Reduction Of P700 Research Articles

Range of photosynthetic control of postillumination P700+ reduction rate in sunflower leaves

The kinetics of the postillumination reduction of P700(+) which reflects the rate constant for plastoquinol (PQH2) oxidation was recorded in sunflower leaves at different photon absorption densities (PAD), CO2 and O2 concentrations. The P700 oxidation state was calculated from the leaf transmittance at 830 nm logged at 50 μs intervals. The P700(+) dark reduction kinetics were fitted with two exponents with time constants of 6.5 and about 45 ms at atmospheric CO2 and O2 concentrations. The time constant of the fast component, which is the major contributor to the linear electron transport rate (ETR), did not change over the range of PADs of 14.5 to 134 nmol cm(-2) s(-1) in 21% O2, but it increased up to 40 ms under severe limitation of ETR at low O2 and CO2. The acceptor side of Photosystem I (PS I) became reduced in correlation with the downregulation of the PQH2 oxidation rate constant. It is concluded that thylakoid pH-related downregulation of the PQH2 oxidation rate constant (photosynthetic control) is not present under normal atmospheric conditions but appears under severe limitation of the availability of electron acceptors. The measured range of photosynthetic control fits with the maximum variation of ETR under natural stress in C3 plants. Increasing the carboxylase/oxygenase specificity would lead to higher reduction of the PS I acceptor side under stress.

Mathematical modelling of free-pool and channelled electron transport in photosynthesis: evidence for a functional supercomplex around photosystem 1

A mathematical model of photosynthesis (Laisk & Eichelmann (Phil. Trans. R. Soc. Lond. B 323, 369 (1989))) is further developed which aims to reproduce experimental data from parallel measurements of photosynthetic rate and the reduction state of P700 (the donor pigment of photosystem 1 (PS1)) in leaves. In the model, the electron transport chain comprises photosystem 2, plastoquinone, cytochrome b/f, plastocyanin, photosystem 1, ferredoxin and NADPH. In a first version, low-molecular carriers are assumed to be freely diffusible. Carbon metabolism is described in detail. Most of the known regulatory factors are included. Under conditions of upstream (cytochrome b/f) limitation, the computed quantum yield of photosynthetic O$_{2}$ evolution is proportional to the fraction of reduced P700, in agreement with experiment. Under downstream limitation (electron accumulation due to low ATP:NADPH ratio), photosynthesis does not decline until P700 becomes almost completely reduced. This is in contradiction to experiments. Introduction of the kinetics of channelled electron transport in a second version of the model causes photosynthetic rate to decline proportionally with excess reduction of P700 under downstream limitation, in agreement with experimental data. Such a result supports the proposed existance of functional supercomplexes of cytochrome b/f, plastocyanin, photosystem 1, ferredoxin and ferredoxin-NADP reductase which can carry out channelled electron transport during photosynthesis.

Substitution of lanthanides at the calcium site(s) in photosystem II affects electron transport from tyrosine Z to P680 +

Substitution of lanthanides for Ca 2+ in Photosystem II results in an inhibition of oxygen-evolution activity. In addition to blocking electron transport from the Mn-complex to TyrZ +, the presence of the lanthanides also affects electron transport from TyrZ to P680 +. This latter effect depends on two factors: (i) The ionic radius of the trivalent lanthanide ion; lanthanide ions with an ionic radius smaller than that of Ca 2+, exert a more pronounced inhibitory effect. (ii) The pH of the system; although the presence of lanthanides has a strong influence on the reduction of P680 + by TyrZ at pH 6.0, no such effect is observed at pH 7.5. A pH titration has attributed the pH effect to an amino acid residue with a p K of 6.5.

Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus. I. Is the psaF gene product required for oxidation of cytochrome c-553?

Photosystem I (PS I) reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus with nonionic detergents, digitonin or sucrose monolaurate, contained eight small subunit polypeptides. Two of the small polypeptides were identified by analysis of their N-terminal amino-acid sequences as the psaF and psaE gene products. Treatment with a cationic detergent, cetyltrimethylammonium bromide, resulted in depletion of five small subunits including the psaF gene product. Five PS I complexes isolated with an anionic detergent, sodium dodecylsulfate, contained zero to four small subunits but were all depleted of the psaF polypeptide. The function of the psaF gene product was examined by measuring reduction kinetics of flash-oxidized P-700 in the presence of different concentrations of cytochrome c-553. Oxidized P-700 was rapidly reduced by the reduced cytochrome in all the PS I complexes that contained, at least, the psaC and psaD polypeptides and the second-order rate constants of electron transfer from cytochrome c-553 to P-700 were essentially the same between PS I complexes that contained the psaF polypeptide and those that lost this polypeptide. Thus, the psaF polypeptide is not required for the bimolecular reaction between P-700 and cytochrome c-553. Mg 2+ had a moderate stimulating effect on the rate of P-700 reduction whether PS I complexes were associated with the psaF gene product or not. The function of this subunit polypeptide is discussed.

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium Synechococcus sp. PCC 7002 as Determined by the Reduction of P700+

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium <italic>Synechococcus</italic> sp. PCC 7002 as Determined by the Reduction of P700⁺

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO2. It was found that at the first maximum, light-and CO2-saturated photosynthesis can be much faster than steady-state photosynthesis. Both QA in the reaction center of PS II and P700 in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P700 oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force FA, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(Pi), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO2, the assimilatory force FA declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P700. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.

Electron transfer from cytochrome f to photosystem I in green algae

The time course of P700(+) reduction and cytochrome f oxidation following a single-turnover flash excitation of photosystem I was measured under various conditions in different strains of green algae. P700(+) was reduced with a half-time of 4 μs. The rate of cytochrome f oxidation was found to depend widely on physiological factors. Reversible transitions are described from a 'slow-oxidation' state (t 1/2=500 μs) to a 'fast-oxidation' state (t 1/2=80 μs). The addition of ionophore strongly favours and stabilizes the 'fast-oxidation' state. We suggest that these transitions reflect either reversible association between the cytochrome bf complex and the reaction center of photosystem I or changes in the mobility of oxidized plastocyanin. The transitions might be under the control of the membrane potential or the intracellular ATP content. The relation of these reversible transitions with the 'light state' transitions, and their possible involvement in a switch from linear to cyclic electron transfer, are discussed.

Detection of photosynthetic energy storage in a photosystem I reaction center preparation by photoacoustic spectroscopy

Thermal emission and photochemical energy storage were examined in photosystem I reaction center/core antenna complexes (about 40 Chl a/P700) using photoacoustic spectroscopy. Satisfactory signals could only be obtained from samples bound to hydroxyapatite and all samples had a low signal-to-noise ratio compared to either PS I or PS II in thylakoid membranes. The energy storage signal was saturated at low intensity (half saturation at 1.5 W m(-2)) and predicted a photochemical quantum yield of >90%. Exogenous donors and acceptors had no effect on the signal amplitudes indicating that energy storage is the result of charge separation between endogenous components. Fe(CN)6 (-3) oxidation of P700 and dithionite-induced reduction of acceptors FA-FB inhibited energy storage. These data are compatible with the hypothesis that energy storage in PS I arises from charge separation between P700 and Fe-S centers FA-FB that is stable on the time scale of the photoacoustic modulation. High intensity background light (160 W m(-2)) caused an irreversible loss of energy storage and correlated with a decrease in oxidizable P700; both are probably the result of high light-induced photoinhibition. By analogy to the low fluorescence yield of PS I, the low signal-to-noise ratio in these preparations is attributed to the short lifetime of Chl singlet excited states in PS I-40 and its indirect effect on the yield of thermal emission.

Selective destruction of iron-sulfur centers by heat/ethylene glycol treatment and isolation of Photosystem I core complex

Heat treatment of spinach thylakoids or Photosystem I particles in the presence of ethylene glycol caused the selective destruction of iron sulfur centers F A, F B and F X as judged by low-temperature EPR and flash spectroscopy. Incubation of thylakoids or Photosystem I particles in the presence of 50% ethylene glycol for 5 min selectively destroyed F A and F B at temperatures between 50 and 55°C, and F X between 60 and 70°C. P-700 was destroyed only above 70°C, and electron transport from ascorbate-dichlorophenolindophenol to methyl viologen was inhibited between 35 and 50°C by the same treatment. The destruction of F A and F B occurred at the same temperature range and resulted in the decrease of the decay phase with a 30 ms half-time in re-reduction kinetics of flash-oxidized P-700. This was accompanied by the appearance of the 1 ms decay phase, which reflects reduction of P-700 + by F − X. The 1 ms phase disappeared when F X was destroyed. Photosystem I core complex was isolated by solubilizing the heat/ethylene glycol-treated Photosystem I particles with Triton X-100, followed by sucrose density gradient ultracentrifugation. The complex obtained from 60°C/ethylene glycol-treated preparation lacked the 8 kDa F A/F B polypeptide and was composed of the polypeptides of apparent molecular weight 63, 60 and 5 kDa. This complex showed a small and broadened g = 1.77 F X signal. It is concluded that the heat/ethylene glycol treatment gives a simple and selective degradation-isolation method of Photosystem I reaction center complex.

Silicomolybdate substitutes for the function of a primary electron acceptor and stabilizes charge separation in the photosystem II reaction center complex

Effects of silicomolybdate on the charge recombination between P680 + and the reduced pheophytin were studied by absorption and electron paramagnetic resonance spectroscopies in the photosystem II D1/D2/cytochrome b-559 reaction center complex. This preparation lacks the primary and secondary quinone acceptors, Q A and Q B, and exhibits the charge recombination which produces the triplet state of P680 to a large extent. In the presence of silicomolybdate, the light-induced triplet signal of P680 was almost completely eliminated at cryogenic temperatures as well as at 4°C. Under these conditions, two types of signals, one reversible and the other irreversible, which are ascribable to P680 + and the cation radical of antenna chlorophyll a, respectively, were generated upon illumination at cryogenic temperatures. These results indicate that silicomolybdate, which is known to be an artificial electron acceptor of Q A, rapidly receives electrons from the reduced pheophytin even at cryogenic temperatures and thus suppresses the radical pair recombination which occurs in the time range of nanosecond. P680 + formed by flash excitation in the presence of silicomolybdate relaxed mainly with a long half decay time of 74 ms at 4°C. This indicates that the reduction of P680 + by the secondary electron donor, Z, is significantly decreased.

Relationship between the Quantum Efficiencies of Photosystems I and II in Pea Leaves

The irradiance dependence of the efficiencies of photosystems I and II were measured for two pea (Pisum sativum [L.]) varieties grown under cold conditions and one pea variety grown under warm conditions. The efficiencies of both photosystems declined with increasing irradiance for all plants, and the quantum efficiency of photosystem I electron transport was closely correlated with the quantum efficiency of photosystem II electron transport. In contrast to the consistent pattern shown by efficiency of the photosystems, the redox state of photosystem II (as estimated from the photochemical quenching coefficient of chlorophyll fluorescence) exhibited relationships with both irradiance and the reduction of P-700 that varied with growth environment and genotype. This variability is considered in the context of the modulation of photosystem II quantum efficiency by both photochemical and nonphotochemical quenching of excitation energy.

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids.

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.

Flash-induced absorption change at 703nm in intact leaves of kidney bean plants.

The absorption change at 703 nm induced by Hash actinic light in intact leaves of kidney bean plants was measured. The dependency of the absorption change upon the intensity of the detecting light and intensity and wavelength of actinic flash was examined in order to investigate photosystems and electron transport system in vivo. The light saturation of P700 oxidation and reduction by flash light was represented by hyperbolic curves, while that of P700 oxidation by detecting light was represented by an exponential curve. The action spectra of P700 oxidation and reduction at shorter wavelengths than 600 nm fluctuated in almost the same pattern, showing a minimum at 481 nm and a peak around 450 nm. Apparent differences between the two were observed above 630 nm. The action spectrum of P700 oxidation showed a peak at 621 nm, decreased at 683 nm and showed another peak at 700 nm, while the spectrum of P700 reduction decreased above a wavelength of 660 nm. Based on the analysis by the present method, the photoreactions observed in leaves with dense pigments were discussed.

Picosecond kinetics of fluorescence and absorbance changes in photosystem II particles excited at low photon density

Oxygen-evolving photosystem II particles (from Synechococcus) with about 80 chlorophyll molecules per primary electron donor (P(680)) were used for a correlated study of picosecond kinetics of fluorescence and absorbance changes, detected by the single-photon-timing technique and by a pump-probe apparatus, respectively. Chlorophyll fluorescence decays were biexponential with lifetimes tau(1) = 80 +/- 20 ps and tau(2) = 520 +/- 120 ps in open reaction centers and tau(1) = 220 +/- 30 ps and tau(2) = 1.3 +/- 0.15 ns in closed reaction centers. The corresponding fluorescence yield ratio F(max)/F(o) was 3-4. Absorbance changes were monitored in the spectral range of 620-700 nm after excitation at 675 nm with 10-ps pulses sufficiently weak (<7 x 10(12) photons/cm(2) per pulse) to avoid singlet-singlet annihilation. With open reaction centers, the absorbance changes could be fit to the sum of three exponentials. The associated absorbance difference spectra were attributed to (i) exciton trapping and charge separation (tau = 100 +/- 20 ps), (ii) the electron-transfer step P(680) (+) I(-) Q(A) --> P(680) (+) I Q(A) (-) (where I is the primary electron acceptor and Q(A) is the first quinone acceptor) (tau = 510 +/- 50 ps), and (iii) the reduction of P(680) (+) by the intact donor side (tau > 10 ns). With closed reaction centers, the absorbance changes were biexponential with lifetimes tau(1) = 170-260 ps and tau(2) = 1.6-1.75 ns. The results are explained in terms of a kinetic model that assumes P(680) to constitute a shallow trap. The results show that Q(A) reduction in these photosystem II particles decreases both the apparent rate and the yield of the primary charge separation by a factor of 2-3 and increases the mean lifetime of excitons in the antenna by a factor of 3-4. Thus, we conclude that the long-lived, nanosecond chlorophyll fluorescence is not charge-recombination luminescence but rather emission from equilibrated excited states of antenna chlorophylls.

Study of the PS I acceptor side by double and triple flash experiments

Electron transfer from P-700 to the PS I electron acceptors has been studied by flash absorption spectroscopy after double or triple excitation at room temperature, in the presence of glycerol, in pea chloroplasts and in PS I particles supplemented with purified plastocyanin. After two flashes, the reduction of P-700 + occurs with t 1 2 = 250 μ s when the excitations are separated by less than 10 ms. This reduction is attributed to the recombination reaction between P-700 + and the reduced iron-sulfur center X. Under conditions where bound plastocyanin can rapidly reduce P-700 + after the second excitation, a third flash induces an oxidation of P-700 which decays with a t 1 2 of 30 μs. This new kinetic phase is tentatively attributed to a back-reaction between P-700 + and an electron acceptor more primary than the iron-sulfur center X. Addition of glycerol to the samples is shown to inhibit the reduction of the iron-sulfur center A at room temperature. Data are compared with results obtained under background illumination and reducing conditions.

The site and mechanism of duroquinol oxidation by the cytochrome b6- f complex in Synechococcus sp.

Duroquinol (tetramethyl- p-hydroquinone) served as an excellent electron donor to Photosystem I and promoted methyl viologen photoreduction in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned cells of the thermophilic cyanobacterium Synechococcus sp. at a rate comparable to or, more likely, higher than that of the photoreduction with water as electron donor. The restored activity was inhibited by 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB). The inhibition is competitive and partially reversed by increasing duroquinol concentration. Spectrophotometric experiments with flashes demonstrated that duroquinol accelerates the reduction of P-700, cytochrome c-533 and cytochrome f in the DBMIB-poisoned cells similarly and in a way just opposite to the inhibitory action of DBMIB: the electron donor specifically increased the fraction of the three electron carriers that was reduced with the 2 ms half-time. The results indicate that duroquinol does not serve as a reductant of the plastoquinone pool, nor of P-700 and cytochrome c-533, but directly donates its electrons to the cytochrome b 6- f complex by binding to the plastoquinone-binding site and that DBMIB inhibits the quinol oxidation by binding competitively to the same site.

