Energy transducing roles of antiporter-like subunits in Escherichia coli NDH-1 with main focus on subunit NuoN (ND2)

Abstract

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys(395)) is conserved in NuoL (LLys(399)) but is replaced by glutamic acid (MGlu(407)) in NuoM. Our mutation study on NLys(395) suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu(407) is also essential and most likely interacts with conserved LArg(175). Glutamic acids, NGlu(133), MGlu(144), and LGlu(144), are corresponding residues. Unlike mutants of MGlu(144) and LGlu(144), mutation of NGlu(133) scarcely affected the energy-transducing activities. However, a double mutant of NGlu(133) and nearby KGlu(72) showed significant inhibition of these activities. This suggests that NGlu(133) bears a functional role similar to LGlu(144) and MGlu(144) but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that NLys(158) is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of NTM14 interacts with the Mβ sheet located on the opposite side of helix HL. Taken together, the mechanism of H(+) translocation in NDH-1 is discussed.

Keywords: Bioenergetics; Electron Transport System (ETS); Membrane Energetics; Membrane Enzymes; NADH Dehydrogenase; Site-directed Mutagenesis.

FIGURE 1.

Comparison of the amino acid sequences among the NuoN (ND2) subunits and other homologous antiporters. The alignment around helices that contain conserved charged residues presumably involved in H⁺ translocation was carried out by using the Clustal W program (54). Helices are depicted above the alignment based on the three-dimensional structure of E. coli NuoN (17), highlighting the candidates of essentially charged residues for energy-coupled NDH-1 activities (dark colored) and prolines in discontinuous helices (P). Black boxes with white letters show identical residues, whereas dark gray boxes with white letters illustrate similar residues among at least eight listed organisms. Dashes represent gaps to facilitate alignment. Amino acids mutated in this study are marked by arrows with the numbering in E. coli NuoN. Sequence sources and their UniProtKB/Swiss-Prot accession numbers are: E.c-NuoN, E. coli K-12 NuoN subunit (P0AFF0); P.a-NuoN, Pseudomonas aeruginosa NuoN subunit (Q9I0I9); T.t-Nqo14, Thermus thermophilus Nqo14 subunit (Q56229); P.d-Nqo14, Paraccocus denitrificans Nqo14 subunit (A1B479); R.c-NuoN, Rhodobacter capsulatus NuoN subunit (P50973);, N.t-ND2, Nicotiana tabacum GN Nad2 subunit (Q5MA39); B.t-ND2, Bos taurus ND2 subunit (P03892); H.s-ND2, Homo sapiens ND2 subunit (B1NU62); X.l-ND2, Xenopus laevis ND2 subunit (P03894); Y.l-ND2, Yarrowia lipolytica ND2 subunit (Q9B6C8);, C.e-ND2, Caenorhabditis elegans ND2 subunit (P24889); E.c-NuoM, E. coli K-12 NuoM subunit (P0AFE8); E.c-NuoL, E. coli K-12 NuoL subunit (P33607); M.b-EchA, Methanosarcina barkeri EchA subunit (O59652); B.s-MrpA, Bacillus subtilis MrpA subunit (Q9K2S2); B.s-MrpD, B. subtilis MrpD subunit (O05229).

FIGURE 2.

A schematic representation of membrane subunits of E. coli NDH-1 illustrating amino acids investigated in this work. Amino acids proposed to participate in energy transducing activities are listed at the bottom part, highlighting residues studied in this paper (blue rectangles). NuoN residues involved in connection to the other subunits (green ovals) and prolines in the discontinuous helices (TM7 and TM12) in NuoN, NuoM, and NuoL (orange pentagons) are listed in the upper part. Positively and negatively charged residues are shown in blue and red, respectively.

FIGURE 3.

SDS-PAGE and immunoblotting of membrane preparations from NDH-1 mutants. E. coli membranes were loaded on a 4–20% Tris glycine gel. After electrophoresis, the proteins were transferred onto PVDF membranes and Western blotting was carried out. Antibodies specific to NuoB, NuoCD, NuoE, NuoF, NuoG, NuoI, NuoK, NuoL, and NuoM were used.

