Binding of dicyclohexylcarbodiimide to aspartate-155 or glutamate-166 of cytochrome b6 in a cytochrome bf complex isolated from spinach thylakoids.
In a recent study [Wang & Beattie (1991) Arch. Biochem. Biophys. 291, 363-370], we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bf complex reconstituted into proteoliposomes and was bound selectively to cytochrome b6. To establish the site of binding of DCCD on cytochrome b6, the cytochrome bf complex labeled with [14C]DCCD was selectively digested with chymotrypsin and trypsin. A 17-kDa fragment containing radioactive DCCD and the heme moiety was obtained after chymotrypsin digestion, while a 12.5-kDa fragment containing both radioactive DCCD and the heme moiety was obtained after trypsin digestion, suggesting that the site of DCCD binding might be on aspartate-140, aspartate-155, or glutamate-166. Extensive digestion of cytochrome b6 isolated from a [14C]DCCD-labeled cytochrome bf complex with trypsin followed by isolation and sequencing of two radioactive peptides obtained revealed that DCCD is bound at either residue aspartate-155 or residue glutamate-166 localized in amphipathic extramembranous helix IV. In addition, the cytochrome bf complex labeled with [14C]DCCD was reconstituted into liposomes and digested with trypsin. Three fragments of 9.3, 10.5, and 11.5 kDa were obtained, suggesting that the four-helix model for the topography of cytochrome b6 in the membrane is correct.
Wang
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Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506.
Beattie
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000737:chemistry*; D003573:Cytochrome b Group / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D003580:Cytochromes / Q000302:isolation & purification / Q000378:metabolism*; D045348:Cytochromes f; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D008081:Liposomes; D008566:Membranes / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010944:Plants / Q000737:chemistry; D017385:Sequence Homology
D003573:Cytochrome b Group; D003580:Cytochromes; D008081:Liposomes; D010446:Peptide Fragments; D004024:Dicyclohexylcarbodiimide; D045346:Cytochrome b6f Complex; D045348:Cytochromes f