Abstract Photoactivation of the water splitting enzyme was performed with 13 different synthetic manganese complexes and characterized by oxygen evolution yield, thermoluminescence and chlorophyll fluorescence induction kinetics. The efficiency of different compounds in photoactivation correlated with the rate of linear electron transport in the presence of these com pounds. The organic ligands, associated with the manganese ions, do not prevent the photoactivation of the water splitting complex (WOC). Photoactivation with different manganese complexes depended on the number of the Mn-ions in the complex, their valence state and the nature of their donor atoms. The most efficient restorations were achieved by using tetram eric complexes having a dimer+dimer structure, complexes containing Mn(II) ions, and having 4-6 oxygen and 0-2 nitrogen atoms as donor atoms. Further, the effectiveness of photoactivation depended largely on the structure of the complexes. Our data support the notion that WOC in intact thylakoids requires the cooperation and well determined arrangement of all four manganese ions, and argue against the hypothesis that two manganese ions are sufficient for water splitting. Photoactivation by some complexes led to anomalous flashoxygen patterns, which are explained by a modified/perturbed water splitting complex.

The effects of potassium-(picrate)-(18-crown-6) on the electron transport of photosystem II was investigated in isolated pea thylakoids. Low concentrations of the compound inhibited the fast decay of fluorescence yield associated with electron transfer between the primary (Q(A)) and secondary (Q(B)) quinone electron acceptor and increased the intermediary level of fluorescence to the F-max level. The decay half-time of fluorescence yield measured in the presence of DCMU (S(2)Q(A)(-) charge recombination) decreased from about 1.8 s to about 0.3 s in thylakoids treated with potassium-(picrate)-(18-crown-6). While the inhibition of electron transport by DCMU gave rise to the appearance of a thermoluminescence band at about +10 degrees C (S(2)Q(A)(-)charge recombination) addition of potassium-(picrate)-(18-crown-6) resulted in a thermoluminescence band at about -10 degrees C. Increasing concentrations of potassium-(picrate)-(18-crown-6) diminished the fluorescence yield and the -10 degrees C TL band and abolished the Signal IIs and Signal IIf EPR signals of the tyrosine-D and tyrosine-Z electron donors, respectively. The phenolic-type inhibitor, potassium picrate had the same effect on thermoluminescence and on the tyrosine EPR signals. It is concluded that potassium-(picrate)-(18-crown-6) is a phenolic type inhibitor owing to its picrate constituent. At low concentrations picrate and potassium-(picrate)-18-crown) not only block the electron transport between Q(A), and Q(B) but they probably decrease the midpoint redox potential of Q(A), as well. At high concentrations they also inhibit the light-induced oxidation of the tyrosine-D and tyrosine-Z donors.

Cultivation of the green algae, Chlamydobotrys stellata and Chlamydomonas reinhardtii in CO2-depleted medium at moderate light intensity resulted in an inhibition of Photosystem II electron transport from water to diaminodurene (acceptor at the plastoquinone pool) but only slightly affected the electron flow from water to 2,6-dichlorobenzoquinone (acceptor at the primary quinone electron acceptor, Q(A)). The intermediary fluorescence level, F-i was raised to the maximum level of fluorescence, F-m. The initial level of fluorescence, F-0 was considerably enhanced. The observations suggest that illumination of CO2-depleted green alga cells results in the development of an inhibition of electron transport between the primary (Q(A)) and secondary (Q(B)) quinone electron acceptor. Prolonged photoinhibitory illumination of the CO2-depleted cells led to an irreversible loss of variable fluorescence and electron transport. The photoinactivation developed slower in the CO2-depleted than in the CO2-containing cells. Consequently, in the bicarbonate-depleted redox state the photosystem II reaction center is less susceptible to photoinhibition than in the bicarbonate-containing state.

