Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
Biochemistry. 2012 Jun 5;51(22):4453-62. doi: 10.1021/bi3003204. Epub 2012 May 24.
IscR is an 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 cluster in vivo. However, upon anaerobic isolation of IscR, some clusters became oxidized to the 2Fe-2S form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that the cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mössbauer, and nuclear magnetic resonance 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 His107 residue 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.
IscR 是一种含有 Fe-S 簇的转录因子，参与一种体内平衡机制，该机制控制大肠杆菌中 Fe-S 簇的生物发生。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起作用，但 IscR 执行此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，以深入了解该传感器的活性。穆斯堡尔研究表明，IscR 体内主要含有还原的 2Fe-2S簇。然而，在厌氧分离 IscR 时，一些簇被氧化为 2Fe-2S形式。然而，簇的氧化并没有改变 IscR 在体外与其在 iscR 启动子上的结合位点的亲和力，这表明簇的氧化状态对 DNA 结合的调节并不重要。此外，使用共振拉曼、穆斯堡尔和核磁共振光谱学对厌氧分离的 IscR 的特性进行表征，导致提出 [2Fe-2S]簇没有完全的半胱氨酸连接。突变研究表明，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 His107 残基对于簇的连接是必需的。因此，这些数据表明，IscR 以一种非典型的三个半胱氨酸和一个组氨酸的连接方案结合簇，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。
