The prokaryotic proton-translocating NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzyme that contains 14 subunits (NuoA-NuoN or Nqo1-Nqo14). All subunits have their counterparts in the eukaryotic enzyme (complex I). NDH-1 consists of two domains: the peripheral arm (NuoB, -C, -D, -E, -F, -G, and -I) and the membrane arm (NuoA, -H, -J, -K, -L, -M, and -N). In Escherichia coli NDH-1, the hydrophilic subunits NuoC/Nqo5/30k and NuoD/Nqo4/49k are fused together in a single polypeptide as the NuoCD subunit. The NuoCD subunit is the only subunit that does not bear a cofactor in the peripheral arm. While some roles for inhibitor and quinone association have been reported for the NuoD segment, structural and functional roles of the NuoC segment remain mostly elusive. In this work, 14 highly conserved residues of the NuoC segment were mutated and 21 mutants were constructed using the chromosomal gene manipulation technique. From the enzymatic assays and immunochemical and blue-native gel analyses, it was found that residues Glu-138, Glu-140, and Asp-143 that are thought to be in the third α-helix are absolutely required for the energy-transducing NDH-1 activities and the assembly of the whole enzyme. Together with available information for the hydrophobic subunits, we propose that Glu-138, Glu-140, and Asp-143 of the NuoC segment may have a pivotal role in the structural stability of NDH-1.