A hyperthermophilic plant-type [2Fe-2S] ferredoxin from Aquifex aeolicus is stabilized by a disulfide bond
A hyperthermophilic plant-type [2Fe-2S] ferredoxin from Aquifex aeolicus is stabilized by a disulfide bond
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DOI：
10.1021/bi015981m
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发表时间：
2002-03-05
期刊：
影响因子：
2.9
通讯作者：
Wittung-Stafshede, P
中科院分区：
文献类型：
--
作者：
Meyer, J;Clay, MD;Wittung-Stafshede, P
通过在大肠杆菌中异源表达[2Fe-2S]铁氧还蛋白(FD1)，从嗜热菌Aquifex aeolicus中获得了一个新的铁氧还蛋白。序列比较表明，该蛋白属于植物型和哺乳动物型铁氧还蛋白的扩展家族，但也表明它与植物型和哺乳动物型亚家族并不是很相似。相反，它似乎与这个家族的新成员有一些相似之处，特别是参与体内铁-硫簇组装的ISC型铁氧还蛋白。用紫外可见光谱、共振拉曼光谱、EPR光谱、变温磁圆二色谱和穆斯堡尔谱研究了铁氧化还蛋白[2Fe2S](2+/+)金属中心的两个氧化还原能级。对穆斯堡尔谱进行了全自旋哈密顿分析。总体而言，光谱数据揭示了与植物型和哺乳动物型铁氧还蛋白的差异，这与序列比较一致。与正常氢电极相比，[2Fe-2S](2+/+)对的中点电位为-375 mV，比哺乳类铁氧还蛋白的中点电位更负，处于植物型铁氧还蛋白覆盖范围的上端。除了作为[2Fe-2S]簇配体的四个半胱氨酸外，黄曲霉铁氧还蛋白还含有两个半胱氨酸。这两个残基已经通过化学修饰和定点突变在天然蛋白质中形成了一座二硫键。虽然胱氨酸单元在铁氧化还蛋白的特殊热稳定性中起着重要作用(在pH 7时T-m=121℃，而在二硫键被移除的分子变体中T-m=113℃)，但它对[2Fe-2S](2+/+)生色团的性质没有影响。这一观察结果与预测将铁硫发色团与二硫键分离的大距离(约20埃)是一致的。
A [2Fe-2S] ferredoxin (Fd1) from the hyperthermophilic bacterium Aquifex aeolicus has been obtained by heterologous expression of the encoding gene in Escherichia coli. Sequence comparisons show that this protein belongs to the extended family of plant- and mammalian-type [2Fe-2S] ferredoxins but also indicate that it is not closely similar to either the plant-type or mammalian-type subfamilies. Instead, it appears to bear some similarity to novel members of this family, in particular the Isc-type ferredoxins involved in the assembly of iron-sulfur clusters in vivo. The two redox levels of the [2Fe2S](2+/+) metal site of A. aeolicus ferredoxin have been studied by UV-visible, resonance Raman, EPR, variable temperature magnetic circular dichroism, and Mossbauer spectroscopies. A full-spin Hamiltonian analysis is given for the Mossbauer spectra. In aggregate, the spectroscopic data reveal differences with both the plant-type and mammalian-type ferredoxins, in keeping with the sequence comparisons. The midpoint potential of the [2Fe-2S](2+/+) couple, at -375 mV versus the normal hydrogen electrode, is more negative than those of mammalian-type ferredoxins and at the upper end of the range covered by plant-type ferredoxins. A. aeolicus ferredoxin contains two cysteines in addition to the four that are committed as ligands of the [2Fe-2S] cluster. These two residues have been shown by chemical modification and site-directed mutagenesis to form a disulfide bridge in the native protein. While that cystine unit plays a significant role in the exceptional thermostability of A. aeolicus ferredoxin (T-m = 121 degreesC at pH 7 versus T-m = 113 degreesC in a molecular variant where the disulfide bridge has been removed), it does not bear on the properties of the [2Fe-2S](2+/+) chromophore. This observation is consistent with the large distance (ca. 20 Angstrom) that is predicted to separate the iron-sulfur chromophore from the disulfide bridge.