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 bc1 complex. Preincubation of the bc1 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 bc1 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 bc1 complex. Moreover, addition of the reduced ubiquinols to the bc1 complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc1 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 bc1 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 bc1 complex had no effect on the binding of NCD-4 to the bc1 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.