Studies on the Mechanism of Catalysis of Iron-Sulfur Cluster Transfer from IscU[2Fe2S] by HscA/HscB Chaperones
Studies on the Mechanism of Catalysis of Iron-Sulfur Cluster Transfer from IscU[2Fe2S] by HscA/HscB Chaperones
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DOI：
10.1021/bi801565j
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发表时间：
2008-12-02
期刊：
影响因子：
2.9
通讯作者：
Vickery, Larry E.
中科院分区：
文献类型：
--
作者：
Bonomi, Francesco;Iametti, Stefania;Vickery, Larry E.
HscA/HscB分子伴侣/辅分子伴侣系统以ATP依赖性方式加速铁硫簇从FeS-支架蛋白IscU（IscU（2）[Fe 2S]，holo-IscU）转移到受体蛋白。我们采用可见光区圆二色性（CD）测量来监测分子伴侣催化的IscU（2）[2Fe 2S]团簇从holo-IscU到脱铁铁氧还蛋白的转移，并研究分子伴侣诱导的IscU（2）[2Fe 2S]团簇性质的变化。HscA介导的[2Fe 2S]簇转移的加速表现出对HscB和ATP的绝对需求。缺乏ATP酶活性的突变形式HscA（T212 V）不能加速簇转移，这表明HscA伴侣蛋白中伴随ATP（T-状态）到ADP（R-状态）转变的ATP水解和构象变化是催化所需的。加入HscA和HscB到IscU（2）[2Fe 2S]中并不影响[2Fe 2S]团簇的性质，但随后加入ATP会引起可见光区CD光谱的瞬时变化，表明IscU结合的团簇发生了畸变。所观察到的CD变化的衰减速率对ATP浓度的依赖性和缺乏HscA（T212 V）突变体的影响与HscA与ATP水解偶联的簇中的构象变化一致。在HscA、HscB和ATP浓度有限的条件下进行的实验进一步表明，1：1：1的HscA-HscB-IscU（2）[2Fe 2S]复合物的形成和单个ATP水解步骤足以引发分子伴侣对[2Fe 2S]簇的全部作用。这些结果表明，铁硫簇转移的加速涉及在HscA伴随ATP水解的T双右箭头R跃迁期间IscU（2）[2Fe 2S]复合物的结构变化。
The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T double right arrow R transition of HscA accompanying ATP hydrolysis.