Program in Molecular and Cellular Biology and Biochemistry and ‡Department of Chemistry, Boston University , Boston, Massachusetts 02215, United States.
Biochemistry. 2013 Jul 9;52(27):4687-96. doi: 10.1021/bi400540m. Epub 2013 Jun 26.
While its biological function remains unclear, the three-cysteine, one-histidine ligated human [2Fe-2S] cluster containing protein mitoNEET is of interest because of its interaction with the anti-diabetes drug pioglitazone. The mitoNEET [2Fe-2S] cluster demonstrates proton-coupled electron transfer (PCET) and marked cluster instability, which have both been linked to the single His ligand. Highly conserved hydrogen bonding networks, which include the His-87 ligand, exist around the [2Fe-2S] cluster. Through a series of site-directed mutations, PCET of the cluster has been examined, demonstrating that multiple sites of protonation exist in addition to the His ligand, which can influence redox potential. The mutations also demonstrate that while replacement of the His ligand with cysteine results in a stable cluster, the removal of Lys-55 also greatly stabilizes the cluster. We have also noted for the first time that the oxidation state of the cluster controls stability: the reduced cluster is stable, while the oxidized one is much more labile. Finally, it is shown that upon cluster loss the mitoNEET protein structure becomes less stable, while upon in vitro reconstitution, both the cluster and the secondary structure are recovered. Recently, two other proteins have been identified with a three-Cys(sulfur), one-His motif, IscR and Grx3/4-Fra2, both of which are sensors of iron and redox homeostatsis. These results lead to a model in which mitoNEET could sense the cellular oxidation state and proton concentration and respond through cluster loss and unfolding.
虽然其生物学功能尚不清楚，但含有三半胱氨酸和一组氨酸配体的人类 [2Fe-2S] 簇的 mitoNEET 蛋白因其与抗糖尿病药物吡格列酮相互作用而受到关注。mitoNEET [2Fe-2S] 簇表现出质子偶联电子转移（PCET）和明显的簇不稳定，这两者都与单个 His 配体有关。高度保守的氢键网络，包括 His-87 配体，存在于 [2Fe-2S] 簇周围。通过一系列定点突变，已经检查了该簇的 PCET，表明除 His 配体外还存在多个质子化部位，这可能会影响氧化还原电位。这些突变还表明，虽然用半胱氨酸替代 His 配体可导致稳定的簇，但去除 Lys-55 也可大大稳定该簇。我们还首次注意到，簇的氧化状态控制其稳定性：还原态簇稳定，而氧化态簇则不稳定。最后，结果表明，簇丢失后，mitoNEET 蛋白结构变得不稳定，而体外重新组装时，簇和二级结构均得到恢复。最近，又发现了另外两种具有三半胱氨酸（硫）和一组氨酸基序的蛋白质 IscR 和 Grx3/4-Fra2，它们都是铁和氧化还原稳态的传感器。这些结果提出了一个模型，即 mitoNEET 可以感知细胞的氧化状态和质子浓度，并通过簇丢失和展开来响应。
