Multiple turnover transfer of [2Fe2S] clusters by the iron-sulfur cluster assembly scaffold proteins IscU and IscA
Multiple turnover transfer of [2Fe2S] clusters by the iron-sulfur cluster assembly scaffold proteins IscU and IscA
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DOI：
10.1074/jbc.m504344200
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发表时间：
2005-08-19
影响因子：
4.8
通讯作者：
Vickery, LE
中科院分区：
文献类型：
--
作者：
Bonomi, F;Iametti, S;Vickery, LE
IscU/Isu 和 IscA/Isa（以及相关的 NifU 和 SufA 蛋白）已被提议用作预组装 [FeS] 簇的分子支架，用于铁硫蛋白的生物合成。证明预制支架-[FeS]复合物向脱辅基蛋白受体转移的体外研究为这一假设提供了实验支持，但迄今为止的研究仅产生了单簇转移事件。我们描述了一种体外测定系统，该系统允许使用圆二色光谱实时监测[FeS]簇的形成，并用它来研究从头[FeS]簇的形成以及从大肠杆菌IscU和IscA到apo-铁氧还蛋白的转移。发现 IscU 和 IscA 都能够进行 [2Fe2S] 簇形成和转移的多个循环，表明这些支架蛋白能够发挥“催化”作用。动力学研究进一步表明，簇转移表现出 Michaelis-Menten 行为，表明 Holo-IscU 和 Holo-IscA 与脱铁铁氧还蛋白形成复合物，并且与直接 [FeS] 簇转移机制一致。然而，对簇转移速率依赖性的分析表明，在支架与受体蛋白的低比率下，效率提高，这表明在转移过程中存在短暂的、不稳定的支架-[FeS]物种。
IscU/Isu and IscA/Isa (and related NifU and SufA proteins) have been proposed to serve as molecular scaffolds for preassembly of [FeS] clusters to be used in the biogenesis of iron-sulfur proteins. In vitro studies demonstrating transfer of preformed scaffold-[FeS] complexes to apoprotein acceptors have provided experimental support for this hypothesis, but investigations to date have yielded only single-cluster transfer events. We describe an in vitro assay system that allows for real-time monitoring of [ FeS] cluster formation using circular dichroism spectroscopy and use this to investigate de novo [FeS] cluster formation and transfer from Escherichia coli IscU and IscA to apo-ferredoxin. Both IscU and IscA were found to be capable of multiple cycles of [2Fe2S] cluster formation and transfer suggesting that these scaffold proteins are capable of acting "catalytically." Kinetic studies further showed that cluster transfer exhibits Michaelis-Menten behavior indicative of complex formation of holo-IscU and holo-IscA with apoferredoxin and consistent with a direct [FeS] cluster transfer mechanism. Analysis of the dependence of the rate of cluster transfer, however, revealed enhanced efficiency at low ratios of scaffold to acceptor protein suggesting participation of a transient, labile scaffold-[FeS] species in the transfer process.