Department of Molecular and Experimental Medicine, MEM-256, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 2013 Aug 23;288(34):24705-16. doi: 10.1074/jbc.M113.482968. Epub 2013 Jul 17.
The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys(395)) is conserved in NuoL (LLys(399)) but is replaced by glutamic acid (MGlu(407)) in NuoM. Our mutation study on NLys(395) suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu(407) is also essential and most likely interacts with conserved LArg(175). Glutamic acids, NGlu(133), MGlu(144), and LGlu(144), are corresponding residues. Unlike mutants of MGlu(144) and LGlu(144), mutation of NGlu(133) scarcely affected the energy-transducing activities. However, a double mutant of NGlu(133) and nearby KGlu(72) showed significant inhibition of these activities. This suggests that NGlu(133) bears a functional role similar to LGlu(144) and MGlu(144) but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that NLys(158) is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of NTM14 interacts with the Mβ sheet located on the opposite side of helix HL. Taken together, the mechanism of H(+) translocation in NDH-1 is discussed.
质子移位的 NADH-醌氧化还原酶（复合物 I/NDH-1）包含一个外周和一个膜域。膜域中的三个类似转运蛋白的亚基，NuoL、NuoM 和 NuoN（分别为 ND5、ND4 和 ND2），在结构上是相似的。我们分析了 NuoN 在大肠杆菌 NDH-1 中的作用。NuoN 位置 395 上的赖氨酸残基（NLys（395））在 NuoL 中保守（LLys（399）），但在 NuoM 中被谷氨酸（MGlu（407））取代。我们对 NLys（395）的突变研究表明，该残基参与质子转运。此外，我们发现 MGlu（407）也是必需的，并且很可能与保守的 LArg（175）相互作用。谷氨酸、NGlu（133）、MGlu（144）和 LGlu（144）是相应的残基。与 MGlu（144）和 LGlu（144）的突变体不同，NGlu（133）的突变几乎不影响能量传递活性。然而，NGlu（133）和附近的 KGlu（72）的双突变体显示出这些活性的显著抑制。这表明 NGlu（133）具有类似于 LGlu（144）和 MGlu（144）的功能作用，但它的突变可以被附近的羧基残基部分补偿。位于 NuoL、NuoM 和 NuoN 的不连续跨膜螺旋环中的保守脯氨酸被证明在能量传递活性中发挥类似的作用。似乎 NuoL、NuoM 和 NuoN 通过类似的机制泵质子。我们的数据还表明，NLys（158）是与 NuoL 中的螺旋 HL 相互作用的关键相互作用点之一。截断研究表明，NTM14 的 C 端两性片段与位于螺旋 HL 对面的 Mβ 片层相互作用。综上所述，讨论了 NDH-1 中 H+转运的机制。
