Department of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy.
Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, Italy.
Int J Mol Sci. 2020 Dec 17;21(24):9619. doi: 10.3390/ijms21249619.
Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich's ataxia. Frataxin's actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin . Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe and Fe might play a relevant role in the context of the proposed FXN physiological functions.
铁蛋白是一种高度保守的蛋白质，其缺乏会导致神经退行性疾病弗里德里希共济失调。铁蛋白的实际生理功能长期以来一直存在争议，尚未达成共识；然而，人们普遍认为该蛋白参与生物合成铁-硫簇（ISC）机制，并且有几位作者指出它还参与铁稳态。在这项工作中，我们使用定点自旋标记与电子顺磁共振（SDSL EPR）结合，为铁和亚铁结合对人铁蛋白特性的影响提供新的信息。使用 SDSL EPR 并将结果与通常用于研究铁与 FXN 结合的荧光实验相关联，我们提供了证据表明，三价铁以浓度依赖的方式引起可逆聚集，而没有优先界面，起始于相对较低的浓度（微摩尔范围），而二价铁结合不会诱导聚集。此外，我们的实验表明，亚铁结合不会导致蛋白质构象发生变化。本研究报告的数据表明，目前报道的结合化学计量应谨慎对待。使用对还原具有抗性的自旋标记以及比较野生型和铁蛋白病理性 D122Y 变体中 Fe 的结合效应，使我们能够表征不同蛋白质部位的 Fe 结合特性，并突出 D122Y 取代对周围残基的影响。我们认为，Fe 和 Fe 可能在拟议的 FXN 生理功能背景下发挥相关作用。
