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Abstract
Complex I pumps protons across the membrane by using downhill redox energy. Here, to investigate the proton pumping mechanism by complex I, we focused on the largest transmembrane subunit NuoL (Escherichia coli ND5 homolog). NuoL/ND5 is believed to have H(+) translocation site(s), because of a high sequence similarity to multi-subunit Na(+)/H(+) antiporters. We mutated thirteen highly conserved residues between NuoL/ND5 and MrpA of Na(+)/H(+) antiporters in the chromosomal nuoL gene. The dNADH oxidase activities in mutant membranes were mostly at the control level or modestly reduced, except mutants of Glu-144, Lys-229, and Lys-399. In contrast, the peripheral dNADH-K(3)Fe(CN)(6) reductase activities basically remained unchanged in all the NuoL mutants, suggesting that the peripheral arm of complex I was not affected by point mutations in NuoL. The proton pumping efficiency (the ratio of H(+)/e(-)), however, was decreased in most NuoL mutants by 30-50%, while the IC(50) values for asimicin (a potent complex I inhibitor) remained unchanged. This suggests that the H(+)/e(-) stoichiometry has changed from 4H(+)/2e(-) to 3H(+) or 2H(+)/2e(-) without affecting the direct coupling site. Furthermore, 50 μm of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor for Na(+)/H(+) antiporters, caused a 38 ± 5% decrease in the initial H(+) pump activity in the wild type, while no change was observed in D178N, D303A, and D400A mutants where the H(+) pumping efficiency had already been significantly decreased. The electron transfer activities were basically unaffected by EIPA in both control and mutants. Taken together, our data strongly indicate that the NuoL subunit is involved in the indirect coupling mechanism.
Highlights
Between NADH and ubiquinone (UQ),4 as a proton motive force across the inner membrane [1,2,3]
Mitochondrial complex I is recognized as one of the most elaborate membranebound iron-sulfur proteins with a total mass close to 1,000 kDa, and it is composed of 45 different protein subunits [4], seven of which are encoded by mitochondrial DNA and all others by nuclear DNA [5, 6]
The x-ray crystallographic structure [10], photoaffinity labeling/cross-linking studies [24, 25], extensive mutagenesis studies of subunits 49 kDa and PSST in Y. lipolytica complex I [26, 27] and the NuoH subunit [28] support that the primary Q-binding site is in the pocket surrounded by the 49 kDa, PSST, and ND1 subunits, and that Hϩ translocation is directly coupled to electron transfer from cluster N2 in PSST to UQ in membrane close to the interface region
Summary
Materials—The pCRScript cloning kit, QuikChange II XL site-directed mutagenesis kit and Herculase-enhanced DNA polymerase were obtained from Stratagene (Cedar Creek, TX). Preparation of nuoL Knock-out and Mutant Cells—The E. coli MC4100 strain was used to generate knock-out and sitespecific mutations of nuoL employing the pKO3 system with the modification described previously by Kao et al [14]. Activity Analyses—The dNADH oxidase assays were performed spectrophotometrically at 37 °C using a Shimadzu UV1800 spectrophotometer with 50 g/ml of membrane samples in 10 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA. The dNADHK3Fe(CN) reductase activities were performed in the same conditions, except that the reaction buffer contained 10 mM KCN and 1 mM K3Fe(CN) at 420 nm. To try to observe proton-pumping activity of complex I alone, we measure the activity in the presence of exogenous quinone, decylubiquinone (DQ) as an electron acceptor and 5 mM KCN, which inhibits the bo oxidase that exists in the downstream of the E. coli respiratory chain. Any variations from the procedures and other details are described in the figure legends
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