di Rago J P, Bruel C, Graham L A, Slonimski P, Trumpower B L
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 1996 Jun 28;271(26):15341-5. doi: 10.1074/jbc.271.26.15341.
A cDNA carrying the Rip1 gene, which encodes the Rieske iron-sulfur protein of Schizosaccharomyces pombe, has been cloned by complementing the respiratory deficiency of a Saccharomyces cerevisiae strain in which the endogenous copy of the RIP1 gene has been deleted. The deduced amino acid sequences of the S. pombe and S. cerevisiae iron-sulfur proteins are 50% identical, with the highest region of identity being in the C termini of the proteins, where the 2Fe:2S cluster is bound. When expressed in the S. cerevisiae deletion strain, the S. pombe iron-sulfur protein restores 25-30% of the ubiquinol-cytochrome c reductase activity. The kinetics of cytochrome c reduction, the effects of inhibitors which act at defined sites in the cytochrome bc1 complex, and the optical properties of cytochrome b in membranes from the S. cerevisiae deletion strain complemented with S. pombe iron-sulfur protein indicate that the S. pombe protein interacts with cytochrome b to restore an apparently normal ubiquinol oxidase site, but that interaction between the iron-sulfur protein and cytochrome c1 is partially impaired. This is the first heterologous replacement of an electron transfer protein in a respiratory enzyme complex in S. cerevisiae.
通过互补酿酒酵母菌株中RIP1基因内源性拷贝已缺失的呼吸缺陷，克隆了一个携带Rip1基因的cDNA，该基因编码粟酒裂殖酵母的 Rieske 铁硫蛋白。粟酒裂殖酵母和酿酒酵母铁硫蛋白的推导氨基酸序列有50% 相同，相同程度最高的区域位于蛋白质的C末端，即2Fe:2S簇结合的位置。当在酿酒酵母缺失菌株中表达时，粟酒裂殖酵母铁硫蛋白可恢复泛醇 - 细胞色素c还原酶活性的25% - 30%。细胞色素c还原的动力学、作用于细胞色素bc1复合体特定位点的抑制剂的影响，以及用粟酒裂殖酵母铁硫蛋白互补的酿酒酵母缺失菌株膜中细胞色素b的光学性质表明，粟酒裂殖酵母蛋白与细胞色素b相互作用以恢复一个明显正常的泛醇氧化酶位点，但铁硫蛋白与细胞色素c1之间的相互作用部分受损。这是酿酒酵母呼吸酶复合体中电子传递蛋白的首次异源替代。
