Photosystem II: Structure and mechanism of the water:plastoquinone oxidoreductase

This mini-review briefly summarizes our current knowledge on the reaction pattern of light-driven water splitting and the structure of Photosystem II that acts as a water:plastoquinone oxidoreductase. The overall process comprises three types of reaction sequences: (a) light-induced charge separation leading to formation of the radical ion pair P680^+•Q_A^−•; (b) reduction of plastoquinone to plastoquinol at the Q_B site via a two-step reaction sequence with Q_A^−• as reductant and (c) oxidative water splitting into O₂ and four protons at a manganese-containing catalytic site via a four-step sequence driven by P680^+• as oxidant and a redox active tyrosine Y_Z acting as mediator. Based on recent progress in X-ray diffraction crystallographic structure analysis the array of the cofactors within the protein matrix is discussed in relation to the functional pattern. Special emphasis is paid on the structure of the catalytic sites of PQH₂ formation (Q_B-site) and oxidative water splitting (Mn₄O _x Ca cluster). The energetics and kinetics of the reactions taking place at these sites are presented only in a very concise manner with reference to recent up-to-date reviews. It is illustrated that several questions on the mechanism of oxidative water splitting and the structure of the catalytic sites are far from being satisfactorily answered.     

Photophysical processes of energy conversion in thylakoid membranes of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> mutants D1-R323H,D1-R323D,and D1-R323L

Chlamydomonas reinhardtii mutants D1-R323H, D1-R323D, and D1-R323L showed elevated chlorophyll fluorescence yields, which increased with decline of oxygen evolving capacity. The extra step K ascribed to the disturbance of electron transport at the donor side of PS II was observed in OJIP kinetics measured in mutants with a PEA fluorometer. Fluorescence decay kinetics were recorded and analyzed in a pseudo-wild type (pWt) and in mutants of C. reinhardtii with a Becker and Hickl single photon counting system in pico- to nanosecond time range. The kinetics curves were fitted by three exponentials. The first one (rapid, with lifetime about 300 ps) reflects energy migration from antenna complex to the reaction center (RC) of photosystem II (PS II); the second component (600–700 ps) has been assigned to an electron transfer from P680 to Q_A, while the third one (slow, 3 ns) assumingly originates from charge recombination in the radical pair [P680^+• Pheo^−•] and/or from antenna complexes energetically disconnected from RC II. Mutants showed reduced contribution of the first component, whereas the yield of the second component increased due to slowing down of the electron transport to Q_A. The mutant D1-R323L with completely inactive oxygen evolving complex did not reveal rapid component at all, while its kinetics was approximated by two slow components with lifetimes of about 2 and 3 ns. These may be due to two reasons: a) disconnection between antennae complexes and RC II, and b) recombination in a radical pair [P680^+• Pheo^−•] under restricted electron transport to Q_A. The data obtained suggest that disturbance of oxygen evolving function in mutants may induce an upshift of the midpoint redox potential of Q_A/Q_A⁻ couple causing limitation of electron transport at the acceptor side of PS II.     

Theoretical study of hydrogenation of the doubly aromatic B 7 − cluster

We have studied the influence of hydrogenation on the relative stability of the low-lying isomers of the anionic B₇⁻ cluster, computationally. It is known that the pure-boron B₇⁻ cluster has a doubly (σ- and π-) aromatic C_6v (³A₁) quasi-planar wheel-type triplet global minimum (structure 1), a low-lying σ-aromatic and π-antiaromatic quasi-planar singlet C_2v (¹A₁) isomer 2 (0.7 kcal mol⁻¹ above the global minimum), and a planar doubly (σ- and π-) antiaromatic C_2v (¹A₁) isomer 3 (7.8 kcal mol⁻¹ above the global minimum). However, upon hydrogenation, an inversion in the stability of the species occurs. The planar B₇H₂⁻ (C_2v, ¹A₁) isomer 4, originated from the addition of two hydrogen atoms to the doubly antiaromatic B₇⁻ isomer 3, becomes the global minimum structure. The second most stable B₇H₂⁻ isomer 5, originated from the quasi-planar triplet wheel isomer 1 of B₇⁻, was found to be 27 kcal mol⁻¹ higher in energy. The inversion in stability occurs due to the loss of the doubly aromatic character in the wheel-type global minimum isomer (C_6v, ³A₁) of B₇⁻ upon H₂−addition. In contrast, the planar isomer of B₇⁻ (C_2v, ¹A₁) gains aromatic character upon addition of two hydrogen atoms, which makes it more stable. Figure The B₇H₂-global minimum structure and its σ-aromatic and π-antiaromatic MOs Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday.     

Toxic effects of antimony on photosystem II of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. as probed by in vivo chlorophyll fluorescence

It has been demonstrated that antimony (Sb) at concentrations ranging from 1.0 to 10.0 mg L⁻¹ inhibits O₂ evolution. Deeper insight into the influence of Sb on PSII was obtained with measurements of in vivo chlorophyll fluorescence. The donor and the acceptor sides of PSII were shown to be the target of Sb. Sb treatment induces inhibition of electron transport from Q_A⁻ to Q_B/Q_B⁻ and accumulation of P₆₈₀⁺. S₂(Q_AQ_B)⁻ charge recombination and oxidation by PQ9 molecules became more important in Q_A⁻ reoxidation as the electron transfer in PSII was inhibited. Sb exposure caused a steady increase in the proportion of PSII_X and PSII_β. These changes resulted in increased fluxes of dissipated energy and decreased index of photosynthesis performance, of maximum quantum yield, and of the overall photosynthetic driving force of PSII.     

The aprotic electrochemistry of quinones

Quinones play important roles in biological electron transfer reactions in almost all organisms, with specific roles in many physiological processes and chemotherapy. Quinones participate in two-electron, two-proton reactions in aqueous solution at equilibrium near neutral pH, but protons often lag behind the electron transfers. The relevant reactions in proteins are often sequential one electron redox processes without involving protons. Here we report the aprotic electrochemistry of the two half-couples, Q/Q^.- and Q^.?/Q⁼, of 11 parent quinones and 118 substituted 1,4-benzoquinones, 91 1,4-naphthoquinones, and 107 9,10-anthraquinones. The measured redox potentials are fit quite well with the Hammett para sigma (σ_para) parameter. Occasional exceptions can involve important groups, such as methoxy substituents in ubiquinone and hydroxy substituents in therapeutics. These can generally be explained by reasonable conjectures involving steric clashes and internal hydrogen bonds. We also provide data for 25 other quinones, 2 double quinones and 15 non-quinones, all measured under similar conditions.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoinactivation of the photosynthetic electron transport chain by accumulation of over-saturating light pulses given to dark adapted pea leaves

The effect of cumulative over-saturating pulses (OSP) of white light (1 s, >10 000 μmol photons m⁻² s⁻¹), applied every 20 min on pea leaves, was investigated during a complete diurnal cycle of 24 h. In dark-adapted leaves, this treatment leads to a progressive decline of the optimum Photosystem II (PS II) quantum yield. Continuous low background light (except far-red light) had a protective effect against this OSP-induced photoinactivation. The lack of far-red effect could be due to its absorption mainly in PS I and not in PS II, but could be also due to the general low absorption in this wavelength region. The photoinactivation was enhanced in leaves that had been previously infiltrated with chloramphenicol. The quantum yield of CO₂ assimilation, but not its maximal capacity, was inhibited by the OSP treatment. The most spectacular effects observed, in addition to an irreversible quenching of Fm, was a strong inhibition of Q_A ⁻ reoxidation revealed by a large increase in the Fs level and consequently by a decrease of ΔF/Fm′. Under such conditions, we observed that the electron flow deduced from ΔF/Fm′ underestimated the real electron flow to CO₂. Time-resolved Chlorophyll a fluorescence measurements showed that the reduced capacity of Q_A ⁻ reoxidation in OSP treated leaves was accompanied by the appearance of a 4.7 ns component attributed to PS II charge recombination. We suggest that a modification at the Q_B site may influence the redox potential of Q_A/Q_A ⁻, facilitating the reversion of the primary charge separation. In addition, a 1.2 ns fluorescence component accumulated, which appeared to be responsible for the underestimation of PS II electron flow. The observed photoinactivation seemed to be different from the photoinhibition often described in the literature, which occurs under continuous light. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II

Chlorophyll a fluorescence induction (FI) is widely used as a probe for studying photosynthesis. On illumination, fluorescence emission rises from an initial level O to a maximum P through transient steps, termed J and I. FI kinetics reflect the overall performance of photosystem II (PSII). Although FI kinetics are commonly and easily measured, there is a lack of consensus as to what controls the characteristic series of transients, partially because most of the current models of FI focus on subsets of reactions of PSII, but not the whole. Here we present a model of fluorescence induction, which includes all discrete energy and electron transfer steps in and around PSII, avoiding any assumptions about what is critical to obtaining O J I P kinetics. This model successfully simulates the observed kinetics of fluorescence induction including O J I P transients. The fluorescence emission in this model was calculated directly from the amount of excited singlet-state chlorophyll in the core and peripheral antennae of PSII. Electron and energy transfer were simulated by a series of linked differential equations. A variable step numerical integration procedure (ode15s) from MATLAB provided a computationally efficient method of solving these linked equations. This in silico representation of the complete molecular system provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the O J I P kinetics and fluorescence emission at these points. Simulations based on this model showed that J corresponds to the peak concentrations of Q _A ⁻ Q_B (Q_A and Q_B are the first and second quinone electron acceptor of PSII respectively) and Q _A ⁻ Q _B ⁻ and I to the first shoulder in the increase in concentration of Q _A ⁻ Q _B ²⁻ . The P peak coincides with maximum concentrations of both Q _A ⁻ Q _B ²⁻ and PQH₂. In addition, simulations using this model suggest that different ratios of the peripheral antenna and core antenna lead to differences in fluorescence emission at O without affecting fluorescence emission at J, I and P. An increase in the concentration of Q_B-nonreducing PSII centers leads to higher fluorescence emission at O and correspondingly decreases the variable to maximum fluorescence ratio (F _v/F _m).     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient light harvesting through carotenoids

