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Direct Evidence for the Interaction of Stigmatellin with a Protonated Acidic Group in the bc1 Complex from Saccharomyces cerevisiae As Monitored by FTIR Difference Spectroscopy and 13C Specific Labeling.

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Palsdottir,  Hildur
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Hunte,  Carola
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Ritter, M., Palsdottir, H., Abe, M., Mäntele, W., Hunte, C., Miyoshi, H., et al. (2004). Direct Evidence for the Interaction of Stigmatellin with a Protonated Acidic Group in the bc1 Complex from Saccharomyces cerevisiae As Monitored by FTIR Difference Spectroscopy and 13C Specific Labeling. Biochemistry, 43, 8439-8446. doi:10.1021/bi049649x.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-DAD2-4
Abstract
In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Qo site inhibitor of the cytochrome bc1 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 13C labeled. Electrochemically induced FTIR difference spectra of the inhibitors in CH3OD 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 bc1 complex was monitored. Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the ν(CO) 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].