Abstract
Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible changes in the cd helix resulting from the binding of exogenous ubiquinol analogues to the bc 1 complex. Preincubation of the bc 1 complex with the reduced substrate analogues, DQH2, DBH2, and Q6H2 resulted in 20–40% increase in the fluorescence emission intensity of NCD-4 and a 10–20% increase in the binding of [14C]DCCD to the bc 1 complex. By contrast, preincubation with the oxidized analogues DQ, DB, and Q6 resulted in a 20–40% decrease in the fluorescence emission intensity of NCD-4 and a 20–40% decrease in the binding of [14C]DCCD to the bc 1 complex. Moreover, addition of the reduced ubiquinols to the bc 1 complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc 1 complex reconstituted into proteoliposomes with both reduced and oxidized ubiquinol analogues resulted in changes in the quenching of NCD-4 fluorescence by CAT-16, the spin-label probe that intercalates at the membrane surface. These results indicate that the addition of exogenous ubiquinol to the bc 1 complex may result in changes in the cd helix leading to a more hydrophobic environment surrounding the NCD-4 binding site. By contrast, preincubation with the inhibitors of electron transfer through the bc 1 complex had no effect on the binding of NCD-4 to the bc 1 complex or on the fluorescent emission spectra, which suggests that the binding of the inhibitors does not result in changes in the environment of the NCD-4 binding site.
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Wang, Y., Bruel, C., Yan, L. et al. Exogenous Ubiquinol Analogues Affect the Fluorescence of NCD-4 Bound to Aspartate-160 of Yeast Cytochrome b . J Bioenerg Biomembr 30, 455–464 (1998). https://doi.org/10.1023/A:1020590113470
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DOI: https://doi.org/10.1023/A:1020590113470