Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 2013 May 17;288(20):14310-14319. doi: 10.1074/jbc.M113.467803. Epub 2013 Mar 29.
ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.
泛醌氧化还原酶（复合物 I）利用向下的氧化还原能量将质子泵过膜。大肠杆菌复合物 I 由 13 个不同的亚基组成，这些亚基由 nuo 操纵子编码，分别命名为 nuoA-N。由于该蛋白质的丰度较低，并且其约 15kb 操纵子的遗传操作存在一些困难，因此大肠杆菌复合物 I 的纯化在技术上具有挑战性。在这里，我们在操纵子中的 nuoE 上游插入了一个组氨酸序列，从而产生了一个新的菌株。这使我们能够通过高效的亲和纯化来制备大量高纯度和高活性的复合物 I。纯化的复合物 I 含有 0.94±0.1mol 的 FMN、29.0±0.37mol 的铁和 1.99±0.07mol 的泛醌/1mol 复合物 I。分离的复合物 I 的消光系数在 274nm 时为 495mM(-1)cm(-1)，在 410nm 时为 50.3mM(-1)cm(-1)。使用 HEPES-Bis-Tris 丙烷，pH7.5，NADH:铁氰化物活性为 219±9.7μmol/min/mg。详细的 EPR 分析除了先前分配的信号外，还显示了另外两个铁硫簇信号 N6a 和 N6b。此外，我们发现了小但明显的半醌信号，这些信号仅在牛复合物 I 中报道过。线宽约为 12G，表明其为中性半醌形式。超过 90%的半醌信号来自于 P½（半饱和功率水平）=1.85 毫瓦的单一实体。当使用作为一种有效的复合物 I 抑制剂的asimicin 时，半醌信号减少了 60%。对半醌的功能作用和簇 N6a/N6b 的 EPR 分配进行了讨论。
