Copper Induced Radical Dimerization of α-Synuclein Requires Histidine

Aggregation of the neuronal protein a-synuclein (alpha S) is a critical factor in the pathogenesis of Parkinson's disease. Analytical methods to detect post-translational modifications of alpha S are under development, yet the mechanistic underpinnings of biomarkers like dityrosine formation within aS have yet to be established. In our work, we demonstrate that Cu-I-bound N-terminally acetylated alpha S ((NAc)alpha S) activates O-2 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/O-2 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/O-2 reactivity. The aggregation propensity of (NAc)H50Q-Cu-I is also enhanced in comparison to (NAc)alpha S-Cu-I, suggesting a potential functional role for both copper and intermolecular cross-linking in attenuating (NAc)alpha S fibrillization.