The Ubiquinone-binding Site in NADH:Ubiquinone Oxidoreductase from Escherichia coli

• Published in: The Journal of biological chemistry 2003-07, Vol.278 (28), p.25731-25737
• Main Authors: Gong, Xing, Xie, Tong, Yu, Linda, Hesterberg, Micaela, Scheide, Dierk, Friedrich, Thorsten, Yu, Chang-An
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Azides - metabolism; Binding Sites; Cell Membrane - metabolism; Chromatography, High Pressure Liquid; Cytoplasm - metabolism; Detergents - pharmacology; Dose-Response Relationship, Drug; Electron Transport Complex I; Electrophoresis, Polyacrylamide Gel; Endopeptidase K - metabolism; Escherichia coli - enzymology; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Molecular Sequence Data; NADH Dehydrogenase; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - metabolism; Peptides - chemistry; Protein Structure, Secondary; Protein Structure, Tertiary; Time Factors; Ubiquinone - analogs & derivatives; Ubiquinone - chemistry; Ubiquinone - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302361200

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 °C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184–193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.