Unique constitution of photosystem I with a novel subunit in the cyanobacterium Gloeobacter violaceus PCC 7421

Constitution of the photosystem I complex isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 was investigated by tricine-urea-SDS-PAGE, followed by peptide mass fingerprinting or N-terminal sequencing. Eight subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL and PsaM) were identified as predicted from the genome sequence. A novel subunit (PsaZ) was discovered, but PsaI, PsaJ, PsaK and PsaX were absent. PsaB has a C-terminal extension with 155 amino acids in addition to the conserved region and this domain is similar to the peptidoglycan-binding domain. These results suggest that PS I complexes of G. violaceus have unique structural properties.     

The low energy emitting states of the Lhca4 subunit of higher plant photosystem I

The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.     

Evidence that histidine forms a coordination bond to the A0A and A0B chlorophylls and a second H-bond to the A1A and A1B phylloquinones in M688HPsaA and M668HPsaB variants of Synechocystis sp. PCC 6803

The axial ligands of the acceptor chlorophylls, A_0A and A_0B, in Photosystem I are the Met sulfur atoms of M688_PsaA and M668_PsaB. To determine the role of the Met, His variants were generated in Synechocystis sp. PCC 6803. Molecular dynamics simulations on M688H_PsaA show that there exist low energy conformations with the His coordinated to A_0A and possibly H-bonded to A_1A. Transient EPR studies on M688H_PsaA indicate a more symmetrical electron spin distribution in the A_1A phyllosemiquinone ring consistent with the presence of an H-bond to the C1 carbonyl. Ultrafast optical studies on the variants show that the 150 fs charge separation between P₇₀₀ and A₀ remains unaffected. Studies on the ns timescale show that 57% of the electrons are transferred from A_0A⁻ to A_1A in M688H_PsaA and 48% from A_0B⁻ to A_1B in M668H_PsaB; the remainder recombine with P₇₀₀⁺ with 1/e times of 25 ns and 37 ns, respectively. Those electrons that reach A_1A and A_1B in the branch carrying the mutation are not transferred to F_X, but recombine with P₇₀₀⁺ with 1/e times of ~ 15 μs and ~ 5 μs, respectively. Hence, the His is coordinated to A₀ in all populations, but in a second population, the His may be additionally H-bonded to A₁. Electron transfer from A₀ to A₁ occurs only in the latter, but the higher redox potentials of A₀ and A₁ as a result of the stronger coordination bond to A₀ and the proposed second H-bond to A₁ preclude electron transfer to the Fe/S clusters.     

Spectroelectrochemistry of P700 in native photosystem I particles and diethyl ether-treated thylakoid membranes from spinach and Thermosynechococcus elongatus

The redox potentials (E(composite function')) of P700 in intact and diethyl ether-treated thylakoid membranes as well as native photosystem (PS) I particles from spinach and Thermosynechococcus elongatus have been measured by a spectroelectrochemistry with an error range of +/-2-3 mV. Stepwise removal of antenna pigments by ether treatment caused distinct shifts of the E( composite function') value with increasing degree of water saturation in ether; negatively from +471 to +428 mV for spinach, but positively from +423 to +436 mV for T. elongatus. Such a contrasting behavior is discussed by invoking the mode of action of ether on the microenvironments around P700.     

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⁻.     

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.     

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.     

Photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina

Fourier transform infrared spectroscopy (FTIR) difference spectroscopy in combination with deuterium exchange experiments has been used to study the photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina. Comparison of (P740(+)-P740) and (P700(+)-P700) FTIR difference spectra show that P700 and P740 share many structural similarities. However, there are several distinct differences also: 1), The (P740(+)-P740) FTIR difference spectrum is significantly altered upon proton exchange, considerably more so than the (P700(+)-P700) FTIR difference spectrum. The P740 binding pocket is therefore more accessible than the P700 binding pocket. 2), Broad, "dimer" absorption bands are observed for both P700(+) and P740(+). These bands differ significantly in substructure, however, suggesting differences in the electronic organization of P700(+) and P740(+). 3), Bands are observed at 2727(-) and 2715(-) cm(-1) in the (P740(+)-P740) FTIR difference spectrum, but are absent in the (P700(+)-P700) FTIR difference spectrum. These bands are due to formyl CH modes of chlorophyll d. Therefore, P740 consists of two chlorophyll d molecules. Deuterium-induced modification of the (P740(+)-P740) FTIR difference spectrum indicates that only the highest frequency 13(3) ester carbonyl mode of P740 downshifts, indicating that this ester mode is weakly H-bonded. In contrast, the highest frequency ester carbonyl mode of P700 is free from H-bonding. Deuterium-induced changes in (P740(+)-P740) FTIR difference spectrum could also indicate that one of the chlorophyll d 3(1) carbonyls of P740 is hydrogen bonded.     

