The Structure of the Dimeric State of IscU Harboring Two Adjacent [2Fe-2S] Clusters Provides Mechanistic Insights into Cluster Conversion to [4Fe-4S]
The Structure of the Dimeric State of IscU Harboring Two Adjacent [2Fe-2S] Clusters Provides Mechanistic Insights into Cluster Conversion to [4Fe-4S]
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DOI：
10.1021/acs.biochem.1c00112
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发表时间：
2021-05-03
期刊：
影响因子：
2.9
通讯作者：
Takahashi, Yasuhiro
中科院分区：
文献类型：
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作者：
Kunichika, Kouhei;Nakamura, Ryosuke;Takahashi, Yasuhiro
IscU作为支架，在将[2Fe-2S]簇递送到受体蛋白之前进行从头组装。也有人提出，在细菌IscU的一个二聚体上，两个[2Fe-2S]簇可以转化为一个[4Fe-4S]簇。然而，缺乏关于IscU二聚体状态的结构信息阻碍了我们对潜在机制的理解。在本研究中，我们从嗜热古菌Methanothrix thermoacetophila中测定了IscU的x射线晶体结构，并证明了IscU的二聚体结构，其中两个[2Fe-2S]簇在二聚体界面上彼此靠近。我们的结构也首次揭示了Asp40作为[2Fe-2S]簇的第四个配体，每个单体中有三个Cys配体，与之前的光谱数据一致。我们通过EPR光谱分析证实，在溶液中，野生型二聚体中两个相邻的[2Fe-2S]簇通过还原耦合转化为[4Fe-4S]簇。此外，我们发现H106A取代消除了向[4Fe-4S]簇的还原转化而没有结构改变，这表明His106在功能上参与了这一过程。总的来说，这些发现为Fe-S簇在IscU上的组装和转化提供了结构上的解释，并强调了通过IscU二聚体的结合和解离来推进的动态过程。
IscU serves as a scaffold for the de novo assembly of a [2Fe-2S] cluster prior to its delivery to recipient protein. It has also been proposed that on one dimer of bacterial IscU, two [2Fe-2S] clusters can be converted into a single [4Fe-4S] cluster. However, lack of structural information about the dimeric state of IscU has hindered our understanding of the underlying mechanisms. In this study, we determine the X-ray crystal structure of IscU from the thermophilic archaeon Methanothrix thermoacetophila and demonstrate a dimer structure of IscU in which two [2Fe-2S] clusters are facing each other in close proximity at the dimer interface. Our structure also reveals for the first time that Asp40 serves as a fourth ligand to the [2Fe-2S] cluster with three Cys ligands in each monomer, consistent with previous spectroscopic data. We confirm by EPR spectroscopic analysis that in solution two adjacent [2Fe-2S] clusters in the wild-type dimer are converted to a [4Fe-4S] cluster via reductive coupling. Furthermore, we find that the H106A substitution abolishes the reductive conversion to the [4Fe-4S] cluster without structural alteration, suggesting that His106 is functionally involved in this process. Overall, these findings provide a structural explanation for the assembly and conversion of Fe-S clusters on IscU and highlight a dynamic process that advances via association and dissociation of the IscU dimer.