In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Q(o) site inhibitor of the cytochrome bc(1) complex from Saccharomyces cerevisiae. Natural stigmatellin A induced clear shifts in the redox-induced FTIR difference spectra. For data interpretation a stigmatellin derivative (UST) with the conjugated trienes replaced by an aliphatic tail was synthesized, and the carbonyl group shown in crystal structures to interact with His181, the [2Fe-2S] ligand of the Rieske, was specifically (13)C labeled. Electrochemically induced FTIR difference spectra of the inhibitors in CH(3)OD were obtained and revealed signals characteristic for the oxidized and reduced forms of the labeled and unlabeled compounds. On the basis of signals from the inhibitors alone, the binding of the inhibitor to the bc(1) complex was monitored. Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the nu(C=O) vibrational mode of about 10 cm(-1). In addition, redox-dependent reorganizations of the protein were identified, including protonation changes of acidic residues at 1746 and 1734 cm(-1). The conformational changes observed upon inhibitor binding are discussed with respect to the crystal structures and proposed mechanistic models [Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669-684; Palsdottir, H., Lojero, C. G., Trumpower, B. L., and Hunte, C. (2003) J. Biol. Chem. 278, 31303-31311].
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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