Inhibition of photosynthetic oxygen evolution by protonophoric uncouplers

The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K₃[Fe(CN)₆] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I₅₀ values were approx. 1–2, 4–6 and 0.04–0.10 M, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Q_p) which is also the electron donor for K₃ [Fe(CN)₆] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y - ANT2p- 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene - CCCP- carbonyl cyanide m-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl 6-isopropyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT- 2-iodo-6-isopropyl-3-methyl 2,4,4-trinitrodiphenyl ether - DPC- 1,5-diphenylcarbazide - DPIP- 2,6-dichlorophenolindophenol - FCCP- carbonyl cyanide p-trifuoromethoxyphenylhydrazone - FeCy- potassium ferricyanide - HQNO- 2-n-heptyl-4-hydroxyquinoline N-oxide - (MN)₄- the tetranuclear Mn cluster of water oxidizing complex - P680- photoactive Chl of the reaction center of Photosystem II - PCP- 2,3,4,5,6-pentachlorophenol - PS- photosystem - Q_A and Q_B- primary and secondary plastoquinones of PS II - Q_C and Q_Z- plastoquinone binding sites in the cytochrome blf complex - Q_p- membrane pool of plastoquinone - SiMo- sodium silicomolybdate - TMPD- N,N,N-tetramethyl-p-phenylenediamine - TTFB- 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - WOC- water oxidixing complex - Y_Z- tyrosine-161 of the Photosystem II D1 polypeptide