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

Abstract

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.

Keywords

Superoxide dismutase X-ray absorption spectroscopy Differential scanning calorimetry 

Abbreviations

CuZnSOD

Copper- and zinc-containing superoxide dismutase

DFT

Density functional theory

DSC

Differential scanning calorimetry

EPR

Electron paramagnetic resonance

ESI-MS

Electrospray ionization mass spectrometry

EXAFS

Extended X-ray absorption fine structure

FeSOD

Iron-containing superoxide dismutase

LIC

Ligation-independent cloning

MnSOD

Manganese-containing superoxide dismutase

NHE

Normal hydrogen electrode

Ni–NTA

Nickel nitrilotriacetic acid

NiSOD

Nickel-containing superoxide dismutase

PCR

Polymerase chain reaction

SOD

Superoxide dismutase

Tris

Tris(hydroxymethyl)aminomethane

XANES

X-ray absorption near-edge spectroscopy

XAS

X-ray absorption spectroscopy

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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