Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina

We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P⁺Q⁻, between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (Q_A). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, Chl_D1. It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Steady-state and transient polarized absorption spectroscopy of photosytem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosytem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(−), 683-685(−), 696-697(−), and 711(−) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700⁺A₁⁻ or ³P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P_A, whereas the cation is localized most likely on P_B.     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0 reoxidation dynamics in Photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P₇₀₀, and a sequence of electron acceptors, A, A₀ and A₁, bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A₀, are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A₀⁻ (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A₀⁻ on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A₀⁻ is transient, and that reoxidation of A₀⁻ occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A₁. This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A₀ in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.     

Effect of the P700 pre-oxidation and point mutations near A0 on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii

Time-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700⁺). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A₀, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700⁺. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼ 7 ps and ∼ 25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A₀: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A₀. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700⁺. The same charge is proposed to be responsible for the fast A⁺A₀⁻ → AA₀ charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A₀ axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A⁺A₀⁻.     

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q_A. The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8 ± 0.5 kJ mol⁻¹ and 12.5 ± 0.7 kJ mol⁻¹ have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol⁻¹ between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.     

Formation and decay of P680 (PD1–PD2)PheoD1 radical ion pair in photosystem II core complexes

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo⁻) at 460 nm is observed with rise time of ~ 11 ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (Pheo_D1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680⁺Pheo_DI⁻. The subsequent electron transfer from Pheo_D1⁻ to primary plastoquinone electron acceptor (Q_A) was accompanied by relaxation of the 460-nm band and occurred within ~ 250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⁺ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo_DI⁻, which is very similar to that measured earlier by accumulation method. The spectrum of Pheo_DI⁻ formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo_D1. According to previous measurements in the femtosecond–picosecond time range this Chl-670 was ascribed to Chl_D1 [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45–50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~ 1 ps and ~ 14 ps. These components appear to reflect formation of P680⁺Chl_D1⁻ and P680⁺Pheo_D1⁻, respectively, as found earlier. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.     

Assignment of a kinetic component to electron transfer between iron-sulfur clusters FX and FA/B of Photosystem I

We studied the kinetics of reoxidation of the phylloquinones in Chlamydomonas reinhardtii Photosystem I using site-directed mutations in the PhQ_A-binding site and of the residues serving as the axial ligand to ec3_A and ec3_B chlorophylls. In wild type PS I, these kinetics are biphasic, and mutations in the binding region of PhQ_A induced a specific slowing down of the slow component. This slowing allowed detection of a previously unobserved 180-ns phase having spectral characteristics that differ from electron transfer between phylloquinones and F_X. The new kinetic phase thus reflects a different reaction that we ascribe to oxidation of F_X⁻ by the F_A/B FeS clusters. These absorption changes partly account for the differences between the spectra associated with the two kinetic components assigned to phylloquinone reoxidation. In the mutant in which the axial ligand to ec3_A (PsaA-Met688) was targeted, about 25% of charge separations ended in P₇₀₀⁺A₀⁻ charge recombination; no such recombination was detected in the B-side symmetric mutant. Despite significant changes in the amplitude of the components ascribed to phylloquinone reoxidation in the two mutants, the overall nanosecond absorption changes were similar to the wild type. This suggests that these absorption changes are similar for the two different phylloquinones and that part of the differences between the decay-associated spectra of the two components reflect a contribution from different electron acceptors, i.e. from an inter-FeS cluster electron transfer.     

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.     

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.     

Molecular dynamics study of the primary charge separation reactions in Photosystem I: Effect of the replacement of the axial ligands to the electron acceptor A0

Molecular dynamics (MD) calculations, a semi-continuum (SC) approach, and quantum chemistry (QC) calculations were employed together to investigate the molecular mechanics of ultrafast charge separation reactions in Photosystem I (PS I) of Thermosynechococcus elongatus. A molecular model of PS I was developed with the aim to relate the atomic structure with electron transfer events in the two branches of cofactors. A structural flexibility map of PS I was constructed based on MD simulations, which demonstrated its rigid hydrophobic core and more flexible peripheral regions. The MD model permitted the study of atomic movements (dielectric polarization) in response to primary and secondary charge separations, while QC calculations were used to estimate the direct chemical effect of the A_0A/A_0B ligands (Met or Asn in the 688/668 position) on the redox potential of chlorophylls A_0A/A_0B and phylloquinones A_1A/A_1B. A combination of MD and SC approaches was used to estimate reorganization energies λ of the primary (λ₁) and secondary (λ₂) charge separation reactions, which were found to be independent of the active branch of electron transfer; in PS I from the wild type, λ₁ was estimated to be 390 ± 20 mV, while λ₂ was estimated to be higher at 445 ± 15 mV. MD and QC approaches were used to describe the effect of substituting Met688_PsaA/Met668_PsaB by Asn688_PsaA/Asn668_PsaB on the energetics of electron transfer. Unlike Met, which has limited degrees of freedom in the site, Asn was found to switch between two relatively stable conformations depending on cofactor charge. The introduction of Asn and its conformation flexibility significantly affected the reorganization energy of charge separation and the redox potentials of chlorophylls A_0A/A_0B and phylloquinones A_1A/A_1B, which may explain the experimentally observed slowdown of secondary electron transfer in the M688N_PsaA variant. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700 induction kinetics

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA₈₁₀, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700⁻ oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700⁺ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700⁺ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q_r site of the cytb₆f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

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).     

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.     

Bidirectional electron transfer in photosystem I: Replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone (A1B) with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures

A conserved tryptophan residue located between the A_1B and F_X redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A_1A and the A_1B phyllo(semi)quinones. The kinetics of A₁⁻ reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F_X is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F_X leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F_A and F_B by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A_1B.     

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.     

The structure of genetically modified iron-sulfur cluster Fx in photosystem I as determined by X-ray absorption spectroscopy

Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster F_X, located on the core subunits PsaA/B to iron-sulfur clusters F_A/B on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of F_X cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of F_X was determined in PS I lacking clusters F_A/B by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 Å and 2.69 Å, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 Å. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to F_X is discussed.     

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.