Effects of inhibitors on the activity of the cytochrome b(6)f complex: Evidence for the existence of two binding pockets in the lumenal site

The effects of two inhibitors of electron transfer in the cytochrome b(6)f complex have been studied in whole cells of Chlamydomonas reinhardtii. DNP- INT affected equally the two steps of the concerted oxidation of plastoquin ol at the Q(o) site; it decreased the rates of both cytochrome f reduction and cytochrome bs turnover, without affecting the amplitude of their redox signals. On the contrary, DBMIB inhibited only the rate of cytochrome S red uction while reducing, at the same time, the amplitude of cytochrome bs sig nals. The accessibility of DNP-INT to the Q(o) site was unaffected by preil lumination, while that of DBMIB was greatly enhanced, even after a single t urnover of the cytochrome b(6)f complex. Similar results were obtained with a mutant strain (FUD2) where the Q(o) site has an affinity for plastoquino l that is diminished by a factor of similar to 50 [Finazzi, G., et al. (199 7) Biochemistry 36, 2867-2874]. However, the binding of the two inhibitors was differentially influenced by the mutation: a factor of similar to 250 w as calculated for DNP-INT and a factor of only similar to 5 for DBMIB. This suggests that they bind within the Q(o) site in two distinct pockets, whic h are differentially involved in the process of quinol oxidation, in agreem ent with a recent model where two distinct positions for the reduced and se mireduced quinones are considered [Crofts, A. R., and Berry, E. A. (1998) C urr. Opin. Struct. Biol. 8, 501-509].