A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y_D^ox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S₂Q_A⁻ and S₂Q_B⁻ charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y_D⁺Q_A⁻ recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S₁-to-S₂-transition and S₂-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q_A to Q_B electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

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.     

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

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

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F_V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg²⁺. It is also demonstrated that the increase in F_V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

Molecular interference of Cd with Photosystem II

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

Wavelength dependence of the fluorescence emission under conditions of open and closed Photosystem II reaction centres in the green alga Chlorella sorokiniana

The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open (F₀) and closed (F_M) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo, in the green alga Chlorella sorokiniana. The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, F_V = F_M − F₀, over F_M, a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the F_V/F_M ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of F_V/F_M principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F₀ and F_M indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo.     

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: Accumulation and function of QB-nonreducing centers

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in F_m relative to F_o, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of Q_B-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of Q_B-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.     

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+.     

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.     

Luminostat operation: a tool to maximize microalgae photosynthetic efficiency in photobioreactors during the daily light cycle?

The luminostat regime has been proposed as a way to maximize light absorption and thus to increase the microalgae photosynthetic efficiency within photobioreactors. In this study, simulated outdoor light conditions were applied to a lab-scale photobioreactor in order to evaluate the luminostat control under varying light conditions. The photon flux density leaving the reactor (PFD_out) was varied from 4 to 20 μmol photons m⁻² s⁻¹and the productivity and photosynthetic efficiency of Chlorella sorokiniana were assessed.Maximal volumetric productivity (1.22 g kg⁻¹ d⁻¹) and biomass yield on PAR photons (400-700 nm) absorbed (1.27 g mol⁻¹) were found when PFD_out was maintained between 4 and 6 μmol photons m⁻² s⁻¹. The resultant photosynthetic efficiency was comparable to that already reported in a chemostat-controlled reactor. A strict luminostat regime could not be maintained under varying light conditions. Further modifications to the luminostat control are required before application under outdoor conditions.     

Comparison of the electron transport properties of the psbo1 and psbo2 mutants of Arabidopsis thaliana

Genome sequence of Arabidopsis thaliana (Arabidopsis) revealed two psbO genes (At5g66570 and At3g50820) which encode two distinct PsbO isoforms: PsbO1 and PsbO2, respectively. To get insights into the function of the PsbO1 and PsbO2 isoforms in Arabidopsis we have performed systematic and comprehensive investigations of the whole photosynthetic electron transfer chain in the T-DNA insertion mutant lines, psbo1 and psbo2.The absence of the PsbO1 isoform and presence of only the PsbO2 isoform in the psbo1 mutant results in (i) malfunction of both the donor and acceptor sides of Photosystem (PS) II and (ii) high sensitivity of PSII centers to photodamage, thus implying the importance of the PsbO1 isoform for proper structure and function of PSII. The presence of only the PsbO2 isoform in the PSII centers has consequences not only to the function of PSII but also to the PSI/PSII ratio in thylakoids. These results in modification of the whole electron transfer chain with higher rate of cyclic electron transfer around PSI, faster induction of NPQ and a larger size of the PQ-pool compared to WT, being in line with apparently increased chlororespiration in the psbo1 mutant plants. The presence of only the PsbO1 isoform in the psbo2 mutant did not induce any significant differences in the performance of PSII under standard growth conditions as compared to WT. Nevertheless, under high light illumination, it seems that the presence of also the PsbO2 isoform becomes favourable for efficient repair of the PSII complex.     

In intact leaves, the maximum fluorescence level (FM) is independent of the redox state of the plastoquinone pool: A DCMU-inhibition study

The effects of DCMU (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea) on the fluorescence induction transient (OJIP) in higher plants were re-investigated. We found that the initial (F₀) and maximum (F_M) fluorescence levels of DCMU-treated leaves do not change relative to controls when the treatment is done in complete darkness and DCMU is allowed to diffuse slowly into the leaves either by submersion or by application via the stem. Simultaneous 820 nm transmission measurements (a measure of electron flow through Photosystem I) showed that in the DCMU-treated samples, the plastoquinone pool remained oxidized during the light pulses whereas in uninhibited leaves, the F_M level coincided with a fully reduced electron transport chain. The identical F_M values with and without DCMU indicate that in intact leaves, the F_M value is independent of the redox state of the plastoquinone pool. We also show that (i) the generally observed F₀ increase is probably due to the presence of (even very weak) light during the DCMU treatment, (ii) vacuum infiltration of leaf discs leads to a drastic decrease of the fluorescence yield, and in DCMU-treated samples, the F_M decreases to the I-level of their control (leaves vacuum infiltrated with 1% ethanol), (iii) and in thylakoid membranes, the addition of DCMU lowers the F_M relative to that of a control sample.     

Putrescine stimulates chemiosmotic ATP synthesis

Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a ΔpH and (iv) a pool of putrescine.     

Localization of GABA high-affinity binding sites in the pancreas of neonatal rat

Summary The localization of gamma-aminobutyric acid (GABA) high-affinity binding sites was investigated in the exocrine and endocrine pancreas of neonatal rats by means of ³H-GABA autoradiography. GABA-binding was identified on Schwann cells and on the cells of the intralobular excretory ducts. In the endocrine part of the pancreas, no labelling was observed except in peripheral islet cells which, on the basis of their scarcity and distribution, could be somatostatin cells. Furthermore, peri-insular innervation showed considerable labelling.     

On the advantages of using green light to study fluorescence yield changes in leaves

In photosynthetic chains, the kinetics of fluorescence yield depends on the photochemical rates at the level of both Photosystem I and II and thus on the absorption cross section of the photosynthetic units as well as on the coupling between light harvesting complexes and photosynthetic traps. A new set-up is described which, at variance with the commonly used set-ups, uses of a weakly absorbed light source (light-emitting diodes with maximum output at 520 nm) to excite the photosynthetic electron chain and probe the resulting fluorescence yield changes and their time course. This approach optimizes the homogeneity of the exciting light throughout the leaf and we show that this homogeneity narrows the distribution of the photochemical rates. Although the exciting light is weakly absorbed, the possibility to tune the intensity of the light emitting diodes allows one to reach photochemical rates ranging from 10⁴ s^− 1 to 0.25 s^− 1 rendering experimentally accessible different functional regimes. The variations of the fluorescence yield induced by the photosynthetic activity are qualitatively and quantitatively discussed. When illuminating dark-adapted leaves by a weak light, the kinetics of fluorescence changes displays a pronounced plateau which precedes the fluorescence increase reflecting the full reduction of the plastoquinone pool. We ascribe this plateau to the time delay needed to reduce the photosystem I electron acceptors.     

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

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

Role of specific residues in coenzyme binding, charge-transfer complex formation, and catalysis in Anabaena ferredoxin NADP-reductase

Two transient charge-transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP⁺ reductase (FNR) with NADP⁺/H, FNR_ox-NADPH (CTC-1), and FNR_rd-NADP⁺ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP⁺/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.     

Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.     

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.