Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

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 picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, 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 decay 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.     

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2°K

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2°K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are $\text{D = 0.02794 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, $\text{E = 0.00382 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, i.e. equal to those of monomeric chlorophyll $\text{a}$ to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperature 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.     

Isolation and characterisation of metribuzin-resistant Chlamydomonas reinhardii cells

Chlamydomonas reinhardii cells were treated with 5-fluorodeoxyuridine and ethylmethanesulfonate to induce mutagenesis. The mutant cells were analyzed for resistance against metribuzin (4-amino-6-(t-butyl)-3-methylthio-1,2,4-triazine-5-one). Clones with normal growth were isolated and the mutant cells further characterized. The photosynthetic rates of the mutant cells were about 20% lower than those of wild-type cells. The mutant cells were not only resistant against metribuzin (pI₅₀ lowered from 6.65 to 3.41) but also against bromacil, atrazine, phenisopham and tolerant against 3-(3,4-dichlorophenyl)-1,1-dimethylurea. However, the mutant was more susceptible to phenolic electron-transport inhibitors like bromonitrothymol, ioxynil and i-dinoseb. 2,4-Dinitrophenyl-2′-iodo-3′-methyl-4′-nitro-6′-isopropyl phenyl ether inhibited the wild-type thylakoids more than the mutant. The analysis of the electron transport with artificial electron donors and acceptors showed that only Photosystem II was affected by the mutation and not Photosystem I. Binding experiments with isolated thylakoids of resistant and susceptible cells using [¹⁴C]metribuzin and [³H]-i-dinoseb revealed that metribuzin did not bind specifically to the thylakoids of the mutant cells, but that i-dinoseb did bind to the thylakoids of the mutant, and even better than to the thylakoids of the wild-type cells. Fluorescence studies confirmed these results.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

The kinetic relationship between the C-550 absorbance change,the reduction of Q(ΔA320) and the variable fluorescence yield change in chloroplasts at room temperature

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolated, envelopefree chloroplasts in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isosbestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differential extinction coefficient Δ?(540–550) is calculated to be approximately 5 mM^?1 · cm^?1. The kinetics of the C-550 absorbance change, occurring upon the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical conditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The light-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers.It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Thus, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at room temperature.     

Possible effects of the detachment of stromal lamellae from granal stacks on salt-induced changes in spillover. A study by sonication of chloroplasts

Salt-induced chlorophyll fluorescence and spillover changes in control and briefly sonicated chloroplasts have been studied under conditions where Photosystem II traps are closed. In a low-salt medium containing 10 mM KCl, control envelope-free chloroplasts exhibited good spillover, as measured by low chlorophyll fluorescence yield at room temperature, a high ratio of the fluorescence peaks $\text{F}{}_{735}\text{F}{}_{685}$ at 77 K, and increased Photosystem I activity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and Photosystem II light. In contrast, when stacked chloroplasts were briefly sonicated and subsequently diluted into a low-salt medium, a high fluorescence yield at room temperature and a low ratio of $\text{F}{}_{735}\text{F}{}_{685}$ at 77 K persisted. When unstacked chloroplasts were sonicated and then diluted into a high-salt medium, the room temperature fluorescence yield remained low. The results are interpreted in terms of a model relating the changes in chlorophyll fluoresecence with the lateral diffusion of Photosystem I and Photosystem II chlorophyll-protein complexes in the plane of the thylakoid membrane creating randomized or segregated domains, depending on the degree of electrostatic screening of surface charges (Barber, J. (1980) FEBS Lett. 188, 1–10). It is argued that brief sonication of stacked chloroplasts separates stromal membranes from granal stacks, thus limiting the inter-mixing of the photosystems via lateral diffusion even when the ionic composition of the medium is varied. Consequently energy transfer from Photosystem II to Photosystem I is relatively poor and chlorophyll fluorescence from Photosystem II is enhanced. The loss of the salt effect on sonicated unstacked membranes can also be accommodated by the model. In this case it seems that the generation of small membrane fragments does not allow the normal salt-induced phase separation of the pigment-protein complexes to occur.     

Isolation and characterization of Photosystem I and II membrane particles from the blue-green alga, Synechococcus cedrorum

Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10–30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose.The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 μmol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains β-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N₂ fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome $\text{b-559}$ ratio of 50 : 1. The Photosystem I particle is highly enriched in $\text{P-700}$, with a chlorophyll to $\text{P-700}$ ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.     

Herbicide and plastoquinone-binding proteins of Photosystem II reaction center complexes from the thermophilic cyanobacterium,Synechococcus sp.

Photoaffinity labeling of Synechococcus Photosystem (PS) II preparations with radioactive azido-derivatives of three herbicides and of plastoquinone was carried out to identify herbicide and plastoquinone-binding proteins. [¹⁴C]Azido-atrazine and [¹⁴C]azido-monuron specifically labeled the 28 kDa polypeptide of the PS II reaction center complex, which is sensitive to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). No specific labeling of this polypeptide with azido-atrazine was found in CP2-b (PS II reaction center lacking the 40 kDa subunit) which is insensitive to DCMU. [³H]Azido-dinoseb reacted with the 28 kDa polypeptide and the 47 kDa chlorophyll-carrying protein. The labeling with [³H]azido-plastoquinone resulted in the incorporation of the radioactivity exclusively into the 47 kDa polypeptide. It is concluded that the 28 kDa polypeptide is the herbicide-binding protein of the cyanobacterium and that the 47 kDa polypeptide has a binding site for plastoquinone and for phenol-type herbicides.     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

The far red induced slow component of delayed light from chloroplasts is emitted from Photosystem II

In spinach chloroplasts illuminated with far red light, the relative intensity maximum during the decay of delayed light is emitted at 680–690 nm. This finding supports previous models predicting emission from Photosystem II, and contradicts earlier attributions to Photosystem I.Due to self absorption, the emission spectrum of the relative maximum is shifted to longer wavelengths and displays apparent Photosystem I characteristics in chloroplast samples of higher concentration or in leaves. This may have caused earlier investigators to ascribe the emission to Photosystem I.A differences between the spectral width of the emission spectra of delayed fluorescence and the relative maximum indicates that these two phenomena represent emission from different sub-populations of Photosystem II centers.Abbreviations PS I Photosystem I - PS II Photosystem II - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-Q_A-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, K_i, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the Q_B niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.Abbreviations BSA bovine seroalbumin - Chl chlorophyll - DCBQ 2,6-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2-(N-morpholino)-ethanesulphonic acid - Pheo pheophytin - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - PS Photosystem - RC reaction center - Tricine N-[Tris(hydroxymethyl)-methyl]-glycine     

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA₈₂₀) in the microsecond time range. The extent of ΔA₈₂₀ induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg²⁺ free medium (plus 15 mM K⁺) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg²⁺ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.     

Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts IV. The effects of electron-transport conditions on violaxanthin availability

1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electrontransport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than for cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear r flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity.2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (≤ μM) had no effect on availability, whereas low levels of 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50% decrease at 1 μM). Furthermore, availability in 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone.3. Violaxanthin availability was zero in the dark and increased in the light to a maximum of 67% of the total violaxanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.     

Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts

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 (> 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 in the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

The yield of chlorophyll a fluorescence as a means to test the various deexcitation mechanisms in the antenna system of green plants

With the aid of measurements of the fluorescence yield, the efficiency of the various deexcitation mechanisms of an exciton in the light-harvesting system has been determined. For this purpose, the fluorescence of dark-adapted as well as of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated and preilluminated leaves of Zea mays L. was excited by single ultrashort laser pulses of different energies. The experimental results have served for the fitting of solutions of rate equations, which describe the deexcitation by linear relaxation processes like fluorescence and radiationless transitions, by annihiation of excitons, and by traps both in the ground state and in an excited state. We have obtained the following results: a ratio of antenna chlorophyll molecules to Photosystem II traps of 600:1, an annihilation constant γ = 2·10^?8 cm³·s^?1, a mean trapping time of $\text{t}\text{?}\text{=0.5}\text{ns}$, a trapping probability for traps in the ground state of 2·10^?8 cm³·s^?1, and 6·10^?9 cm³·s^?1 for traps in an excited state.     

Determination and modification of the redox state of the secondary acceptor of Photosystem II in the dark

The redox state of the secondary electron acceptor B of Photosystem II was studied using fluorescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given $\text{B}\text{B}{}^{\mathrm{?}}$ ratio in the dark-adapted samples.We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in Chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts.Benzoquinone treatment modifies this distribution in Chlorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark.The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B^? is generated and stabilized in the dark.