Analysis of absorption changes in the ultraviolet related to charge-accumulating electron carriers in photosystem II of chloroplasts.
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Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.
Dark adapted spinach chloroplasts and Chlorella with variably reduced plastoquinone pools were given 1 or 2 saturating flashes. Under these conditions the rate of deactivation of state S2 of the oxygen evolving site of Photosystem II (B, Kok, B, Forbush, M. McGloin (1970) Photochem. Photobiol. 11, 457-475) is highly dependent on the pool redox state, undergoing a nearly 10-fold acceleration upon transforming the plastoquinone pool (A) from 100% oxidized to 90% reduced. Deactivation of state S3 is unaffected by the same variation of the pool redox state. These observations are attributed to a high concentration of Photosystem II reduced primary electron acceptor, Q-, coincident with the formation of S2 and a low concentration coincident with the formation of S3, under the conditions of highly reduced plastoquinone pool. Simultaneous determination of Q- and A2- result in an estimated equilibrium constant of 15-20 for reaction Q-B in equilibrium QB- and a value greater than 50 for equilibrium Q-B- in equilibrium QB2-, where B is the secondary electron acceptor described by B. Bouges-Bocquet ((1973) Biochim. Biophys. Acta. 314, 250-256) and B.R. Velthuys and J. Amesz ((1974) Biochim. Biophys. Acta. 333, 85-94). It is proposed that doubly reduced B becomes protonated in the last reaction.
The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.
The MgCl2-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3',4'-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a pico-second time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp --At1/2, where A was found to be 0.052 ps-1/2, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl2 produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence deday law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.
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A study was made of the inactivation by Tris of O2 evolution in chloroplasts and the subsequent reactivation of O2 evolution. We conclude: 1. At concentrations of Tris sufficient to inhibit O2 evolution directly, a slow rate (t 1/2 approximately 20--25 min) of inactivation occurs; 2. Inactivation is accelerated (t 1/2 approximately 2 min) by weak light absorbed by system II and is rate limited by a dark step with a half-time of about 200 s; 3. Minimally one quantum event within System II is sufficient to inactive 50--70% of the O2 evolving centers; 4. This process is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive but is inhibited by reduced dichlorophenol indophenol and phenazine methosulfate, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, 2-(3-chloro-4-trifluoromethyl)-anilino-3,5-dinitrothiophene and tetraphenylboron; 5. Partial reactivation of inactive O2 evolving centers is affected by the use of the same reagents inhibiting the light induced inactivations; 6. The life-time (t 1/2 approximately 1 to 3 h) of the activable state is correlated with diffusion across thylakoids of the larger manganese pool released from binding sites and remaining in thylakoids following inactivation of O2 evolution.
1. The induction of Photosystem II chlorophyll fluorescence from chloroplasts blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and uncoupled with gramicidin has been measured. 2. In agreement with other authors it was found that the addition of cations to chloroplasts suspended in a low-cation medium not only stimulated the intensity of fluorescence but also changed the shape of the induction from being nearly exponential to being sigmoid. 3. A new theory of the photosynthetic unit of Photosystem II (Paillotin, G. (1976) J. Theor. Biol. 58, 237--252) was used to analyse the fluorescence inductions. 4. A comparison of the results of the Paillotin model with the experimental data suggests that excitation energy is not able to migrate between all the photosynthetic units of a photosynthetic domain. However, it is concluded that excitation energy may migrate from one photosynthetic unit to another, and that the energy migration is in competition with other processes leading to the decay of the excitation within Photosystem II. 5. It is suggested that the size of the "functional" photosynthetic unit, defined as the number of chlorophyll molecules that may communicate with a reaction centre, is variable.
1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period. 2. Two distinct decay components of delayed luminescence were measured a "fast" component (from approximately 1 ms to approximately 6 ms) and a "slow" component (at approximately 6 ms). 3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change. 4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation. 5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics. 6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity. 7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant. 8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the "lake" model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the "w" hypothesis) were in closer agreement with the results.
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1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O2 evolution by envelope-free chloroplasts and oxaloacetate-dependent O2 evolution by intact chloroplasts. The half maximal concentration (C1/2) for stimulation of oxygen evolution in both cases is approximately 4 micrometer . 100 microgram chlorophyll . ml-1. 2. Ionophore A23187 also induces inhibition of CO2 and 3-phosphoglycerate-dependent O2 evolution by intact chloroplasts in the presence of 3 mM MgCl2. The half maximal concentrations (C1/2) for inhibition of O2 evolution are 3 micrometer and 5 micrometer respectively . 100 microgram-1 chlorophyll . ml-1. 3. A very high concentration of ionophore A23187 (10 microgram . 20 microgram-1 chlorophyll . ml-1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes. 4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.
