Identification of the ubiquinone-binding domain in QPs1 of succinate-ubiquinone reductase.
An azidoubiquinone 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 ubiquinone-binding proteins in bovine heart mitochondrial succinate-ubiquinone reductase. When the reductase was incubated with [3H]azido-Q and illuminated with long wavelength UV light, the decrease in the enzymatic activity correlated with the amount of azido-Q incorporated into the protein. When the illuminated, [3H]azido-Q-treated reductase was extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found primarily in the QPs1 subunit. The [3H]azido-Q-labeled QPs1 was purified from labeled reductase by a procedure involving ammonium sulfate fractionation, dialysis, organic solvent extraction, lyophilization, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cold acetone precipitation. The purified, [3H]azido-Q-labeled QPs1 protein was subjected to reductive carboxymethylation prior to digestion by trypsin. One azido-Q-linked peptide, with a retention time of 66.9 min, was obtained by high performance liquid chromatographic separation. The partial amino-terminal sequence of this peptide is GLTISQL-, indicating that this tryptic peptide comprises amino acid residues 113-140 of the revised amino acid sequence of QPs1. The Q-binding domain, using the proposed structure of QPs1, is probably located in the stretch connecting transmembrane helices 2 and 3 that extrude from the surface of the M side of the inner membrane.
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078.
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D042963:Electron Transport Complex II; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D013379:Substrate Specificity; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D014316:Tritium; D014451:Ubiquinone; C048123:3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase