Copper Induced Radical Dimerization of α-Synuclein Requires Histidine

Aggregation of the neuronal protein α-synuclein (αS) is a critical factor in the pathogenesis of Parkinson's disease. Analytical methods to detect post-translational modifications of αS are under development, yet the mechanistic underpinnings of biomarkers like dityrosine formation within αS have yet to be established. In our work, we demonstrate that Cu^I-bound N-terminally acetylated αS (^NAcαS) activates O₂ resulting in both intermolecular dityrosine cross-linking within the fibrillar core as well as intramolecular cross-linking within the C-terminal region. Substitution of the H50 residue with a disease relevant Q mutation abolishes intermolecular dityrosine cross-linking and limits the Cu^I/O₂ promoted cross-linking to the C-terminal region. Such a dramatic change in reaction behavior establishes a previously unidentified role for H50 in facilitating intermolecular cross-linking. Involvement of H50 in the reaction profile implies that long-range histidine coordination with the upstream Cu^I coordination site is necessary to stabilize the transition of Cu^I to Cu^II as is a required mechanistic outcome of Cu^I/O₂ reactivity. The aggregation propensity of ^NAcH50Q-Cu^I is also enhanced in comparison to ^NAcαS-Cu^I, suggesting a potential functional role for both copper and intermolecular cross-linking in attenuating ^NAcαS fibrillization.