PMID:12730198
| 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 |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.