Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli

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 Mg , Zn and the Na /H antipor...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2016-10, Vol.160 (4), p.205-215
Main Authors: Narayanan, Madhavan, Sakyiama, Joseph A, Elguindy, Mahmoud M, Nakamaru-Ogiso, Eiko
Format: Article
Language:English
Subjects:
Amiloride - analogs & derivatives
Amiloride - chemistry
Cell Membrane - enzymology
Cell Membrane - genetics
Electron Transport Complex I - antagonists & inhibitors
Electron Transport Complex I - chemistry
Electron Transport Complex I - genetics
Electron Transport Complex I - metabolism
Escherichia coli - enzymology
Escherichia coli Proteins - antagonists & inhibitors
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Mutation
NADH Dehydrogenase - antagonists & inhibitors
NADH Dehydrogenase - chemistry
NADH Dehydrogenase - genetics
NADH Dehydrogenase - metabolism
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Summary: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 Mg , Zn 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 into Escherichia coli double knockout (DKO) membrane vesicles lacking complex I and the NADH dehydrogenase type 2. We found that Mg was critical for proton pumping activity of complex I. At 2 µM Zn , 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 Zn 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.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvw027