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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 into Escherichia coli double 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. [ABSTRACT FROM AUTHOR]

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 with Escherichia coli complex I reconstituted in proteoliposomes, we successfully resolved and characterized three distinct SQ species by EPR. These species include: fast-relaxing SQ (SQ Nf ) with P 1/2 (half-saturation power level) > 50 mW and a wider linewidth (12.8 G); slow-relaxing SQ (SQ Ns ) with P 1/2 = 2–3 mW and a 10 G linewidth; and very slow-relaxing SQ (SQ Nvs ) with P 1/2 = ~ 0.1 mW and a 7.5 G linewidth. The SQ Nf signals completely disappeared in the presence of the uncoupler gramicidin D or squamotacin, a potent E. coli complex I inhibitor. The pH dependency of the SQ Nf signals correlated with the proton-pumping activities of complex I. The SQ Ns signals were insensitive to gramicidin D, but sensitive to squamotacin. The SQ Nvs signals were insensitive to both gramicidin D and squamotacin. Our deuterium exchange experiments suggested that SQ Nf is neutral, while SQ Ns and SQ Nvs are anion radicals. The SQ Ns signals 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. [ABSTRACT FROM AUTHOR]