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

J Biol Inorg Chem. 2010 Jun;15(5):795-807. doi: 10.1007/s00775-010-0645-y. Epub 2010 Mar 24.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biocatalysis
  • Catalytic Domain
  • Cysteine / chemistry*
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Electron Spin Resonance Spectroscopy
  • Enzyme Stability
  • Ligands
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Nickel / metabolism*
  • Protein Structure, Quaternary
  • Streptomyces coelicolor / enzymology
  • Superoxide Dismutase / chemistry*
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase / metabolism*
  • X-Ray Absorption Spectroscopy

Substances

  • Ligands
  • Nickel
  • Superoxide Dismutase
  • Cysteine