ESR Spectroscopy Suggests Unequal Contributions of the Three Histidine Residues to Cu(II) Binding in Amyloid- β at Physiological PH

Abstract

We provide precise information about the contribution of each of the three histidine residues, His6, His13, and His14, to Cu(II) binding in the equimolar Cu(II)-Aβ(1-16) complex at physiological pH by employing electron spin resonance spectroscopy. Based on our experimental results, we propose probable Cu(II)-coordination environments for the major and the minor component of the Cu(II)-Aβ(1-16) complex. Three-pulse electron spin-echo envelope modulation (ESEEM) experiments conducted on nonlabeled and 15N-labeled Cu(II)-Aβ(1-16) complexes at a pH of 7.4 reveal that the contributions of the three residues to the Cu(II) coordination are in the order of His14 ≈ His6 > His13 in the major component of the Cu(II)-Aβ(1-16) complex. Also, the ESEEM spectra of diethylenetriamine (DETA)-Cu(II)-Aβ(1-16) complexes suggest that the relative Cu(II)-binding affinities of the three histidine residues are in essentially the same order as in the major component. The corresponding hyperfine sublevel correlation spectra show that multiple histidine residues coordinate to Cu(II) and suggest the presence of axially bound water in the Cu(II)-coordination environment. In addition, a broader interpretation of our results suggests a significant contribution from the simultaneous coordination of His13 and His14 in the major component, which has been underappreciated, and indicates the possibility of intermolecular bridges through His13 in the minor component. Our strategy and experimental results are meaningful in that the subtle difference in the three histidine residues at physiological pH is traced by 15N-labeling and introduction of DETA, a tridendate ligand, without any side chain modification, amino acid residue replacement, or pH change, each of which might lead to an alteration in the peptide structure or the coordination environment.