Secondary sphere interactions modulate peroxynitrite scavenging by the E2 domain of amyloid precursor protein

Peroxynitrite (ONOO^-) is a highly reactive nitrogen species that can cause significant damage to proteins, lipids, and DNA. Various enzymes, including metalloenzymes, play crucial roles in reducing ONOO^- concentrations to protect cellular components. While the interaction of ONOO^- with heme proteins is well known, the reduction by Cu-containing proteins is less studied. Amyloid precursor protein (APP), implicated in Alzheimer's disease, has an E2 domain that binds copper ions with a dissociation constant of K_D ∼ 10^-12 M and is proposed to be involved in iron homeostasis, copper trafficking, and oxidative stress response. Our recent studies using EXAFS, UV-Vis, and EPR spectroscopy revealed a previously unidentified labile water ligand in the Cu(II) site of the E2 domain, suggesting reactivity with anionic substrates like ONOO^-. Experimental data showed that Cu(I)-E2 reduces ONOO^- at a significant rate (1.1 × 10⁵ M^-1 s^-1), comparable to native peroxynitrite scavengers, while maintaining active site integrity through multiple redox cycles. This study further investigates the mechanism of ONOO^- reduction by Cu(I)-E2 using the Griess assay, demonstrating that reduction occurs via single electron transfer, forming nitrite and nitrate. This process aligns with previous findings that Cu(I)-E2 is oxidized to Cu(II)-E2 upon ONOO^- reduction. Mutations at Lys435, affecting secondary sphere interactions, revealed that factors beyond electrostatics are involved in substrate recruitment. MD simulations suggest that steric hindrance from a newly formed hydrogen bond also plays a role. Understanding ONOO^- reduction by the E2 domain of APP expands our knowledge of copper proteins in mitigating oxidative stress and elucidates their physiological and pathological roles, particularly in Alzheimer's disease.