Department of Chemistry, Purdue University, West Lafayette, Indiana 47906, United States.
Department of Physics and Astronomy, Purdue University, West Lafayette, Indiana 47907, United States.
Inorg Chem. 2023 Jul 10;62(27):10780-10791. doi: 10.1021/acs.inorgchem.3c01336. Epub 2023 Jun 27.
Amyloid precursor protein (APP) is the biological precursor of β-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 10 M s. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M s, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O) and peroxynitrite (ONOO) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O at a rate of 1.6 × 10 M s, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO achieved a rate of 1.1 × 10 M s, comparable to native ONOO scavenger peroxiredoxins (10-10 M s). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O scavenger, and an ONOO remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.
淀粉样前体蛋白 (APP) 是 β-淀粉样蛋白的生物前体，β-淀粉样蛋白是与阿尔茨海默病 (AD) 相关的已知组织病理学标志。APP 的功能引起了广泛关注，但仍不明确。APP 的一个细胞外结构域，即 E2 结构域，被提出具有亚铁氧化酶活性并影响神经元铁稳态。然而，已有相互矛盾的证据报道，其确切作用仍不确定。在这里，我们使用扩展 X 射线吸收精细结构 (EXAFS)、紫外-可见和电子顺磁共振 (EPR) 研究了 E2 结构域的 Cu 结合位点，发现除了四个已知的组氨酸外，一个新的不稳定水配体与 Cu(II)辅因子配位。我们通过与二价铁的反应探索了 Cu(II)-E2 结构域的拟亚铁氧化酶活性，并观察到高达 1.0×10 M s 的单轮二价铁氧化活性。Cu(I)-E2 与分子氧的反应速率仅为 5.3 M s，这将限制任何潜在的多轮亚铁氧化酶活性的这种缓慢速率，并阻止在多轮条件下观察到活性。蛋白质的正静电势表面表明其可能与带负电荷的小分子底物（如超氧化物自由基 (O) 和过氧亚硝酸盐 (ONOO)) 反应，而过氧亚硝酸盐是细胞外环境中普遍存在的氧化应激的主要贡献者。我们的测定表明，Cu(I)-E2 可以以 1.6×10 M s 的速率去除 O，这比天然 SOD 的速率慢。然而，Cu(I)-E2 与 ONOO 的反应速率达到 1.1×10 M s，与天然 ONOO 清除剂过氧化物酶 (10-10 M s) 相当。因此，APP 的 E2 结构域可以作为一个酶切位点，在底物限制条件下可能作为亚铁氧化酶、补充性 O 清除剂以及细胞铁外排通道附近的 ONOO 清除剂发挥作用，从而保护神经元细胞免受活性氧 (ROS) 和活性氮 (RNS) 损伤。
