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Sharp RE, Gibney BR, Palmitessa A, White JL, Dixon JA, Moser CC, Daldal F, Dutton PL: Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc (1) complex. Biochemistry. 1999 Nov 9;38(45):14973-80.
A key issue concerning the primary conversion (Q (O)) site function in the cytochrome bc (1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q (O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc (1) complex, no electron density was identified in the Q (O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc (1) complex Q (O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q (O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q (O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q (O) site inhibitors of the cytochrome bc (1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc (1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g (x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q (O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc (1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, 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 results are quite consistent with the Q (O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q (O) site ubiquinones. |
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