Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.

中文翻译：

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引起疾病的Ile67Asn替代对BOLA3蛋白的生化影响。

铁-硫（Fe-S）簇的生物合成涉及多种功能不同的蛋白质的作用，其中大多数蛋白质在进化上是保守的。这些Fe-S支架和运输蛋白中的突变可引起疾病，例如多线粒体功能障碍综合征（MMDS），铁粒幼细胞性贫血和线粒体脑病。在本文中，我们调查了Ile67Asn替代在BOLA3蛋白中导致MMDS2表型的影响。尽管尚不清楚BOLA3在Fe-S簇生物合成中的确切功能作用，但有人提出BOLA3与[2Fe-2S]桥接的复合物GLRX5（另一种Fe-S蛋白）是下游靶标的可行中间簇载体。我们的研究表明，Ile67Asn取代会削弱BOLA3结合其生理伴侣GLRX5的能力，导致无法形成[2Fe-2S]桥联的复合物。尽管野生型和突变型BOLA3 1H-15N HSQC以及离子淌度天然质谱实验证明，取代并没有使BOLA3发生剧烈的结构变化，但这种取代似乎影响了导致疾病表型的下游蛋白质的簇重构。相反，形成了BOLA3的全同二聚体形式的取代衍生物，并且对簇交换具有活性。