Yang X, Yu L, He D, Yu C A
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
J Biol Chem. 1998 Nov 27;273(48):31916-23. doi: 10.1074/jbc.273.48.31916.
When purified ubiquinone (Q)-depleted succinate-ubiquinone reductase from Escherichia coli is photoaffinity-labeled with 3-azido-2-methyl-5-methoxy-[3H]6-geranyl-1,4-benzoquinone ([3H]azido-Q) followed by SDS-polyacrylamide gel electrophoresis, radioactivity is found in the SdhC subunit, indicating that this subunit is responsible for ubiquinone binding. An [3H]azido-Q-linked peptide, with a retention time of 61.7 min, is obtained by high performance liquid chromatography of the protease K digest of [3H]azido-Q-labeled SdhC obtained from preparative SDS-polyacrylamide gel electrophoresis on labeled reductase. The partial N-terminal amino acid sequence of this peptide is NH2-TIRFPITAIASILHRVS-, corresponding to residues 17-33. The ubiquinone-binding domain in the proposed structural model of SdhC, constructed based on the hydropathy plot of the deduced amino acid sequence of this protein, is located at the N-terminal end toward the transmembrane helix I. To identify amino acid residues responsible for ubiquinone binding, substitution mutations at the putative ubiquinone-binding region of SdhC were generated and characterized. E. coli NM256 lacking genomic succinate-Q reductase genes was constructed and used to harbor the mutated succinate-Q reductase genes in a low copy number pRKD418 plasmid. Substitution of serine 27 of SdhC with alanine, cysteine, or threonine or substitution of arginine 31 with alanine, lysine, or histidine yields cells unable to grow aerobically in minimum medium with succinate as carbon source. Furthermore, little succinate-ubiquinone reductase activity and [3H]azido-Q uptake are detected in succinate-ubiquinone reductases prepared from these mutant cells grown aerobically in LB medium. These results indicate that the hydroxyl group, the size of the amino acid side chain at position 27, and the guanidino group at position 31 of SdhC are critical for succinate-ubiquinone reductase activity, perhaps by formation of hydrogen bonds with carbonyl groups of the 1,4-benzoquinone ring of the quinone molecule. The hydroxyl group, but not the size of the amino acid side chain, at position 33 of SdhC is also important, because Ser-33 can be substituted with threonine but not with alanine.
当用3-叠氮基-2-甲基-5-甲氧基-[3H]6-香叶基-1,4-苯醌（[3H]叠氮基-Q）对纯化的来自大肠杆菌的泛醌（Q）缺失型琥珀酸-泛醌还原酶进行光亲和标记，随后进行SDS-聚丙烯酰胺凝胶电泳时，在SdhC亚基中发现放射性，这表明该亚基负责泛醌结合。通过对从制备性SDS-聚丙烯酰胺凝胶电泳得到的[3H]叠氮基-Q标记的SdhC进行蛋白酶K消化产物的高效液相色谱分析，获得了保留时间为61.7分钟的[3H]叠氮基-Q连接肽。该肽的部分N端氨基酸序列为NH2-TIRFPITAIASILHRVS-，对应于第17至33位残基。基于该蛋白质推导氨基酸序列的亲水性图谱构建的SdhC结构模型中，泛醌结合结构域位于靠近跨膜螺旋I的N端。为了鉴定负责泛醌结合的氨基酸残基，在SdhC假定的泛醌结合区域产生并表征了取代突变。构建了缺乏基因组琥珀酸-Q还原酶基因的大肠杆菌NM256，并用于在低拷贝数pRKD418质粒中携带突变的琥珀酸-Q还原酶基因。将SdhC的丝氨酸27替换为丙氨酸、半胱氨酸或苏氨酸，或将精氨酸31替换为丙氨酸、赖氨酸或组氨酸，会导致细胞在以琥珀酸为碳源的基本培养基中无法有氧生长。此外，在这些在LB培养基中好氧生长的突变细胞制备的琥珀酸-泛醌还原酶中，几乎检测不到琥珀酸-泛醌还原酶活性和[3H]叠氮基-Q摄取。这些结果表明，SdhC的第27位羟基、氨基酸侧链大小以及第31位胍基对于琥珀酸-泛醌还原酶活性至关重要，可能是通过与醌分子1,4-苯醌环的羰基形成氢键。SdhC第33位的羟基而非氨基酸侧链大小也很重要，因为丝氨酸33可以被苏氨酸替换但不能被丙氨酸替换。
