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 ∼ 10 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 × 10 M s), 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 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.