Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H1 and H6 or H9 or H12) binding sites Structure Stability and Biological Activity

Abstract

Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H1GVSGH6GQA9GVA12G, H6A/H12A H1GVSGA6GQH9GVA12G and H6A/H9A H1GVSGA6GQA9GVH12G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 – 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H1,NIm-H6 or 9 or 12} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H1 and the imidazole nitrogen atoms of H6 or H9 or H12 can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other.The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H1,NIm-H6} (CuL) and 3N{NH2,N−,NIm-H6} and/or 4N{NH2,NIm-H1,N−,NIm-H6} (CuH−1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.