title: Features of subunit NuoM (ND4) in Escherichia coli NDH-1: TOPOLOGY AND IMPLICATION OF CONSERVED GLU144 FOR COUPLING SITE 1.
abstract: The bacterial H(+)-pumping NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzymatic complex. Escherichia coli NDH-1 is composed of 13 subunits (NuoA-N). NuoM (ND4) subunit is one of the hydrophobic subunits that constitute the membrane arm of NDH-1 and was predicted to bear 14 helices. We attempted to clarify the membrane topology of NuoM by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. From the data, we propose a topology model containing 12 helices (helices I-IX and XII-XIV) located in transmembrane position and two (helices X and XI) present in the cytoplasm. We reported previously that residue Glu(144) of NuoM was located in the membrane (helix V) and was essential for the energy-coupling activities of NDH-1 (Torres-Bacete, J., Nakamaru-Ogiso, E., Matsuno-Yagi, A., and Yagi, T. (2007) J. Biol. Chem. 282, 36914-36922). Using mutant E144A, we studied the effect of shifting the glutamate residue to all sites within helix V and three sites each in helix IV and VI on the function of NDH-1. Twenty double site-directed mutants including the mutation E144A were constructed and characterized. None of the mutants showed alteration in the detectable levels of expressed NuoM or on the NDH-1 assembly. In addition, most of the double mutants did not restore the energy transducing NDH-1 activities. Only two mutants E144A/F140E and E144A/L147E, one helix turn downstream and upstream restored the energy transducing activities of NDH-1. Based on these results, a role of Glu(144) for proton translocation has been discussed.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Jesus
authors.initials: J
authors.lastname: Torres-Bacete
authors.forename: Prem Kumar
authors.initials: PK
authors.lastname: Sinha
authors.forename: Norma
authors.initials: N
authors.lastname: Castro-Guerrero
authors.forename: Akemi
authors.initials: A
authors.lastname: Matsuno-Yagi
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; D018698:Glutamic Acid; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase; C476247:NuoM protein, E coli