title: The membrane subunit NuoL(ND5) is involved in the indirect proton pumping mechanism of Escherichia coli complex I.
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.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. eikoo@mail.med.upenn.edu
authors.forename: Eiko
authors.initials: E
authors.lastname: Nakamaru-Ogiso
authors.forename: Mou-Chieh
authors.initials: MC
authors.lastname: Kao
authors.forename: Han
authors.initials: H
authors.lastname: Chen
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Sinha
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ohnishi
mesh_terms: D000595:Amino Acid Sequence; D017920:Antiporters; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005663:Furans / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D020128:Inhibitory Concentration 50; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009245:NADH Dehydrogenase / Q000378:metabolism*; D017494:Proton Pumps; D011522:Protons; D017386:Sequence Homology, Amino Acid
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D005663:Furans; D017494:Proton Pumps; D011522:Protons; C061022:bullatacin; D009245:NADH Dehydrogenase; C479743:NuoL protein, E coli
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