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 Q(O) 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 b(L) reduction in the cytochrome b subunit. In both cases, the kinetic inhibition constant (K-i) is 25 +/- 10 mu M. 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 Q(O) 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 Q(O) site. Nevertheless, at higher concentrations (>10 mM), DPA can interfere with the Q(O) site ubiquinone occupancy, leading to a [2Fe-2S] cluster EPR spectrum characteristic of the presence of only one ubiquinone in the Q(O) 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.