Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA.
Université de Lorraine, INRAE, IAM, F-54000 Nancy, France.
Int J Mol Sci. 2020 Dec 3;21(23):9237. doi: 10.3390/ijms21239237.
Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S] cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S] cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S] cluster assembly on mitochondrial ferredoxin and [4Fe-4S] cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S] clusters, via two-electron reductive coupling of two [2Fe-2S] clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S] to [4Fe-4S] cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.
铁硫（Fe-S）蛋白对于许多细胞功能至关重要，特别是涉及电子转移和代谢反应的功能。一种必需的单硫谷胱甘肽还原酶 GRXS15 在植物线粒体 Fe-S 蛋白的成熟中起着关键作用。然而，根据已知特性，其特定的分子功能可能与更具特征的酵母和人类同源物不同。因此，我们在此报告了使用体内和体外方法详细表征 GRXS15 与 ISCA 蛋白之间的相互作用。酵母双杂交和双分子荧光互补实验表明，GRXS15 与三种植物线粒体 ISCA1a/1b/2 蛋白中的每一种相互作用。紫外-可见吸收/CD 和共振拉曼光谱表明，ISCA1a 和 ISCA2 的共表达导致每个 ISCA1a/2 异二聚体中含有一个 [2Fe-2S] 簇，但使用纯 [2Fe-2S]-ISCA1a/2 进行簇重建导致一个 [4Fe-4S] 簇结合的 ISCA1a/2 异二聚体。通过紫外-可见吸收和 CD 光谱监测的簇转移反应表明，[2Fe-2S]-GRXS15 在谷胱甘肽过量的情况下介导 [2Fe-2S] 簇在线粒体铁氧还蛋白上的组装和 [4Fe-4S] 簇在 ISCA1a/2 异二聚体上的组装。这表明 ISCA1a/2 是 [4Fe-4S] 簇的组装体，通过两个 [2Fe-2S] 簇的两电子还原偶联。总的来说，这些结果为 GRXS15 和 ISCA1a/2 在促进 [2Fe-2S] 向 [4Fe-4S] 簇转化以成熟植物中含有 [4Fe-4S] 簇的客户蛋白方面的作用提供了新的见解。
