Capturing a Reactive State of Amyloid Aggregates

Background: Association of redox-active Cu2+ with aggregated A in amyloid plaques has been linked with ROS and oxidative stress in AD. Results: Cu2+/Cu+-bound A fibrils undergo a redox cycle reaction with ascorbate and oxygen to produce H2O2. Conclusion: Cu2+/Cu+ ions bound to histidines of A fibril offer enzyme-like reaction centers. Significance: The first site-specific structural evidence is presented on Cu+-bound A fibrils that generate ROS. The interaction of redox-active copper ions with misfolded amyloid (A) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu+/Cu2+ with A aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu+/Cu2+ and A fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of approximate to 60 m hydrogen peroxide (H2O2) from a solution of 20 m Cu2+ ions in complex with A(1-40) in fibrils ([Cu2+]/[A] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (approximate to 1 mm). Furthermore, SSNMR H-1 T-1 measurements demonstrated that during ROS production the conversion of paramagnetic Cu2+ into diamagnetic Cu+ occurs while the reactive Cu+ ions remain bound to the amyloid fibrils. The results also suggest that O-2 is required for rapid recycling of Cu+ bound to A back to Cu2+, which allows for continuous production of H2O2. Both C-13 and N-15 SSNMR results show that Cu+ coordinates to A(1-40) fibrils primarily through the side chain N of both His-13 and His-14, suggesting major rearrangements from the Cu2+ coordination via N-E in the redox cycle. C-13 SSNMR chemical shift analysis suggests that the overall A conformations are largely unaffected by Cu+ binding. These results present crucial site-specific evidence of how the full-length A in amyloid fibrils offers catalytic Cu+ centers.