Topographical organization of cytochrome b in the yeast mitochondrial membrane determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.
In previous studies, we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bc1 complex from yeast mitochondria and was bound selectively to cytochrome b. Extensive trypsin digestion of [14C]DCCD-labeled cytochrome b isolated from a cytochrome bc1 complex treated with DCCD yielded a single radiolabeled 7.0 kDa peptide with the N-terminus VTLWNVG, indicating that trypsin cleavage had occurred at arginines-110 and -178. This segment of cytochrome b contains one acidic residue, aspartate-160, localized in amphiphilic, non-membrane-spanning, helix cd. To explore the environment of amphiphilic helix cd, we employed a fluorescent derivative of DCCD, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide (NCD-4). After incubation of NCD-4 with a cytochrome bc1 complex isolated from yeast mitochondria, a fluorescent compound was formed with a 340 nm excitation peak and a 441 nm emission peak. NCD-4 was selectively bound to cytochrome b and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bc1 complex reconstituted into proteoliposomes. Competition experiments and trypsin digestion of NCD-4-labeled cytochrome b indicated that NCD-4 and DCCD were bound to the same site on cytochrome b. The fluorescence of NCD-4 bound to the cytochrome bc1 complex was quenched equally by CAT-16, an amphiphilic spin-label that intercalates at the membrane surface, and 5-doxylstearic acid, a nitroxide derivative of stearic acid, and to a lesser extent by 7-doxylstearic and 12-doxylstearic acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
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Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506-9142, USA.
Howton
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MM
Beattie
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D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D014450:Electron Transport Complex III / Q000302:isolation & purification; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D008961:Models, Structural; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence
D003573:Cytochrome b Group; D004024:Dicyclohexylcarbodiimide; D014450:Electron Transport Complex III