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.     

Enhancement of YD spin relaxation by the CaMn4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y_D^• of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn₄ cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y_D^• EPR intensity upon the application of a single laser flash to S₁ state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y_D^• by the OEC in the S₂ state, as a consequence of the single laser flash turnover. The post-flash decay reflected S₂ → S₁ back-turnover, as confirmed by their correlations with independent measurements of S₂ multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y_D^• as a probe.     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine_Z (Tyr_Z) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr_Z with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr_Z and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr_Z oxidation at cryogenic temperature in the S₀ and S₁ states was around neutral pH and was essentially pH-independent. The yield of Tyr_Z oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr_Z^· in the S₀ and S₁ states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr_Z oxidation and Tyr_Z^· reduction at cryogenic temperature in the S₀ and S₁ states of the active PSII. Theoretical calculations indicate that Tyr_Z becomes more difficult to oxidize when a H₂O molecule interacts directly with it. It is suggested that Tyr_Z is probably located in a hydrophobic environment with no direct interaction with the substrate H₂O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

Interaction of N,N,N',N'-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the Q(B) niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (muM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S(2)Q(B)(-) and S(3)Q(B)(-) (B-band), S(2)Q(A)(-) (Q-band), and Y(D)(+)Q(A)(-) (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y(n)) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q(B) confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q(B) niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q(B)-site and to reduce the higher S-states of the Mn cluster.     

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.     

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.     

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.     

Molecular origin of the pH dependence of tyrosine D oxidation kinetics and radical stability in photosystem II

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (Tyr_D) at room temperature and the extent of Tyr_D oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK_a of ≈ 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368-14373; Faller et al. 2001 Biochemistry 41, 12914-12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with ¹³C₆-, or ¹³C₁(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced Tyr_D state and not the oxidized Tyr_D state and that Tyr_D does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of Tyr_D oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the Tyr_D and Tyr_D states in the whole pH6-10 range. We show that the interactions between reduced Tyr_D and D2-His189 are modulated by the pH. At pH greater than 7.5, the ν(CO) mode frequency of Tyr_D indicates that Tyr_D is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by Tyr_D is weaker and Tyr_D could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833-13844]. The involvement of Tyr_D in this strong hydrogen-bonding interaction correlates with the ability to oxidize Tyr_D at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of Tyr_D. The IR data point to the role of a protonatable group(s) (with a pK_a of ≈ 7) other than D2-His189 and Tyr_D, in modifying the characteristics of the Tyr_D hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if Tyr_D oxidation involves the same proton transfer route at low and at high pH.     

The S1 split signal of photosystem II; a tyrosine-manganese coupled interaction

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q_A⁻ is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (< 5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680⁺ is observed with the same decay kinetics as the S₁ split signal. Cryogenic donors to P680⁺ seen are: (i) transient (t_1/2 ∼ 150 s) tyrosine related species, including ‘split signals’ (∼ 15% total centers), (ii) reduced cytochrome b₅₅₉ (∼ 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, (∼ 30% centers).     

Low-temperature electron transfer suggests two types of QA in intact photosystem II

The correlation between the reduction of Q_A and the oxidation of Tyr_Z or Car/Chl_Z/Cyt_b559 in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g = 1.95-1.86 Q_A^-•EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q_A^-•, Car^+•/Chl^+• and oxidized Cyt_b559, while enhancing the amplitude of the S₁Tyr_Z• EPR signal in the intact PSII sample. The quantification and stability of the g = 1.95-1.86 EPR signal and signals arising from the oxidized Tyr_Z and the side-path electron donors, respectively, indicate that the EPR-detectable g = 1.95-1.86 Q_A^-• signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl_Z/Cyt_b559), but not of Tyr_Z. These results imply that two types of Q_A^-• probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q_A are discussed.     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b₅₅₉ (cyt b₅₅₉) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP^red) forms of cyt b₅₅₉ exhibit superoxide scavenging activity and interconversion between IP and HP^red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O₂⁻) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O₂⁻ scavengers prevented the light-induced conversion of IP ↔ HP^red cyt b₅₅₉ confirmed that IP and HP^red cyt b₅₅₉ are reduced and oxidized by O₂⁻, respectively. Redox changes in cyt b₅₅₉ by an exogenous source of O₂⁻ reconfirmed the superoxide oxidase and reductase activity of cyt b₅₅₉. Furthermore, the light-induced conversion of IP to HP^red form of cyt b₅₅₉ was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His²² residue generates a favorable condition for the oxidation and reduction of O₂⁻, respectively.     

