Exogenous ubiquinol analogues affect the fluorescence of NCD-4 bound to aspartate-160 of yeast cytochrome b1

Previously, we reported that the carboxyl-reacting reagent DCCD, and its fl uorescent derivative NCD-4 binds covalently to aspartate-160 localized in a mphipathic helix cn of the CD loop connecting membrane-spanning helices C a nd D of cytochrome b (Wang et al., 1995). We have investigated the fluoresc ent properties of NCD-4 to probe possible changes in the ed helix resulting from the binding of exogenous ubiquinol analogues to the hcl complex. Prei ncubation of the bc(1) complex with the reduced substrate analogues, DQH(2) , DBH2, and Q(6)H(2) resulted in 20-40% increase in the fluorescence emissi on intensity of NCD-4 and a 10-20% increase in the binding of [C-14]DCCD to the bc(1) complex. By contrast, preincubation with the oxidized analogues DQ, DB, and Q(6) resulted in a 20-40% decrease in the fluorescence emission intensity of NCD-4 and a 20-40% decrease in the binding of [C-14]DCCD to t he be, complex. Moreover, addition of the reduced ubiquinols to the bc(1) c omplex 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 ubiquino l analogues resulted in changes in the quenching of NCD-4 fluorescence by C AT-16, the spin-label probe that intercalates at the membrane surface. Thes e results indicate that the addition of exogenous ubiquinol to the bc(1) co mplex may result in changes in the ed helix leading to a more hydrophobic e nvironment surrounding the NCD-4 binding site. By contrast, preincubation w ith the inhibitors of electron transfer through the bc(1) complex had no ef fect on the binding of NCD-4 to the be, complex or on the fluorescent emiss ion spectra, which suggests that the binding of the inhibitors does not res ult in changes in the environment of the NCD-4 binding site.