Histidine 103 in Fra2 Is an Iron-Sulfur Cluster Ligand in the [2Fe-2S] Fra2-Grx3 Complex and Is Required for in Vivo Iron Signaling in Yeast
Histidine 103 in Fra2 Is an Iron-Sulfur Cluster Ligand in the [2Fe-2S] Fra2-Grx3 Complex and Is Required for in Vivo Iron Signaling in Yeast
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DOI：
10.1074/jbc.m110.184176
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发表时间：
2011-01-07
影响因子：
4.8
通讯作者：
Outten, Caryn E.
中科院分区：
文献类型：
--
作者：
Li, Haoran;Mapolelo, Daphne T.;Outten, Caryn E.
BolA同源物Fra 2和胞质单硫醇谷氧还蛋白Grx 3和Grx 4一起在调节酿酒酵母中的铁稳态中起关键作用。遗传学研究表明，Grx 3/4和Fra 2调节铁响应转录因子Aft 1和Aft 2的活性，以响应线粒体Fe-S簇的生物合成。我们以前已经表明，Fra 2和Grx 3/4形成一个[2Fe-2S]（2+）-桥接的异二聚体复合物与铁配体提供的活性位点半胱氨酸的Grx 3/4，谷胱甘肽，和组氨酸残基。为了进一步表征这种不寻常的Fe-S结合复合物，使用定点诱变来鉴定Fra 2中影响Fe-S簇结合和调节体内Aft 1活性的特定残基。在这里，我们提出的光谱证据表明，在Fra 2中的His-103是Fra 2-Grx 3复合物中的Fe-S簇配体。该残基的取代并不消除Fe-S簇结合，但它确实导致簇配位的变化和[2Fe-2S]簇的不稳定。体内遗传研究进一步证实，Fra 2 His-103对于控制Aft 1活性以响应细胞铁状态至关重要。使用CD光谱，我们发现近似1 mol eq的apo-Fra 2与[2Fe-2S] Grx 3同二聚体紧密结合形成[2Fe-2S] Fra 2-Grx 3异二聚体，表明体内形成[2Fe-2S] Fra 2-Grx 3异二聚体的机制。综上所述，这些结果表明，组氨酸的协调和稳定性的[2Fe-2S]簇在Fra 2-Grx 3复合物是必不可少的铁调节酵母。
The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. To further characterize this unusual Fe-S-binding complex, site-directed mutagenesis was used to identify specific residues in Fra2 that influence Fe-S cluster binding and regulation of Aft1 activity in vivo. Here, we present spectroscopic evidence that His-103 in Fra2 is an Fe-S cluster ligand in the Fra2-Grx3 complex. Replacement of this residue does not abolish Fe-S cluster binding, but it does lead to a change in cluster coordination and destabilization of the [2Fe-2S] cluster. In vivo genetic studies further confirm that Fra2 His-103 is critical for control of Aft1 activity in response to the cellular iron status. Using CD spectroscopy, we find that similar to 1 mol eq of apo-Fra2 binds tightly to the [2Fe-2S] Grx3 homodimer to form the [2Fe-2S] Fra2-Grx3 heterodimer, suggesting a mechanism for formation of the [2Fe-2S] Fra2-Grx3 heterodimer in vivo. Taken together, these results demonstrate that the histidine coordination and stability of the [2Fe-2S] cluster in the Fra2-Grx3 complex are essential for iron regulation in yeast.