Symposium 05 Bioenergetics of Photosynthetic Electron Flow
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The Contribution of Light-Dependent Bicarbonate Uptake in Thylakoid Membrane Energization *Zolotareva EK, Polishchuk OV, Semenikhin AV, Onoiko EB M.G. Kholodny Institute of botany of the National Academy of Sciences of Ukraine Tereschenkivska str., 2, 01601, Kyiv-1, Ukraine. *Corresponding author. E-mail:
[email protected].
Abstract: Illumination of the well stirred suspension of isolated spinach chloroplasts induced CO2 uptake (up to 320 nmol CO2/mg chlorophyll) from air phase over suspension. The process started immediately after onset of illumination, developed during 15–20 sec and completely reversed for the same time after switching off the light. Uncouplers (gramicidine D, NH4Cl) inhibited the light-induced CO2 uptake. The value of light-induced CO2 uptake was dependent on carbonic anhydrase (CA) activity and inhibited by CA inhibitors – lipophylic ethoxyzolamide (EZ) and water-soluble acetazolamide. The effect of exogenic bicarbonate and inhibitors of carbonic anhydrase on the rate of photophosphorylation was examined in the pH range 7.0–8.2. It was shown that exogenic bicarbonate (3–6 mmol) effectively stimulated photophosphorylation. The bicarbonate-induced enhancement of photophosphorylation showed a marked pH dependence, with the – greatest response occurring at pH near 7.0. Both EZ and AZ reduced the stimulating effect of HCO3 on the rate of photophosphorylation. It is concluded that light-induced ATP synthesis depends not only on exogenic bicarbonate, but also on the activity of carbonic anhydrase that rapidly converts the forms of carbonic acid thereby facilitating protons removal from sites of their evolution. Keywords: Proton transfer; Bicarbonate; Photophosphorylation; Carbonic anhydrase; Thylakoid membrane
Introduction Thylakoid membranes contain a rather considerable amount (up to 1 mol/mg Chl) of bicarbonate, bound with different affinity (Stemler, 1977; Klimov and Baranov, 2001). Tightly bound bicarbonate is absolutely necessary for retaining the functional activity of photosystem II (PS II), its removal leads to inhibiting the electron transfer reactions, as at the donor, as at the acceptor side of PS II. Localization point of the tightly bound bicarbonate, by a whole number of works, is PS II site between primary and secondary plastoquinone QA and QB acceptors. The functional role of the loosely bound – HCO3 pool is not known. The value of the proton motive force (PMF) in chloroplasts of higher plants under steady-state conditions is mainly determined by the difference of hydrogen ions concentration between the internal volume of thylakoids and the stromal space (Kramer
et al., 1999; Dilley, 2000) In the process of photosynthetic electron transport, protons are transferred inside thylakoids, and are also formed during the splitting of water molecules— directly in the intrathylakoid space (Kramer et al., 1999). It is known that illumination of dark-adapted chloroplasts leads to the alkalization of stroma and acidification of thylakoid lumen and it correlates with proton concentration and electrical potential differences across the thylakoid membrane. Massive transmembrane flow, in accordance with the electroneutrality principle, should be compensated by the transfer of other ions, different from + ones. There are some experimental data (Hind et al., 1974) obtained by ion-specific electrodes and indicated that Cl– and Mg2+ fluxes together compensate for most of the charge transferred as H+. Here, with the help of the infrared gas analysis we have demonstrated that the illumination of isolated chloroplasts leads to CO2 uptake of up to 350 nmol/mg
T. Kuang et al., Photosynthesis Research for Food, Fuel and the Future © Zhejiang University Press, Hangzhou and Springer-Verlag Berlin Heidelberg 2013
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Chl from the gas phase above the suspension, which is comparable with the number of protons uptaken during the illuminating of chloroplasts (H+) (Dilley, 2000) Results of present research showed bicarbonate transfer that can compensate light-induced proton uptake by isolated chloroplasts (so-called H+). The aim of this work was investigation of possible role of light-induced CO2 absorption in supporting the proton transport and thylakoid membrane energization.
gas phase. Total CO2 uptake in different experiments varied from 0.1 to 0.35 mol/mg Chl.
Materials and Methods Class C chloroplasts were prepared from market spinach (Spinacea oleracea L.) essentially as previously described (Zolotareva et al., 1997). The analyses were performed in the closed thermostated glass cell at 20 oC by help of infra red gas analyser (S151 type, Qubit Systems Inc., Canada) at the continuous air flow (350 ml/min), which held the 360–380 ppm CO2. Reaction medium contained sorbitol (100 mmol), NaCl (10 mmol), tris-HCl (10 mol, pH 7.5) and chloroplasts equivalent to chlorophyll 10 g/ml. Actinic light was 1,000 mol quanta/m2 s. Photophosphorylation was carried out at room temperature in a mixture containing 20 mmol Tricine, 20 mol MES, 150 mmol KC1, 5 mmol MgCl, 30 mol glucose, 50 nmol phenazine methosulfate or 0.1 mmol methylviologen, 0.250 mmol ADP, 5 mmol Pi, and hexokinase (HK; 1 IU/ml). The mixture was illuminated for 2 min and the amount of ATP was determined enzymatically using glucose-6-phosphate dehydrogenase (G6PDH; 1.125 IU/ml) and NADP (0.5 mmol). The amount of NADPH formed is stoichiometrically equivalent with the amount of ATP and measured spectrophotometerically at 340 nm.
Results and Discussion Fig. 1 shows, how the CO2 flow rate changes at illuminating the chloroplast suspension and after turning off the light. Light saturation of the process was achieved with the help of intensive stirring which was started concurrently with the light turn off. In the absence of exogenic electron acceptor, the value of the light-induced CO2 uptake is somewhat lower than in the presence of MV or PMS. After turning off the light, CO2, in the amounts equivalent to the ones taken up in the light, was released into the
Fig. 1 Light-induced CO2 uptake by isolated chloroplast suspension. Chlorophyll concentration was 1. 0.3 mg/ml; 2. 0.6 mg/ml; 3. 1.1 mg/ml; 4. 3.3 mg/ml.
The effect of 2 uptake by thylakoids was registered only in concentrated suspensions, the content of chorophyll in which constituted 2.5–3.5 mg Chl/ml. In more diluted suspensions CO2 uptakes during illumination were not found. Data represented in Fig. 1 demonstrate how the level of light-dependent CO2 uptake is decreasing in proportion to the suspension being diluted. These results might be explained by the fact that carbon dioxide invades the suspension in the process of simple diffusion, since, according to Fick’s law, diffusion from the gas phase depends on the difference of concentrations between the solution and the ambient gas volume. It is evident that in the darkness, at the constant and temperature, carbon dioxide, diluted in the suspension, is in balance with 2 of the gas phase above the suspension. The illumination causes bicarbonate uptake from the solution, evidently, connected with transmembrane proton gradient formation. Probable correlation of the light-induced uptake and the degree of energization of thylakoid membranes was checked in a series of experiments, the results of which are summed up in Table 1. In test experiments 2 uptake was registered in the presence of the PMS cyclic electron transport mediator. Injection of ionophore antibiotic gramicidin A, which was forming the channels and dissipating the transmembrane proton gradient, was also suppressing the light-induced 2, intake.
Symposium 05 Bioenergetics of Photosynthetic Electron Flow Table 1 The effect of uncouplers on light-dependent CO2 (CO2) and + (+) uptake. Chloroplast concentraton was equivalent to 2.7–2.9 mg Chl/ml. 0,1 mmol methylviologen was present as the electron acceptor. Light-dependent Light-dependent CO2 uptake + uptake Variant (CO2), (+), nmol nmol CO2/mg Chl H+/mg Chl Control 140 ± 20 120 ± 15 +gramicidin A, 0.1 μmol 190 ± 22 80 ± 15 +gramicidin A, 1 μmol 0 0 NH4Cl, 1 mmol 50 ± 12 70 ± 12 NH4Cl, 5 mmol 0 0
Same results were also obtained during the experiments using protonophoric uncoupler NH4Cl. The results allow to conclude that the integrity of the thylakoid membrane and forming a high level of transmembrane proton gradient represent the necessary conditions of the observed effect of CO2 uptake during illumination of chloroplasts. The effect of light-induced proton uptake developing at illumination of thylakoid membranes within the same time range was described about 50 years ago (Neumann and Jagendorf, 1964). This phenomenon, called H+ in the literature, is considered to be the integral part of thylakoids energization process. It is assumed that H+ value depends upon buffer capacity of thylakoid membranes (Walz et al., 1974). Comparison of the two processes—light-dependent proton and carbon dioxide uptakes—enables to detect their similarity. Isolated chloroplasts at illumination of slightly buffered suspension are uptaking 200–500 nmol H+/mg Chl depending on the nature of electron acceptor. It was earlier noted not once that the number of thylakoid protein buffer groups, capable of binding protons in the physiological range, is not large; much smaller than the observed H+ value (Walz et al., 1974). In this respect, the probable role of light-dependent bicarbonate uptake by thylakoids is the increase of the system’s buffer capacity necessary for the stabilization of the level of transmembrane proton gradient under conditions of active electron transport and release of a great number of protons inside thylakoids. The results of this work demonstrate that H+ and CO2 uptakes during the illumination are interconnected processes. Nevertheless, it needs to point out that the experimental registration of CO2 uptake, caused by chloroplasts energization, is possible only in very dense suspensions as distinguished from H+ which is easily registered in solutions of low buffer capacity and is disguised in solutions with a high content of
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buffer groups. Assuming that H+ and CO2 are uptaken simultaneously in parallel processes, it can be considered that 2 diffuses at that from the gas phase, while compensating the changes of soluble carbon dioxide forms in the suspension. In fact, the results of the work show that the light-induced CO2 uptake is detected only under conditions of a low “CO2-capacity”. Table 2 data demonstrate that the value of lightinduced CO2 uptake also depends upon carbonic anhydrase activity. Carbonic anhydrase inhibitors – lipophilic ethoxyzolamide (EZ) and hydrophilic acetazolamide (AZ)—have inhibited 2 uptake caused by illumination of the chloroplasts suspension. The degree of suppressing the reaction was dependent on the duration of the inhibitors activity: short incubation of reaction mixture after the injection of the inhibitor in the reaction medium was leading to partial inhibition, and the prolonged preincubation of thylakoids, that is during 3 h, in the presence of EZ or AZ—to the total inhibition of light-dependent 2 uptake by chloroplasts. Table 2 The effect of carbonic anhydrase inhibitors on lightdependent CO2 uptake in isolated chloroplast suspension.
5 min incubation 3 h incubation at 4 oC
Light-dependent CO2 uptake, nmol/mg Chl + 0.4 + 0.4 mmol Control mmol AZ EZ 260 ± 40 75 ± 10 81 ± 10 230 ± 35 5±2 4±2
In the intact chloroplasts, capable of CO2 photosynthetic uptake, the process of carbon dioxide fixation starts after 5–7 minutes of illumination. This phase of the process is called photosynthesis induction (Walker, 1973). It is shown in this work that class chloroplasts, not capable of CO2 fixation due to the absence of outer shells and loss of the necessary soluble components, also uptake a certain amount of CO2 immediately after the illumination has started. The reaction develops during 30–40 s, and the amount of carbon dioxide uptaken by thylakoids reaches 320 mol CO2/mg Chl. The pool of CO2 uptaken, evidently, remains bound with thylakoid membranes during the whole period of illumination and is released into the gas phase after the light has been turned off. The process of light-induced CO2 uptake is under control by carbonic anhydrase activity and its possible functional role consists in participation in forming of
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the energized state of the thylakoid membrane and the stabilization of the pH level due to membrane buffer capacity increase. This conclusion is not in conflict with the assumption made earlier in a number of works about participation of CA thylakoids in the light-dependent replenishment of the stromal 2 fund, which provides for Rubisco activity (Raven, 1977). As it well-known, light-dependent proton exchange supplies energy for photophosphorylation, forming transembrane proton gradient (), which is converted in stationary conditions into ATP chemical energy in the process of photosynthetic photophosphorylation. The rate of photophosphorylation also depends on presence of bicarbonate in the medium, and accelerates at growing of its concentration in the suspension. (Cohen and MacPeek, 1980). The next part of the work was devoted to investigation of carbonic anhydrase role in stimulation of photosynthetic phosphorylation by exogenic bicardonate. Data on the influence of 3–6 mmol NaHCO3 upon non-cyclic photophosphorylation rate in the presence of MV, as electron acceptor, at different values of media, are given in Table 3. In control conditions, non-cyclic ATP synthesis was registered at > 7.0, and maximum ATP synthesis rate was observed at .2, which corresponds with literary data. Dependency of photophosphorylation on substantially changed, where bicarbonate was added to the reaction medium. At that, maximum ATP synthesis rate did not differ from the control value, and was registered at same values, as at control one. In the presence of 6 mmol NaHCO3 photophosphorylation rate significantly exceeded control values within the range of 6.5–8.0, and at medium < 7.7—by several times. Especially notably photophosphorylation was stimulated at .0 and lower. ATP synthesis rate at these values is very low in controlled conditions and grows by 4–8 imes after addition of NaHCO3 to the concentration of 6 mol (Table 3). Stimulating influence of bicarbonate on photophosphorylation rate was diminishing in proportion to enhancement of reaction medium : at 7.6, addition of 6 mmol NaHCO3 led to increase of ATP synthesis rate approximately by 2 times, and at 8.2—by 1.02–1.1 times. Stimulation of photophosphorylation with 3 mmol bicarbonate was less prominent, compared to the effect of 6 mmol bicarbonate. Photophosphorylation rate in the presence of 3 mmol bicarbonate constituted 130% and lowered to 106 and 109% against the control one with the
addition of AZ and EZ, correspondingly. Stimulation of photophosphorylation with exogenic added bicarbonate was effectively removed after a short-time (during 3 minutes) incubation of chloroplasts in the presence of carbonic anhydrase inhibitors—hydrophilic AZ or lipophilic EZ. Data given in Table 3 show that the effect of carbonic anhydrase inhibitors was most noticeable at 7.6. It is seen that in these conditions photophosphorylation rate, exceeding the control one by 2.3 times in the presence of 6 mmol bicarbonate, after addition of AZ or EZ decreased and amounted to 120% and 125% of the control value, correspondingly. Table 3 Inhibition of pH-dependent bicarbonate stimulation of non-cyclic photophosphorylation by inhibitors of carbonic anhydrase acetazolamine (AA) and ethoxizolamide (EA). Photophosphorylation rate, μmol/mg Chlh
7.0 ± 0.05
7.6 ± 0.05
8.2 ± 0.05
control Without added + EA NaHCO3 0.5 mmol + AA 0.5 mmol + 3 mol control NaHCO3 + EA 0.5 mmol + AA 0.5 mmol + 6 mol control NaHCO3 + EA 0.5 mmol + AA 0.5 mmol
10 ± 1 12 ± 1
90 ± 5 95 ± 5
230 ± 11 225 ± 13
11 ± 1
89 ± 4
220 ± 12
16 ± 1 12 ± 1
116 ± 6 98 ± 5
250 ± 5 245 ± 5
13 ± 1
95 ± 5
240 ± 12
48 ± 3 20 ± 1
210 ± 10 113 ± 6
250 ± 12 228 ± 11
16 ± 1
108 ± 5
220 ± 11
Thus, carbonic anhydrase largely eliminated the stimulation of of photophosphorylation by exogenic bicarbonate. The role of thylakoid carbonic anhydrase, removing the kinetic limitations connected with interconversion of carbon acid forms, in this case consists in retaining a sufficiently high concentration of free bicarbonate, which accepts protons, in the sites of their release. Apparently, binding and transfer of protons by 3/23 pair are most effective when close to carbon acid ionization constant ( ~ 6.36). Thus, if value greatly differs from , proton transfer with participation of bicarbonate ceases to be effective. Evidently, -dependence of carbonate stimulating the active effect on photophosphorylation, observed in this work, is connected with that.
Symposium 05 Bioenergetics of Photosynthetic Electron Flow
As it well-known, light-dependent proton exchange supplies energy for photophosphorylation, forming trans-membrane proton gradient (), which in stationary conditions, in the process of photosynthetic photophosphorylation, is converted into ATP chemical energy. It was possible to demonstrate in this study that photophosphorylation rate, stimulated in the presence of exogenic bicarbonate, depends on carbonic anhydrase activity. Early Shutova and co-authors (Villarejo et al., 2002; Shutova et al., 2008) supposed that thylakoid carbonic anhydrase Cah3 participates in proton transfer on the donor side of PS , easing the removal of protons from water photo-oxidation sites. The data of present work suggest that the intrathylakoid bicarbonate pool is formed during thylakoid membrane light energization and takes part in photophosphorylation acceleration due to its participation in proton transfer from +-generating proton pumps to ATP synthase.
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Kramer DM, Sacksteder CA, Cruz JA (1999) How Acidic Is the Lumen? Photosynth. Res. 60: 151163 Neumann J, Jagendorf AT (1964) Dinitrophenol as an Uncoupler of Photosynthetic Phosphorylation. Biochem Biophys Res Commun. 16: 562-567 Raven JA (1997) CO2-Concentrating Mechanism: a Direct Role for Thylakoid Lumen Acidification? Plant Cell Environ. 20: 147-154 Shutova T, Kenneweg H, Buchta J, Nikitina J, Terentyev V, Chernyshov S, Andersson B, Allakhverdiev SI, Klimov VV, Dau H, Junge W, Samuelsson G (2008) The Photosystem IIAssociated Cah3 in Chlamydomonas Enhances the O2 Evolution Rate by Proton Removal. EMBO J. 27: 782-791 Stemler AJ (1977) The Binding of Bicarbonate Ions to Washed Chloroplast Grana. Biochim. Biophys. Acta. 460: 511-522 Villarejo A, Shutova T, Moskvin , Forssen M, Klimov VV, Samuelsson A (2002) Photosystem II-Associated Carbonic Anhydrase Regulates the Efficiency of Photosynthetic Oxygen Evolution EMBO J. 21: 1930-1938 Walker DA (1973) Photosynthetic Induction Phenomena and the Light Activation of Ribulose Diphosphate Carboxylase. New Phytologist. 72: 209-235 Walz D, Goldstein L, Avron M (1974) Determination and Analysis of the Buffer Capacity of Isolated Chloroplasts in the Light and in the Dark. Eur. J. Biochem. 47: 403-407 Zolotareva EK, Dovbysh EF, Tereshchenko AF (1997) Effect of Alcohols on Inhibition of Photophosphorylation and Electron Transport by N.N'-Dicyclohexylcarbodiimide in Pea Chloroplasts. Biochemistry (Moscow) 62: 631-635
