A theoretical study on effect of the initial redox state of cytochrome b559 on maximal chlorophyll fluorescence level (F(M)): implications for photoinhibition of photosystem II

In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first quinone electron acceptor (Q(A)) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559 accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal fluorescence, F(M), increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the reactions occurring in the model control the F(M) level and how this control depends on the initial redox state of cyt b559. The simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also experimental data, which might be used to validate our theory, are presented and discussed.     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q_A) of photosystem (PS) II [C. Vernotte, A.L. Etienne, J.-M. Briantais, Quenching of the system II chlorophyll fluorescence by the plastoquinone pool, Biochim. Biophys. Acta 545 (1979) 519-527]. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 μs to 5 s). The about 20% lowering of the maximum fluorescence yield F_M, observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH₂ by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH₂ starts getting reoxidised by PS I activity. NAD(P)H-dependent restoration of F_M was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl₂ that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F_M. Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F₀ allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F₀ level (Q₀) and to compare it with the fractional quenching at the F_M level (Q_M). The experimentally determined Q₀/Q_M ratios were found to be equal to the corresponding F₀/F_M ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Damage and protection of the photosynthetic apparatus from UV-B radiation. I. Effect of ascorbate

In this work, the effect of the exogenously added ascorbate (Asc) against the UV-B inhibition of the photosystem II (PSII) functions in isolated pea thylakoid membranes was studied. The results reveal that Asc decreases the UV-B induced damage of the donor and the acceptor side of PSII during short treatment up to 60 min. The exogenous Asc exhibits a different UV-protective effect on PSII centers in grana and stroma lamellae, as the effect is more pronounced on the PSIIβ centers in comparison to PSIIα centers. Data also suggest that one of the possible protective roles of the Asc in photosynthetic membranes is the modification of the oxygen-evolving complex by influence on the initial S₀–S₁ state distribution in the dark.     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q_B. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O₂ evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q_A and Q_B were found in the crystallized PSII. We propose that the extra quinones are located in the Q_B cavity and serve as a PSII intrinsic pool of electron acceptors.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680 and the quinone acceptors QA to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q_A and Q_B, and the S₂ state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q_A⁻ reoxidation.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.     

Molecular interference of Cd with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd²⁺ is known to exchange, with high affinity in a slow reaction, for the Ca²⁺ cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd²⁺ binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd²⁺-binding to those sites, we have studied how Cd²⁺ affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd²⁺ with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd²⁺ were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y_Z to P₆₈₀⁺ was inhibited; (ii) Q_A⁻ to Q_B electron transfer was slowed down; (iii) the S₂ state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q_A⁻Fe²⁺ was modified but its amplitude was not sensitive to the presence of Cd²⁺. In addition, the presence of both Ca²⁺ and DCMU abolished Cd²⁺-induced effects partially and in different sites. The number of sites for Cd²⁺ binding and the possible nature of these sites are discussed.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or Q_B). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680⁺, the yield of its reduction by Q_B or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.     

Disentangling two non-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77 K

Diatoms, which are primary producers in the oceans, can rapidly switch on/off efficient photoprotection to respond to fast light-intensity changes in moving waters. The corresponding thermal dissipation of excess-absorbed-light energy can be observed as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Fluorescence-induction measurements on Cyclotella meneghiniana diatoms show two NPQ processes: qE₁ relaxes rapidly in the dark while qE₂ remains present upon switching to darkness and is related to the presence of the xanthophyll-cycle pigment diatoxanthin (Dtx). We performed picosecond fluorescence measurements on cells locked in different (quenching) states, revealing the following sequence of events during full development of NPQ. At first, trimers of light-harvesting complexes (fucoxanthin–chlorophyll a/c proteins), or FCPa, become quenched, while being part of photosystem II (PSII), due to the induced pH gradient across the thylakoid membrane. This is followed by (partial) detachment of FCPa from PSII after which quenching persists. The pH gradient also causes the formation of Dtx which leads to further quenching of isolated PSII cores and some aggregated FCPa. In subsequent darkness, the pH gradient disappears but Dtx remains present and quenching partly pertains. Only in the presence of some light the system completely recovers to the unquenched state.     

The redox state of the plastoquinone pool directly modulates minimum chlorophyll fluorescence yield in Chlamydomonas reinhardtii

The effect of the plastoquionone (PQ) pool oxidation state on minimum chlorophyll fluorescence was studied in the green alga Chlamydomonas reinhardtii. In wild type and a mutant strain that lacks both photosystems but retains light harvesting complexes, oxygen depletion induced a rise in minimum chlorophyll fluorescence. An increase in minimum fluorescence yield is also observed when the PQ pool becomes reduced in the presence of oxygen and after application of an ionophore that collapses the transmembrane proton gradient. Together these results indicate that minimum chlorophyll fluorescence is modulated by the PQ oxidation state.     

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 2. Multi-frequency phase and modulation analysis evidences a loosely connected PSII pigment-protein complex

We report the first direct decomposition of the fluorescence lifetime heterogeneity during multiphasic fluorescence induction in dark-adapted leaves by multi-frequency phase and modulation fluorometry (PMF). A very fast component, assigned to photosystem I (PSI), was found to be constant in lifetime and yield, whereas the two slow components, which are strongly affected by the closure of the reaction centers by light, were assigned to PSII. Based on a modified “reversible radical pair” kinetic model with three compartments, we showed that a loosely connected pigment complex, which is assumed to be the CP47 complex, plays a specific role with respect to the structure and function of the PSII: (i) it explains the heterogeneity of PSII fluorescence lifetime as a compartmentation of excitation energy in the antenna, (ii) it is the site of a conformational change in the first second of illumination, and (iii) it is involved in the mechanisms of nonphotochemical quenching (NPQ). On the basis of the multi-frequency PMF analysis, we reconciled two apparently antagonistic aspects of chlorophyll a fluorescence in vivo: it is heterogeneous with respect to the kinetic structure (several lifetime components) and homogeneous with respect to average quantities (quasi-linear mean τ-Φ relationship).     

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700 induction kinetics

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA₈₁₀, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700⁻ oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700⁺ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700⁺ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q_r site of the cytb₆f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.     

Photosynthetic electron transport activity in heat-treated barley leaves: The role of internal alternative electron donors to photosystem II

Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼ F_400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial Q_A reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating Q_A⁻ accumulation) occurs in the 0.2-2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this Q_A⁻ accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3-500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t_1/2 ∼ 30 ms. This alternative electron donor is most probably ascorbate.     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe− to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (ΔG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 °C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA−] radical pair to be −0.43 ± 0.025 eV, at 10 μs after the photon absorption event for PSII in its S₃-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA−] is calculated to be −0.50 ± 0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34 ± 0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA−/QA redox potential of −0.08 V [Krieger et al., 1995, Biochim. Biophys. Acta 1229, 193], the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 μs); −0.42 V for Phe−/Phe; −0.58 V for P680*/P680+.     

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.     

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O₂ photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O₂ photoreduction on the acceptor side of PSII, there is light-induced O₂ consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O₂ uptake on the donor side of PSII is related to interaction of O₂ with radicals produced by photooxidation of organic molecules. The study of flash-induced O₂ uptake finds that removal of Mn from the WOC leads to O₂ photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O₂ evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O₂ uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10 μM, corresponding to 2-4 Mn per RC) of Mn²⁺, while at higher concentrations (> 100 μM) Mn²⁺ inhibits the O₂ photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn²⁺) leads to both suppression of flash-induced O₂ uptake and disappearance of the Mn-induced activation of the O₂ photoconsumption. We assume that the light-induced O₂ uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O₂ or its reactive forms in the formation of the inorganic core of the WOC.     

Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements — Method, rationale, and results on the activation energy of dioxygen formation

The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 μs to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 μs (at 20 °C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 °C, pH 6.4). In the second part of the study, the temperature dependence (− 2.7 to 27.5 °C) of the rate constant of dioxygen formation (600/s at 20 °C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.