Features of Subunit NuoM (ND4) in Escherichia coli NDH-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.The proton-translocating NADH-quinone oxidoreductase (designated complex I and NDH-1 for mitochondrial and bacterial enzymes, respectively) constitutes the first energy-coupling site in the respiratory chain in most eukaryotic and prokaryotic cells. It catalyzes the reduction of quinone using NADH as electron donor. This activity is coupled to the translocation of protons through the inner mitochondrial/cytoplasmic membranes (1-4). Complex I is one of the largest and most intricate proteins of the respiratory chain, e.g. bovine complex I was reported to contain 45 different subunits (5, 6). In contrast, NDH-1 has relatively simpler structure and seems suitable to study the coupling site 1. In case of Escherichia coli, NDH-1 is made up of only 13 subunits (designated NuoA-N), which are homologous to the 14 subunits that compose the central core of mitochondrial complex I (7, 8). Electron microscopic images of both mitochondrial complex I and bacterial NDH-1 have revealed a characteristic L-shaped form with two distinct domains, a hydrophilic peripheral arm projected into the mitochondrial matrix (or bacterial cytoplasm) and a transmembrane hydrophobic arm (9). The hydrophilic domain catalyzes electron transfer from NADH to quinone and carries all of the cofactors (one FMN and eight or nine iron-sulfur clusters) (10 -13). In contrast, the membrane domain is considered to participate in proton translocation and quinone binding (14 -18).T...

Structural Contribution of C-terminal Segments of NuoL (ND5) and NuoM (ND4) Subunits of Complex I from Escherichia coli

“…The distal location of antiporter-like subunits NuoM and NuoL (7,43) might suggest that their removal would not affect the activity of the peripheral domain. However, our results with the deletion mutants demonstrated a complete absence of the NADH-quinone reductase activity if either was absent (25,44). The null activity of these mutants could be explained by the incorrect NDH-1 assembly and the reduced levels of other subunits examined.…”

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

“…Like MrpA-Glu 140 and MrpD-Glu 137 , the three lysines that are here implicated in antiport roles are conserved among the NuoL, -M, and -N proteins of Complex I as well as across Mrp systems. Mutations in these positions of the respiratory protein homologues have been shown to reduce activity of the proton pumping complex (37,40,41). MrpA-G392R and MrpE-T113Y were also included in Group 3 because of their very low antiport level.…”

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L

“…Three of the pathways involve subunits N, M, and L. Each pathway appears to be formed by two offset vertical half-channels, connected by a horizontal channel near the middle of the membrane. Mutagenesis of residues along these pathways has confirmed their importance in enzyme activity (Amarneh and Vik 2003; Euro et al 2008; Michel et al 2011; Nakamaru-Ogiso et al 2010; Sato et al 2013; Torres-Bacete et al 2007; Torres-Bacete et al 2009). From the crystal structure of the entire Complex I (Baradaran et al 2013), a fourth proton pathway (the E-channel) was proposed that is formed by the remaining bacterial membrane subunits A, H, J, and K. Subunit K has 100 amino acids and 3 TM helices, with the N-terminus localized to the periplasm, and the C-terminus, consisting of about 18 amino acids, in the cytoplasm.…”