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

Abstract

Nickel-dependent superoxide dismutase (Ni-SOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O 2 ·− ). 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 O 2 ·− , 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 Ni 2+ 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 Ni 2+ 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 Ni 2+ 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.

Dipeptide-Based Models of Nickel Superoxide Dismutase: Solvent Effects Highlight a Critical Role to Ni–S Bonding and Active Site Stabilization

“…Recent mutagenesis studies by Maroney and Brunold on Ni-SOD at Cys2, Cys6 and Cys2/Cys6 (double mutant) have shown that even the absence of one cysteine thiolate promotes high-spin ( S =1) aquated Ni(II) complexes at the Ni-SOD active site with no evidence of the remaining Ni-SCys bond in the single mutants . 21,23 This finding in combination with the results reported here suggests more than redox-modulation/H-storage roles for cysteine in Ni-SOD. We propose that both Cys6 and Cys2 ligands in Ni-SOD are crucial for proper active site assembly and stabilization of the low-spin square-planar Ni(II) state.…”

“…In fact, site-directed mutagenesis of the Cys-S ligands of Ni-SOD suggest that even mutation of one cysteine to serine promotes a high-spin ( S = 1) octahedral Ni(II) site with no S-ligands (replaced by H 2 O). 21,23 When one mol-equiv of the corresponding thiolate is added to these D 2 O solutions, however, the NMR became well-resolved with defined peaks that are consistent with monomeric ( S = 0) [Ni(GC-OMe)(SR)] − complexes (Figure 1 for 2 , Supporting Information for 3–5 ). Indeed, ESI-MS − analysis of as-isolated 2 and 4 dissolved in protic solvents revealed ions corresponding to [Ni 2 (GC-OMe) 2 (SR)] − , [Ni 3 (GC-OMe) 3 (SR)] − and [Ni 4 (GC-OMe) 4 (SR)] − in addition to those of the parent ion (Figures S20–27).…”

Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and Its H-Bonding Network

“…Such high-spin Ni(II) centers have also been observed in Cys-to-Ser mutants of NiSOD, which are catalytically inactive. (14, 27) The formation of a high-spin six-coordinate complex for the disordered “Ni-hook” domain may also sterically prevent folding into the “Ni-hook” structure that features a His1 imidazole-Glu17 H-bond, a possible explanation for why exogenous imidazole does not complement the loss of the His1 imidazole ligand.…”

Superoxide Dismutases and Superoxide Reductases

“…The role of the nickel ligands in NiSOD in those features has been addressed using a mutagenic approach, which has resulted in a number of mutant proteins with lower catalytic activity. 81 , 89 , 96 The activities of mutant NiSODs have been reviewed. 81 , 97…”