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：

---

引起疾病的 Ile67Asn 替代对 BOLA3 蛋白的生化影响。

铁硫 (Fe-S) 簇生物合成涉及多种功能不同的蛋白质的作用，其中大多数在进化上是保守的。这些 Fe-S 支架和运输蛋白的突变可导致多种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等疾病。在此，我们研究了导致 MMDS2 表型的 BOLA3 蛋白中 Ile67Asn 取代的影响。尽管 BOLA3 在 Fe-S 簇生物合成中的确切功能作用尚不清楚，但已提出 BOLA3 与另一种 Fe-S 蛋白 GLRX5 的 [2Fe-2S]-桥接复合物作为下游靶标的可行中间簇载体。我们的研究表明，Ile67Asn 的替代削弱了 BOLA3 结合其生理伙伴 GLRX5 的能力，导致无法形成[2Fe-2S]-桥接络合物。虽然 BOLA3 的结构没有发生剧烈的变化，但野生型和突变体 BOLA3 1H-15N HSQC 和离子迁移率天然质谱实验证明了这一点，但这种替代似乎会影响导致疾病表型的下游蛋白质的簇重组。相比之下，BOLA3 的全同二聚体形式的取代衍生物形成并保持对簇交换的活性。