NMR insights into the behavior of bis-His motifs toward copper ions: A study using mono-N-methylated histidines

The bis-histidine (bis-His) motif, formed by two adjacent histidines, plays a central role in metal coordination within biological systems. Its versatility stems from the imidazole ring of histidine, which offers two distinct nitrogen donors (Nδ/Nπ and Nε/Nτ). This motif contributes to enzymatic activity, redox processes, and metal homeostasis in proteins and peptides. To investigate its coordination properties, we developed a four-point comparative model based on the Aβ_12-16 pentapeptide, introducing selective mono-N-methylation of histidine residues. This strategy enabled a detailed NMR-based characterization of the bis-His motif in the presence of Cu (II) and Cu (I). For Cu(II), distinct paramagnetic relaxation effects were observed depending on the methylation site, reflecting differences in metal-ligand coordination sphere. Similarly, variations in chemical shift induced by Ag(I), used as a probe for Cu(I), confirmed differential interactions with the imidazole nitrogens. The His-His pair was found to act as a structurally flexible binding site able to accommodate both copper oxidation state. Our findings highlight a clear preference for Nπ coordination, with specific patterns of interaction depending on the oxidation state and histidine modification. Reactivity studies in the presence of the endogenous antioxidant glutathione (GSH) further confirmed the role of histidine coordination within the bis-His motif in Cu(II)/Cu(I) redox cycling. Notably, the redox behavior was influenced by the specific imidazole nitrogen involved in metal coordination, with distinct reactivity patterns observed depending on whether the Nπ or Nτ nitrogen was engaged.