Ubiquinone binding capacity of the Rhodobacter capsulatus cytochrome bc(1)complex: Effect of diphenylamine, a weak binding Q(o) 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 u bihydroquinone:cytochrome c oxidoreductase, where it inhibits not only redu ction of the [2Fe-2S](2+) cluster in the FeS subunit and subsequent cytochr ome c reduction but also heme b(L) reduction in the cytochrome b subunit. I n both cases, the kinetic inhibition constant (K-i) is 25 +/- 10 mu M. A no vel aspect of the mode of action of DPA is that complete inhibition is esta blished without disturbing the interaction between the reduced [2Fe-2S](+) cluster and the Q(O) site ubiquinone complement, as observed from the elect ron 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] c luster EPR spectrum characteristic of the presence of only one ubiquinone i n the Q(O) site. Evidently, DPA can displace the more weakly bound of the t wo ubiquinones in the site, but this is not requisite for its inhibiting. a ction.