Characterization of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR.
Characterization of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR.
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DOI：
10.1021/bi3003204
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发表时间：
2012-06-05
期刊：
影响因子：
2.9
通讯作者：
Kiley PJ
中科院分区：
文献类型：
--
作者：
Fleischhacker AS;Stubna A;Hsueh KL;Guo Y;Teter SJ;Rose JC;Brunold TC;Markley JL;Münck E;Kiley PJ
IscR是一种含Fe-S簇的转录因子，参与控制大肠杆菌中Fe-S簇生物发生的稳态机制。尽管已经提出IscR作为细胞对Fe-S簇生物发生需求的传感器，但IscR执行这一功能的机制尚不清楚。在这项研究中，我们研究了IscR的Fe-S簇的生化特性，以深入了解所提出的传感活性。Mössbauer研究表明，在体内，IscR主要含有一个还原的[2Fe-2S]1+簇。然而，在厌氧分离IscR后，一些簇被氧化成[2Fe-2S]2+形式。然而，在体外实验中，簇氧化并没有改变IscR对其启动子内结合位点的亲和力，这表明簇氧化状态对DNA结合的调节并不重要。此外，利用共振拉曼、Mössbauer和核磁共振光谱对厌氧分离的IscR进行表征，提出[2Fe-2S]簇没有完整的半胱氨酸连接。诱变研究表明，除了先前鉴定的三个半胱氨酸残基（Cys92、Cys98和Cys104）外，高度保守的残基His107对于簇连接至关重要。因此，这些数据表明，IscR通过三种半胱氨酸和一种组氨酸的非典型连接方案结合该簇，这一特征可能与IscR作为细胞Fe-S簇状态传感器的功能有关。
IscR is a Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mössbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S]1+ cluster in vivo. However, upon anaerobic isolation of IscR some clusters became oxidized to the [2Fe-2S]2+ form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mössbauer, and NMR spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved residue His107 is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status.
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影响因子：
2.9
作者：
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通讯作者：
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