A proposed pathway of proton translocation through the bc complexes of mitochondria and chloroplasts.
The cytochrome bc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50-70% with minimal effect on electron transfer in the cytochrome bc1 and cytochrome bf complexes reconstituted into liposomes. Subsequent binding studies with cytochrome bc1 and cytochrome bf complexes indicate that DCCD specifically binds to the subunit b and subunit b6, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochrome b and aspartate 155 or glutamate 166 of spinach cytochrome b6. Moreover, similar inhibition of proton translocating activity and binding to cytochrome b and cytochrome b6 were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochrome b and cytochrome b6 is localized near the surface of the membrane but shielded from the external medium.
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506-9142.
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Beattie
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D003580:Cytochromes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045348:Cytochromes f; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology; D004563:Electrochemistry; D004579:Electron Transport / Q000187:drug effects; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008081:Liposomes; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011522:Protons; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D002234:Carbodiimides; D003580:Cytochromes; D005656:Fungal Proteins; D008081:Liposomes; D010940:Plant Proteins; D011522:Protons; D004024:Dicyclohexylcarbodiimide; D045348:Cytochromes f; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide; D014450:Electron Transport Complex III