The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.

中文翻译：

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通过 NMR 和 EPR 光谱探索铁与人 frataxin 和选定的 Friedreich 共济失调突变体的结合。


神经退行性疾病弗里德赖希共济失调是由线粒体蛋白 frataxin 缺乏引起的。大多数患者在 frataxin 基因的两个等位基因的第一个内含子中都有 GAA 扩增，而少数患者的扩增是杂合的，并且在另一个等位基因中含有突变。 Frataxin 据称参与铁稳态和 FeS 簇的生物合成，但其在这两种途径中的作用尚未明确定义。在这项工作中，我们结合了不同的先进光谱分析来探索人 frataxin 的铁结合特性，如分离的和在 FeS 簇组装机器上。我们首次使用 EPR 光谱来解决这个关键问题，提供了金属离子低对称配位络合物形成的明确证据。通过 2D NMR，我们证实铁可以在两种氧化态下结合，这是一个有争议的问题，此外，我们还能够指出 frataxin 与 ISCU（支架）的 N 端 6his 标记变体的短暂相互作用FeS簇组装机器的蛋白质。为了深入了解与了解疾病分子机制相关的结构/功能关系，我们将研究扩展到四种临床 frataxin 突变体。所有变体在体外激活 FeS 簇组装机器的能力均表现出中度至强烈的损害，同时保持与野生型蛋白相同的铁结合特征。这支持了 frataxin 的多功能性质及其突变的复杂生化后果。