Destabilization of the Oxygen Evolving Complex of Photosystem II by Al3+

The inhibitory effect of Al³⁺ on photosynthetic electron transport was investigated in isolated thylakoid membranes of spinach. A combination of oxygen evolution, chlorophyll fluorescence induction (FI) and decay and thermoluminescence measurements have been used to characterize photosystem II (PSII) electron transport in the presence of this toxic metal cation. Our results show that below 3 mm , Al³⁺ already caused a destabilization of the Mn₄O₅Ca cluster of the oxygen evolving complex (OEC). At these concentrations, an increase in the relative amplitude of the first phase (OJ) of FI curve and retardation of the fluorescence decay kinetics following excitation with a single turnover flash were also observed. A transmembrane structural modification of PSII polypeptides due to the interaction of Al³⁺ at the OEC is proposed to retard electron transfer between the quinones Q_A and Q_B. Above 3 mm , Al³⁺ strongly retarded fluorescence induction and significantly reduced F_v/F_m together with the maximal amplitude of chlorophyll fluorescence induced by a single turnover flash. This chlorophyll fluorescence quenching was attributed to the formation of P680⁺ due to inhibition of electron transfer between tyrosine 161 of D1 subunit and P680.     

Phosphatidylcholine-induced reactivation of photosystem II membranes pretreated with Triton X-100

Triton X-100-induced inactivation and phosphatidylcholine-induced reactivation of photosystem II (PSII) membranes were investigated using oxygen electrode, variable fluorescence and spectroscopic techniques including absorption and circular dichroism spectroscopy. Incubation of the PSII membrane with Triton X-100 reduced the oxygen-evolving rate, modified the variable chlorophyll fluorescence kinetics, changed the protein secondary structures, altered the chlorophyll binding state to proteins and decreased the excitonic interaction of chlorophyll molecules. Phosphatidylcholine addition did not change the protein secondary structures, but could partially reactivate the reduced oxygen-evolving rate, and partly reversed the variable fluorescence parameters, the chlorophyll binding state and the excitonic interaction of the chlorophyll molecules. The results indicate that the phosphatidylcholine environment can optimize the tertiary structures of PSII.     

Period-four Modulation of Photosystem II Primary Quinone Acceptor (QA) Reduction/Oxidation Kinetics in Thylakoid Membranes

Photosystem II (PSII), a multiprotein complex mainly coded by the chloroplast genome in higher plants and algae, contains the oxygen-evolving complex with four manganese atoms responsible for the oxidation of water. After each absorption of a light quantum by pigment molecules in the light harvesting complexes of PSII, the Mn cluster advances in its oxidation states denoted from S₀ to S₄. The S₄ state decays to S₀ in the dark with the concurrent release of molecular oxygen. Therefore, the oxygen production in PSII exposed to successive single turnover excitations follows a period-four oscillation pattern. The intensity of chlorophyll a fluorescence of PSII is also known to be influenced by the oxidation state of the Mn cluster. In the present work, fluorescence induction kinetics was measured in isolated thylakoids with various initial S-state populations settled by preflashes. The shape of the fluorescence induction traces was strongly affected by preflashes. O-J and J-I phases of the induction followed a period-four oscillation pattern. The results indicate that these changes reflect the influence of the oxidation rate of the Mn cluster on the reduction/oxidation kinetics of the primary quinone acceptor (Q_A) of PSII.     

QSPR Modeling of Potentiometric Mg2+/Ca2+ Selectivity for PVC‐plasticized Sensor Membranes

The development of novel ionophores for ion‐selective sensors is a time‐consuming and tedious process requiring synthesis of candidate substances, preparation of plasticized polymeric membranes, and their thorough characterization with traditional protocols to assess sensitivity, selectivity and detection limits for target ions. The vast amount of literature data accumulated on various ion‐selective sensors allows for significant facilitation of the development through in silico experiments. In this report, we performed the feasibility study on the prediction of potentiometric Mg²⁺/Ca²⁺ selectivity for various amide ligands using quantitative structure‐property relationship (QSPR) modeling. The approach proved to be promising for ionophore screening purposes with achieved precision in prediction of the selectivity coefficient logK(Mg²⁺/Ca²⁺) of 0.5 in the range from ?1.7 to +2.3. The study also shows a route for prediction of new potential ionophores with high selectivity values.     

