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: I. Photosystem II Cyclic Photophosphorylation Catalyzed by p-Phenylenediamine

Incubation of spinach chloroplast membranes for 90 minutes in the presence of 50 mm KCN and 100 mum HgCl(2) produces an inhibition of photosystem I activity which is stable to washing and to storage of the chloroplasts at -70 C. Subsequent exposure of these preparations to NH(2)OH and ethylenediaminetetraacetic acid destroys O(2) evolution and flow of electrons from water to oxidized p-phenylenediamine, but two types of phosphorylating cyclic electron flow can still be observed. In the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazinemethosulfate catalyzes ATP synthesis at a rate 60% that observed in uninhibited chloroplasts. C-Substituted p-phenylenediamines will also support low rates of photosystem I-catalyzed cyclic photophosphorylation, but p-phenylenediamine is completely inactive. When photosystem II is not inhibited, p-phenylenediamine will catalyze ATP synthesis at rates up to 90 mumol/hr.mg chlorophyll. This reaction is unaffected by anaerobiosis, and an action spectrum for ATP synthesis shows a peak at 640 nm. These results are interpreted as evidence for the existence of photosystem II-dependent cyclic photophosphorylation in these chloroplast preparations.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Photoreduction and Photophosphorylation with Tris-Washed Chloroplasts

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e₂ ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.     

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.     

Direct Spectrophotometric Measurement of Photosystem I and Photosystem II Activities of Photosynthetic Membrane Preparations from Cyanophora paradoxa, Phormidium laminosum, and Spinach

Vesicles prepared with the French press from membranes of cyanelles of Cyanophora paradoxa retain O₂ evolution activity with rates up to 500 micromoles 2,6-dichlorophenolindophenol reduced per hour per milligram chlorophyll. This activity is immediately lost when the vesicles are transferred from the sucrose-phosphate-citrate preparation buffer into dilute phosphate buffer. Similar preparations from Phormidium laminosum, a thermophilic cyanobacterium retain activity under such conditions. Photosystem I activities of both cyanobacterial vesicle preparations were determined by direct spectrophotometric measurement of N,N,N′,N′-tetramethyl-p-phenylenediamine photooxidation in the presence of anthraquinone-2-sulfonate. The rates so determined were compared with rates of O₂ taken up in the presence of methyl viologen or anthraquinone-2-sulfonate as electron acceptors. The predicted stoichiometry of two was observed for moles of N,N,N′,N′-tetramethyl-p-phenylenediamine oxidized per mole of oxygen taken up. Anthraquinone-2-sulfonate was the better electron acceptor, and maximal rates of 943 micromoles per hour per milligram chlorophyll for O₂ uptake were observed for Phormidium laminosum preparations in the presence of superoxide dismutase. For purposes of comparison, spinach chloroplasts were assayed for similar activities. All preparations were readily assayed for photosystem I activity by the direct spectrophotometric method, which has advantages of simplicity and freedom from errors introduced by photoxidation of other substrates by photosystem I when O₂ uptake is measured.     

Photophosphorylation Associated with Photosystem II: IV. KINETIC ANALYSES OF PHOTOSYSTEM II CYCLIC PHOTOPHOSPHORYLATION ACTIVITY: EVIDENCE FOR TWO CYCLIC REACTIONS

Photosystem II-dependent cyclic photophosphorylation activity produced by addition of p-phenylenediamines to KCN-Hg-NH₂OH-inhibited chloroplasts is the product of two separate reactions when a proton/electron donor is the catalyst. The activity observed with an electron donor as catalyst consists of a single reaction. One of the cyclic reactions, evoked by low (≤40 micromolar) concentrations of a proton/electron donor is sensitive to dibromothymoquinone and to perturbation of membrane organization by sonication. The second reaction, requiring higher catalyst concentrations, is less sensitive to either dibromothymoquinone or membrane perturbation. These results indicate that at low concentrations, proton/electron or electron donor catalysts act to produce a photosystem II cyclic reaction which is dependent on membrane-bound electron carriers. High concentrations of proton/electron donors, on the other hand, can produce a phosphorylation reaction in which the catalyst itself is largely responsible for cyclic activity.     

Studies on Effect of Certain Quinones: I. Electron Transport, Photophosphorylation, and CO(2) Fixation in Isolated Chloroplasts

The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.     

Inhibition of the Hill Reaction by Tris and Restoration by Electron Donation to Photosystem II

Experiments in which chloroplasts were washed with tris and tricine buffers at different pH's indicated that the non-protonated (uncharged) form of tris was inhibitory to the Hill reaction while the protonated form of tris and the zwitterionic forms of tricine were non-inhibitory. Buffers analogous to tris and tricine gave similar results. Photoreduction of NADP could be restored to the inhibited chloroplasts by adding the reduced forms of p-hydroquinone, p-aminophenol, p-phenylenediamine, benzidine, semicarbazide, and dihydroxydiphenyl, all of which donated electrons to photosystem II. Photoreduction of ferricyanide was shown with those donor systems (benzidine and semicarbazide) which did not react chemically with ferricyanide. Photophosphorylation was also restored with all of the electron donors except semicarbazide.     

Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites

The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H₂O→methyl viologen, H₂O→silicomolybdic acid H₂O→ferricyanide, and H₂O→dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H₂O→silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition.     

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.     

