Characterization of the Membrane Domain Subunit NuoK (ND4L) of the NADH-Quinone Oxidoreductase from Escherichia coli

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• 作者：Mou-Chieh Kao ; Eiko Nakamaru-Ogiso ; Akemi Matsuno-Yagi ; and Takao Yagi
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：July 12, 2005
• 年：2005
• 卷：44
• 期：27
• 页码：9545 - 9554
• 全文大小：430K
• 年卷期：v.44,no.27(July 12, 2005)
• ISSN：1520-4995

文摘

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 structuralroles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinoneoxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of theNDH-1 operon by using the homologous recombination technique. The amino acid residues we targetedincluded two highly conserved glutamic acids that are presumably located in the middle of the membraneand several arginine residues that are predicted to be on the cytosolic side. All point mutants examinedhad fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutationsof nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with aconcomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activitieswas also observed with mutations of another highly conserved residue, Glu-72. These results may suggestthat 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 residueson a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and otherconserved residues in the NuoK subunit for NDH-1 function were discussed.