Duroquinol (tetramethylhydroquinone) was found to function as an electron donor in chloroplasts. Non-cyclic electron transfer from duroquinol to electron acceptors such as oxygen proceeded at high rates, was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) but was sensitive to the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB). The electron transport from duroquinol was coupled to the synthesis of ATP. Spectroscopic studies of chloroplast electron carriers in the dark indicated the high-potential "Rieske" iron-sulfur center, cytochrome f, plastocyanin and P-700 were all reduced by duroquinol. The dark reduction of the "Rieske" iron-sulfur center and cytochrome f were inhibited by DBMIB but not by DCMU. These results have been interpreted in terms of a linear sequence of electron carriers in the non-cyclic electron transport chain which includes plastoquinone, the "Rieske" iron-sulfur center, cytochrome f, plastocyanin and P-700.
The heat-induced changes of chlorophyll fluorescence excitation and emission properties were studied in isolated chloroplasts of Larrea divaricata Cav. An analysis of the temperature dependency of fluorescence, under Fo and Fmax conditions, of temperature-jump fluorescence induction kinetics, and of 77 degrees K emission spectra of preheated chloroplasts revealed two major components in the heat-induced fluorescence changes: (1) a fluorescence rise, reflecting the block of Photosystem II reaction centers; and (2) a fluorescence decrease, caused by the functional separation of light-harvesting pigment protein complex from the rest of the pigment system. Preferential excitation of chlorophyll a around 420 nm, produced a predominant fluorescence rise. Preferential excitation of chlorophyll b, at 480 nm, gives a predominant fluorescence decrease. It is proposed that the overlapping of the fluorescence decrease on the somewhat faster fluorescence rise, results in the biphasic fluorescence rise kinetics observed in isolated chloroplasts. Both the rise component and the decay component are affected by the thermal stability of the chloroplasts, acquired during growth of the plants in different thermal environments. Mg2+ enhances the stability against heat-damage expressed in the decrease component, but has no effect on the rise component. Heat pretreatment leads to a decrease of the variable fluorescence in the light-induced 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) rise curve, but no change in half-rise time is observed. It is concluded that the block of Photosystem II reaction centers precedes the loss of the light-harvesting pigment protein complex. However, the approximately antiparallel heat-induced Fmax decrease and Fo increase suggest a common cause for the two events. A heat-induced perturbation of the thylakoid membrane is discussed.
1. The decay of delayed fluorescence from chloroplasts blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and uncoupled with gramicidin has been measured in the time range 0.75--45 ms by use of a laser phosphoroscope. 2. The decays have been analysed as the sum of three first-order components of approximate half-lives 0.2, 2.5 and 300 ms by a computer-assisted least-squares fit procedure. 3. The prompt fluorescence yield of the chloroplasts was manipulated by changing the cation concentration of the chloroplast-suspending medium. 4. Analysis of the concentration dependence of the components of the delayed fluorescence decay and of the prompt fluorescence inductions indicates that the emission yield of the intermediate (tau approximately 2.5 ms) component of the decay is equal to the fluorescence yield of a Photosystem II photosynthetic unit with an open trap, and that for the slow (tau approximately 300 ms) component the emission yield is equal to the total Photosystem II prompt fluorescence yield. 5. It is concluded that the delayed fluorescence yield in the time range studied is a complex function of time, which may be due to there being different mechanisms leading to delayed fluorescence production at short and long times after cessation of illumination.
Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The sepration was obtained by partitionin an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.
The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N',N'-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP+ as acceptor, the illuminated steady-state concentration of P-700+ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo. The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg2+ or Ca2+, had no effect on the signal amplitude during cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700+ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg+. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.
Antimycin has been used to study the role of cyclic electron transport in isolated intact chloroplasts maintained under aerobic conditions. At all light intensities, antimycin inhibits CO2 fixation when assay conditions are optimal. When turnover of the Calvin cycle is inhibited, antimycin stimulates bicarbonate-dependent O2 evolution. Energy-dependent processes such as chlorophyll a and 9-aminoacridine fluorescence quenching, and light-scattering (apparent absorption) changes are inhibited by antimycin. The results suggest that cyclic electron transport contributes to photophosphorylation under aerobic conditions and is obligatory as a source of ATP during the most active periods of CO2 fixation in vivo. Cyclic electron transport can be stimulated either by inhibiting Photosystem II activity or increasing the turnover of Photosystem I relative to Photosystem II. These effects are interpreted in terms of the need for correct redox poising of carriers in the pathway in order to sustain maximum rates of cyclic electron flow. Binding studies indicate the presence of a high affinity antimycin binding site on chloroplast membranes. The stoichiometry and dissociation constant of the high affinity site are consistent with the idea that antimycin inhibits cyclic electron transport by binding to a b-type cytochrome in the thylakoid membrane.
A hypothesis is proposed to explain the changes in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase. Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149--166). Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.