Effect of High Cation Concentrations on Photosystem II Activities

The effects of wide concentration ranges of NaCl, KCl, and MgCl₂ on ferricyanide reduction and the fluorescence induction curve of isolated spinach (Spinacia oleracea) chloroplasts were investigated. Concentrations of the monovalent salts above 100 mm and MgCl₂ above 25 mm produced a decrease in the rate of ferricyanide reduction by thylakoids uncoupled with 2.5 mm NH₄Cl which cannot be attributed to changes in the primary photochemical capacity of photosystem II. Salt-induced decreases in the effective concentration of the secondary electron acceptor of photosystem II, plastoquinone, reduce the capacity for secondary photochemistry of photosystem II and this could contribute to the reduction in ferricyanide reduction by uncoupled thylakoids at high salinities. The rate of ferricyanide reduction by coupled thylakoids is little affected by salinity changes, indicating that the rate-limiting phosphorylation mechanism in electron flow from water to ferricyanide in coupled thylakoids is salt-tolerant, whereas the rate-limiting reaction in uncoupled ferricyanide reduction is considerably affected by salinity changes. Salt-induced changes in the fluorescence induction curve are interpreted in terms of changes in the rate constants for excitation decay by radiationless transitions, exciton transfer from photosystem II chlorophylls to other associated chlorophyll species, and photochemistry.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

Stimulation of electron transport from Photosystem II to Photosystem I in spinach chloroplasts

Electron transport from Photosystem II to Photosystem I of spinach chloroplasts can be stimulated by bicarbonate and various carbonyl or carboxyl compounds. Monovalent or divalent cations, which have hitherto been implicated in the energy distribution between the two photosystems, i.e., spillover phenomena at low light intensities, show a similar effect under high light conditions employed in this study. A mechanism for this stimulation of forward electron transport from Photosystem II to Photosystem I could involve inhibition of two types of Photosystem II partial reactions, which may involve cycling of electrons around Photosystem II. One of these is the DCMU-insensitive silicomolybdate reduction, and the other is ferricyanide reduction by Photosystem II at pH 8 in the presence of dibromothymoquinone. Greater stimulation of forward electron transport reactions is observed when both types of Photosystem II cyclic reactions are inhibited by bicarbonate, carbonyl and carboxyl-type compounds, or by certain mono- or divalent cations.Abbreviations used: DCMU, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea; DCIP, 2,6-dichloroindophenol; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; FeCN, potassium ferricyanide; MV, methylviologen; PS I, photosystem I; PS II, photosystem II; SM, silicomolybdic acid.     

Photophosphorylation in isolated maize bundle sheath chloroplasts and cells

Isolated maize bundle sheath chloroplasts showed substantial rates of noncyclic photophosphorylation. A typical rate of phosphorylation coupled to whole-chain electron transport (methylviologen or ferricyanide as acceptor) was 60 μmol per hour per milligram chlorophyll) with a coupling efficiency (P/e₂) of 0.6. Partial electron transport reactions driven by photosystem I or II supported phosphorylation with P/e₂ values of 0.2 to 0.3. Thus, two sites of phosphorylation seem to be associated with the photosynthetic chain in much the same way as in spinach chloroplasts.     

Evidence for Cyclic Electron Flow around Photosystem II in Chlorella pyrenoidosa

Electron flow around photosystem II was investigated in Chlorella pyrenoidosa. Using a bare platinum O₂ electrode, simultaneous measuremnts were made of steady-state photosynthesis in continuous light, the yield of oxygen (Y_o2) produced by a superimposed saturating xenon flash, and the change in fluorescence yield of a weak flash triggered before and 70 microseconds after the saturating flash. Throughout most of the continuous photosynthesis-irradiance curve, normalized O₂ flash yields (Y_o2/Y_o2max) and normalized variable fluorescence yields (Δ/Δ′) were linearly correlated with a slope of 1.0. As photosynthetic rates reached light saturation, however, the variable fluorescence yields remained relatively constant while O₂ flash yields decreased. These results strongly suggest that there is a cyclic electron flow around photosystem II in unpoisoned intact cells at light saturation and supraoptimal light intensities.     

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O₂ in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b ₆-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b ₆-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and poises the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO₂ can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone - LHC light harvesting chlorophyll - LHCP II light harvesting chlorophyll protein of Photosystem II - PQ plastoquinone - PS I, II Photosystem I, II - SHAM salicyl hydroxamic acid - TBT Tri-n-butyltin CIW/DPB Publication No. 1146     

Partial Reactions of Photosynthesis in Briefly Sonicated Chlamydomonas: II. Photophosphorylation Activities

Briefly sonicated Chlamydomonas reinhardi cells are capable of both cyclic and noncyclic photophosphorylation and, in each case, the maximum rates approach those reported for higher plant chloroplasts. Photophosphorylation coupled to ferricyanide reduction occurs with a P/2e ratio approaching unity.     

Hydrogen Photoevolution Indicates an Increase in the Antenna Size of Photosystem I in Chlamydobotrys stellata during Transition from Autotrophic to Photoheterotrophic Nutrition

The changes in the light-harvesting antenna size of photosystem I were investigated in the green alga Chlamydobotrys stellata during transition from autotrophic to photoheterotrophic nutrition by measuring the light-saturation behavior of hydrogen evolution following single turnover flashes. It was found that during autotrophic-to-photoheterotrophic transition the antenna size of photosystem I increased from 180 to 250 chlorophyll. The chlorophyll (a + b)/P700 ratio decreased from 800 to 550. The electron transport of photosystem I measured from reduced 2,6-dichloro-phenolindophenol to methylviologen was accelerated 1.4 times. In the 77K fluorescence spectra, the photosystem II fluorescence yield was considerably lowered relative to the photosystem I fluorescence yield. It is suggested that the increased light-harvesting capacity and redistribution of absorbed excitation energy in favor of photosystem I is a response of photoheterotrophic algae to meet the ATP demand for acetate metabolism by efficient photosystem I cyclic electron transport when the noncyclic photophosphorylation is inhibited by CO₂ deficiency.