Nickel superoxide dismutase: structural and functional roles of Cys2 and Cys6 |
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Authors: | Kelly C Ryan Olivia E Johnson Diane E Cabelli Thomas C Brunold Michael J Maroney |
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Institution: | (1) Department of Chemistry, University of Massachusetts at Amherst, 104 Lederle Graduate Research Tower A, 710 North Pleasant Street, Amherst, MA 01003, USA;(2) Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA;(3) Department of Chemistry, Building 555A Brookhaven National Laboratory, P.O. Box 5000, Upton, NY 11973, USA; |
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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. |
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