Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level F_j are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_i initial fluorescence level immediately after shutter open - F_pt intermediate plateau fluorescence level - F_m maximum fluorescence level emitted when all PS II centers are closed - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Light-dependent modification of Photosystem II in spinach leaves

In dark-adapted spinach leaves approximately one third of the Photosystem II (PS II) reaction centers are impaired in their ability to transfer electrons to Photosystem I. Although these inactive PS II centers are capable of reducing the primary quinone acceptor, Q_A, oxidation of Q_A ^– occurs approximately 1000 times more slowly than at active centers. Previous studies based on dark-adapted leaves show that minimal energy transfer occurs from inactive centers to active centers, indicating that the quantum yield of photosynthesis could be significantly impaired by the presence of inactive centers. The objective of the work described here was to determine the performance of inactive PS II centers in light-adapted leaves. Measurements of PS II activity within leaves did not indicate any increase in the concentration of active PS II centers during light treatments between 10 s and 5 min, showing that inactive centers are not converted to active centers during light treatment. Light-induced modification of inactive PS II centers did occur, however, such that 75% of these centers were unable to sustain stable charge separation. In addition, the maximum yield of chlorophyll fluorescence associated with inactive PS II centers decreased substantially, despite the lack of any overall quenching of the maximum fluorescence yield. The effect of light treatment on inactive centers was reversed in the dark within 10–20 mins. These results indicate that illumination changes inactive PS II centers into a form that quenches fluorescence, but does not allow stable charge separation across the photosynthetic membrane. One possibility is that inactive centers are converted into centers that quench fluorescence by formation of a radical, such as reduced pheophytin or oxidized P680. Alternatively, it is possible that inactive PS II centers are modified such that absorbed excitation energy is dissipated thermally, through electron cycling at the reaction center.Abbreviations A518 absorbance change at 518 nm, reflecting the formation of an electric field across the thylakoid membrane - A_FL1 amplitude of the fast (<100 ms) phase of A518 induced by the first of two saturating, single-turnover flashes spaced 30 ms apart - A_FL2 amplitude of the fast (<100 ms) phase of A518 induced by the second of two saturating, single-turnover flashes spaced 50 ms apart - DCBQ 2,6-dichloro-p-benzoquinone - Fo yield of chlorophyll fluorescence when Q_A is fully oxidized - Fm yield of chlorophyll fluorescence when Q_A is fully reduced - Fx yield of chlorophyll fluorescence when Q_A is fully reduced at inactive PS II centers, but fully oxidized at active PS II centers - Pheo pheophytin - P680 the primary donor of Photosystem II - PPFD photosynthetic photon flux density - Q_A Primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Properties of inactive Photosystem II centers

A fraction (usually in the range of 10–25%) of PS II centers is unable to transfer electrons from the primary quinone acceptor Q_A to the secondary acceptor Q_B. These centers are inactive with respect to O₂ evolution since their reopening after photochemical charge separation to the S₂O_A ^- state involves predominantly a back reaction to S₁Q_A in the few seconds time range (slower phases are also occurring). Several properties of these centers are analyzed by fluorescence and absorption change experiments. The initial rise phase F_o-F_pl of fluorescence induction under weak illumination reflects both the closure of inactive centers and the modulation of the fluorescence yield by the S-states of the oxygen-evolving system: We estimate typical relative amplitudes of these contributions as, respectively, 65 and 35% of the F_o-F_pl amplitude. The half-rise time of this phase is significantly shorter than for the fluorescence induction in the presence of DCMU (in which all centers are involved). This finding is shown to be consistent with inactive centers sharing the same light-harvesting antenna as normal centers, a view which is also supported by comparing the dependence of the fluorescence yield on the amount of closed active or inactive centers estimated through absorption changes. It is argued that the exponential kinetics of the F_o-F_pl phase does not indicate absence of excitation energy transfer between the antennas of inactive and active centers. We show that the acceptor dichlorobenzoquinone does not restore electron transfer in inactive centers, in disagreement with previous suggestions. We confirm, however, the enhancement of steady-state electron flow caused by this quinone and suggest that it acts by relieving a blocking step involved in the reoxidation of a fraction of the plastoquinone pool. Part of the discrepancies between the present results and those from previous literature may arise from the confusion of inactive centers characterized on a single turnover basis and PS II centers that become blocked under steady-state conditions because of deficient reoxidation of their secondary acceptors.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - PS photosystem     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

