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PMID:12730198

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Citation

Gong, X, Xie, T, Yu, L, Hesterberg, M, Scheide, D, Friedrich, T and Yu, CA (2003) The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli. J. Biol. Chem. 278:25731-7

Abstract

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.

Links

PubMed Online version:10.1074/jbc.M302361200

Keywords

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

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:NUOM

part_of

GO:0045272: plasma membrane respiratory chain complex I

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:NUOM

enables

GO:0048039: ubiquinone binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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