Semiquinone intermediates are involved in the energy coupling mechanism of E. coli complex I.
Semiquinone intermediates are involved in the energy coupling mechanism of E. coli complex I.
复制标题
半醌中间体参与大肠杆菌复合体 I 的能量耦合机制。
DOI：
10.1016/j.bbabio.2015.04.004
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Nakamaru-Ogiso,Eiko
中科院分区：
文献类型：
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作者：
Narayanan,Madhavan;Leung,StevenA;Inaba,Yuta;Elguindy,MahmoudM;Nakamaru-Ogiso,Eiko
复合物 I（NADH：醌氧化还原酶）是细胞有氧能量代谢的核心，其缺乏与许多人类线粒体疾病有关。复合物 I 利用电子转移能量使质子穿过膜。催化过程中出现的半醌（SQ）中间体被认为是复合物 I 偶联机制的关键。然而，由于检测不稳定和低强度的 SQ 信号极其困难，SQ 的存在仍然存在争议。在这里，我们首次利用蛋白脂质体中重组的大肠杆菌复合物 I，成功地通过 EPR 解析并表征了三种不同的 SQ 物种。这些种类包括：具有 P1/2（半饱和功率水平）> 50 mW 和更宽线宽 (12.8 G) 的快速松弛 SQ (SQNf)；慢弛豫 SQ (SQN)，P1/2= 2–3 mW 和 10 G 线宽；以及非常慢弛豫 SQ (SQNvs)，其中 P1/2= ~ 0.1 mW 和 7.5 G 线宽。当存在解偶联剂短杆菌肽 D 或强效的鳞霉素 E 时，SQNf 信号完全消失。大肠杆菌复合物 I 抑制剂。 SQNf信号的pH依赖性与复合物I的质子泵浦活性相关。SQNs信号对短杆菌肽D不敏感，但对squamotacin敏感。 SQNvs信号对短杆菌肽D和squamotacin均不敏感。我们的氘交换实验表明SQNfi是中性的，而SQNs和SQNv是阴离子自由基。 SQNs信号在缺失转运蛋白模块亚基NuoL和NuoM的ΔNuoL突变体中丢失。讨论了SQ中间体在耦合机制中的作用和关系。
Complex I (NADH:quinone oxidoreductase) is central to cellular aerobic energy metabolism, and its deficiency is involved in many human mitochondrial diseases. Complex I translocates protons across the membrane using electron transfer energy. Semiquinone (SQ) intermediates appearing during catalysis are suggested to be key for the coupling mechanism in complex I. However, the existence of SQ has remained controversial due to the extreme difficulty in detecting unstable and low intensity SQ signals. Here, for the first time withEscherichia colicomplex I reconstituted in proteoliposomes, we successfully resolved and characterized three distinct SQ species by EPR. These species include: fast-relaxing SQ (SQNf) withP1/2(half-saturation power level) > 50 mW and a wider linewidth (12.8 G); slow-relaxing SQ (SQNs) withP1/2= 2–3 mW and a 10 G linewidth; and very slow-relaxing SQ (SQNvs) withP1/2= ~ 0.1 mW and a 7.5 G linewidth. The SQNfsignals completely disappeared in the presence of the uncoupler gramicidin D or squamotacin, a potentE. colicomplex I inhibitor. The pH dependency of the SQNfsignals correlated with the proton-pumping activities of complex I. The SQNssignals were insensitive to gramicidin D, but sensitive to squamotacin. The SQNvssignals were insensitive to both gramicidin D and squamotacin. Our deuterium exchange experiments suggested that SQNfis neutral, while SQNsand SQNvsare anion radicals. The SQNssignals were lost in the ΔNuoL mutant missing transporter module subunits NuoL and NuoM. The roles and relationships of the SQ intermediates in the coupling mechanism are discussed.