Meyer Jacques, Clay Michael D, Johnson Michael K, Stubna Audria, Münck Eckard, Higgins Catherine, Wittung-Stafshede Pernilla
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble, France.
Biochemistry. 2002 Mar 5;41(9):3096-108. doi: 10.1021/bi015981m.
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 2Fe-2S metal site of A. aeolicus ferredoxin have been studied by UV-visible, resonance Raman, EPR, variable temperature magnetic circular dichroism, and Mössbauer spectroscopies. A full-spin Hamiltonian analysis is given for the Mössbauer 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 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 degrees C at pH 7 versus T(m) = 113 degrees C in a molecular variant where the disulfide bridge has been removed), it does not bear on the properties of the 2Fe-2S chromophore. This observation is consistent with the large distance (ca. 20 A) that is predicted to separate the iron-sulfur chromophore from the disulfide bridge.
通过在大肠杆菌中对编码基因进行异源表达，获得了嗜热细菌嗜热栖热菌的一种[2Fe-2S]铁氧化还原蛋白（Fd1）。序列比较表明，该蛋白属于植物和哺乳动物型[2Fe-2S]铁氧化还原蛋白的扩展家族，但也表明它与植物型或哺乳动物型亚家族均无密切相似性。相反，它似乎与该家族的新成员有一些相似之处，特别是与体内铁硫簇组装中涉及的Isc型铁氧化还原蛋白。通过紫外可见光谱、共振拉曼光谱、电子顺磁共振光谱、变温磁圆二色光谱和穆斯堡尔光谱对嗜热栖热菌铁氧化还原蛋白2Fe-2S金属位点的两个氧化还原水平进行了研究。对穆斯堡尔光谱进行了全自旋哈密顿分析。总的来说，光谱数据揭示了与植物型和哺乳动物型铁氧化还原蛋白的差异，这与序列比较结果一致。2Fe-2S电对的中点电位相对于标准氢电极在-375 mV，比哺乳动物型铁氧化还原蛋白的更负，处于植物型铁氧化还原蛋白覆盖范围的上限。嗜热栖热菌铁氧化还原蛋白除了作为[2Fe-2S]簇配体的四个半胱氨酸外，还含有两个半胱氨酸。通过化学修饰和定点诱变已表明，这两个残基在天然蛋白中形成二硫键。虽然该胱氨酸单元在嗜热栖热菌铁氧化还原蛋白的异常热稳定性中起重要作用（在pH 7时Tm = 121℃，而在去除二硫键的分子变体中Tm = 113℃），但它与2Fe-2S发色团的性质无关。这一观察结果与预计将铁硫发色团与二硫键分隔开的大距离（约20 Å）一致。