Short-term illumination of the green algae, Chlamydobotrys stellata and Chlamydomonas reinhardtii in CO2-depleted cultivation medium under low photon flux density (50 and 150 μmol m−2 s−1, respectively) resulted in an inhibition of Photosystem II electron transport from water to diaminodurene, but only slightly affected the electron flow from water to 2,6-dichlorobenzoquinone. The intermediary fluorescence level, Fi was raised to the maximum level of fluorescence, Fm. The initial level of fluorescence, Fo was considerably enhanced. The development of the Fo rise was facilitated by low pH, but inhibited in the presence of an acceptor, dichlorobenzoquinone, or by chemical cross-linking of proteins with glutaraldehyde. The uninhibited electron transport and the original Fo level were restored by readdition of CO2 or by dark adaptation of algae. The observations suggest that in green alga cells CO2-depletion in the light results in a reversible inhibition of steady-state electron flow between the primary (QA) and secondary quinone electron acceptor (QB). Following the inhibition of electron transport a long-lived but reversible state of singly-reduced and probably protonated QA is formed which manifests itself as an apparent Fo rise. Prolonged photoinhibitory illumination of the CO2-depleted green alga cells resulted in an irreversible loss of variable fluorescence and electron transport. The photoinactivation developed more slowly in the CO2-depleted than in the CO2-containing cells. It is concluded that in the bicarbonate-depleted redox state, which is accompanied with an enhanced Fo level of fluorescence, the Photosystem II reaction center is less susceptible to photoinhibition than in the bicarbonate-containing state.

The photosynthetic characteristics of the previously obtained psbB, psbC, and psbE/F mutants of the cyanobacterium Synechocystis sp. PCC 6803 incapable of photoautotrophic growth were investigated. Based on their variable fluorescence, EPR spectra, thermoluminescence, and photosynthetic oxygen evolution, all the mutants, except the psbB mutant NF2, completely lack Photosystem II activity but retain Photosystem I activity. The psbB mutant NF2 cells characterized by substitution of arginine for tryptophan in position 167 of protein CP47, has Photosystem II activity of 30% or less than the wild-type level. The impaired capacity for autotrophic growth in mutant NF2 is presumably due to enhanced photoinhibition which, in turn, results from the incorporation of the mutant protein CP47 into the Photosystem II complex.

The origin of the low temperature thermoluminescence (TL) bands appearing at about -55-degrees-C and -25-degrees-C were investigated in chloroplasts and in subchloroplast particles enriched with photosystem 2 (PS 2). In the thermoluminescence glow curves five bands were distinguished: Z (-160-degrees-C), Z (-75-degrees-C), TL-55, TL-25 and TL-10. By using various artificial electron donors and acceptors as well as Inhibitors of electron transport a correlation was found between the intensity of the TL-55, TL-25 and TL-10 bands and the photochemical activity of PS 2 particias. Addition of the histidine modifier, diethylpyrocarbonate (DEPC) resulted in the disappearance of the band peaking at around -25-degrees-C (TL-25). The tyrosine modifier, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) abolished the TL band at -55-degrees-C (TL-55). It is concluded that the TL-55 and TL-25 bands are probably associated with radiative charge recombination of the [Tyr+ Pheo-] and [His+.Q(A)- redox couples, respectively. On the basis of the data a scheme, including a redox active histidine component, is suggested for the composition of the PS 2 reaction center.

Following illumination at 200 K, the charge recombination reactions and the origin of the thermoluminescence (TL) bands appearing at about 0 degree C (Q band) and +50 degrees C (C band) in the glow curve were investigated by comparative TL and EPR measurements in DCMU-treated photosystem II particles. Decay half-time measurements carried out at -25 degrees C and +25 degrees C, respectively, suggest that the S2 state (multi-line signal) undergoes charge recombination with the g = 1.9 form of the semiquinone-iron complex, QA-.Fe2+, resulting in the appearance of the Q band, and that the g = 1.82 form of QA-.Fe2+ back-reacts with the oxidized tyrosine, YD+ (Signal IIs), accounting for the generation of the C band.

We investigated the influence of CO2/HCO3−-depletion and of the presence of acetate and formate on the in vivo photosynthetic electron transport in the two green algae Chlamydobotrys stellata and Chlamydomonas reinhardtii by means of thermoluminescence technique and mathematical glow curve analysis. The main effects of the removal of CO2 from the algal cultures was: (1) A shift of the glow curve peak position to lower temperatures resulting from a decrease of the B band and an increase of the Q band. (2) Treatment of CO2-deficient Chl. stellata with DCMU yielded two thermoluminescence bands in the Q band region peaking at around +12°C and +5°C; in case of Chl. reinhardtii DCMU treatment induced only one band with an emission maximum at +5°C. The presence of acetate or formate in CO2-depleted algal cultures lowered the intensities of all of the individual TL bands but that of a HT band (TL+37). The effects of CO2-depletion and of the presence of anions were fully reversible.

We investigated the influence of CO/HCO-depletion and of the presence of acetate and formate on the in vivo photosynthetic electron transport in the two green algae and by means of thermoluminescence technique and mathematical glow curve analysis. The main effects of the removal of CO from the algal cultures was: (1) A shift of the glow curve peak position to lower temperatures resulting from a decrease of the B band and an increase of the Q band. (2) Treatment of CO-deficient with DCMU yielded two thermoluminescence bands in the Q band region peaking at around +12°C and +5°C; in case of DCMU treatment induced only one band with an emission maximum at +5°C. The presence of acetate or formate in CO-depleted algal cultures lowered the intensities of all of the individual TL bands but that of a HT band (TL+37). The effects of CO-depletion and of the presence of anions were fully reversible.

The changes in the light-harvesting antenna size of photosystem I were investigated in the green alga Chlamydobotrys stellata during transition from autotrophic to photoheterotrophic nutrition by measuring the light-saturation behavior of hydrogen evolution following single turnover flashes. It was found that during autotrophic-to-photoheterotrophic transition the antenna size of photosystem I increased from 180 to 250 chlorophyll. The chlorophyll (a + b)/P700 ratio decreased from 800 to 550. The electron transport of photosystem I measured from reduced 2,6-dichloro-phenolindophenol to methylviologen was accelerated 1.4 times. In the 77K fluorescence spectra, the photosystem II fluorescence yield was considerably lowered relative to the photosystem I fluorescence yield. It is suggested that the increased light-harvesting capacity and redistribution of absorbed excitation energy in favor of photosystem I is a response of photoheterotrophic algae to meet the ATP demand for acetate metabolism by efficient photosystem I cyclic electron transport when the noncyclic photophosphorylation is inhibited by CO2 deficiency.

We investigated the effects of various chemical agents, modifying the His. Tyr residues in the isolated D1-D2-Cyt b559 complex from pea, on thermoluminescence (TL). The complex was shown to have four TL bands Z (-160-degrees-C), Z(V) (-75-degrees-C), TL-55 (-55-degrees-C) and TL+30 (+30-degrees-C). In the presence of various artificial acceptors and donors. the Z(V) and TL-55 bands were found to correlate with the photochemical activity of the D1-D2-Cyt b559 complexes. TL-55 did not change after the modification of His residues with diethylpyrocarbonate (DEPC). In the presence of 7-chloro-nitrobenz-2-oxa-1.3-diazole (NBD) which modified the Tyr residue, the TL-55 band was completely abolished. It is concluded that the TL-55 band probably arises from Z+ znd Pheo- (pheophytin) and electron component Z - the secondary donor for P680+ - is really a Tyr residue.

In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) abolishes the TL band appearing around -55-degrees-C (TL-55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around -30-degrees-C (TL-30). NBD treatment also abolishes the EPR signal II(fast) of oxidized tyrosine donor, Y(z), and inhibits the electron transport from diphenylcarbazide to 2,6-dichlorophenol-indophenol. It is concluded that the TL-55 and TL-30 bands can be assigned to oxidized tyrosine (Y(z)+) and histidine (His+) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680+.

During photoinhibitory light treatment of the green alga, Chlamydobotrys stellata and Pisum sativum leaves the amplitude of the B thermoluminescence band (S2OB - charge recombination) decreased faster than that of the Q band (S2QA - charge recombination). Consistently, in the alga cells the electron transport rate from water to oxidized diaminodurene (electron acceptor after Q B) was also diminished faster than that measured from water to phenyl- p-benzoquinone (electron acceptor after QA). These observations suggest that in intact photosynthetizing organisms at high light intensities on the acceptor side of photosystem II photoinhibition is initiated at the binding site of the secondary quinone acceptor, QB.

Thermoluminescence and delayed luminescence investigations of the autotrophically and photoheterotrophically cultivated green alga, Chlamydobotrys stellata, demonstrated that both the thermoluminescence and delayed luminescence yields are much lower in the photoheterotophic algae than in the autotrophic ones due to an efficient luminescence quenching of unknown mechanism. The relative contributions of the so called Q recombinations) thermoluminescence bands to the glow curves as well as the Q(A)(S2Q(A)-charge recombination) and Q(B) (S2Q(B)- and S3Q(B)-charge recombinations) delayed luminescence components to the delayed luminescence decay of autotrophically and photoheterotrophically cultivated Chl. stellata were compared using a computer assisted curve resolution method.It was found that, while in the autotrophic cells the area of the B band was considerably larger than of the Q band, in photoheterotrophic cells the Q band was more effectively charged than the B band.In the delayed luminescence decay curves measured in the seconds to minutes time region the amplitude of the Q(A) component relative to that of the Q(B) component was larger in the photoheterotrophic cells than in the autotrophic ones.These observations demonstrate that, after light-induced charge separation in the photosystem II reaction centers of autotrophic cells, electrons are "quasipermanently" stored mainly in the secondary quinone acceptor pool, Q(B) but in the nonquenched photosystem II reaction centers of photoheterotrophic cells the main reservoir of electrons is the primary quinone acceptor, Q(A). This behaviour indicates an inhibition of electron transport in the photoheterotrophic alga at the level of the secondary quinone acceptor, Q(B).

The obligate phototrophic green alga Chlamydobotrys stellata does not evolve oxygen when grown in CO2-free atmosphere on acetate. With the application of the lipophilic acceptor 2,6-dichloro-p-benzoquinone it was investigated whether this phenomenon is caused by the inactivation of the water-splitting system or by an inhibition of the electron transport chain. It was found that in the presence of DCQ, the photoheterotrophic alga exhibited a normal period-4 flash oxygen pattern, but the steady state yield was only 25% of that measured in the autotrophic cells. After DCQ addition, the initial distribution of S-states and the values of the transition probabilities proved to be the same in the autotrophic and photoheterotrophic algae. These results indicate that photoheterotrophic growth conditions inhibit the electron transport of Chl. stellata behind the acceptor site of DCQ, but the water-splitting system remains active with a reduced oxygen evolving capacity.

In Synechococcus sp. cells AS-1 cyanophage infection gradually inhibits the photosystem II mediated photosynthetic electron flow whereas the activity of photosystem I is apparently unaffected by the cyanophage infection. Transient fluorescence induction and flash-induced delayed luminescence decay studies revealed that the inhibition may occur at the level of the secondary acceptor, QB of photosystem II. In addition, the breakdown of D1-protein is inhibited, comparable to DCMU-induced protection of D1-protein turnover, in AS-1-infected cells.

The toxicity of heavy metals on photosystem 2 photochemistry, was investigated by monitoring Hill activity, fluorescence, and thermoluminescence properties of photosystem 2 (PS 2) in pea (Pisum sativum L. cv. Bombay) chloroplasts. In Co2+‐, Ni2+‐ or Zn2+‐treated chloroplasts 2,6‐dichlorophenolindophenol‐Hill activity was markedly inhibited. Addition of hydroxylamine which donates electrons close to PS 2 reaction center did not restore the PS 2 activity. Co2+‐, Ni2+ or Zn2+ also inhibited PS 2 activity supported by hydroxylamine in tris (hydroxymethyl)aminomethane (Tris)‐inactivated chloroplasts. These observations were confirmed by fluorescence transient measurements. This implies that the metal ions inhibit either the reaction center or the components of PS 2 acceptor side. Flash‐induced thermoluminescence studies revealed that the S2Q−A charge recombination was insensitive to metal ion addition. The S2Q−B charge recombination, however, was inhibited with increase in the level of Co2+, Ni2+ or Zn2+. The observed sensitivity of S2−B charge recombination in comparison to the stability of S2Q−A recombination suggests that the metal ions inhibit at the level of secondary quinone electron acceptor. QB. We suggest that Co2+, Ni2+ or Zn2+ do not block the electron flow between the primary and secondary quinone electron acceptor, but possibly, directly modify QB site, leading to the loss of PS 2 activity.