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     

Evidence for slow turnover in a fraction of Photosystem II complexes in thylakoid membranes

We used two different techniques to measure the recovery time of Photosystem II following the transfer of a single electron from P-680 to Q_A in thylakoid membranes isolated from spinach. Electron transfer in Photosystem II reaction centers was probed first by spectroscopic measurements of the electrochromic shift at 518 nm due to charge separation within the reaction centers. Using two short actinic flashes separated by a variable time interval we determined the time required after the first flash for the electrochromic shift at 518 nm to recover to the full extent on the second flash. In the second technique the redox state of Q_A at variable times after a saturating flash was monitored by measurement of the fluorescence induction in the absence of an inhibitor and in the presence of ferricyanide. The objective was to determine the time required after the actinic flash for the fluorescence induction to recover to the value observed after a 60 s dark period. Measurements were done under conditions in which (1) the electron donor for Photosystem II was water and the acceptor was the endogenous plastoquinone pool, and (2) Q₄₀₀, the Fe²⁺ near Q_A, remained reduced and therefore was not a participant in the flash-induced electron-transfer reactions. The electrochromic shift at 518 nm and the fluorescence induction revealed a prominent biphasic recovery time for Photosystem II reaction centers. The majority of the Photosystem II reaction centers recovered in less than 50 ms. However, approx. one-third of the Photosystem II reaction centers required a half-time of 2–3 s to recover. Our interpretation of these data is that Photosystem II reaction centers consist of at least two distinct populations. One population, typically 68% of the total amount of Photosystem II as determined by the electrochromic shift, has a steady-state turnover rate for the electron-transfer reaction from water to the plastoquinone pool of approx. 250 e⁻ / s, sufficiently rapid to account for measured rates of steady-state electron transport. The other population, typically 32%, has a turnover rate of approx. 0.2 e⁻ / s. Since this turnover rate is over 1000-times slower than normally active Photosystem II complexes, we conclude that the slowly turning over Photosystem II complexes are inconsequential in contributing to energy transduction. The slowly turning over Photosystem II complexes are able to transfer an electron from P-680 to Q_A rapidly, but the reoxidation of Q⁻_A is slow (t1/2 = 2 s). The fluorescence induction measurements lead us to conclude that there is significant overlap between the slowly turning over fraction of Photosystem II complexes and PS II_β reaction centers. One corollary of this conclusion is that electron transfer from P-680 to Q_A in PS II_β reaction centers results in charge separation across the membrane and gives rise to an electrochromic shift.     

Interaction of exogenous quinones with membranes of higher plant chloroplasts: modulation of quinone capacities as photochemical and non-photochemical quenchers of energy in Photosystem II during light-dark transitions

Light modulation of the ability of three artificial quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duroquinone), to quench chlorophyll (Chl) fluorescence photochemically or non-photochemically was studied to simulate the functions of endogenous plastoquinones during the thermal phase of fast Chl fluorescence induction kinetics. DBMIB was found to suppress by severalfold the basal level of Chl fluorescence (F_o) and to markedly retard the light-induced rise of variable fluorescence (F_v). After irradiation with actinic light, Chl fluorescence rapidly dropped down to the level corresponding to F_o level in untreated thylakoids and then slowly declined to the initial level. DBMIB was found to be an efficient photochemical quencher of energy in Photosystem II (PSII) in the dark, but not after prolonged irradiation. Those events were owing to DBMIB reduction under light and its oxidation in the dark. At high concentrations, DCBQ exhibited quenching behaviours similar to those of DBMIB. In contrast, duroquinone demonstrated the ability to quench F_v at low concentration, while F_o was declined only at high concentrations of this artificial quinone. Unlike for DBMIB and DCBQ, quenched F_o level was attained rapidly after actinic light had been turned off in the presence of high duroquinone concentrations. That finding evidenced that the capacity of duroquinone to non-photochemically quench excitation energy in PSII was maintained during irradiation, which is likely owing to the rapid electron transfer from duroquinol to Photosystem I (PSI). It was suggested that DBMIB and DCBQ at high concentration, on the one hand, and duroquinone, on the other hand, mimic the properties of plastoquinones as photochemical and non-photochemical quenchers of energy in PSII under different conditions. The first model corresponds to the conditions under which the plastoquinone pool can be largely reduced (weak electron release from PSII to PSI compared to PSII-driven electron flow from water under strong light and weak PSI photochemical capacity because of inactive electron transport on its reducing side), while the second one mimics the behaviour of the plastoquinone pool when it cannot be filled up with electrons (weak or moderate light and high photochemical competence of PSI).     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Inhibition of photosynthetic oxygen evolution by protonophoric uncouplers

