Exogenous Ubiquinol Analogues Affect the Fluorescence of NCD-4 Bound to Aspartate-160 of Yeast Cytochrome b

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.