Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.
Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.
复制标题
亚基 NuoL 在 NADH 质子泵耦合机制中的作用：大肠杆菌泛醌氧化还原酶（复合物 I）。
DOI：
10.1093/jb/mvw027
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发表时间：
2016
影响因子：
2.7
通讯作者：
Nakamaru-Ogiso,Eiko
中科院分区：
文献类型：
--
作者：
Narayanan,Madhavan;Sakyiama,JosephA;Elguindy,MahmoudM;Nakamaru-Ogiso,Eiko
呼吸复合物I具有由负责电子转移的亲水臂和包含质子泵送机构的膜臂形成的L形结构。在这里，获得亚基NuoL的作用机理的见解，我们研究了Mg 2+，Zn 2+和Na+/H+逆向转运抑制剂5-（N-乙基-N-异丙基）-阿米洛利（EIPA）对质子泵活性的各种分离NuoL突变体复合物I重组成大肠杆菌双敲除（DKO）膜囊泡缺乏复合物I和NADH脱氢酶2型。发现Mg ~（2+）对配合物I的质子泵活性起着关键作用。在2 µM Zn 2+时，野生型的质子泵被选择性抑制，而不影响电子转移;未观察到D178 N和D400 A的质子泵抑制，表明这些残基参与了Zn 2+结合。15微摩尔的EIPA导致野生型、D303 A和D400 A/E的质子泵活性降低高达40%，而在D178 N中没有检测到显著变化，表明其可能参与EIPA结合。此外，当使用富含甲基萘醌的DKO膜时，与NuoL突变体相比，野生型中的质子泵送效率显著降低（约50%），这强烈表明NuoL参与复合物I中的高效泵送机制。
Respiratory complex I has an L-shaped structure formed by the hydrophilic arm responsible for electron transfer and the membrane arm that contains protons pumping machinery. Here, to gain mechanistic insights into the role of subunit NuoL, we investigated the effects of Mg2+, Zn2+and the Na+/H+antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on proton pumping activities of various isolated NuoL mutant complex I after reconstitution intoEscherichia colidouble knockout (DKO) membrane vesicles lacking complex I and the NADH dehydrogenase type 2. We found that Mg2+was critical for proton pumping activity of complex I. At 2 µM Zn2+, proton pumping of the wild-type was selectively inhibited without affecting electron transfer; no inhibition in proton pumping of D178N and D400A was observed, suggesting the involvement of these residues in Zn2+binding. Fifteen micromolar of EIPA caused up to ∼40% decrease in the proton pumping activity of the wild-type, D303A and D400A/E, whereas no significant change was detected in D178N, indicating its possible involvement in the EIPA binding. Furthermore, when menaquinone-rich DKO membranes were used, the proton pumping efficiency in the wild-type was decreased significantly (∼50%) compared with NuoL mutants strongly suggesting that NuoL is involved in the high efficiency pumping mechanism in complex I.