The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K₃[Fe(CN)₆] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I₅₀ values were approx. 1–2, 4–6 and 0.04–0.10 M, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Q_p) which is also the electron donor for K₃ [Fe(CN)₆] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y - ANT2p- 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene - CCCP- carbonyl cyanide m-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl 6-isopropyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT- 2-iodo-6-isopropyl-3-methyl 2,4,4-trinitrodiphenyl ether - DPC- 1,5-diphenylcarbazide - DPIP- 2,6-dichlorophenolindophenol - FCCP- carbonyl cyanide p-trifuoromethoxyphenylhydrazone - FeCy- potassium ferricyanide - HQNO- 2-n-heptyl-4-hydroxyquinoline N-oxide - (MN)₄- the tetranuclear Mn cluster of water oxidizing complex - P680- photoactive Chl of the reaction center of Photosystem II - PCP- 2,3,4,5,6-pentachlorophenol - PS- photosystem - Q_A and Q_B- primary and secondary plastoquinones of PS II - Q_C and Q_Z- plastoquinone binding sites in the cytochrome blf complex - Q_p- membrane pool of plastoquinone - SiMo- sodium silicomolybdate - TMPD- N,N,N-tetramethyl-p-phenylenediamine - TTFB- 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - WOC- water oxidixing complex - Y_Z- tyrosine-161 of the Photosystem II D1 polypeptide     

High misses after odd flashes in oxygen evolution in thoroughly dark-adapted thylakoids from pea and Chenopodium album

In Photosystem II (PS II), water is oxidized to molecular oxygen and plastoquinone is reduced to plastoquinol. The oxidation of water requires the accumulation of four oxidizing equivalents, through the so-called S-states of the oxygen evolving complex; the production of plastoquinol requires the accumulation of two reducing equivalents on a bound plastoquinone, Q_B. It has been generally believed that during the flash-induced transition of each of the S-states (S_n S_n+1, where n=0, 1, 2 and 3), a certain small but equal fraction of the PS II reaction centers are unable to function and, thus, miss being turned over. We used thoroughly dark-adapted thylakoids from peas (Pisum sativum) and Chenopodium album (susceptible and resistant to atrazine) starting with 100% of the oxygen evolving complex in the S₁ state. Thylakoids were illuminated with saturating flashes, providing a double hit parameter of about 0.07. Our experimental data on flashnumber dependent oscillations in the amount of oxygen per flash fit very well with a binary pattern of misses: 0, 0.2, 0, 0.4 during S₀ S₁, S₁ S₂, S₂ S₃ and S₃ S₀ transitions. Addition of 2 mM ferricyanide appears to shift this pattern by one flash. These results are consistent with the bicycle model recently proposed by V. P. Shinkarev and C. A. Wraight (Oxygen evolution in photosynthesis: From unicycle to bicycle, 1993, Proc Natl Acad Sci USA 90: 1834–1838), where misses are due to the presence of P⁺ or Q_A ^- among the various equilibrium states of PS II centers.Abbreviations miss parameter - double hit parameter - PS II Photosystem II - Q_A primary one-electron acceptor of PS II, a plastoquinone molecule - Q_B secondary plastoquinone two-electron acceptor of PS II - S-states (S_n, where n=0, 1, 2, 3 or 4) redox states of the oxygen evolving complex     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

Increases in peroxide formation by the Photosystem II oxygen evolving reactions upon removal of the extrinsic 16, 22 and 33 kDa proteins are reversed by CaCl2 addition

