Holton B, Wu X, Tsapin A I, Kramer D M, Malkin R, Kallas T
Department of Biology and Microbiology, University of Wisconsin, Oshkosh 54901, USA.
Biochemistry. 1996 Dec 3;35(48):15485-93. doi: 10.1021/bi961367c.
The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.
Rieske 2Fe-2S蛋白是叶绿体和蓝藻类囊体膜中光合电子传递细胞色素b6f复合物的一个独特亚基。我们构建了质粒，用于在大肠杆菌中过量表达来自蓝藻念珠藻属Nostoc sp. PCC 7906的Rieske（PetC）蛋白的融合、全长和截短形式。一种谷胱甘肽S-转移酶/Rieske融合蛋白被用于制备针对Rieske蛋白的特异性鸡卵黄抗体。非融合petC基因在T7 RNA聚合酶启动子载体中的表达产生了大量的全长Rieske蛋白，主要以包涵体形式存在。从包涵体中高度富集的Rieske蛋白已在盐酸胍中变性、复性，并在还原条件下与铁和硫化物孵育，在体外重建了特征性的2Fe-2S簇。通过在Whatman DE52纤维素上进行色谱纯化以及通过截留分子量为30000的膜进行超滤，得到了纯的且主要为单体的Rieske蛋白。重建的Rieske制剂在15 K时显示出强烈且高度特征性的gx = 1.74、gy = 1.89和gz = 2.03的“Rieske型”电子顺磁共振信号。两种重建方法得到的Rieske制剂中，通过EPR自旋定量测定，20 - 60%的蛋白含有2Fe-2S簇。重建的Rieske蛋白在含有非离子去污剂的缓冲液中于4℃可溶且稳定，通过光学圆二色性（CD）光谱测定，在pH 7.0时显示出+321 mV的氧化还原中点电位。这些数据证明了半胱氨酸和组氨酸配位的2Fe-2S簇在体外的重建，并为光合放氧中PetC Rieske蛋白的突变和结构分析提供了基础。