Selective detection of the structural changes upon photoreactions of several redox cofactors in photosystem II by means of light-induced ATR-FTIR difference spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was applied for the first time to detect the structural changes upon photoreactions of redox cofactors in photosystem II (PSII). The PSII-enriched membranes from spinach were adsorbed on the surface of a silicon prism, and FTIR measurements of various redox cofactors were performed for the same sample but under different conditions by exchanging buffers in a flow cell. Light-induced FTIR difference spectra upon redox reactions of the oxygen-evolving Mn cluster, the primary quinone electron acceptor QA, the redox-active tyrosine YD, the primary electron acceptor pheophytin, and the primary electron donor chlorophyll P680 were successively recorded in buffers including different redox reagents and inhibitors. All of these cofactors remained active in the PSII membranes on the silicon surface, and the resultant spectra were basically identical to those previously recorded by the conventional transmission method. These ATR-FTIR measurements enable accurate comparison between reactions of different active sites in a single PSII sample. The present results demonstrated that the ATR-FTIR spectroscopy is a useful technique for investigation of the reaction mechanism of PSII.     

Polymer-Modified Single-Walled Carbon Nanotubes Affect Photosystem II Photochemistry,Intersystem Electron Transport Carriers and Photosystem I End Acceptors in Pea Plants

Single-walled carbon nanotubes (SWCNT) have recently been attracting the attention of plant biologists as a prospective tool for modulation of photosynthesis in higher plants. However, the exact mode of action of SWCNT on the photosynthetic electron transport chain remains unknown. In this work, we examined the effect of foliar application of polymer-grafted SWCNT on the donor side of photosystem II, the intersystem electron transfer chain and the acceptor side of photosystem I. Analysis of the induction curves of chlorophyll fluorescence via JIP test and construction of differential curves revealed that SWCNT concentrations up to 100 mg/L did not affect the photosynthetic electron transport chain. SWCNT concentration of 300 mg/L had no effect on the photosystem II donor side but provoked inactivation of photosystem II reaction centres and slowed down the reduction of the plastoquinone pool and the photosystem I end acceptors. Changes in the modulated reflection at 820 nm, too, indicated slower re-reduction of photosystem I reaction centres in SWCNT-treated leaves. We conclude that SWCNT are likely to be able to divert electrons from the photosynthetic electron transport chain at the level of photosystem I end acceptors and plastoquinone pool in vivo. Further research is needed to unequivocally prove if the observed effects are due to specific interaction between SWCNT and the photosynthetic apparatus.     

Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells

Calcium (Ca²⁺) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca²⁺]_i is the use of fluorescent Ca²⁺ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca²⁺ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca²⁺-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca²⁺-free and Ca²⁺-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca²⁺-free and Ca²⁺-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca²⁺ sensitivity is reduced. Nevertheless, quantitative [Ca²⁺]_i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.

Functional Models for the Oxygen-Evolving Complex of Photosystem II

In the last ten years, a number of advances have been made in the study of the oxygen-evolving complex (OEC) of photosystem II (PSII). Along with this new understanding of the natural system has come rapid advance in chemical models of this system. The advance of PSII model chemistry is seen most strikingly in the area of functional models where the few known systems available when this topic was last reviewed has grown into two families of model systems. In concert with this work, numerous mechanistic proposals for photosynthetic water oxidation have been proposed. Here, we review the recent efforts in functional model chemistry of the oxygen-evolving complex of photosystem II.     

Spectroscopic study of the complexation of an aza-15-crown-5 containing chromofluoroionophore with Ba2+ and Ca2+ cations

The complexation of 1-[(4-benzothiazolyl)phenyl]-4,7,10,13-tetraoxa-1-aza-cyclopenta-decane with Ba²⁺ and Ca²⁺ cations was investigated spectrophotometrically and spectrofluorometrically. The stability constants of the complexes formed are: for Ba²⁺ logK _st=3.17±0.01 (absorption) and logK _st=2.95±0.03 (fluorescence); for Ca²⁺ logK _st=3.71±0.02 (absorption) and logK _st=3.58±0.05 (fluorescence). Protonation of the ligand leads to fluorescence quenching. AM1 and PPP quantum chemical calculations were used to predict molecular geometry, proton affinities and the spectra of the compounds studied.Dedicated to Prof. Dr. Karl-Heinz Drexhage on the occasion of his 60th birthday     

Engineering a subcellular targetable, red-emitting, and ratiometric fluorescent probe for Ca2+ and its bioimaging applications

A new, visible-light-excited and red-emitting fluorescent Ca²⁺ probe, STDBT, was synthesized, which consists of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid as a Ca²⁺-chelating moiety and two benzothiazolium hemicyanine dyes as fluorophores. The spectral profiles of its free and Ca²⁺-bound forms were studied. Upon addition of Ca²⁺, the fluorescence spectra of STDBT displayed a significant enhancement (about 48-fold) in fluorescence intensity and a 20-nm blueshift (from 600 to 580 nm) in the emission spectrum. Both the absorption and the excitation spectra of STDBT showed a very large (more than 100 nm) hypsochromic shift in the long-wavelength maxima upon binding with Ca²⁺. Interestingly, in contrast with the commonly used Ca²⁺ indicator Fluo-3, when the acetoxymethyl ester of STDBT enters into cells, it distributes both in the cytosol and the nucleus, but displays a very clear boundary between the two compartments. This allows STDBT to be used as a double targetable Ca²⁺ probe that can be used to report cytoplasmic Ca²⁺ and nuclear Ca²⁺ simultaneously.     

Comparative Time-Resolved Photosystem II Chlorophyll a Fluorescence Analyses Reveal Distinctive Differences between Photoinhibitory Reaction Center Damage and Xanthophyll Cycle-Dependent Energy Dissipation*

Abstract— The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important pho-toprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (ΔpH) and the carotenoid pigment interconversions of the xanthophyll cycle. Co-incidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chi a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chi a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhi-bition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from ～2 ns to ～0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonra-diative decay path for the charge-separated state of the PSII reaction center. In contrast, the ΔpH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et ah, 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the ΔpH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.     

PHOTOSENSITIZED INHIBITION OF PHOTOSYNTHETIC 14 CO2 FIXATION BY α-TERTHIENYL and ULTRAVIOLET-A*

Abstract –A variety of naturally occurring photosensitizers of plant origin were tested for their ability to cause ultraviolet-A (UVA)-dependent inhibition of photosynthetic ¹⁴CO₂ fixation in leaf disks of Pisum sativum L. At 0.1 mM concentrations and 60 min UVA irradiation, α-terthienyl was strongly inhibitory, harmine and sanguinarine inhibited to a lesser degree, and curcumin, 8-methoxypsoralen and nordihydroguaiaretic acid had no effect under the conditions tested. Alpha-terthienyl + UVA treatments that fully inhibited ¹⁴CO₂ fixation had no effect on fresh weight, chlorophyll or protein content of the tissue. Chloroplast malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were inhibited 45% and 29%, respectively, by α-terthienyl + UVA treatment. Electron transport from H₂O to the reducing side of photosystem I was inhibited to a similar extent, suggesting interference with the reductive activation of chloroplast enzymes. Alpha-terthienyl + UVA-treated tissue exhibited a seven-fold increase in leakage of labeled photosynthate into the external medium. Treated leaf disks showed no ability to recover ¹⁴CO₂ fixing ability over a 24 h period. These results indicate photosensitized damage at the level of the thylakoid membranes resulting in partial loss of electron transport capability and more general damage to chloroplast and cell membranes.     

Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+‐regulated Photoproteins of Different Organisms

Upon binding their metal ion cofactors, Ca²⁺‐regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca²⁺‐regulated photoproteins—aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata—demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca²⁺‐regulated photoproteins.     

Function of ROC4 in the Efficient Repair of Photodamaged Photosystem II in Arabidopsis†

ROC4 is the only cyclophilin in the chloroplast stroma. Here, we used the T‐DNA knockout mutant of roc4 to study the physiological role of ROC4 in vivo in Arabidopsis thaliana. Our results showed that ROC4 is not required for the biogenesis and functional operation of photosystem II (PSII). However, growth in greenhouse and PSII activity, as detected by photoinhibition measurements showed increased sensitivity to high light irradiance in the mutant. In the presence of chloroplast protein synthesis inhibitor lincomycin, which blocks de novo protein synthesis and thus the repair of PSII, wild‐type and mutant plants showed a similar extent of inactivation of PSII activity. The recovery of PSII activity in roc4 leaves from photoinhibition is also impaired compared with that of wild‐type plants. Immunoblot analysis showed that the degradation of PSII reaction center proteins occurred at a similar rate in the presence of lincomycin in wild‐type and mutant plants. Thus, these results suggest that ROC4 functions in the repair of photodamaged PSII.     

Overall kinetics of photosystem II: pH Dependence and deuterium isotope effect

The overall kinetics of photosystem II was scanned by means of a double flash technique. Oxygen evolution by pea thylakoids provided with an artificial electron acceptor was measured under a regime of double flashes of variant intervals. The pH optimum in H₂O lies near p¹H 7.2 (p¹H meaning the pH in H₂O), with a first-order rate constant of 800 s⁻¹ at 20°C. In deuterium oxide (D₂O), a plateau of maximum reaction rate was found between p²H 6.6 and 7.8 (p²H meaning the “pH” in D₂O), the highest rate constant being 550 s⁻¹. The apparent kinetic deuterium isotope effect is therefore 1.45. Outside this plateau region, there seems to exist another isotope effect of 1.2 to 1.3. These effects are small but may nevertheless reflect the fact that more than one step of the photosystem II reaction sequence is involved in the splitting of a bond to hydrogen. However, the effects may also be solvent effects or located on the acceptor side of the photosystem.     

Ca2+ Responsive Microgel-Stabilized Pickering Emulsions

As an ionic cross-linker that can change the size of poly(N-isopropylacrylamide-co-acrylic acid) microgel, Ca²⁺ is applied as a trigger to demulsify microgel-stabilized oil/water Pickering emulsions. The influence of Ca²⁺ induced intra-particle ionic cross-linking and inter-particle aggregation on the stability of microgel-stablized “Pickering” emulsion is described. At low and mediate concentration of Ca²⁺, ionic cross-linking can change the internal elasticity of the microgel, and cause the coarsening of the oil droplets. At high concentration of Ca²⁺, microgels flocculate due to the salt out effect and the emulsion is destabilized. This work provide a facile method to control the stability of the Pickering emulsions at ambient condition.     

A Novel Dual Channel Fluorescent Probe for Ca2+ and Zn2+ Based on a Coumarin Schiff Base

A novel coumarin Schiff base fluorescent probe ethyl 7‐hydroxycoumarin‐3‐carboxylate‐8‐formaldehyde benzoyl hydrazone ( EBH ) has been designed and synthesized which shows solvent dependent dual sensing, viz., recognition of Ca²⁺ in DMF‐H₂O (9∶1, V /V ) solution based on C = N isomerization, photoinduced electron transfer (PET ) inhibition and chelation‐enhanced fluorescence (CHEF ) mechanism as well as detection of Zn²⁺ in H₂O‐CH₃OH (9∶1, V /V ) solution by excited‐state intramolecular proton transfer (ESIPT ) and CHEF processes. The structure of the probe EBH has been confirmed by single‐crystal X‐ray diffraction analysis. Meanwhile, the probe was also used to image intracellular Zn²⁺ ions in MCF ‐7 cells with a good performance.     

