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 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.

Ubiquinone binding capacity of the Rhodobacter capsulatus cytochrome bc1 complex: effect of diphenylamine, a weak binding QO site inhibitor.

Diphenylamine (DPA), a known inhibitor of polyene and isoprene biosynthesis, is shown to inhibit flash-activatable electron transfer in photosynthetic membranes of Rhodobacter capsulatus. DPA is specific to the QO site of ubihydroquinone:cytochrome c oxidoreductase, where it inhibits not only reduction of the [2Fe-2S]2+ cluster in the FeS subunit and subsequent cytochrome c reduction but also heme bL reduction in the cytochrome b subunit. In both cases, the kinetic inhibition constant (Ki) is 25 +/- 10 microM. A novel aspect of the mode of action of DPA is that complete inhibition is established without disturbing the interaction between the reduced [2Fe-2S]+ cluster and the QO site ubiquinone complement, as observed from the electron paramagnetic resonance (EPR) spectral line shape of the reduced [2Fe-2S] cluster, which remained characteristic of two ubiquinones being present. These observations imply that DPA is behaving as a noncompetitive inhibitor of the QO site. Nevertheless, at higher concentrations (>10 mM), DPA can interfere with the QO site ubiquinone occupancy, leading to a [2Fe-2S] cluster EPR spectrum characteristic of the presence of only one ubiquinone in the QO site. Evidently, DPA can displace the more weakly bound of the two ubiquinones in the site, but this is not requisite for its inhibiting action.

Non-inhibiting perturbation of the primary energy conversion site (Qo site) in Rhodobacter capsulatus ubihydroquinone: cytochrome c oxidoreductase (cytochrome bc1 complex).

Ethanol added to Rhodobacter capsulatus chromatophore membranes containing the cytochrome bc1 complex effectively uncouples the sensitivity of the [2Fe-2S] cluster EPR spectrum to the number and redox state of ubiquinone/ubihydroquinone within the Qo site. Ethanol has no effect upon the rate of catalysis, leading to a non-inhibiting perturbation of cytochrome bc1 function. We suggest that displacement occurs by ethanol acting from the aqueous phase to successfully compete with the Qo site ubiquinones and water to hydrogen bond the N(epsilon)H atom(s) of the coordinating [2Fe-2S] cluster histidines.