CHARACTERIZATION OF UNCOUPLING , ENERGY TRANSFER INHIBITION , AND PROTON UPTAKE REACTIONS ASSOCIATED WITH PHOTOSYSTEM II CYCLIC PHOTOPHOSPHORYLATION

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCI, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncydic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N',N'-tetramethyl-pphenylenediamine stimulated proton uptake in KCN-Hg-NH2OH-inhibited spinach (Spinacia oleracca L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm iumination, indicating that photosystem IIcatalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrne particpate in coupling of ATP synthesis to photosystem II cyclic electron transport. Studies by Saha et al. (16) and Trebst and Reimer (18) have shown that ATP synthesis chloroplasts is site-specific. Electron transport from water to ferricyanide produces ATP synthesis with a P/e2l exceeding 1. Inhibition of this reaction (by KCN or DBMIB) and subsequent restoration of electron transport with quinonediimines or p-benzoquinones produce a DCMU-sensitive photophosphorylation reaction with a P/e2 of 0.3 to 0.5 (14, 17). Measurement of PSI noncyclic electron transport to methylviologen, catalyzed by oxidation of a proton/electron donor (DAD), also produces a P/e2 of about 0.5 after correction of electron transport rates for superoxide ion formation (13). l Abbreviations: PD: p-phenylenediamine; TMPD: N,N,N' ,N'-tetramethyl-p-phenylenediamine; PMS: phenazinemethosulfate; DBMIB: 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCCD: N,N'-dicyclohexylcarbodiimide; CCCP: carbonylcyanide 3-chlorophenyl-hydrazone; DNP: 2,4-dinitrophenol; P/e2: ratio of ATP synthesis to electron pairs transferred. These experiments have led to the hypothesis that noncyclic electron transport in isolated chloroplasts traverses two distinct sites of energy conservation. One site (site II) is presumed to be associated with PSII, while the other (site I) is proposed to function near PSI. Experiments with electron and proton/electron donors to PSII in NH20H-EDTA-inhibited chloroplasts by Izawa and Ort (6) strongly suggest that proton liberation near the oxidizing side of PSII represents the energy-'coupling reaction ascribed to site II. Yocum and Guikema (23) and Yocum (21, 22) have described conditions under which appropriately inhibited chloroplasts will catalyze cyclic photophosphorylation dependent upon PSII activity. This reaction is unlike PSI cycles catalyzed by DAD or reduced PMS in that electron donors (TMPD, ferrocyanide) will support ATP synthesis which is completely sensitive to inhibition by DCMU, and partially sensitive to DBMIB and antimycin A. These findings suggest that optimal coupling of ATP synthesis to PSII cyclic electron transport involves the activity of endogenous electron carriers in the chloroplast membrane. The results presented in this communication demonstrate that PSII cyclic photophosphorylation is susceptible to uncoupling and energy transfer inhibition. The combination of valinomycin and KCl is shown to be an inhibitor of the PSII cycle, but this inhibition does not appear to be due to the collapse of a membrane potential. Data are also presented which show that PSII cyclic electron transport promotes proton uptake. These results are interpreted as support for the hypothesis that PSII cyclic photophosphorylation requires the operation of membrane-bound electron transport carriers to support proton accumulation and ATP synthesis. MATERIALS AND METHODS Chloroplasts, KCN-Hg-inhibited chloroplasts, and KCN-HgNH2OH-inhibited chloroplasts from spinach (Spinacia oleracea L.) were prepared as described earlier (23). PSII cyclic and noncyclic photophosphorylation activities were assayed at 25 C in a reaction mixture (1.6 ml) which contained 50 mm Tricine (pH 8), 50 mm NaCl, 3 mM MgCl2, 1 mm ADP, 5 mM NaH232PO4 (106/cpm/ml), and 20 to 30 ,g Chl (21, 23). In addition, the noncyclic reaction mixtures contained 0.25 mm PD plus 2.5 mm ferricyanide. Noncyclic electron transport was measured polarographically with a Clark electrode (YSI). All reaction mixtures were illuminated with white light (106 ergs.cm-2-sec-1) for 1 min. Reactions were terminated by the addition of 0.2 ml of 30% trichloroacetic acid, and ATP synthesis was determined by gas flow counting of dried samples from which unreacted phosphate had been extracted. Uncou-