EPR kinetic studies of the S−1 state in spinach thylakoids

The Y_Z decay kinetics in a formal S₋₁ state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S₋₁ state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S₋₁ state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the Y_Z re-reduction rate by the S₋₁ state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S₋₁ to S₀ transition, which is in the order of the S₃ to S₀ transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S₋₁ to S₀ transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples.     

Species-dependence of the redox potential of the primary quinone electron acceptor QA in photosystem II verified by spectroelectrochemistry

The redox potentials E_m(Q_A/) of the primary quinone electron acceptor Q_A in oxygen-evolving photosystem II complexes of three species were determined by spectroelectrochemistry. The E_m(Q_A/) values were experimentally found to be −162 ± 3 mV for a higher plant spinach, −171 ± 3 mV for a green alga Chlamydomonas reinhardtii and −104 ± 4 mV vs. SHE for a red alga Cyanidioschyzonmerolae. On the basis of possible deviations for the experimental values, as estimated to differ by 9-29 mV from each true value, plausible causes for such remarkable species-dependence of E_m(Q_A/) are discussed, mainly by invoking the effects of extrinsic subunits on the delicate structural environment around Q_A.     

A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2-2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100-1800 μmol photons m⁻² s⁻¹). Simultaneous measurements of FI and 820-nm transmission kinetics (I₈₂₀) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2-0.4 s) is accompanied by a decrease of I₈₂₀, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4-2 s) of the dip is not paralleled by the change in I₈₂₀. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I-P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I₈₂₀ kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin-NADP⁺-oxidoreductase or Mehler-peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.     

Substitution of chloride by bromide modifies the low-temperature tyrosine Z oxidation in active photosystem II

Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl⁻ replacement by Br⁻ in active PSII. In Br⁻ substituted samples, Cl⁻ is effectively replaced by Br⁻ in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. The following results have been obtained. i) The oxygen-evolving activities of the Br⁻-PSII samples are significantly lower than that of the Cl⁻-PSII samples; ii) The same S₂ multiline EPR signals are observed in both Br⁻ and Cl⁻-PSII samples; iii) The amplitudes of the visible light induced S₁Tyr_Z^• and S₂Tyr_Z^• EPR signals are significantly decreased after Br⁻ substitution; the S₁Tyr_Z^• EPR signal is up-shifted about 8 G, whereas the S₂Tyr_Z^• signal is down-shifted about 12 G after Br⁻ substitution. These results imply that the redox properties of Tyr_Z and spin interactions between Tyr_Z^• and Mn-cluster could be significantly modified due to Br⁻ substitution. It is suggested that Cl⁻/Br⁻ probably coordinates to the Ca²⁺ ion of the Mn-cluster in active photosystem II.     

Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn₄ cluster are known in all S states except S₄. Many signals are insufficiently understood and the S₀, S₁, and S₃ states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S₀, S₁, and S₃ states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S₀, split S₁, split S₃ and S₂ state multiline EPR signals. We demonstrate that quantification of the S₁, S₃ and S₀ states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S₁ → S₂ transition proceeds without misses following a saturating flash at 1 °C, whilst substantial misses occur in the S₂ → S₃ transition following the second flash.     

Low-barrier hydrogen bond plays key role in active photosystem II — A new model for photosynthetic water oxidation

The function and mechanism of Tyr_Z in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr_Z, and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr_Z function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr_Z oxidation and Tyr_Z^· reduction during photosynthetic water oxidation. Upon the oxidation of Tyr_Z, the hydrogen bond between Tyr_Z and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr_Z^· reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr_Z oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr_Z oxidation and Tyr_Z^· reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr_Z and Tyr_D in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr_Z. This type of change arising from radiation damage has been confirmed in other enzyme systems.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.