Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.
In a recent study [Wang & Beattie (1992) Biochemistry 31, 8445-8459], we reported that dicyclohexylcarbodiimide (DCCD) was bound to either aspartate-155 or glutamate-166 localized in an amphiphilic, non-membrane-spanning, helix of cytochrome. Moreover, DCCD inhibits proton translocation in a cytochrome bf complex reconstituted into proteoliposomes without significant inhibition of electron transfer, suggesting that the helix containing aspartate-155 and glutamate-166 may play a role in proton movements. In order to explore the environment of this amphiphilic helix, we employed a fluorescent derivative of DCCD, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide (NCD-4). After incubation of NCD-4 with a cytochrome bf complex isolated from spinach chloroplasts, a fluorescent compound was formed with a 331-nm excitation peak and 440-nm emission peak. NCD-4 was selectively bound to cytochrome b6 and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bf complex reconstituted into proteoliposomes. Exhaustive digestion of the NCD-4-labeled cytochrome b6 with trypsin resulted in the formation of a single 6-kDa fluorescent peptide with similar properties to the peptide labeled with radioactive DCCD. The fluorescence of NCD-4 bound to the cytochrome bf complex reconstituted into proteoliposomes was quenched by CAT-16, an amphiphilic spin label that intercalates at the membrane surface, as well as by nitroxide derivatives of stearic acid in the order 5-doxylstearic acid > 7-doxylstearic acid > 12-doxylstearic acid. At higher concentrations, the hydrophilic membrane-impermeant quenchers, CAT-1 and D-569, also quenched the fluorescence of NCD-4.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
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Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506-9142.
Beattie
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DS
D000595:Amino Acid Sequence; D002234:Carbodiimides / Q000737:chemistry; D002736:Chloroplasts / Q000648:ultrastructure*; D003573:Cytochrome b Group / Q000737:chemistry*; D045346:Cytochrome b6f Complex; D003580:Cytochromes / Q000737:chemistry; D045348:Cytochromes f; D004579:Electron Transport; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D010944:Plants; D011510:Proteolipids / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002234:Carbodiimides; D003573:Cytochrome b Group; D003580:Cytochromes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010940:Plant Proteins; D011510:Proteolipids; C020485:proteoliposomes; D045346:Cytochrome b6f Complex; D045348:Cytochromes f; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide