Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl₂ at a molar ratio of HgCl₂ to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ? was similarly sensitive towards HgCl₂, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ? and light-induced increase in fluorescence yield were enhanced by the HgCl₂ treatment of chloroplasts.     

Sulfhydryl reagents inhibit electron transport in Photosystem II of spinach chloroplasts

A 120 min incubation period with sulfhydryl reagents, such as p-chloromercuribenzoic acid, shows greater than 50% loss of electron-transport activity in Photosystem (PS) II of spinach chloroplasts. Since p-chloromercuriphenylsulfonic acid, a nonpenetrating sulfhydryl reagent, and 4,4′-dithiopyridine, a bifunctional sulfhydryl reagent, show greater inhibition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate reduction than of dibromothymoquinone-insensitive indophenol reduction, it is postulated that two different sulfhydryl reagent-sensitive sites are involved in the PS II electron-transport chain of spinach chloroplasts.     

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.     

Site-specific inhibition of photophosphorylation in isolated spinach chloroplasts by HgCl2. II. Evidence for three sites of energy conservation associated with non-cyclic electron transport

Energy transfer inhibition by HgCl₂ has been demonstrated to be selective for certain System I partial reactions. On the basis of different HgCl₂ effects on the System I reactions, reduced 2,6-dichlorophenolindophenol → methylviologen, diaminodurene → methylviologen and N-phenazine methosulfate cyclic, two sites of energy conservation associated with System I are proposed. Furthermore, these sites are in parallel with each other, in series with the site closely associated with Photosystem II and are shared between non-cyclic and cyclic electron transport.     

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).     

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa II. Reduction kinetics and electric field increase in the 10 ms range

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC⁺ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern.The reduction of PC⁺ and cytochrome f⁺ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267–284). Both need the presence of a compound R′ in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R′ as terminal reducant, PC⁺ and cytochrome f⁺ as terminal oxidants, would account for this field generation.Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Cation-induced,inhibitor-resistant photosystem II reactions in cyanobacterial membranes

Ferricyanide-supported oxygen evolution in sonic vesicles from the cyanobacterium $\text{Spirulina}\text{platensis}$ is only partially sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and addition of cations to inhibited membranes stimulates the rate of oxygen evolution. The order of cation effectiveness (M³⁺ > M²⁺ > M⁺) suggests that this stimulation is due at least in part to surface charge screening effects which permit freer access of anionic ferricyanide to the vesicle membrane surface; La³⁺, Ca²⁺, and K⁺ are most effective in this regard. Ferricyanide photoreduction is completely sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and neither mono- nor divalent cations affect this inhibition. Addition of La³⁺, on the other hand, causes a nearly complete restoration of ferricyanide-supported oxygen evolution. We conclude that the membrane surfaces of these vesicles are uniquely different from those o higher plants; sites of ferricyanide reduction associated with the interphotosystem chain are surface localized, and the primary acceptor region of photosystem II is susceptible to a trivalent cation-specific reaction in which ferricyanide may directly oxidize the primary acceptor.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl₂, an inhibitor of cytochrome b₆f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b₆f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Characterization of wave phenomena in the relaxation of flash-induced chlorophyll fluorescence yield in cyanobacteria

Fluorescence yield relaxation following a light pulse was studied in various cyanobacteria under aerobic and microaerobic conditions. In Synechocystis PCC 6803 fluorescence yield decays in a monotonous fashion under aerobic conditions. However, under microaerobic conditions the decay exhibits a wave feature showing a dip at 30–50 ms after the flash followed by a transient rise, reaching maximum at ~ 1 s, before decaying back to the initial level. The wave phenomenon can also be observed under aerobic conditions in cells preilluminated with continuous light. Illumination preconditions cells for the wave phenomenon transiently: for few seconds in Synechocystis PCC 6803, but up to one hour in Thermosynechocystis elongatus BP-1. The wave is eliminated by inhibition of plastoquinone binding either to the Q_B site of Photosystem-II or the Q_o site of cytochrome b₆f complex by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, respectively. The wave is also absent in mutants, which lack either Photosystem-I or the NAD(P)H-quinone oxidoreductase (NDH-1) complex. Monitoring the redox state of the plastoquinone pool revealed that the dip of the fluorescence wave corresponds to transient oxidation, whereas the following rise to re-reduction of the plastoquinone pool. It is concluded that the unusual wave feature of fluorescence yield relaxation reflects transient oxidation of highly reduced plastoquinone pool by Photosystem-I followed by its re-reduction from stromal components via the NDH-1 complex, which is transmitted back to the fluorescence yield modulator primary quinone electron acceptor via charge equilibria. Potential applications of the wave phenomenon in studying photosynthetic and respiratory electron transport are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

A seconds range component of the reoxidation of the primary Photosystem II acceptor,Q. Effects of bicarbonate depletion in chloroplasts

Broken chloroplasts depleted of bicarbonate (HCO^?₃) show 30–50% inhibition of the Hill reaction in low-intensity light. Also, photoreactions excited by repetitive flashes measured by oxygen evolution, ESR signal IIvf, and absorption changes at 680 and 334 nm show inhibition of 30–50%. An effect of HCO^?₃ was sought to explain these phenomena. The decay of chlorophyll a fluorescence yield in the millisecond and seconds range, following a single flash, was observed to be multiphasic with a very slow component of 1–2 s half-time. In HCO^?₃ -depleted samples this component is enhanced 2- or 3-fold. Since this occurs even after one flash, it is suggested that HCO^?₃ affects the Q^? B → QB^? reaction. In this work it is shown that 40% inhibition of oxygen flash yield is relieved to a great extent if the excitation flash rate is decreased from 2 to 0.33 Hz. A measurement of 520 nm absorption change in the presence of ferricyanide, which is proportional to Photosystem II charge separation, shows a similar inhibition that is dependent on flash rate. The maximum amplitude of variable fluorescence yield and 520 nm absorption change after a single flash are unaffected by HCO^?₃ depletion. The dark distribution of oxygen-evolution S-states is found to be shifted to a more reduced configuration in depleted samples. It is concluded that normal charge separation occurs in HCO^?₃ -depleted Photosystem II reaction centers but that a large fraction of Q^? decays so slowly that not all Q^? is reoxidized between flashes given at a rate of 1 or 2 Hz. Thus, a portion of the Photosystem II centers would be closed to photochemistry. There is a reversible effect of HCO^?₃ depletion on the oxygen-evolution system that is observed as a shift in the dark distribution of S-states.     

Electron transfer and proton pumping under excitation of dark-adapted chloroplasts with flashes of light

Proton release inside thylakoids, which is linked to the action of the water-oxidizing enzyme system, was investigated spectrophotometrically with the dye neutral red under conditions when the external phase was buffered. Under excitation of dark-adapted chloroplasts with four short laser flashes in series, the pattern of proton release as a function of the flash number was recorded and interpreted in the light of the generally accepted scheme for consecutive transitions of the water-oxidizing enzyme system: S₀ → S₁, S₁ → S₂, S₂ → S₃, S₃ → S₄, S₀. It was found that the proton yield after the first flash varied in a reproducible manner, being dependent upon the dark pretreatment given. In terms of the proton-electron reaction during these transitions, the pattern was as follows. In strictly dark-adapted chloroplasts (frozen chloroplasts thawed in darkness and kept for at least 7 min in the dark after dilution), it was fitted well by a stoichiometry of 1:0:1:2. In a less stringently dark-adapted preparation (as above but thawed under light), it was fitted by 0:1:1:2. Mechanistically this is not yet understood. However, it is a first step towards resolving controversy over this pattern among different laboratories. Under conditions where the 1:0:1:2 stoichiometry was observed, proton release was time resolved. Components with half-rise times of 500 and 1000 μs could be correlated with the S₂ → S₃ and S₃ → S₄ transitions, respectively. Proton release during the S₀ → S₁ transition is more rapid, but is less well attributable to the transitions due to error proliferation. A distinct component with a half-rise time of only 100 μs was observed after the second flash. Since it did not fit into the expected kinetics (based on literature data) for the S_i → S_i+1 transitions, we propose that it reflects proton release from a site which is closer to the reaction center of Photosystem (PS) II than the water-splitting enzyme system. This is supported by the observation of rapid proton release under conditions where water oxidation is blocked. Related experiments on the pattern of proton uptake at the reducing side of PS II indicated that protons act as specific counterions for semiquinone anions without binding to them.     

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m²·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

ATP-induced increase in chlorophyll fluorescence. Characterization of rapid and slow induction phases

The biphasic rise of chlorophyll fluorescence induced in the dark (following activation of the latent ATP-ase) upon ATP-hydrolysis was investigated in detail, yielding the following main results: (1) The rapid phase is independent of artificial reductants or redox mediators. On the contrary, the slow phase requires such additions. (2) The slow phase is selectively eliminated by substances which collapse the transmembrane proton gradient, while the rapid phase may even be stimulated. (3) The ratio of rapid-to-slow phase is favored by a high degree of chloroplast integrity. The same factors which favor the rapid phase appear to be essential for a pronounced ‘slow electrogenic reaction’ in the flash-induced P 515 absorbance change. (4) For the rapid phase of the ATP-induced fluorescence increase, neither a ΔpH nor a Δψ are obligatory intermediates. (5) Hydroxylamine at about 5 · 10^?3 M causes a preferential stimulation of the rapid phase by about a factor 2. (6) There is selective inhibition of the slow phase by DBMIB, dinitrophenylether of iodonitrothymol, Bathocuproine and HQNO (2-heptyl-4-hydroxy quinoline-N-oxide) which are known to block at the level of the Cyt $\text{b}\text{f}$ FeS-complex. (7) The rapid phase is not affected by presence of 5 mM ferricyanide; however, there is substantial suppression if in addition a lipophilic redox mediator, like diamino-durene, is present. It is concluded that the two components of the reverse coupling reactions, reflected by the biphasic ATP-induced fluorescence rise, involve different coupling intermediates and different types of reverse electron flow. The rapid component appears to reflect close interaction between the coupling factor and a redox component in the vicinity of Photosystem II.     

Metastable proton pools in thylakoids and their importance for the stability of Photosystem II

After isolated chloroplast thylakoids have been transferred to a medium which is more alkaline than their storage medium, they retain considerable amounts of unequilibrated protons for often longer than 10 min. Essentially all of these protons are released upon uncoupler addition when the thylakoids are osmotically swollen, but only a portion of them when they are in a shrunken state. Osmotic swelling also greatly accelerates the inactivation of the water-oxidizing system enzyme of Photosystem II, and its depletion of functional Cl^?, at alkaline pH. Analyses of the mestable proton gradient in terms of stoichiometry, temperature dependence, and effect on fluorescent amine probes, suggest that most of the protons involved are bound and exchange readily with the bulk phases only when the thylakoids are swollen. It is concluded that, in shrunken thylakoids, the water-oxidizing enzymes are buried in special H⁺-sequestering domains which probably are formed by cavities in the inner surface of the thylakoid membrane. An observed cooperative action of alkaline pH and divalent cations during Cl^?-extraction from Photosystem II is interpreted as revealing an involvement of both a negatively charged surface region and positively charged groups in maintaining the functional integrity of the site of water oxidization.