LaCl3提高菠菜光系统Ⅱ活性的作用机制

The effect of LaCl3 on the K3Fe(CN)6 (FeCy) reduction rate and the oxygen-evolving rate of PSU particles of spinach, and the spectral characterization of the D1/D2/Cytb559 of a PSII reaction center complex consisting of three polypeptides from spinach were studied. The experimental results showed that LaCl3 could significantly accelerate the transformation from light energy to electric energy, the electron transport, water photolysis and oxygen evolution of PSII of spinach, which was related to the spectral characterization of the D1/D2/Cytb559 complex.Soret band and Q band of Chl-a of UV-vis spectrum of D1/D2/Cytb559 complex were blue shifted, and the fluorescence emission peak was blue shifted in LaCl3 treated spinach compared with that in the control. The EXAFS (extended X-ray absorption fine structure spectroscopy) revealed that La^3 was coordinated with 8 nitrogen or oxygen atoms in the first coordination shell with La-N or La-O bond length of 0.254 nm, and with 6 nitrogen or oxygen atoms in the second coordination shell with La-N or La-O bond length of 0.321 nm in the D1/D2/Cytb559 complex. The CD suggested that the secondary structure of D1/D2/Cytb559 complex have been litfie affected by the treatment of LaCl3.     

Investigation of the electron transfer site of p-benzoquinone in isolated photosystem II particles and thylakoid membranes using alpha- and beta-cyclodextrins

The electron transfer sites of p-benzoquinone (pBQ) and 2,6-dichloro-p-benzoquinone (DCBQ) were investigated in thylakoid membranes and isolated photosystem II (PSII) particles from barley (Hordeum vulgare) using alpha- and beta-cyclodextrins (CD) at concentrations up to 16 mM. In CD-treated thylakoid membranes incubated with DCBQ the electron transport through PSII, estimated as oxygen evolution (OE), is largely enhanced according to a S-shaped (sigmoidal) dose-response curve displaying a sharp inflection point, or transition. The maxima percent OE enhancement at cyclodextrin concentrations above 14 mM are about 100% (alpha-CD) and 190% (beta-CD). On the contrary, in thylakoid membrane preparations incubated with pBQ as electron acceptor one observes an OE inhibition of about 30% which might result from the depletion of the thylakoid membrane of its plastoquinone content. It was also found that in isolated PSII particles incubated with either pBQ or DCBQ the cyclodextrins induce only a small OE enhancement. Moreover, the observation in CD-treated thylakoid membranes incubated with pBQ of a residual, non-inhibited oxygen-evolving activity of about 70% puts a twofold question. That is, either the plastoquinone depletion was not complete, or, pBQ binds to electron acceptor sites of different nature. From this and data published in the literature, it is concluded that in the thylakoid membrane (i) DCBQ binds to Q(B), as is generally accepted, and (ii) pBQ binds to the plastoquinol molecules in the PQ pool and most likely also to Q(B), thereby in accord with Satoh et al.'s model [K. Satoh, M. Ohhashi, Y. Kashino, H. Koike, Plant Cell Physiol. 36 (1995) 597-605]. An attractive alternative hypothesis is the direct interaction of pBQ with the non-haem Fe(2+) between Q(A) and Q(B).     

Function and association of CyanoP in photosystem II of Synechocystis sp. PCC 6803

The CyanoP protein is a cyanobacterial homolog of the PsbP protein, which is an extrinsic subunit of photosystem II (PSII) in green plant species. The molecular function of CyanoP has been investigated in mutant strains of Synechocystis but inconsistent results have been reported by different laboratories. In this study, we generated and characterized a Synechocystis mutant in which entire region of the CyanoP gene was eliminated. After repeated subculture in CaCl₂-depleted medium, growth retardation was clearly observed for a CyanoP knockout mutant of Synechocystis sp. PCC 6803 (?P). The PSII-mediated oxygen-evolving activity of the ?P cells was more susceptible to depletion of CaCl₂ than that of wild-type cells. The 77 K fluorescence emission spectra indicated that energy coupling between phycobilisome and PSII was perturbed in both wild-type and ?P cells under CaCl₂-depleted conditions, and was more evident for the ?P mutant. To examine the association of CyanoP with PSII complexes, we tested several detergents for solubilization of thylakoid membranes and showed that CyanoP was partly included in fractions containing large protein complexes in gel-filtration analysis. These results indicate that CyanoP constitutively stabilizes PSII functionality in vivo.