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.     

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.     

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.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

The induction of CP43′ by iron-stress in Synechococcus sp. PCC 7942 is associated with carotenoid accumulation and enhanced fatty acid unsaturation

Comparative lipid analysis demonstrated reduced amount of PG (50%) and lower ratio of MGDG/DGDG in iron-stressed Synechococcus sp. PCC 7942 cells compared to cells grown under iron sufficient conditions. In parallel, the monoenoic (C:1) fatty acids in MGDG, DGDG and PG increased from 46.8%, 43.7% and 45.6%, respectively in control cells to 51.6%, 48.8% and 48.7%, respectively in iron-stressed cells. This suggests increased membrane dynamics, which may facilitate the diffusion of PQ and keep the PQ pool in relatively more oxidized state in iron-stressed compared to control cells. This was confirmed by chlorophyll fluorescence and thermoluminescence measurements. Analysis of carotenoid composition demonstrated that the induction of isiA (CP43′) protein in response to iron stress is accompanied by significant increase of the relative abundance of all carotenoids. The quantity of carotenoids calculated on a Chl basis increased differentially with nostoxanthin, cryptoxanthin, zeaxanthin and β-carotene showing 2.6-, 3.1-, 1.9- and 1.9-fold increases, respectively, while the relative amount of caloxanthin was increased only by 30%. HPLC analyses of the pigment composition of Chl-protein complexes separated by non-denaturating SDS-PAGE demonstrated even higher relative carotenoids content, especially of cryptoxanthin, in trimer and monomer PSI Chl-protein complexes co-migrating with CP43′ from iron-stressed cells than in PSI complexes from control cells where CP43′ is not present. This implies a carotenoid-binding role for the CP43′ protein which supports our previous suggestion for effective energy quenching and photoprotective role of CP43′ protein in cyanobacteria under iron stress.     

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.     

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.     

A new fluorescence band F689 in photosystem II revealed by picosecond analysis at 4-77 K: Function of two terminal energy sinks F689 and F695 in PS II

We performed picosecond time-resolved fluorescence spectroscopy in spinach photosystem II (PS II) particles at 4, 40, and 77 K and identified a new fluorescence band, F689. F689 was identified in addition to the well-known F685 and F695 bands in both analyses of decay-associated spectra and global Gaussian deconvolution of time-resolved spectra. Its fast decay suggests the energy transfer directly from F689 to the reaction center chlorophyll P680. The contribution of F689, which increases only at low temperature, explains the unusually broad and variable bandwidth of F695 at low temperature. Global analysis revealed the three types of excitation energy transfer/dissipation processes: (1) energy transfer from the peripheral antenna to the three core antenna bands F685, F689, and F695 with time constants of 29 and 171 ps at 77 and 4 K, respectively; (2) between the three core bands (0.18 and 0.82 ns); and (3) the decays of F689 (0.69 and 3.02 ns) and F695 (2.18 and 4.37 ns). The retardations of these energy transfer rates and the slow F689 decay rate produced the strong blue shift of the PS II fluorescence upon the cooling below 77 K.     

Reductive titration of photosystem I and differential extinction coefficient of P700 at 810-950 nm in leaves

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e⁻) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e⁻ carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1-15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch (Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e⁻ generated in PSII by the flash was measured as four times O₂ evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700⁺ and PC⁺ components using the abovementioned oxidative titration method. The P700⁺ component was related to the absolute number of e⁻ that reduced the P700⁺ to calculate the extinction coefficient. The effective differential extinction coefficient of P700⁺ at 810-950 nm was 0.40±0.06 (S.D.)% of transmittance change per μmol P700⁺ m⁻² or 17.6±2.4 mM⁻¹ cm⁻¹. The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (±14% S.D.) is mainly caused by light-scattering properties of the leaf.     

Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana

Thylakoids of the diatom Cyclotella meneghiniana were separated by discontinuous gradient centrifugation into photosystem (PS) I, PSII, and fucoxanthin-chlorophyll protein (FCP) fractions. FCPs are homologue to light harvesting complexes of higher plants with similar function in e.g. brown algae and diatoms. Still, it is unclear if FCP complexes are specifically associated with either PSI or PSII, or if FCP complexes function as one antenna for both photosystems. However, a trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have been described for C. meneghiniana, already. In this study, biochemical and spectroscopical evidences are provided that reveal a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PSII associated Fcp antenna resembles FCPa since it contains Fcp2 and Fcp6, at least three different Fcp polypeptides are associated with PSI. By re-solubilisation and a further purification step Fcp polypeptides were partially removed from PSI and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby a protein related to Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PSI, whereas presumably Fcp5, a subunit of the FCPb complex, is only loosely bound to the PSI core. Thus, an association of FCPb and PSI is assumed.     

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

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