Spectrophotometric measurement of plastoquinone photoreduction in the blue-green alga,Phormidium uncinatum

The redox state of plastoquinone was measured in vivo in the blue-green alga, Phormidium uncinatum by means of a double beam UV-spectrophotometer. The difference in absorbance of the oxidized and the reduced forms of plastoquinone was amplified, and stored and averaged in a computer. The redox state was changed by two alternating actinic light beams. When one actinic wavelength was kept constant at 700 nm (PSI) variation of the other yielded an action spectrum representing photosystem II. The inhibitors of the photosynthetic electron transport chain, DCMU and DBMIB, reduced the difference in absorbance between the oxidized and reduced forms of plastoquinone.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea     

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. I. Isolated intact chloroplasts

We investigated the flash-induced electrochromic absorbance change, A ₅₁₅, of isolated intact chloroplasts in continuous monochromatic background light of different intensities and wavelengths. From the variation of the amplitude of A ₅₁₅ in background illumination the steady-state turnover time of electron transport was found to be around 100 msec and the slowest process could be assigned to a photosystem 1 driven cycle. The change of pH induced by nigericin, ATP, or ADP did not modify substantially the turnover time.In contrast to earlier observations the slow rise (10 msec) of A ₅₁₅ in untreated chloroplasts persists also at high-intensity background illumination exciting both photosystems. The proportion of the slow rise of A ₅₁₅ in nigericin-treated chloroplasts increases in the presence of background light. This result on the slow rise is discussed in terms of two different models existing in the literature.     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

The origin of photosystem-I-mediated electron transport stimulation in heat-stressed chloroplasts

Exposure of isolated chloroplasts of pea (Pisum sativum L.) to temperatures above 35° C leads to a stimulation of photosystem-I-mediated electron transport from dichlorophenolindophenol to methyl viologen. The threshold temperature for this stimulation coincides closely with that for heat-induced inhibition of photosystem-II activity in such chloroplasts. This coincidence is explained in terms of a rearrangement of the thylakoid membrane resulting in the exposure of a new set of donor sites for dichlorophenolindophenol within the cytochrome f/b ₆ complex of the electron-transport chain linking the two photosystems.Abbreviations cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCPIP (H₂) 2,6-dichlorophenolindophenol - EDAC ethyldimethylaminopropyl-carbodiimide - MV methyl viologen - PSI, II photosystem I, II - PCy plastocyanin - PQ(H₂) plastoquinone     

Reduction of cytochromes with menaquinol and sulfide in membranes from green sulfur bacteria

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects oxidant-induced reduction of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.Abbreviations BChl bacteriochlorophyll - cyt cytochrome - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - SQR sulfide-quinone reductase Dedicated to Prof. Dr. Aloys Wild on occasion of his 65th birthday.     

Fractional control of photosynthesis by the QB protein,the cytochrome f/b 6 complex and other components of the photosynthesic apparatus

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the Q_B protein of the electron-transport chain. Fractional control by the cytochrome f/b ₆ complex was insignificant under these conditions. Control by the cytochrome f/b ₆ complex dominated at high energy fluence rates where the contribution to control of the Q_B protein was very small. Uncoupling shifted control from the cytochrome f/b ₆ complex to the Q_B protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b ₆ complex and of the Q_B protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO₂ saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b ₆ complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the Q_B protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2,4-dinitro phenylether of 2-iodo-4-nitrothymol - pCMBS p-chloromercuribenzosulfonate     

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for V_max of 300 and 10 moles reduced/mg bacteriochlorophyll a·h, and for K_m of 5 and 3 M were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc ₁-complex via the ubiquinone pool.Abbreviations BChl a bacteriochlorophyll a - DAD diaminodurene - decyl-UQ decyl-ubiquinone - LED light emitting diode - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PQ-1 plastoquinone 1 - SQR sulfide-quinone reductase (E.C. 1.8.5.'.) - UQ ubiquinone 10 - Q_c the quinone reduction site on the cytochrome b ₆ f/bc ₁, complex (also termed Q_i or Q_r or Q_n) - Q_s the quinone reduction site on SQR - Q_z quinol oxidation site on the b ₆ f/bc ₁, complex (also termed Q_o or Q_p)     

The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage

The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b ₆ and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2-monophosphoadenosine-5-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.Abbreviations A^max maximal photothermal signal - Cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES photochemical energy storage - FNR ferredoxin NADP⁺ reductase - HQNO 2-(n-heptyl)-4-hydroxyquinoline N-oxide - NEM N-ethylmaleimide - P₇₀₀ reaction-center pigment of PSI - PADR 2-monophosphoadenosine-5-diphosphoribose - pBQ p-benzoquinone - PMA phenylmercuric acetate We are very grateful to Dr. M.-H. Montane (Cadarache, Saint-Paul-lez-Durance, France) for her advice in the electroporation experiments.     

Cyclic electron flow around photosystem I in unicellular green algae

Cyclic electron flow around PSI, or cyclic photophosphorylation, is the photosynthetic process which recycles the reducing equivalents produced by photosystem I in the stroma towards the plastoquinone pool. Through the activity of cytochrome b ₆ f, which also transfers protons across the membrane, it promotes the synthesis of ATP. The literature dealing with cyclic electron flow in unicellular algae is far less abundant than it is for plants. However, in the chloroplast of algae such as Chlorella or Chlamydomonas, an efficient carbohydrate catabolism renders the redox poise much more reducing than in plant chloroplasts. It is therefore worthwhile highlighting the specific properties of unicellular algae because cyclic electron flow is highly dependent upon the accumulation of these stromal reducing equivalents. Such an increase of reducing power in the stroma stimulates the reduction of plastoquinones, which is the limiting step of cyclic electron flow. In anaerobic conditions in the dark, this reaction can lead to a fully reduced plastoquinone pool and induce state transitions, the migration of 80% of light harvesting complexes II and 20% of cytochrome b ₆ f complex from the PSII-enriched grana to the PSI-enriched lamella. These ultrastructural changes have been proposed to further enhance cyclic electron flow by increasing PSI antenna size, and forming PSI-cyt b ₆ f supercomplexes. These hypotheses are discussed in light of recently published data.     

Quantitation of plastoquinone photoreduction in spinach chloroplasts

The question of plastoquinone (PQ) concentration and its stoichiometry to photosystem I (PSI) and PSII in spinach chloroplasts is addressed here. The results from three different experimental approaches were compared. (a) Quantitation from the light-induced absorbance change at 263 nm (A₂₆₃) yielded the following ratios (mol:mol); Chl:PQ=70:1, PQ:PSI=9:1 and PQ:PSII=7:1. The kinetics of PQ photoreduction were a monophasic but non-exponential function of time. The deviation of the semilogarithmic plots from linearity reflects the cooperativity of several electron transport chains at the PQ pool level. (b) Estimates from the area over the fluorescence induction curve (A_fl) tend to exaggerate the PQ pool size because of electron transfer via PSI to molecular oxygen (Mehler reaction) resulting in the apparent increase of the pool of electron acceptors. The reliability of the A_fl method is increased substantially upon plastocyanin inhibition by KCN. (c) Quantitation of the number of electrons removed from PQH₂ by PSI, either under far-red excitation or after the addition of DCMU to preilluminated chloroplasts, is complicated due to the competitive loss of electrons from PQH₂ to molecular oxygen. The latter is biphasic reaction occurring with half-times of about 2 s (30–40% of PQH₂) and of about 60 s (60–70% of PQH₂).Abbreviations A_fl area over the fluorescence induction curve - Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PQ plastoquinone - PS photosystem - P700 reaction center of PSI - Q primary quinone acceptor of PSII - Tricine N-tris (hydroxymethyl) methyl glycine - Triton X-100 octyl phenoxy polyethoxyethanol     

Inhibition of chlororespiration by myxothiazol and antimycin A in Chlamydomonas reinhardtii

Myxothiazol and antimycin A are shown to suppress the oxygen transient previously attributed to the flash-induced inhibition of chlororespiration in Chlamydomonas reinhardtii (Peltier et al. 1987, Biochim Biophys Acta 893: 83–90). However, these two compounds do not affect the photosynthetic electron transport chain as inferred by the insensitivity of the CO₂-dependent photosynthetic O₂ evolution and of the flash-induced electrochromic effect. Chlorophyll fluorescence induction measurements carried out in dark-adapted cells of a mutant of Chlamydomonas lacking photosystem 1, show that myxothiazol and antimycin A significantly increase the redox state of the photosystem 2 acceptors. We conclude from these results that chlororespiration is inhibited by myxothiazol and antimycin A and that the site of inhibition is located on the dark oxidation pathway of the plastoquinone pool. This inhibition is interpreted through the involvement of a myxothiazol and antimycin A sensitive cytochrome in the chlororespiratory chain.Abbreviations cyt cytochrome - PQ plastoquinone - PS photosystem     

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (–196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 mol O₂ per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable fluorescence - LHC light-harvesting complex - PpBQ phenyl-p-benzoquinone - PQ plastoquinone pool - P700 reaction center of PS I - PS I, PS II Photosystem I, II - Q_A first bound plastoquinone accepter - RC reaction centre     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

Inhibition of photosynthetic electron transport and formation of inactive chlorophyll in winter stressed Pinus silvestris

Kinetics of fluorescence at room temperature, electron transport and photooxidation of P700 and cytochrome f have been studied in chloroplasts isolated from active and winter stressed Pinus silvestris. The winter stress induced block in the electron transport chain between the two photosystems is close to the site of plastoquinone, since winter stress and DCMU caused the same type of inhibition of the reoxidation of the primary electron acceptor Q of photosystem II. No winter inhibition of the electron transport between cytochrome f and P700 was observed. Time course studies of P700 photooxidation in chloroplasts of active and winter stressed pine have shown that the photosynthetic unit size must be about equal in the two types of chloroplasts. An apparent increase of the photosynthetic unit size was induced by winter stress, as revealed by the high chlorophyll/P700 ratio of winter stressed pine. The phenomenon is explained by the formation of photosynthetically inactive chlorophyll. Low-temperature fluorescence emission spectra were recorded when either chlorophyll a (433 nm) or chlorophyll b (477 nm) were preferentially excited. Winter stress induced the formation of a chlorophyll a fraction emitting at 673 nm. This chlorophyll is most likely derived from the chlorophyll a antennae of the two photosystems, and it probably contributes to the photosynthetically inactive pool of chlorophyll in winter stressed pine. The light harvesting chlorophyll a/b complex is relatively resistant to winter stress.     

Inhibition of photosynthetic reactions by light

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m^-2 red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO₂-dependent O₂ evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI photosystem I - PSII photosystem II     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft