Formation of [4Fe-4S] Clusters in the Mitochondrial Iron-Sulfur Cluster Assembly Machinery
Formation of [4Fe-4S] Clusters in the Mitochondrial Iron-Sulfur Cluster Assembly Machinery
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DOI：
10.1021/ja507822j
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发表时间：
2014-11-19
影响因子：
15
通讯作者：
Banci, Lucia
中科院分区：
文献类型：
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作者：
Brancaccio, Diego;Gallo, Angelo;Banci, Lucia
在酵母和人类细胞中，线粒体中[4Fe-4S]簇的产生关键依赖于两个A-型ISC蛋白(在哺乳动物中称为ISCA1和ISCA2)，它们在体内发挥非冗余的功能，形成一个异源复合体。ISCA1和ISCA2蛋白的分子功能，即这些蛋白如何帮助产生[4Fe-4S]簇，目前仍不清楚。在这项工作中，我们对人ISCA2的Fe/S簇结合性质进行了结构表征，并在体外研究了当人ISCA1和ISCA2与生理的[2Fe-2S](2+)簇供体人GRX5相互作用时，是否以及如何组装了[4Fe-4S]簇。我们发现：(I)ISCA2以二聚态结合[2Fe-2S]或[4Fe-4S]团簇，(Ii)[2Fe-2S](2+)GRX5的两个分子将其团簇贡献给异二聚体ISCA1/ISCA2络合物。该络合物充当[4Fe-4S]团簇的“集合器”，即两个GRX5捐赠的[2Fe-2S](2+)团簇生成一个[4Fe-4S](2+)团簇。也观察到相同的[4Fe-4S](2+)簇结异二聚体的形成，首先将一个[2Fe-2S](2+)簇从GRX5转移到每个单独的ISCA1和ISCA2蛋白上，形成[2Fe-2S](2+)ISCA2和[2Fe-2S](2+)ISCA1，然后将它们混合在一起。这些发现表明，这种异二聚体复合体是线粒体中的功能单位，接收来自hGRX5的[2Fe-2S]簇，并在它们转移到最终目标载脂蛋白之前组装[4Fe-4S]簇。
The generation of [4Fe-4S] clusters in mitochondria critically depends, in both yeast and human cells, on two A-type ISC proteins (in mammals named ISCA1 and ISCA2), which perform a nonredundant functional role forming in vivo a heterocomplex. The molecular function of ISCA1 and ISCA2 proteins, i.e., how these proteins help in generating [4Fe-4S] clusters, is still unknown. In this work we have structurally characterized the Fe/S cluster binding properties of human ISCA2 and investigated in vitro whether and how a [4Fe-4S] cluster is assembled when human ISCA1 and ISCA2 interact with the physiological [2Fe-2S](2+) cluster-donor human GRX5. We found that (i) ISCA2 binds either [2Fe-2S] or [4Fe-4S] cluster in a dimeric state, and (ii) two molecules of [2Fe-2S](2+) GRX5 donate their cluster to a heterodimeric ISCA1/ISCA2 complex. This complex acts as an "assembler" of [4Fe-4S] clusters; i.e., the two GRX5-donated [2Fe-2S](2+) clusters generate a [4Fe-4S](2+) duster. The formation of the same [4Fe-4S](2+) cluster-bound heterodimeric species is also observed by having first one [2Fe-2S](2+) cluster transferred from GRX5 to each individual ISCA1 and ISCA2 proteins to form [2Fe-2S](2+) ISCA2 and [2Fe-2S](2+) ISCA1, and then mixing them together. These findings imply that such heterodimeric complex is the functional unit in mitochondria receiving [2Fe-2S] clusters from hGRX5 and assembling [4Fe-4S] clusters before their transfer to the final target apo proteins.