The impact of the histidyl residue position on the formation and stability of Cu(ii) complexes and their ability of ROS generation

Abstract

The coordination process of Cu(II) ions to the terminally blocked Ac-H¹WKGPLR-NH₂ (L1), Ac-EH²KA-NH₂ (L2), and Ac-KEH³K-NH₂ (L3) peptides containing a histidyl residue at different positions of the peptide chain (the first, second, or third, respectively) was studied by potentiometry, spectroscopic techniques (UV-Vis, CD and EPR), and mass spectrometry. L3 peptide after binding Cu(II) ions (1 : 1 M : L molar ratio) at pH 7.2 (colon and intracellular brain pH) forms the most stable complex with the 3N {N_Im, 2N⁻} coordination mode. The binding process of the metal ion by L1 and L2 ligands proceeds towards the C-terminus, while for L3 peptide towards the N-terminus. The formation of bis-complexes for L2 and L3 ligands at 1 : 2 metal-to-ligand molar ratio was noted. The Cu(II) bis-complexes with 2N {2N_Im} binding sites are characterized by similar stability. The ability of Cu(II) complexes in the presence of hydrogen peroxide (H₂O₂) or ascorbic acid (Asc) to produce reactive oxygen species (ROS) was also investigated. The generation of hydroxyl radicals (˙OH) was monitored by luminescence with terephthalic acid (TA) as the detector. Moreover, the rate of ascorbate consumption by the studied compounds and their ability to produce ˙OH radicals were compared. Gel electrophoresis was also carried out to determine the type of ROS formed. It was confirmed that all selected Cu(II) peptide complexes in the presence of H₂O₂ or Asc produce ˙OH, while the complex with the L1 ligand additionally generates ¹O₂ in the system. Moreover, the produced ROS cause single- and double-strand DNA damage.