Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.
Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.
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通过 N-环己基-N-[4-(二甲基氨基)萘基]碳二亚胺荧光研究确定菠菜叶绿体类囊体膜中细胞色素 b6 的拓扑结构。
DOI：
10.1021/bi00088a010
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Beattie,DS
中科院分区：
文献类型：
--
作者：
Wang,Y;Beattie,DS
Revised Manuscript Received July 8, 1993® abstract: 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 reconstitutedinto 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, 7V-cyclohexyl-Ar-[4-(dimethylamino) naphthyl] carbodiimide (NCD-4). After incu-bation 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 emissionpeak. NCD-4 was selectively bound to cytochrome bf and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bf complex reconstitutedinto proteoliposomes. Exhaustive digestion of the NCD-4-labeled cytochromebf with trypsin resulted in the formation of a single 6-kDa fluorescent peptide with similar propertiesto the peptide labeled with radioactive DCCD. The fluorescence of NCD-4 bound to the cytochrome bf complex reconstitutedinto 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.The cytochrome bf complex, an intrinsic membrane-bound protein complex localized in the thylakoid membrane of chloroplasts of higher plants, mediates electron transfer between photosynthesis I and II (Cramer et al., 1987). As electrons move from plastoquinol through the cytochrome bf complex to plastocyanine, protons are translocated across the thylakoid membrane into the lumen of the thylakoid such that an electrogenic proton gradient is generated across the membrane (Hauska et al., 1983). A similar function is catalyzed by the cytochrome bc\complex of the mitochondrial inner membrane, which transfers electrons from ubiquinol to cytochrome c with the translocation of protons in the opposite direction across the mitochondrial membrane to the cytosolic side of the inner membrane (Hauska et al., 1983). Recently, we reported that dicyclohexylcarbodiimide (DC-
