistance in EPR and ESI-MS data collection.

Supplementary material

775_2010_645_MOESM1_ESM.pdf (120 kb)
Supplementary material 1 (PDF 120 kb)


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Copyright information

© SBIC 2010

Authors and Affiliations

  • Kelly C. Ryan
    • 1
  • Olivia E. Johnson
    • 2
  • Diane E. Cabelli
    • 3
  • Thomas C. Brunold
    • 2
  • Michael J. Maroney
    • 1
  1. 1.Department of ChemistryUniversity of Massachusetts at AmherstAmherstUSA
  2. 2.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Department of ChemistryBuilding 555A Brookhaven National LaboratoryUptonUSA

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