We review the factors that control the efficiency of carotenoid-chlorophyll excitation transfer in photosynthetic light harvesting. For this we summarize first the recently developed theory that describes electronic couplings between carotenoids and chlorophylls and we outline in particular the influence of length of conjugated system and of symmetry breaking on the couplings. We focus hereby on the structurally solved lycopene-BChl system of LH 2 from Rhodospirillum molischianum and the peridinin-Chl a system of PCP from Amphidinium carterae. In addition, we review recent spectroscopic data for neurosporene, spheroidene and lycopene, three carotenoids with different lengths of conjugated systems. On the basis of the measured energies, emission lineshapes, solution and protein environment lifetimes for their 2A⁻ _g and 1B_u ⁺ states as well as of the theoretically determined couplings, we conclude that the transfer efficiencies from the 2A_g ⁻ state are controlled by the Car(2A_g ⁻)–BChl(Q_g) electronic couplings and the 2A_g ⁻ → 1A_g ⁻ internal conversion rates. We suggest that symmetry breaking and geometry rather than length of conjugated system dominate couplings involving the 2A_g ⁻ state. Differences in transfer efficiencies from the 1B_u ⁺ state in LH 2 and PCP are found to be dominated by the differences in spectral overlap. The role of the 1B_u ⁺ state is likely to be influenced by a lower-lying (in longer polyenes), optically forbidden 1B_u ⁻ state. This revised version was published online in June 2006 with corrections to the Cover Date.     

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P⁺B_A⁻ in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P⁺/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P⁺B_A⁻ stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm⁻¹ reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B_A⁻ at 1020 nm is registered in RCs of both mutants. The formation of the B_A⁻ band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm⁻¹. Only a partial stabilization of the P⁺B_A⁻ state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P⁺ and B_A⁻ is proposed to explain rapid stabilization of the P⁺B_A⁻ state in native RCs. Small oscillatory components at ∼330–380 cm⁻¹ in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P⁺B_A⁻ free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P⁺ and B_A⁻ is assumed to be another potential contributor to the mechanism of P⁺B_A⁻ stabilization.     

Relation Between the Heat-Induced Increase of F0 Fluorescence and a Shift in the Electronic Equilibrium at the Acceptor Side of Photosystem 2

F₀ fluorescence and thermoluminescence (TL) were recorded simultaneously on various dark-adapted leaf samples. Above 40 °C, a sharp peak of TL coincided with the onset of the heat-induced F₀ rise. It results from a back-transfer of an electron from the secondary Q_B ^-to the primary acceptor Q_A of photosystem 2, followed by a luminescence-emitting recombination with Tyr-D¹. This demonstrates that the critical temperature at which the F₀ starts rising also corresponds to a shift towards the left of the Q_A↔Q_B ^- equilibrium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Voltage changes involving photosystem II quinone–iron complex turnover

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q_A⁻ and the oxidized non-heme iron Fe³⁺ on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Δψ (negative charging of the proteoliposome interior) indicates that the iron–quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (τ ∼ 0.1 μs) of Δψ generation related to electron transfer between the redox-active tyrosine Y_Z and Q_A, an additional phase in the submillisecond time domain (τ ∼ 0.1 ms at 23°C, pH 7.0) and relative amplitude ∼ 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q_A and the secondary quinone Q_B. The rate of the additional electrogenic phase is decreased by about one-half in the presence of D₂O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

Period four oscillations in chlorophyll a fluorescence

The discovery of period four oscillations of the fluorescence yield under flashing light demonstrated that not only the redox state of the Photosystem II (PS II) electron acceptor Q_A, but also the oxygen evolving cycle (described by the S states) modulates the fluorescence yield of chlorophyll (Chl). The positive charges accumulated on the donor side of PS II act on the fluorescence yield (measured in the Q_A ⁻ state during a strong flash) through the concentration of the quencher P₆₈₀ ⁺, the oxidized form of PS II reaction center Chl a. However, the period four oscillations of the fluorescence yield detected 1 s after a strong flash (in the P₆₈₀Q_A state) have not yet been fully explained. This revised version was published online in June 2006 with corrections to the Cover Date.     

pH dependent stabilization of S2QA − and S2QB − charge pairs studied by thermoluminescence

The pH dependence of emission peak temperature and decay time of thermoluminescence arising from S₂Q_B ^– and S₂Q_A ^– recombinations demonstrates that a stabilization of S₂Q_B ^– occurs at low pH whereas stabilization of S₂Q_A ^– occurs at high pH. Based on comparative analysis of thermoluminescence parameters of the two types of recombination, we suggest that in the pH range between 5.3 and 7.5, E_m(S₂/S₁) and E_m(Q_A/Q_A ^–) are constant, but E_m(Q_B/Q_B ^–) gradually increases with decreasing pH, while in the pH range between 7.5 and 8.5, an unusual change occurs on S₂Q_A ^– charge pair, which is interpreted as either a decrease in E_m(S₂/S₁) or an increase in E_m(Q_A/Q_A ^–).     

Photosynthetic response of clusterbean chloroplasts to UV-B radiation: Energy imbalance and loss in redox homeostasis between QA and QB of photosystem II

The effects of ultraviolet-B (UV-B: 280-320 nm) radiation on the photosynthetic pigments, primary photochemical reactions of thylakoids and the rate of carbon assimilation (P_n) in the cotyledons of clusterbean (Cyamopsis tetragonoloba) seedlings have been examined. The radiation induces an imbalance between the energy absorbed through the photophysical process of photosystem (PS) II and the energy consumed for carbon assimilation. Decline in the primary photochemistry of PS II induced by UV-B in the background of relatively stable P_n, has been implicated in the creation of the energy imbalance_. The radiation induced damage of PS II hinders the flow of electron from Q_A to Q_B resulting in a loss in the redox homeostasis between the Q_A to Q_B leading to an accumulation of Q_A⁻. The accumulation of Q_A⁻ generates an excitation pressure that diminishes the PS II-mediated O₂ evolution, maximal photochemical potential (F_v/F_m) and PS II quantum yield (Φ_{PS II}). While UV-B radiation inactivates the carotenoid-mediated protective mechanisms, the accumulation of flavonoids seems to have a small role in protecting the photosynthetic apparatus from UV-B onslaught. The failure of protective mechanisms makes PS II further vulnerable to the radiation and facilitates the accumulation of malondialdehyde (MDA) indicating the involvement of reactive oxygen species (ROS) metabolism in UV-B-induced damage of photosynthetic apparatus of clusterbean cotyledons.     

Molecular mechanism of high-temperature-induced inhibition of acceptor side of Photosystem II

High-temperature-induced inhibition of the acceptor side of Photosystem II (PS II) was studied in tobacco thylakoids using oxygen evolution, chlorophyll a (Chl a) fluorescence and redox potential measurements. When thylakoids were heated at 2 °C/min from 25 to 50 °C, the oxygen evolving complex became inhibited between 32 and 45 °C, whereas the acceptor side of PS II tolerated higher temperatures. Variable Chl a fluorescence decreased more slowly than oxygen evolution, suggesting that transitions between some S-states occurred even after heat-induced inhibition of the oxygen evolving activity. 77 K emission spectroscopy reveals that heating does not cause detachment of the light-harvesting complex II from PS II, and thus the heat-induced increase in the initial F₀ fluorescence is due to loss of exciton trapping in the heated PS II centers. Redox titrations showed a heat-induced increase in the midpoint potential of the Q_A/Q_A ^-) couple from the control value of –80 mV to +40 mV at 50 °C, indicating a loss of the reducing power of Q_A ^-). When its driving force thus decreased, electron transfer from Q_A ^-) to Q_B in the PS II centers that still could reduce Q_A became gradually inhibited, as shown by measurements of the decay of Chl a fluorescence yield after a single turnover flash. Interestingly, the heat-induced loss of variable fluorescence and inhibition of electron transfer from Q_A ^-) to Q_B could be partially prevented by the presence of 5 mM bicarbonate during heating, suggesting that high temperatures cause release of the bicarbonate bound to PS II. We speculate that both the upshift in the redox potential of the Q_A/Q_A ^-) couple and the release of bicarbonate may be caused by a heat-induced structural change in the transmembrane D1 or D2 proteins. This structural change may, in turn, be caused by the inhibition of the oxygen evolving complex during heating.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q_B site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q_B sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q_A to Q_B electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q_B site inhibitors, DCMU and atrazine. In the sensitive centers, the S₂Q_A⁻ state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S₂Q_A⁻ state than the S₁Q_A state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S₂-multiline electron paramagnetic resonance (EPR) signal and the ‘split’ EPR signal, originating from the S₂Y_Z state showed no significant changes upon binding of phenolic inhibitors at the Q_B site. We thus propose a working model where Q_A redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q_B niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q_B site.