Abstract：
Mitochondria harbor the bacteria-inherited iron-sulfur cluster assembly (ISC) machinery to generate [2Fe-2S; iron-sulfur (Fe-S)] and [4Fe-4S] proteins. In yeast, assembly of [4Fe-4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe-4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe-4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe-4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe-4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.
摘要：
线粒体拥有细菌遗传的铁-硫簇组装（ISC）机制，以产生[2Fe-2S;铁-硫（Fe-S）]和[4Fe-4S]蛋白质。在酵母中，[4Fe-4S]蛋白的组装特别涉及ISC蛋白Isa1，Isa2，Iba57，Bol3和Nfu1。人类等效物的功能缺陷会导致多种线粒体功能障碍综合征，具有广泛临床谱的严重疾病。据描述，细菌Iba57祖先YgfZ需要四氢叶酸（THF）才能在选定的[4Fe-4S]蛋白成熟中发挥功能。YgfZ和Iba57在结构上都与催化THF依赖性单碳转移反应的酶家族相关,包括甘氨酸裂解系统的GcvT。在此基础上,提出了在ISC依赖性[4Fe-4S]蛋白质生物合成中普遍保守的叶酸需求。为了测试线粒体Iba57的想法，我们在酿酒酵母中进行了遗传和生化研究，我们从嗜热真菌嗜热Chaetomium中解析了Iba57的晶体结构。我们为Iba57催化的[4Fe-4S]蛋白质组装途径的THF独立性提供了三条证据。首先,缺乏叶酸的酵母突变体在线粒体[4Fe-4S]蛋白成熟中没有缺陷。第二,Iba57的3D结构缺乏许多与GcvT中定义的THF的侧链接触，并且THF-结合袋收缩。第三，对于GcvT中的THF依赖性催化必不可少的保守Iba57残基中的突变不会损害体内Iba57功能，与不变式的交换相反，表面暴露的半胱氨酸残基。我们得出结论，线粒体Iba57，尽管与YgfZ和THF结合蛋白的结构相似，不利用叶酸的功能。
