Sazanov Leonid A, Carroll Joe, Holt Peter, Toime Laurence, Fearnley Ian M
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 2XY, United Kingdom.
J Biol Chem. 2003 May 23;278(21):19483-91. doi: 10.1074/jbc.M208959200. Epub 2003 Mar 10.
NADH-ubiquinone oxidoreductase (complex I or NDH-1) was purified from the BL21 strain of Escherichia coli using an improved procedure. The complex was effectively stabilized by addition of divalent cations and lipids, making the preparation suitable for structural studies. The ubiquinone reductase activity of the enzyme was fully restored by addition of native E. coli lipids. Two different two-dimensional crystal forms, with p2 and p3 symmetry, were obtained using lipids containing native E. coli extracts. Analysis of the crystals showed that they are formed by fully intact complex I in an L-shaped conformation. Activity assays and single particle analysis indicated that complex I maintains this structure in detergent solution and does not adopt a different conformation in the active state. Thus, we provide the first experimental evidence that complex I from E. coli has an L-shape in a lipid bilayer and confirm that this is also the case for the active enzyme in solution. This suggests strongly that bacterial complex I exists in an L-shaped conformation in vivo. Our results also indicate that native lipids play an important role in the activation, stabilization and, as a consequence, crystallization of purified complex I from E. coli.
使用改进的方法从大肠杆菌BL21菌株中纯化出NADH-泛醌氧化还原酶（复合体I或NDH-1）。通过添加二价阳离子和脂质有效地稳定了该复合体，使得该制剂适合进行结构研究。通过添加天然大肠杆菌脂质，该酶的泛醌还原酶活性得以完全恢复。使用含有天然大肠杆菌提取物的脂质获得了具有p2和p3对称性的两种不同的二维晶体形式。对晶体的分析表明，它们是由处于L形构象的完全完整的复合体I形成的。活性测定和单颗粒分析表明，复合体I在去污剂溶液中保持这种结构，在活性状态下不会采用不同的构象。因此，我们提供了第一个实验证据，即大肠杆菌的复合体I在脂质双层中呈L形，并证实溶液中的活性酶也是如此。这强烈表明细菌复合体I在体内以L形构象存在。我们的结果还表明，天然脂质在从大肠杆菌中纯化的复合体I的激活、稳定以及结晶过程中起着重要作用。