The nature of Cu(2+) inhibition of photosystem II (PSII) photochemistry in pea (Pisum sativum L.) thylakoids was investigated monitoring Hill activity and light emission properties of photosystem II. In Cu(2+)-inhibited thylakoids, diphenyl carbazide addition does not relieve the loss of Hill activity. The maximum yield of fluorescence induction restored by hydroxylamine in Tris-inactivated thylakoids is markedly reduced by Cu(2+). This suggests that Cu(2+) does not act on the donor side of PSII but on the reaction center of PSII or on components beyond. Thermoluminescence and delayed luminescence studies show that charge recombination between the positively charged intermediate in water oxidation cycle (S(2)) and negatively charged primary quinone acceptor of pSII (Q(A) (-)) is largely unaffected by Cu(2+). The S(2)Q(B) (-) charge recombination, however, is drastically inhibited which parallels the loss of Hill activity. This indicates that Cu(2+) inhibits photosystem II photochemistry primarily affecting the function of the secondary quinone electron acceptor, Q(B). We suggest that Cu(2+) does not block electron flow between the primary and secondary quinone acceptor but modifies the Q(B) site in such a way that it becomes unsuitable for further photosystem II photochemistry.

Recently considerable progress has been achieved in the elucidation of the origin of thermoluminescence in chloroplasts. The assignment of 2 of the thermoluminescence bands, peaking at around +5°C (Q or D band) and +30°C (B band), to the recombination of charges, originating from the oxidzed species of the oxygen evolving complex (the so‐called S states) and to the reduced primary and secondary quinone acceptors Q A and Q B , respectively, has aided in the investigation of reactions involving both the electron donor and acceptor sides of photosystem II. In this paper we review recent thermoluminescence results concerning the deactivation of S states, temperature and pH dependence of S state transition, and the activity of the water oxidizing system after removal of Cl − , manganese or the 33 kDa protein. Reports on the use of thermoluminescence in investigations on the sites of action of herbicides and redox changes of Q B conferred by herbicide resistance are also discussed. The effect of pH, bicarbonate, and Acceleration of Deactivation Reaction of enzyme “Y” (ADRY) reagents on the photosystem II reactions are presented in the light of thermoluminescence observations. Further possible applications of thermoluminescence promising better understanding of the photosynthetic processes are suggested.

Abstract The effect of photoinhibition on the primary (QA) and secondary (QB) quinone acceptors of photosystem I I was investigated in isolated spinach thylakoids by the methods of thermoluminescence and delayed luminescence. The amplitudes of the Q (at about 2 °C) and B (at about 30 °C) thermoluminescence bands which are associated with the recombination of the S2QA - and S2QB charge pairs, respectively, exhibited parallel decay courses during photoinhibitory treatment. Similarly, the amplitudes of the flash-induced delayed luminescence components ascribed to the recombination of S20A and S2OB charge pairs and having half life-times of about 3 s and 30 s, respectively, declined in parallel with the amplitudes of the corresponding Q and B thermoluminescence bands. The course of inhibition of thermoluminescence and delayed luminescence intensity was parallel with that of the rate of oxygen evolution. The peak positions of the B and Q thermoluminescence bands as well as the half life-times of the corresponding delayed luminescence components were not affected by photoinhibition. These results indicate that in isolated thylakoids neither the amount nor the stability of the reduced OB acceptor is preferentially decreased by photoinhibition. We conclude that either the primary target of photodamage is located before the O b binding site in the reaction center of photosystem II or QA and OB undergo simultaneous damage.