Photophosphorylation Associated with Photosystem II: III. 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, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic 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-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed 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 membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.     

Cyclic photophosphorylation reactions catalyzed by ferredoxin,methyl viologen and anthraquinone sulfonate. Use of photochemical reactions to optimize redox poising

The flavin analogue 5-deazariboflavin is a convenient catalyst for the photoreduction of low-potential redox compounds. In an anaerobic medium with Tricine buffer as the electron donor, 5-deazariboflavin is capable of photoreducing both ferredoxin and methyl viologen. We have used this method to conduct a comparative study of the Photosystem I photophosphorylation activities supported by the reduced forms of ferredoxin, methyl viologen and anthraquinone sulfonate. All of these catalysts are capable of generating high rates (200–500 μmol ATP/h per mg chlorophyll) of cyclic photophosphorylation, but only the activity dependent on ferredoxin exhibits sensitivity to antimycin A. This finding suggests that the size of the catalyst and its ability to approach the thylakoid membrane, rather than low-redox potential, governs antimycin A sensitivity. Ferredoxin-catalyzed activity is, however, less sensitive to inhibition by dibromothymoquinone than are the activities supported by methyl viologen and anthraquinone sulfonate. This discrepancy is due to binding of the inhibitor by ferredoxin.     

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Photosystem II - mediated cyclic photophosphorylation.

DCMU-sensitive synthesis of ATP can be shown to continue in KCN-treated chloroplasts after cessation of O₂ evolution. The catalyst for this reaction, $\text{p}$-phenylenediamine, also stimulates synthesis of ATP in NH₂OH-treated chloroplasts, but at much higher rates. This ATP synthesis can be observed in the presence of the quinone antagonist dibromothymoquinone, and under the appropriate conditions it is completely sensitive to DCMU. Since neither uptake nor evolution of O₂ can be observed during illumination, these results are interpreted as evidence for catalysis of cyclic photophosphorylation by photosystem II.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).     

Anaerobic hydrogenase activity in Anacystis nidulans H2-dependent photoreduction and related reactions

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H₂-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors.2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 × g, 3 h).3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark.4. Evolution of H₂ by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents.5. Both uptake and evolution of H₂ were sensitive to O₂, CO, and thiol-blocking agents. The H₂-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H₂-dependent reactions.6. The results present evidence for a membrane-bound ‘photoreduction’ hydrogenase in H₂-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H₂ to various low-potential acceptors including endogenous ferredoxin.     

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO₂ or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP⁺ is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O₂ to H₂O₂. Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed.At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a₆₈₀ and carotenoid pigments and a dampening of the sequence of O₂ evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination.Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity.

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanide-sensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 microM and 20 microM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured. Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity. The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

Inhibition by dibromothymoquinone of photosynthetic electron transfer in chloroplasts of differing ultrastructure

The effect of the plastoquinone antagonist, dibromothymoquinone, on the photoreduction of ferricyanide and plastocyanin by maize mesophyll, maize bundle-sheath and Euglena gracilis chloroplasts has been investigated. Maximum inhibition of FeCN and plastocyanin reduction by mesophyll chloroplasts was obtained at dibromothymoquinone concentrations of 5 × 10^?7m. At higher concentrations dibromothymoquinone acted as an electron shuttle, increasing the rate of reduction of both substrates. In contrast, little inhibition of FeCN and plastocyanin reduction by bundle-sheath chloroplasts occurred at 5 × 10?7 m dibromothymoquinone, and above this concentration of inhibitor, the extent of inhibition increased, with no shuttle effect being observed. Euglena chloroplasts showed a response intermediate between that of mesophyll and bundle-sheath chloroplasts.The presence of a shuttle effect caused by dibromothymoquinone appears to be directly related to the presence of a proton pump in the chloroplast preparations. Plastocyanin is reduced by photosystem 2 alone and shows some of the properties of a class III electron acceptor, although the rates of reduction observed were much lower than those observed with lipophilic class III acceptors.     

A structural investigation of cytochrome c binding to photosynthetic reaction centers in reconstituted membranes

Mammalian cytochrome c can effectively replace bacterial cytochrome c₂ as the electron donor to the bacterial photosynthetic reaction center in either the natural chromatophore or a reconstituted reaction center/phospholipid membrane. In this paper, the reconstituted membrane was used to describe the nature of cytochrome c binding to the reaction center, the location of bound cytochrome c in the membrane profile and the perturbation of the reaction center and phospholipid profile structures induced by cytochrome c binding. These structural studies utilized the combined techniques of X-ray and neutron diffraction.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured.Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity.The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

On an Inhibitor of Ferredoxin Catalyzed Cyclic Photophosphorylation in Thylakoid Membranes*

Dibromo- and diiodo-naphthoquinones are shown to be inhibitors of the cytochrome b₆/f complex in isolated thylakoid membranes from spinach chloroplasts. Dibromo-naphthoquinone inhibits ferredoxin catalyzed cyclic photophosphorylation at 0.1 μM concentrations, but non cyclic e-flow only at 10 μM. It does not inhibit cyclic systems with artifical cofactors, nor non-cyclic electron flow from duroquinol through photosystem I via the cytochrome b₆/f complex. Dibromo-naphthoquinone does however, lower the stoichiometry for ATP formation in the duroquinol donor system. This inhibitory pattern is quite different from that of DBMIB, but very similar to that of antimycin. This antimycin-like behaviour of these inhibitors is interpreted to indicate a) the existence of a Q_c site in the cytochrome b₆/f complex and its obligate function in ferredoxin catalyzed cyclic electron flow and b) a non-essential role of the Q_c site in non-cyclic electron flow, but which — when operative — pumps an extra proton across the thylakoid membrane increasing the ATP yield.     

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O₂ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP⁺ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.     

Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase

The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO₂ (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously.