Effect-of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex

A key issue concerning the primary conversion (Q(O)) site function in the c ytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommo date two ubiquinone molecules, the double occupancy model [Ding, H., Robert son, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158 ]; In the recently reported crystal structures of the cytochrome bc(1) comp lex, no electron density was identified in the Q(O) site that could be ascr ibed to ubiquinone. To provide further insight into this issue, we have man ipulated the cytochrome bet complex Q(O) site occupancy in photosynthetic m embranes from Rhodobacter capsulatus by using inhibitor titrations and ubiq uinone extraction to modulate the amount of ubiquinone bound in the site. T he nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to m odulation:of Q(O) site: occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bet complex . Addition of stoichiometric concentrations of MOA-stilbene or excess DPA t o cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x) resonance broaden s and shifts from 1.800 to 1.783. This is exactly the same signal as that o btained when there is only one ubiquinone present in the Q(O) site. Further more, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex deple ted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line sha pes, which remain indicative of one ubiquinone or zero ubiquinones in the Q (O) site, with broad g(x) resonances at 1.783 or 1.765, respectively. The r esults are quite consistent with the Q(O) site double occupancy model, in w hich MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q (O) site ubiquinones.