Identification of Cu–OOH during the Cu–Aβ reaction with oxygen: mechanistic insights into amyloid β peptide fibrillization in Alzheimer's disease

Abstract

The reaction of copper-bound amyloid β (Cu–Aβ) with molecular oxygen is implicated in the fibrillization of Aβ peptides, a hallmark of Alzheimer's disease pathology. Identifying reactive intermediates in Cu–Aβ–O₂ chemistry is essential for understanding how copper ions modulate peptide fibrillization. Here, we employed advanced electron paramagnetic resonance (EPR) spectroscopy to investigate the Cu–Aβ_1–16–O₂ reaction and observed substantial ligand rearrangement at the copper center. Remarkably, a Cu–OOH species was identified for the first time via pulse EPR spectroscopy along with ¹⁷O isotope labeling. ¹⁷O electron spin echo envelope modulation (ESEEM) experiments confirmed the direct coordination of oxygen to copper, and ¹H/²H electron nuclear double resonance (ENDOR) spectroscopy revealed the presence of an exchangeable proton within the Cu–OOH moiety. Isotope labeling in combination with pulse EPR thus provides strong evidence for the formation of the Cu–OOH species. In addition, solvent kinetic isotope effect (KIE) measurements in H₂O and D₂O yielded a KIE < 2, indicating a concerted proton–electron transfer (CPET) mechanism in the formation of the Cu–OOH species. Furthermore, the Cu–OOH species exhibits oxygen-atom-transfer (OAT) reactivity, further substantiating its assignment. This work provides the first detailed structural and kinetic characterization of Cu–OOH formation in the Cu–Aβ_1–16–O₂ system, offering new insights into copper-mediated redox chemistry and its role in Aβ fibrillization.