Dark Reduction of P700&lt;sup&gt;+&lt;/sup&gt; in Photosystem I Particles by Illuminated Chlorophyllide

Illumination of chlorophyllide dissolved in a wet organic solvent generates an unknown species of chlorophyllide which is capable of reducing P700+ in darkness ata considerably high rate in the absence of ascorbate and redox mediators. The formation of this derivative species is accompanied by bleaching of both the red and blue absorption bands of chlorophyllide concomitant with the appearance of a new peak at around 500 nm. The generation of reducing capability is stimulated by the presence of bases but does not require reducing agents. Some of the properties of this reaction are discussed in comparison with the Krasnovskii reaction.

Differential effects of DBMIB and DNP-INT on the kinetic phases of P-700 reduction

In the presence of ferredoxin and NADP, DBMIB abolishes the fast-relaxing portion of P-700 together with the reduction of NADP. The slow-relaxing portion is inhibited at much higher concentrations. Qualitatively similar results have been observed with DNP-INT. However, its action appears to be a light-dependent process. The slow, cyclic turnover of P-700 in the presence of DCMU, ferredoxin and NADPH is inhibited by DBMIB but only slightly by DNP-INT. The data suggest that the inhibitors act at different sites of the electron-transport system.

Restoration by tetramethyl- p-phenylenediamine of photosynthesis in dibromothymoquinone-inhibited cells of the cyanobacterium Synechococcus sp.

Inhibition of electron flow from water to methyl viologen by 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB) was reversed by the addition of N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) to cells of a thermophilic cyanobacterium Synechococcus sp. The restored activity is not a simple Photosystem I reaction because it was suppressed by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). TMPD was also found to restore photosynthetic oxygen evolution in the DBMIB-poisoned cells. That the restored oxygen evolution is coupled with CO 2 reduction was indicated by its sensitivity not only to electron-transport inhibitors such as DCMU and phenylmercuric acetate, but also to an inhibitor of CO 2 fixation (KCN), uncouplers (carbonyl cyanide m-chlorophenylhydrazone, methylamine and ammonium chloride) and an energy-transfer inhibitor ( N, N′-dicyclohexylcarbodiimide). In the DBMIB-poisoned cells, TMPD accelerated only P-700 reduction, while leaving the reduction kinetics of cytochrome c-553 and cytochrome f inhibited, indicating that P-700 is the site of electron entry from TMPD to Photosystem I. It is concluded that TMPD restores photosynthetic electron transport by accepting electrons in the plastoquinone region and in turn donating them directly to P-700, and thereby bypassing cytochrome c-553 and DBMIB-blocked cytochrome b 6- f complexes.

Retardation of electron donation to Photosystem I in aged cyanobacteria and its reversal by metal cations

The dark reduction of photooxidized P-700 in the cyanobacterium Anacystis nidulans becomes slower as a consequence of aging. In cells, of which the envelope has been enzymatically permeabilized to electrolytes, the reduction of P-700 by endogenous electron donors is accelerated by KCl plus valinomycin, but not by KCl alone. The K +-valinomycin system is ineffective, however, in the case of aged but unpermeabilized cells. These results suggest that a consequence of aging in Anacystis is an increase in the negative electric potential at the inner thylakoid surface, which makes electron donation to P-700 by acidic donors (cytochrome c-553 and plastocyanin) less probable. This situation is reversed by the permeant K +-valinomycin system, which suppresses the inner surface potential and collapses the inside-outside potential difference, but not by K + alone, since the thylakoid membrane is impermeable to it.