The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli.
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 degrees 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.
Gong
Xing
X
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Xie
Tong
T
Yu
Linda
L
Hesterberg
Micaela
M
Scheide
Dierk
D
Friedrich
Thorsten
T
Yu
Chang-An
CA
D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D001386:Azides; D003902:Detergents; D029968:Escherichia coli Proteins; D010455:Peptides; D014451:Ubiquinone; C048123:3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; D009247:NADH, NADPH Oxidoreductases; D009245:NADH Dehydrogenase; C476247:NuoM protein, E coli; D019286:Endopeptidase K; D042967:Electron Transport Complex I