Copper(II) complex formation processes of alloferon I with point mutation H1K; combined spectroscopic and potentiometric studies.

Mononuclear and polynuclear complexes of the alloferon I with point mutation (H1K) Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (AlloK) and its acetylated derivative Ac-Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (Ac-AlloK) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1 for AlloK while to 3:1 for Ac-AlloK. At physiological pH 7.4 and the metal-to-ligand 1:1molar ratio the AlloK peptide forms the CuL complex with the 4N {NH(2), N(-), 2N(Im)} binding mode. In the Cu(II)-AlloK 4:1 system in wide pH 6.5-10 range the Cu(4)H(-7)L complex dominates with the 3N {NH(2),2N(-)} 3×{N(Im),2N(-)} coordination mode. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites of Ac-AlloK. For Ac-AlloK and 1:1 metal-to-ligand molar ratio the CuHL complex with the 3N {3N(Im)} binding sites in pH 4.5-7.5 range is present in solution. The amine nitrogen donor and all of the histidine residues can be considered to be independent metal-binding sites in the species formed in the systems studied. As a consequence, tri- (for the Ac-AlloK) and tetra-nuclear (for the AlloK peptide) complexes for the metal-to-ligand 3:1 and 4:1molar ratios, respectively, are present in the solution.