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.     

Effects of Nano-anatase TiO<Subscript>2</Subscript> on Absorption,Distribution of Light,and Photoreduction Activities of Chloroplast Membrane of Spinach

The effects of nano-anatase TiO₂ on light absorption, distribution, and conversion, and photoreduction activities of spinach chloroplast were studied by spectroscopy. Several effects of nano-anatase TiO₂ were observed: (1) the absorption peak intensity of the chloroplast was obviously increased in red and blue region, the ratio of the Soret band and Q band was higher than that of the control; (2) the great enhancement of fluorescence quantum yield near 680 nm of the chloroplast was observed, the quantum yield under excitation wavelength of 480 nm was higher than the excitation wavelength of 440 nm; (3) the excitation peak intensity near 440 and 480 nm of the chloroplast significantly rose under emission wavelength of 680 nm, and F ₄₈₀ / F ₄₄₀ ratio was reduced; (4) when emission wavelength was at 720 nm, the excitation peaks near 650 and 680 nm were obviously raised, and F ₆₅₀ / F ₆₈₀ ratio rose; (5) the rate of whole chain electron transport, photochemical activities of PSII DCPIP photoreduction and oxygen evolution were greatly improved, but the photoreduction activities of PSI were a little changed. Together, the studies of the experiments showed that nano-anatase TiO₂ could increase absorption of light on spinach chloroplast and promote excitation energy to be absorbed by LHCII and transferred to PSII and improve excitation energy from PSI to be transferred to PSII, thus, promote the conversion from light energy to electron energy and accelerate electron transport, water photolysis, and oxygen evolution.     

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.     

Inhibition and uncoupling of photosynthetic electron transport by diterpene lactone amide derivatives

Nine diterpene lactone amide derivatives 1-9 were synthesized from 6-oxovouacapan-7beta,17beta-lactone, which was obtained from 6alpha,7beta-dihydroxyvouacapan-17beta-oic acid isolated from Pterodon polygalaeflorus Benth., and tested for their activity on photosynthetic electron transport. Amide derivatives 3-5 behaved as electron transport chain inhibitors; they inhibited the photophosphorylation and uncoupled non-cyclic electron transport from water to methylviologen (MV). Furthermore, 4 and 5 enhanced the basal electron rate acting as uncouplers. Compound 6 behaved as an uncoupler; it enhanced the light-activated Mg2+-ATPase and basal electron flow, without affecting the uncoupled non-cyclic electron transport. Compounds 1-2 and 7-9 were less active or inactive. Compounds 3-5 did not affect photosystem I (PSI); they inhibited photosystem II (PSII) from water to 2,6-dichlorophenol indophenol (DCPIP). Compound 4 inhibited PSII from water to silicomolybdate (SiMo), but it had no effect on the reaction from diphenylcarbazide (DPC) to DCPIP indicating that its inhibition site was at the water splitting enzyme complex (OEC). Compounds 3 and 5 inhibited PSII from water to DCPIP without any effect from water to SiMo, therefore they inhibited the acceptor site of PSII. Chlorophyll a fluorescence kinetics confirmed the behaviour of 3-5.     

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+.     

Photosynthetic electron transport interaction of xanthorrhizol isolated from Iostephane heterophylla and its derivatives

A bioactivity-guided chemical study of Iostephane heterophylla (Asteraceae) led to the isolation of xanthorrhizol (1) as the compound that causes inhibition of ATP synthesis, H⁺-uptake and electron flow from water to methylviologen (basal, phosphorylating and uncoupled) in freshly lysed spinach chloroplasts, thus acting as an inhibitor of the Hill reaction. Acetyl (2), dihydro (3) and acetyl-dihydro (4) derivatives were synthesized. It was found that 4 was less active than 1 and 2 in ATP synthesis, whereas 3 was the most potent inhibitor of the Hill reaction and was also an inhibitor of H⁺-ATPase. Studies of the photosynthetic partial redox reactions from PQ to MV indicated that 1 partially inhibited the PQ pool, but that 3 did not. However, both inhibited the uncoupled electron transport in PSII from water to DCBQ. Uncoupled electron flow from water to silicomolybdate was completely inhibited by 3 and partially by 1. The reaction from DPC to DCPIP was inhibited by both 1 and 3. These results indicate that the inhibition site is located within PSII for 1 and 3 as was corroborated by fluorescence decay data.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

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.     

Structural characteristics of channels and pathways in photosystem II including the identification of an oxygen channel

Here we use crystal structures to investigate and review channels and pathways for the transfer of substrates (water, plastoquinone (PQ)) and products (electrons, protons, oxygen, reduced PQ (PQH(2))) to, and from, the redox active catalytic sites of photosystem II (PSII). A putative oxygen channel has been identified which is about 21A in length, leading from the water splitting site to the lumen. This channel follows a path along the lumenal surface of CP43, passing across the interface of the large extrinsic loop which joins the fifth and sixth transmembrane helices of this chlorophyll binding protein. In so doing it seems to minimise interactions with the excited states of chlorophylls bound within the PSII complex, especially those that constitute the primary electron donor, P680. Two additional channels leading from the water splitting site, and also exiting at the lumen, were also identified. Their hydrophilic nature suggests that they probably facilitate the delivery of water to, and protons from, the catalytic site. Also discussed are unique features in the electron transfer pathway of PSII, as compared with those of purple photosynthetic bacteria, and structural implications of the PSII Q(B)-site in terms of PQ protonation and PQ/PQH(2) diffusion.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Characteristic features of the interaction between Fe(II) cations and the donor side of the manganese-depleted photosystem II

Ferrous iron cations Fe(II) can effectively bind to the donor side of the manganese-depleted photosystem II (PSII(-Mn)) and in this way block electron transfer from diphenylcarbazide (DPC) to the major donor for P680, Y_Z. The present study was focused on the characteristic features of this process. The oxidation and subsequent binding of Fe(II) cations to PSII(-Mn) may proceed in the absence of an artificial electron acceptor, and therefore we investigated the role of O₂ as a putative endogenous acceptor. Oxygen was shown to participate in the blockade of Y_Z by Fe cations, apparently as a structural element of Fe cluster formed at the donor side of PSII(-Mn). The kinetic study of blocking Y_Z by Fe(II) as dependent on light intensity demonstrated that the quantum efficiency of Fe cations binding to the donor side of PSII(-Mn) considerably exceeded that of Mn cations. We also compared the possibilities of extracting the native Mn cluster and reconstructed Fe cations from PSII and an alternative electron transport from DPC to P680⁺ under the conditions of the Y_Z blockade by Fe cations. Neither an alternative donor for P680, Y_D , nor cytochrome b ₅₅₉ participated in the latter process. As a whole, our evidence shows that many features of binding Fe cation to the donor side of PSII(-Mn) are in common with photoassembling the Mn cluster.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 12–20.Original Russian Text Copyright © 2005 by Lovyagina, Davletshina, Kultysheva, Timofeev, Ivanov, Semin.     

The role of carbon dioxide and oxygen in determining chlorophyll fluorescence quenching during leaf development

Chlorophyll fluorescence emission at 680 nm (F680) and the rate of CO₂ fixation were measured simultaneously in sections along the length of wheat and maize leaves. These leaves possess a basal meristem and show a gradation in development towards the leaf tip. The redox state of the primary electron acceptor, Q, of photosystem II was estimated using a non-invasive method. Distal mature leaf sections displayed typical F680 induction curves which were generally anti-parallel with CO₂ fixation and during which Q became gradually oxidised. In leaf-base sections net assimilation of CO₂ was not detectable, F680 quenched slowly and monotonously without displaying any of the oscillations typical of mature tissue and Q remained relatively reduced. Sections cut from mid-regions of the leaf showed intermediate characteristics. There were no major differences between the wheat and maize leaf in the parameters measured. The results support the hypothesis that generation of the transthylakoid proton gradient and associated ATP production is not a major limitation to photosynthesis during leaf development in either C₃ or C₄ plants. Removal of CO₂ from the mature leaf sections caused little change in steady-state F680 and produced about 50% reduction of Q. When O₂ was then removed, F680 rose sharply and Q became almost totally reduced. In immature tissue unable to assimilate CO₂, removal of O₂ alone caused a similar large rise in F680 and reduction of Q whilst removal of CO₂ had negligible effects on F680 and the redox state of Q. It is concluded that in leaf tissue unable to assimilate CO₂, either because CO₂ is absent or the tissue is immature, O₂ acts as an electron acceptor and maintains Q in a partially oxidised state. The important implication that O₂ may have a role in the prevention of photoinhibition of the photochemical apparatus in the developing leaf is discussed.Abbreviations F680 chlorophyll fluorescence emission at 680 nm - PSI photosystem I - PSII photosystem II - Q PSII primary electron acceptor - pH transthylakoid proton gradient     

Natural diterpenes from <Emphasis Type="Italic">Croton ciliatoglanduliferus</Emphasis> as photosystem II and photosystem I inhibitors in spinach chloroplasts

In our search for new natural photosynthetic inhibitors that could lead to the development of “green herbicides” less toxic to environment, the diterpene labdane-8α,15-diol (1) and its acetyl derivative (2) were isolated for the first time from Croton ciliatoglanduliferus Ort. They inhibited photophosphorylation, electron transport (basal, phosphorylating and uncoupled) and the partial reactions of both photosystems in spinach thylakoids. Compound 1 inhibits the photosystem II (PS II) partial reaction from water to Na⁺ Silicomolibdate (SiMo) and has no effect on partial reaction from diphenylcarbazide (DPC) to 2,6-dichlorophenol indophenol (DCPIP), therefore 1 inhibits at the water splitting enzyme and also inhibits PS I partial reaction from reduced phenylmetasulfate (PMS) to methylviologen (MV). Thus, it also inhibits in the span of P₇₀₀ to Iron sulfur center X (F_X). Compound 2 inhibits both, the PS II partial reactions from water to SiMo and from DPC to DCPIP; besides this, it inhibits the photosystem I (PS I) partial reaction from reduced PMS to MV. With these results, we concluded that the targets of the natural product 2 are located at the water splitting enzyme, and at P₆₈₀ in PS II and at the span of P₇₀₀ to F_X in PS I. The results of compounds 1 and 2 on PS II were corroborated by chlorophyll a fluorescence.     

Efficiency and role of loss processes in light-driven water oxidation by PSII

Its superior quantum efficiency renders PSII a model for biomimetic systems. However, also in biological water oxidation by PSII, the efficiency is restricted by recombination losses. By laser-flash illumination, the secondary radical pair, P680(+)Q(-) (A) (where P680 is the primary Chl donor in PSII and Q(A), primary quinone acceptor of PSII), was formed in close to 100% of the PSII. Investigation of the quantum efficiency (or yield) of the subsequent steps by time-resolved delayed (10 micros to 60 ms) and prompt (70 micros to 700 ms) Chl fluorescence measurements on PSII membrane particles suggests that (1) the effective rate for P680(+) Q(-) (A) recombination is approximately 5 ms(-1) with an activation energy of approximately 0.34 eV, circumstantially confirming dominating losses by reformation of the primary radical pair followed by ground-state recombination. (2) Because of compensatory influences on recombination and forward reactions, the efficiency is only weakly temperature dependent. (3) Recombination losses are several-fold enhanced at lower pH. (4) Calculation based on delayed-fluorescence data suggests that the losses depend on the state of the water-oxidizing manganese complex, being low in the S(0)-->S(1) and S(1)-->S(2) transition, clearly higher in S(2)-->S(3) and S(3)-->S(4)-->S(0). (5) For the used artificial electron acceptor, the efficiency is limited by acceptor-side processes/S-state decay at high/low photon-absorption rates resulting in optimal efficiency at surprisingly low rates of approximately 0.15-15 photons s(-1) (per PSII). The pH and S-state dependence can be rationalized by the basic model of alternate electron-proton removal proposed elsewhere. A physiological function of the recombination losses could be limitation of the lifetime of the reactive donor-side tyrosine radical (Y(.) (Z)) in the case of low-pH blockage of water oxidation.     

Changes in the redox potential of primary and secondary electron-accepting quinones in photosystem II confer increased resistance to photoinhibition in low-temperature-acclimated Arabidopsis

Exposure of control (non-hardened) Arabidopsis leaves for 2 h at high irradiance at 5 degrees C resulted in a 55% decrease in photosystem II (PSII) photochemical efficiency as indicated by F(v)/F(m). In contrast, cold-acclimated leaves exposed to the same conditions showed only a 22% decrease in F(v)/F(m). Thermoluminescence was used to assess the possible role(s) of PSII recombination events in this differential resistance to photoinhibition. Thermoluminescence measurements of PSII revealed that S(2)Q(A)(-) recombination was shifted to higher temperatures, whereas the characteristic temperature of the S(2)Q(B)(-) recombination was shifted to lower temperatures in cold-acclimated plants. These shifts in recombination temperatures indicate higher activation energy for the S(2)Q(A)(-) redox pair and lower activation energy for the S(2)Q(B)(-) redox pair. This results in an increase in the free-energy gap between P680(+)Q(A)(-) and P680(+)Pheo(-) and a narrowing of the free energy gap between primary and secondary electron-accepting quinones in PSII electron acceptors. We propose that these effects result in an increased population of reduced primary electron-accepting quinone in PSII, facilitating non-radiative P680(+)Q(A)(-) radical pair recombination. Enhanced reaction center quenching was confirmed using in vivo chlorophyll fluorescence-quenching analysis. The enhanced dissipation of excess light energy within the reaction center of PSII, in part, accounts for the observed increase in resistance to high-light stress in cold-acclimated Arabidopsis plants.     

Histidine 190-D1 and glutamate 189-D1 provide structural stabilization in photosystem II

In photosynthesis, photosystem II (PSII) conducts the light-driven oxidation of water to oxygen. Tyrosine Z is Tyr 161 of the D1 polypeptide; Z acts as an intermediary electron carrier in water oxidation. In this report, EPR spectroscopy was used to study the effect of His 190 and Glu 189 on Z* yield and reduction kinetics. Neither mutation has a significant impact on the EPR line shape of Z*. At room temperature and pH 7.5, the E189Q-D1 mutation has a single turnover Z* yield that is 84% compared to wild-type. The H190Q-D1 mutation decreases the Z* yield at room temperature by a factor of 2.6 but has a more modest effect (factor of 1.6) at -10 degrees C. The temperature dependence is shown to be primarily reversible. Neither mutation has a dramatic effect on Z* decay kinetics. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on H190Q, reveals perturbations, including an increased spectral contribution from a PSII chlorophyll. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on E189Q, shows perturbations, including a decreased contribution from the carboxylate side chain of a glutamate or aspartate. Temperature-dependent changes in H190Q-D1 and E189Q-D1 Z. yield are attributed to a reversible conformational change, which alters the electron-transfer rate from Z to P(680)(+). On the basis of these results, we conclude that H190 and E189 play a role in the structural stabilization of PSII. We postulate that some or all of the phenotypic changes observed in H190Q and E189Q mutants may be caused by structural alterations in PSII.     

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 PD1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P680, 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 PD1 is not an important determinant of the redox and spectroscopic properties of P680 in T. elongatus.     

Analysis of donors of electrons to photosystem I and cyclic electron flow by redox kinetics of P700 in chloroplasts of isolated bundle sheath strands of maize

Bundle sheath chloroplasts of NADP-malic enzyme (NADP-ME) type C₄ species have a high demand for ATP, while being deficient in linear electron flow and oxidation of water by photosystem II (PSII). To evaluate electron donors to photosystem I (PSI) and possible pathways of cyclic electron flow (CEF1) in isolated bundle sheath strands of maize (Zea mays L.), an NADP-ME species, light-induced redox kinetics of the reaction center chlorophyll of PSI (P700) were followed under aerobic conditions. Donors of electrons to CEF1 are needed to compensate for electrons lost from the cycle. When stromal electron donors to CEF1 are generated during pre-illumination with actinic light (AL), they retard the subsequent rate of oxidation of P700 by far-red light. Ascorbate was more effective than malate in generating stromal electron donors by AL. The generation of stromal donors by ascorbate was inhibited by DCMU, showing ascorbate donates electrons to the oxidizing side of PSII. The inhibitors of NADPH dehydrogenase (NDH), amytal and rotenone, accelerated the oxidation rate of P700 by far-red light after AL, indicating donation of electrons to the intersystem from stromal donors via NDH. These inhibitors, however, did not affect the steady-state level of P700⁺ under AL, which represents a balance of input and output of electrons in P700. In contrast, antimycin A, the inhibitor of the ferredoxin-plastoquinone reductase-dependent CEF1, substantially lowered the level of P700⁺ under AL. Thus, the primary pathway of ATP generation by CEF1 may be through ferredoxin-plastoquinone, while function of CEF1 via NDH may be restricted by low levels of ferredoxin-NADP reductase. NDH may contribute to redox poising of CEF1, or function to generate ATP in linear electron flow to O₂ via PSI, utilizing NADPH generated from malate by chloroplastic NADP-ME.     

UV-B radiation-induced donor- and acceptor-side modifications of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.

We studied the effect of UV-B radiation (280-320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t(1/2) of the decay components assigned to reoxidation of Q(A)(-) by Q(B) from 220 to 330 micros in centers which have the Q(B) site occupied, and from 3 to 6 ms in centers with the Q(B) site empty. In contrast, the t(1/2) of the slow component arising from recombination of the Q(A)Q(B)(-) state with the S(2) state of the water-oxidizing complex decreases from 13 to 1-2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5-0.6 s component, which reflects Q(A)(-) recombination with the S(2) state. After UV-B irradiation, this is partially replaced by much faster components (t(1/2) approximately 800-900 micros and 8-10 ms) arising from recombination of Q(A)(-) with stabilized intermediate photosystem II donors, P680(+) and Tyr-Z(+). Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4-6-fold increase in the half-inhibitory concentration for electron transfer from Q(A) to Q(B). UV-B irradiation in the presence of DCMU reduces Q(A) in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q(A) is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z(+) and P680(+) in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q(B) site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q(A)(-). White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.     

Annonaceous acetogenins: Naturally occurring inhibitors of ATP synthesis and photosystem II in spinach chloroplasts

The effects of squamocin ( 1 ), bullatacin ( 2 ) and motrilin ( 3 ), 3 bis-tetrahydrofuran Annonaceous acetogenins, isolated from Annona purpurea (Annonaceae), were investigated on several photosynthetic activities in spinach thylakoids. The results indicated that compounds 1 – 3 significantly inhibited both ATP synthesis and uncoupled electron transport. In addition, they enhanced light-activated Mg2+-ATPase, and basal electron flow. Therefore, acetogenins 1 – 3 behave as uncouplers and Hill reaction inhibitors. Natural products 1 – 3 did not affect photosystem I (PSI) activity but they inhibited photosystem II (PSII) electron flow. The study of the partial PSII reactions from H2O to DCPIPox, H2O to SiMo and diphenylcarbazide to DCPIP established that the site of inhibition was at the oxygen-evolving complex (OEC). Chlorophyll a fluorescence measurements confirmed the behavior of the Annonaceous acetogenins as water-splitting enzyme inhibitors.