FIGURE 4.

NADH dehydrogenase activity staining (A) and immunoblotting (B) of BN-PAGE gels of membrane preparations from NDH-1 mutants. The location of E. coli NDH-1 bands is marked by arrows. For the extraction of NDH-1 from membrane fractions, 0.5% dodecyl maltoside was used. A, for activity staining, the gels were incubated with p-nitro blue tetrazolium and NADH. B, for immunoblotting, the membrane proteins were electrotransferred onto PVDF membranes after BN-PAGE, and immunostained with the antibody specific to NuoB.

FIGURE 5.

Detection of the membrane potential generated by dNADH oxidation in NDH-1 mutants. The potential changes (ΔΨ) of E. coli membrane samples were monitored by the absorbance changes of oxonol VI at 630–603 nm at 30 °C. The first arrow indicates addition of dNADH, whereas the second arrow indicates addition of FCCP. Representative traces from different groups of mutants: A, conserved charged residue mutants: 1, WT (or _NKO-rev, _NE133A, _NE133A/_KKO-rev, _NK217C, and _NK247R); 2, _NK217A (or _NK217R); 3, _NK247A (or _NK395R); 4, _LR175A (or _LK342A and _LKO); 5, _NK395A (or _ME407A and _NE133A/_KE72A); 6, _NKO (or _NE133A/_KKO); B, conserved proline mutants: 1, WT (or _NP387G); 2, _NP222A (or _NP387A, _MP239A, _MP399A, _LP234A, and _LP390A); and C, structural element residues: 1, WT; 2, _NK158A (or _NH224A and _NV469A); 3, _NAla⁴⁸¹stop; 4, _NK158R; 5, _NIle⁴⁷⁵stop; 6, _NVal⁴⁶⁹stop.

FIGURE 6.

Generation of a pH gradient coupled to dNADH oxidation in NDH-1 mutants. H⁺ translocations of E. coli membrane samples were measured by the quenching of ACMA fluorescence at room temperature with an excitation wavelength of 410 nm and an emission wavelength of 480 nm. Addition of dNADH or FCCP is indicated by the arrows. Representative traces from different group of mutants: A, conserved charged residue mutants: 1, WT (or _NKO-rev, _NE133A, _NE133A/_KKO-rev, _NK217C and _NK247R); 2, _NK217A; 3, _NK247A (or _NK217R and _NK395R); 4, _LR175A (or _LK342A); 5, _NE133A/_KE72A; 6, _LKO; 7, _ME407A; 8, _NK395A; 9, _NKO (or _NE133A/_KKO); B, conserved proline mutants: 1, WT (or _NP387G); 2, _NP222A (or _NP387A, _MP239A, _MP399A, _LP234A, and _LP390A); and C, structural element residues: 1, WT; 2, _NK158A (or _NH224A, _NV469A and _NAla⁴⁸¹stop); 3, _NK158R (or _NIle⁴⁷⁵stop); 4, _NVal⁴⁶⁹stop.

FIGURE 7.

A schematic representation of the membrane domain of E. coli NDH-1 highlighting the candidates of charged residues involved in energy-coupled NDH-1 activity. A, schematics depicting the charged residues at the borders among the membrane subunits. The three-dimensional structure in ribbon was drawn from the coordinate file 3RKO using YASARA version 11.11.2. B, a schematic drawing of membrane subunits displaying their charged residues possibly involved in energy-coupled NDH-1 activity, in red for negatively charged and blue for positively charged residues, respectively. The postulated conformational change driven by horizontal electrochemical transmission among charged residues is illustrated with a green arrow and possible core regions for H⁺ translocation are indicated with blue case arcs. E. coli numbering is displayed in parentheses. The dNADH-oxidase activity of alanine mutants (except _JTyr⁵⁹ which was mutated to Phe) compared with the WT are shown (in %), listed from Tables 1–3 (underlined) and previous reports: a, Ref. ; b, Ref. ; c, Ref. ; d, Ref. ; e, Ref. , highlighting significant decreases in the activities in bright red.