Stabilization of the Nickel Binding Loop in NiSOD and Related Model Complexes: Thermodynamic and Structural Features

Detailed equilibrium and spectroscopic characterization of the complex formation processes of the nickel binding loop in NiSOD and its related fragments is reported in the slightly acidic-alkaline pH range. The N-terminally free and protected nonapeptides HCDLPCGVY-NH₂ (NiSODM¹), HCDLACGVY-NH₂ (NiSODM³), and Ac-HCDLPCGVY-NH₂ (NiSODM²) and the N-terminally shortened analogues HCDL-NH₂ and HCA-NH₂ were synthesized, and their nickel(II) complexes were studied by potentiometric and several spectroscopic techniques. EPR spectroscopy was also used to assign the coordinating donor sites after the in situ oxidation of nickel(II) complexes. The terminal amino groups are the primary metal binding sites for nickel(II) ion in NiSODM¹ and NiSODM³, resulting in the high nickel(II) binding affinity of this peptide via the formation of a square-planar, (NH₂,N^-,S^-,S^-) or (NH₂,N_ImN^-,S^-) coordinated species in a wide pH range. The latter coordination sphere prevents the formation of the active structure of NiSOD under physiological pH, reflecting the crucial role of proline in nickel(II) binding. In situ oxidation of the Ni(II) complexes yielded Ni(III) transient species in the case of nonapeptides. The square-pyramidal coordination environment with axial imidazole ligation provides the active structure of the oxidized form of NiSOD in the case of N-terminally free peptides. Consequently, these ligands are promising candidates for modeling NiSOD. The acylation of the amino terminus significantly reduces the nickel(II) binding affinity of the nonapeptide, while the oxidation results in coordination isomers.