Characterization of the recombinant Rieske [2Fe-2S] proteins HcaC and YeaW from E-coli
Characterization of the recombinant Rieske [2Fe-2S] proteins HcaC and YeaW from E-coli
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DOI：
10.1007/s10534-008-9134-y
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发表时间：
2008-08-01
期刊：
影响因子：
3.5
通讯作者：
Schmidt, Christian L.
中科院分区：
文献类型：
--
作者：
Boxhammer, S.;Glaser, S.;Schmidt, Christian L.
预计大肠杆菌 K12 基因组内的三个基因编码含有典型 Rieske 铁硫簇结合基序的蛋白质。其中两个 hcaC 和 yeaW 在大肠杆菌 BL21 和 Tuner (DE3) pLacI 中过表达。重组蛋白通过紫外/可见光谱和 EPR 光谱进行纯化和分析。 HcaC 和 YeaW 显示铁硫蛋白的典型氧化还原依赖性紫外/可见光谱。还原 HcaC 的 EPR 谱显示了 Rieske 簇的特征 g 值，而 YeaW 的 g 值接近此类铁硫簇的上限。两个铁硫簇都可以被连二亚硫酸盐还原，但不能被抗坏血酸盐还原，这证实了它们根据氨基酸序列被归类为低电位 Rieske 蛋白。这两种蛋白质的系统发育分析表明，HcaC 与 IIB 类加氧酶的 Rieske 铁氧还蛋白明显分离，而 YeaW 的分类仍然值得怀疑。
Three genes within the genome of E. coli K12 are predicted to encode proteins containing the typical Rieske iron-sulfur cluster-binding motifs. Two of these, hcaC and yeaW, were overexpressed in E. coli BL21 and Tuner (DE3) pLacI. The recombinant proteins were purified and analyzed by UV/Vis- and EPR-spectroscopy. HcaC and YeaW display the typical redox-dependent UV/Vis-spectra of iron-sulfur proteins. The EPR spectrum of reduced HcaC shows characteristic g-values of a Rieske cluster whereas the g-values for YeaW are close to the upper limit for this type of iron-sulfur cluster. Both iron-sulfur clusters could be reduced by dithionite, but not by ascorbate, confirming their classification as low-potential Rieske proteins as derived from the amino acid sequences. A phylogenetic analysis of the two proteins reveals that HcaC clearly segregates with the Rieske ferredoxins of class IIB oxygenases whereas the classification of YeaW remains doubtful.