Abstract
A disulfide-bridged peptide containing two Ni2+ binding sites based on the nickel superoxide dismutase protein, {Ni2(SODmds)}, has been prepared. At physiological pH (7.4) it was found that the metal sites are mononuclear with a square planar NOS2 coordination environment with the two sulfur-based ligands derived from cysteinate residues, the nitrogen ligand derived from the amide backbone and a water ligand. Furthermore, S K-edge X-ray absorption spectroscopy indicated that the two cysteinate sulfur atoms ligated to nickel are each protonated. Elevation of the pH to 9.6 results in the deprotonation of the cysteinate sulfur atoms, and yields a binuclear, cysteinate bridged Ni22+ center with each nickel contained in a distorted square planar geometry. At both pH = 7.4 and 9.6 the nickel sites are moderately air sensitive, yielding intractable oxidation products. However, at pH = 9.6 {Ni2(SODmds)} reacts with O2 at an ~3.5-fold faster rate than at pH = 7.4. Electronic structure calculations indicate the reduced reactivity at pH = 7.4 is a result of a reduction in S(3p) character and deactivation of the nucleophilic frontier molecular orbitals upon cysteinate sulfur protonation.
Highlights
Nickel is an essential biological co-factor found at the active-sites of a number of microbial metalloenzymes and proteins (Chart 1) [1,2,3,4,5]
Not without controversy [33], we have provided strong evidence based monomer contains a mononuclear nickel site that is coordinated by two cis-cysteinate sulfur atoms on sulfur K-edge X-ray absorption studies that like nickel containing superoxide dismutase (NiSOD) itself, NiSOD metallopeptide based mimics from Cys2 and+Cys6, an amidate nitrogen atom from Cys2, and the N-terminal amine nitrogen atom possess a Ni–S(H )–Cys moiety at physiological pH as well [18,22]
Considering the only major modification betweenthe peptide from the resin and subsequent aerobic work-up and purification yield purified monomeric and similar NiSOD inspired peptides generated to date is the presence of the 2-mercaptoacetate group, peptides free of disulfide bonds between the two cysteinate sulfur atoms corresponding to Cys2 and we suggest that the disulfide moiety within SODmds results from the oxidative S–S bond formation
Summary
Nickel is an essential biological co-factor found at the active-sites of a number of microbial metalloenzymes and proteins (Chart 1) [1,2,3,4,5]. It has been demonstrated that several redox inactive nickel transport and regulatory proteins possess cysteinate ligands to Ni2+ [6,7,8,9,10,11]. An interesting feature of the nickel-thiolate moiety is its ability to support ligand protonation without subsequent protonolysis [12,13,14]. This is most often observed in (near) square planar Ni2+
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