Assembly of a [2Fe-2S]2+ cluster in a molecular variant of Clostridium pasteurianum rubredoxin.
Assembly of a [2Fe-2S]2+ cluster in a molecular variant of Clostridium pasteurianum rubredoxin.
复制标题
巴氏梭菌红氧还蛋白分子变体中 [2Fe-2S]2 簇的组装。
DOI：
10.1021/bi971775w
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Scott,RA
中科院分区：
文献类型：
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作者：
Meyer,J;Gagnon,J;Gaillard,J;Lutz,M;Achim,C;Munck,E;Petillot,Y;Colangelo,CM;Scott,RA
来自巴氏梭菌的红氧还蛋白含有通过半胱氨酸 6、9、39 和 42 与多肽链结合的单个铁原子。该蛋白的 C42A 变体是通过定点诱变和在大肠杆菌中异源表达该基因而制备的。人们发现突变蛋白含有一种意想不到的发色团，已通过多种技术对其进行了表征。紫外可见吸收和共振拉曼光谱与 [2Fe-2S] 蛋白质的光谱非常相似。在无氧条件下分离的氧化发色团的穆斯堡尔谱表明，反铁磁耦合的高自旋铁离子产生了低温抗磁性。 X 射线吸收精细结构光谱分析得出 Fe−Fe 距离为 2.68 Å。铁和无机硫化物的比色分析表明，这两种元素以 1:1 的比例存在。电喷雾电离质谱显示主要成分为 M= 6190 Da，即 C42A 脱辅基蛋白的分子质量加上两个原子质量的铁和两个原子质量的硫。总而言之，这些数据表明，仅一个点突变就可以稳定通常容纳单核 Fe(Scys)4 位点的蛋白质中的双核 [2Fe-2S] 簇。可能会发生 [2Fe-2S] 簇的组装，因为红氧还蛋白在其金属中心周围呈现与 [2Fe-2S] Rieske 蛋白类似的折叠。或者，也可以考虑C42A红氧还蛋白变体的多肽链的更广泛的结构重排。
The rubredoxin fromClostridium pasteurianumcontains a single iron atom bound to the polypeptide chain by cysteines 6, 9, 39, and 42. The C42A variant of this protein has been prepared by site-directed mutagenesis and heterologous expression of the gene inEscherichia coli. The mutated protein was found to contain an unexpected chromophore that has been characterized by a variety of techniques. UV−visible absorption and resonance Raman spectra were strongly reminiscent of those of [2Fe-2S] proteins. Mössbauer spectra of the oxidized chromophore isolated in oxygen-free conditions indicated low-temperature diamagnetism resulting from antiferromagnetically coupled high-spin ferric ions. Analysis of X-ray absorption fine structure spectra yielded an Fe−Fe distance of 2.68 Å. Colorimetric assays of iron and inorganic sulfide showed that the two elements are present in a 1:1 ratio. Electrospray-ionization mass spectra displayed a major component atM= 6190 Da, i.e. the molecular mass of the C42A apoprotein plus two atomic masses of iron and two atomic masses of sulfur. Taken together, these data show that a mere point mutation allows the stabilization of a binuclear [2Fe-2S] cluster in a protein that normally accommodates a mononuclear Fe(Scys)4site. Assembly of a [2Fe-2S] cluster may occur because rubredoxin assumes a similar fold around its metal center as the [2Fe-2S] Rieske protein. Alternatively, a more extensive structural rearrangement of the polypeptide chain of the C42A rubredoxin variant may be considered as well.