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. Johnson
  • Kelly C. Ryan
  • Michael J. Maroney
  • Thomas C. Brunold
Original Paper

Abstract

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.

Keywords

Nickel-dependent superoxide dismutase Redox-active nickel enzymes Magnetic circular dichroism Density functional theory 

Abbreviations

B3LYP

Becke’s three-parameter hybrid functional for exchange combined with the Lee–Yang–Par correlation functional

CD

Circular dichroism

Cys → Ser

Cysteine to serine

DFT

Density functional theory

EPR

Electron paramagnetic resonance

EXAFS

Extended X-ray absorption fine structure

HOMO

Highest occupied molecular orbital

INDO/S-CI

Intermediate neglect of differential overlap/spectroscopic parameterization with configuration interaction

LF

Ligand field

MCD

Magnetic circular dichroism

MO

Molecular orbital

NiSOD

Nickel-dependent superoxide dismutase

NiSODox

Oxidized nickel-dependent superoxide dismutase

NiSODred

Reduced nickel-dependent superoxide dismutase

SOD

Superoxide dismutase

TD-DFT

Time-dependent density functional theory

Tris

Tris(hydroxymethyl)aminomethane

Supplementary material

775_2010_641_MOESM1_ESM.pdf (398 kb)
Supplementary material 1 (PDF 398 kb)

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Copyright information

© SBIC 2010

Authors and Affiliations

  • Olivia E. Johnson
    • 1
  • Kelly C. Ryan
    • 2
  • Michael J. Maroney
    • 2
  • Thomas C. Brunold
    • 1
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of ChemistryUniversity of Massachusetts at AmherstAmherstUSA

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