Characterization of the NuoM (ND4) subunit in Escherichia coli NDH-1: conserved charged residues essential for energy-coupled activities.
The proton-translocating NADH-quinone (Q) oxidoreductase (NDH-1) from Escherichia coli is composed of two segments: a peripheral arm and a membrane arm. The membrane arm contains 7 hydrophobic subunits. Of these subunits, NuoM, a homolog of the mitochondrial ND4 subunit, is proposed to be involved in proton translocation and Q-binding. Therefore, we conducted site-directed mutation of 15 amino acid residues of NuoM and investigated their properties. In all mutants, the assembly of the whole enzyme seemed intact. Mutation of highly conserved Glu144 and Lys234 leads to almost total elimination of energy-transducing NDH-1 activities as well as increased production of superoxide radicals. Their NADH dehydrogenase activities were almost normal. Because these two residues are predicted to be located in the transmembrane segments of NuoM, the results strongly suggest that they participate in proton translocation. Although it is hypothesized that His interacts with a Q head group, mutations at four His moderately inhibited NDH-1 activities and had almost no effect on the Km values for Q or IC50 values of capsaicin-40, a competitive inhibitor for the Q binding site. The data suggest that these His are not involved in the catalytic Q-binding. Functional roles of NuoM and advantages of NDH-1 research as a model for mitochondrial complex I study have been discussed.
Torres-Bacete
Jesus
J
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Nakamaru-Ogiso
Eiko
E
Matsuno-Yagi
Akemi
A
Yagi
Takao
T
D001665:Binding Sites / Q000502:physiology; D002211:Capsaicin / Q000494:pharmacology; D020134:Catalytic Domain / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011522:Protons; D018689:Sensory System Agents / Q000494:pharmacology; D014451:Ubiquinone / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D011522:Protons; D018689:Sensory System Agents; D014451:Ubiquinone; D009245:NADH Dehydrogenase; C476247:NuoM protein, E coli; D042967:Electron Transport Complex I; D002211:Capsaicin