Zuercher Eli C, Poore Andrew T, Prajapat Devendra, Palazzo Joseph, Thomas Alana, Birthright Caitlin, Lawrence Jack, Chen Ming, Tian Shiliang
Department of Chemistry, Purdue University, West Lafayette, Indiana 47906, USA.
Dalton Trans. 2025 Jan 2;54(2):571-581. doi: 10.1039/d4dt02552k.
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.
过氧亚硝酸根（ONOO）是一种高反应性的氮物种，可对蛋白质、脂质和DNA造成严重损伤。包括金属酶在内的各种酶在降低ONOO浓度以保护细胞成分方面发挥着关键作用。虽然ONOO与血红素蛋白的相互作用已为人所知，但含铜蛋白对其的还原作用研究较少。与阿尔茨海默病相关的淀粉样前体蛋白（APP）有一个E2结构域，它以约10 M的解离常数结合铜离子，并被认为参与铁稳态、铜转运和氧化应激反应。我们最近使用扩展X射线吸收精细结构（EXAFS）、紫外可见光谱和电子顺磁共振（EPR）光谱进行的研究揭示了E2结构域的Cu（II）位点中一个以前未被识别的不稳定水配体，表明其与ONOO等阴离子底物具有反应性。实验数据表明，Cu（I）-E2以显著速率（1.1×10 M s）还原ONOO，与天然过氧亚硝酸根清除剂相当，同时通过多个氧化还原循环保持活性位点的完整性。本研究使用格里斯试剂法进一步研究了Cu（I）-E2还原ONOO的机制，证明还原是通过单电子转移发生的，形成亚硝酸盐和硝酸盐。这一过程与之前的发现一致，即ONOO还原后Cu（I）-E2被氧化为Cu（II）-E2。影响二级球相互作用的赖氨酸435位点的突变表明，除了静电作用外，还有其他因素参与底物募集。分子动力学（MD）模拟表明，新形成的氢键产生的空间位阻也起作用。了解APP的E2结构域对ONOO的还原作用，扩展了我们对铜蛋白在减轻氧化应激方面的认识，并阐明了它们的生理和病理作用，特别是在阿尔茨海默病中的作用。
