Identification of a Mr = 17,000 protein as the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts.
An azidoquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone (azido-Q), was used to study the plastoquinone-protein interaction and to identify the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts. When the lipid- and plastoquinone-deficient cytochrome b6-f complex is incubated with varying concentrations of azido-Q and illuminated with long wavelength UV light for 7 min at 2 degrees C, the enzymatic activity, assayed after reconstitution with lipid, decreases as the concentration of azido-Q increases. Maximum inactivation (45%) is observed when 30 mol of azido-Q is used per mol of cytochrome f. The extent of the decrease in activity upon illumination correlates with the amount of azido-Q incorporated into the protein. The 50% inactivation is in good agreement with that expected based on the amount of plastoquinone deficiency of the isolated enzyme complex. When the photolyzed, [3H]azido-Q-treated sample is extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity is found primarily in the Mr = 17,000 subunit. When the enzyme is pretreated with the electron transfer inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, significantly less radioactive label is observed in the Mr = 17,000 protein, suggesting that the action sites of these inhibitors are the same or near the plastoquinone-binding site. When the deficient complex is reconstituted with glycolipid prior to the addition of azido-Q, less than 5% inactivation is observed upon photolysis, and the amount of radioactive label on the Mr = 17,000 protein decreases greatly, suggesting that the plastoquinone-binding site is easily masked by glycolipid when endogenous plastoquinone is absent. Plastoquinol-2 apparently competes with azido-Q for the plastoquinone-binding site since it decreases the radioactive label on the Mr = 17,000 protein.
Doyle
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Department of Biochemistry, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater 74078.
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D001386:Azides / Q000493:pharmacokinetics; D002352:Carrier Proteins / Q000032:analysis*; D002736:Chloroplasts / Q000201:enzymology*; D003573:Cytochrome b Group / Q000032:analysis*; D045346:Cytochrome b6f Complex; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000494:pharmacology; D008970:Molecular Weight; D010971:Plastoquinone / Q000032:analysis*; D011809:Quinones / Q000032:analysis*; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000493:pharmacokinetics
D001386:Azides; D002352:Carrier Proteins; D003573:Cytochrome b Group; D006017:Glycolipids; D011809:Quinones; D014451:Ubiquinone; D045346:Cytochrome b6f Complex; C048123:3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; D010971:Plastoquinone