Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide

• Published in: Biochemistry (Easton) 1993-09, Vol.32 (37), p.9586-9591
• Main Authors: Wang, Yudong, Beattie, Diana S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbodiimides - chemistry; CATION; CATIONES; CHLOROPLASTE; Chloroplasts - ultrastructure; CITOCHROMO B; CLOROPLASTO; COLORANT; COLORANTES; CYTOCHROME B; Cytochrome b Group - chemistry; Cytochrome b6f Complex; Cytochromes - chemistry; Cytochromes f; Electron Transport; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Hemoproteins; HIDROGENO; Hydrogen-Ion Concentration; HYDROGENE; Intracellular Membranes - ultrastructure; Membrane Proteins - chemistry; Metalloproteins; Molecular Sequence Data; Plant Proteins - chemistry; Plants; PLASTE; PLASTIDIOS; PROPIEDADES OPTICAS; PROPRIETE OPTIQUE; Proteins; Proteolipids - chemistry; REACCIONES QUIMICAS; REACTION CHIMIQUE; Spectrometry, Fluorescence; SPINACIA OLERACEA
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00088a010

In a recent study [Wang and 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. These results suggest that the non-membrane-spanning helix containing aspartate-155 and glutamate-166 is localized within the membrane but near the surface of the membrane where it is shielded from the external aqueous environment