This communication introduces a new spectrophotometric assay for the detection of peroxide generated by Photosystem II (PS II) under steady state illumination in the presence of an electron acceptor. The assay is based on the formation of an indamine dye in a horseradish peroxidase coupled reaction between 3-(dimethylamino)benzoic acid and 3-methyl-2-benzothiazolinone hydrazone. Using this assay, we found that as the O₂ evolution activity of PS II-enriched membrane fragments is decreased by treatments which cause the dissociation of the 33 and/or 23 and 16 kDa extrinsic proteins (i.e., CaCl₂-washing, NaCl-washing, lauroylcholine-treatment and ethylene glycol-treatment), light-induced peroxide formation increases. Both the losses of O₂ evolution and increases in peroxide formation seen under these conditions are reversed by CaCl₂ addition, indicating that the two activities originate from the water-splitting site. However, the increased rates of peroxide formation do not quantitatively match the losses in O₂ evolution activity. We suggest that a rapid consumption of the peroxide takes place via a catalase/peroxidase activity at the water-splitting site which competes with both the O₂ evolution and peroxide formation reactions. The observed peroxide formation is interpreted as arising from enhanced water accessibility to the catalytic site upon perturbation of the extrinsic proteins which then leads to alternate water oxidation side reactions.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichloro)-1,1-dimethylurea - DCPIP 1,6-dichlorophenolindophenol - DMAB 3-(dimethylamino)benzoic acid - DMBQ 2,6-dimethyl-p-benzoquinone - DPC diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazinesulfonic acid - HMD HRP, MBTH, DMAB - HRP horseradish peroxidase - LCC lauroylcholine chloride - MBTH 3-methyl-2-benzothiazolinone hydrazone - MES 4-morpholinoethanesulfonic acid     

Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S₂Q_B ^- (B band) and S₂Q_A ^– (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of Q_A ^- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block Q_A to Q_B electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced Q_B. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the Q_B/DCMU binding site. The preferential loss of dark stable Q_B ^- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.Abbreviations Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A first quinone electron acceptor of PS II - Q_B second quinone electron acceptor of PS II - Tyr-D accessory electron donor of PS II - S₀-S₄ charge storage states of the water-oxidizing complex     

Detection of oxygen-evolving Photosystem II centers inactive in plastoquinone reduction

Quite different estimates of the number of Photosystem II centers present in thylakoid membranes are obtained depending on the technique used in making the determination. By using brief saturating light flashes and measuring the electron transport per flash, we have obtained two values for the number of functional centers. When the electrons produced reduce the intersystem plastoquinone pool, there are about 1.7 mmol of active Photosystem II centers per mol chlorophyll, whereas there are at least 3 mmol of active centers per mol chlorophyll when certain halogenated benzoquinones are being reduced. There are also at least 3 mmol of terbutryn binding sites per mol of chlorophyll when this tightly binding herbicide is employed as a specific inhibitor of Photosystem II. Thus only about 60% of the membrane's total complement of Photosystem II centers are able to transfer electrons to Photosystem I at appreciable rates. Many functional assays requiring significant rates of turnover sample only this more active pool, whereas herbicide-binding studies and measurements of changes in the Photosystem II electron donor Z and electron acceptor Q_A performed by other investigators reveal, in addition, a large population of Photosystem II reaction centers that normally have negligible turnover numbers. However, these normally inactive centers readily transfer electrons to the halogenated benzoquinones and are then counted among the active centers. Therefore, it can be concluded that all of herbicide-binding sites represent centers with operative water-oxidizing reactions. It can also be concluded that there are few, if any, centers capable of binding more than a single herbicide molecule.     

Characterization of the photosystem II centers inactive in plastoquinone reduction by fluorescence induction

In order to characterize the photosystem II (PS II) centers which are inactive in plastoquinone reduction, the initial variable fluorescence rise from the non-variable fluorescence level F_o to an intermediate plateau level F_i has been studied. We find that the initial fluorescence rise is a monophasic exponential function of time. Its rate constant is similar to the initial rate of the fastest phase (-phase) of the fluorescence induction curve from DCMU-poisoned chloroplasts. In addition, the initial fluorescence rise and the -phase have the following common properties: their rate constants vary linearly with excitation light intensity and their fluorescence yields are lowered by removal of Mg⁺⁺ from the suspension medium. We suggest that the inactive PS II centers, which give rise to the fluorescence rise from F_o to F_i, belong to the -type PS II centers. However, since these inactive centers do not display sigmoidicity in fluorescence, they thus do not allow energy transfer between PS II units like PS II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DMQ 2,5-dimethyl-p-benzoquinone - F_o initial non-variable fluorescence yield - F_m maximum fluorescence yield - F_i intermediate fluorescence yield - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II