Characterization of the membrane domain subunit NuoK (ND4L) of the NADH-quinone oxidoreductase from Escherichia coli.
The ND4L subunit is the smallest mitochondrial DNA-encoded subunit of the proton-translocating NADH-quinone oxidoreductase (complex I). In an attempt to study the functional and structural roles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinone oxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of the NDH-1 operon by using the homologous recombination technique. The amino acid residues we targeted included two highly conserved glutamic acids that are presumably located in the middle of the membrane and several arginine residues that are predicted to be on the cytosolic side. All point mutants examined had fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutations of nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with a concomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activities was also observed with mutations of another highly conserved residue, Glu-72. These results may suggest that both membrane-embedded acidic residues are important for the coupling mechanism of NDH-1. Furthermore, a severe impairment of the coupled activities occurred when two vicinal arginine residues on a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and other conserved residues in the NuoK subunit for NDH-1 function were discussed.
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Mou-Chieh
MC
Kao
Eiko
E
Nakamaru-Ogiso
Akemi
A
Matsuno-Yagi
Takao
T
Yagi
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D042967:Electron Transport Complex I / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D017354:Point Mutation; D011199:Potentiometry; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D011808:Quinone Reductases / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D020539:Sequence Analysis, Protein
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C098483:NADH dehydrogenase subunit 4; D021122:Protein Subunits; D018698:Glutamic Acid; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I