Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

Some Photosystem II properties depending on the D1 protein variants in Thermosynechococcus elongatus

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II complex which bears together with PsbD, the D2 protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA₃ gene over the psbA₁ gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at < 10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q_x band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo_D1 Q_x band induced by Q_A⁻ (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t_1/2 > 50 ms) Q_A⁻ yield was ∼ 95% in D1:3, and ∼ 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q_x band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13¹-keto of Pheo_D1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P_D1, in particular Ser153Ala. Photo-induced Q_A⁻ formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P⁺.     

The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II (PSII) complex. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues of PsbA, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this study, we found a new hemoprotein that is expressed when the T. elongatus genome has only the psbA₂ gene for D1. This hemoprotein was found in both the non-membrane proteins and associated to the purified PsbA2-PSII core complex. This protein could be removed by the washing of PSII with Tris-washing or CaCl₂-washing. From MALDI-TOF/TOF spectrometry, N-terminal sequencing and MALDI-MS/MS analysis upon tryptic digestion, the new hemoprotein was identified to be the tll0287 gene product with a molecular mass close to 19 kDa. Until now, tll0287 was registered as a gene encoding a hypothetical protein with an unknown function. From the amino acid sequence and the EPR spectrum the 5th and 6th axial ligands of the heme iron are the His145 and likely either the Tyr93, Tyr159 or Tyr165, respectively. From EPR, the heme containing Tll0287 protein associated to PsbA2-PSII corresponds to approximately 25% of the Cytc₅₅₀ content whereas, from SDS page analysis, the total amount of Tll0287 with and without the heme seems almost in a stoichiometric amount with PsbA2-PSII. Homologous genes to tll0287 are found in several cyanobacteria. Possible roles for Tll0287 are suggested.     

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr_Z were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr_Z/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr_Z by P₆₈₀^+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn₄CaO₅ cluster in the S₂ and S₃ states could weaken the strength of the H-bond interaction between Tyr_Z^● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr_Z.     

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA₁ or the psbA₃ gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491–1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b₅₅₉ (Cyt b₅₅₉) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b₅₅₉ properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b₅₅₉ likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.     

Energetics and proton release in photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA2 or psbA3 gene

In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of Phe_D1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative E_m of Phe_D1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2-PSII by measuring the thermoluminescence from the S₂Q_A⁻/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the E_m of Phe_D1^–/Phe_D1 was decreased by ∼30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S₂Tyr_Z^• to S₃Tyr_Z and S₃Tyr_Z^• → (S₃Tyr_Z^•)’ transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.     

Laboratory experiments on the effects of variable suspended sediment concentrations on the ecophysiology of the porcelain crab Petrolisthes elongatus (Milne Edwards, 1837)

The porcelain crab Petrolisthes elongatus is a particulate suspension feeding species common to coastal areas of New Zealand (NZ). Consistent with the responses of other suspension feeding species, it is likely to be negatively influenced by elevated suspended sediment concentrations. Laboratory experiments were conducted to quantify the effect of temperature (12 °C, 15 °C and 18 °C) and suspended sediment concentration (total particulate matter (TPM): low < 100 mg L^− 1; medium 100-1000 mg L^− 1; high > 1000 mg L^− 1) on the clearance rate (CR in L h^− 1), oxygen uptake rate (VO₂ in mL h⁻¹), net absorption efficiency (AE), and net energy budget (NEB in J h^− 1) of P. elongatus across a range of sizes. Variation in CR and AE was independent of temperature and of body size, but were significantly different (P < 0.05) at low and medium suspended sediment concentrations compared with high suspended sediment concentrations. CR responded in a non-linear manner to changes in TPM, increasing with TPM up to a maximum value at medium-low concentrations (approximately 250 mg L^− 1) and then decreasing thereafter. CR had almost completely shut down at TPM concentrations of > 1000 mg L^− 1 and at particulate organic matter (POM) concentrations of > 250 mg L^− 1. AE was zero at approximate TPM and POM values of 1200 mg L^− 1 and 300 mg L^− 1, respectively. VO₂ was positively correlated with body size and with temperature, but was independent of TPM. NEB values for P. elongatus were low (approx 110 J g^− 1 h^− 1) at low sediment concentrations, were high (approx 320 J g^− 1 h^− 1) at medium sediment concentrations, and were negative (approx − 114 J g^− 1 h^− 1) at high sediment concentrations. These findings indicate that P. elongatus is likely to be food-limited at sediment concentrations of < 100 mg L^− 1, and severely negatively affected at sediment concentrations of > 1000 mg L^− 1, at least for the duration of such events which may persist for 2-3 days in coastal environments where this crab occurs.     

Transcription of a “silent” cyanobacterial psbA gene is induced by microaerobic conditions

Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed.     

Purification, crystallization and X-ray diffraction analyses of the T. elongatus PSII core dimer with strontium replacing calcium in the oxygen-evolving complex

The core complex of photosystem II (PSII) was purified from thermophillic cyanobaterium Thermosynechococcus elongatus grown in Sr²⁺-containing and Ca²⁺-free medium. Functional in vivo incorporation of Sr²⁺ into the oxygen-evolving complex (OEC) was confirmed by EPR analysis of the isolated and highly purified SrPSII complex in agreement with the previous study of Boussac et al. [J. Biol. Chem. 279 (2004) 22809-22819]. Three-dimensional crystals of SrPSII complex were obtained which diffracted to 3.9 Å and belonged to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 133.6 Å, b = 236.6 Å, c = 307.8 Å. Anomalous diffraction data collected at the Sr K-X-ray absorption edge identified a novel Sr²⁺-binding site which, within the resolution of these data (6.5 Å), is consistent with the positioning of Ca²⁺ in the recent crystallographic models of PSII [Ferreira et al. Science 303 (2004) 1831-1838, Loll et al. Nature 438 (2005) 1040-1044]. Fluorescence measurements on SrPSII crystals confirmed that crystallized SrPSII was active in transferring electrons from the OEC to the acceptor site of the reaction centre. However, SrPSII showed altered functional properties of its modified OEC in comparison with that of the CaPSII counterpart: slowdown of the Q_A-to-Q_B electron transfer and stabilized S₂Q_A⁻ charge recombination.     

Environment of TyrZ in photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr_Z^• in the (S₃Tyr_Z^•)′ is slightly modified; (ii) the split EPR signal arising from Tyr_Z^• in the (S₂Tyr_Z^•)′ state induced by near-infrared illumination at 4.2 K of the S₃Tyr_Z state is significantly modified; and (iii) the slow phases of P₆₈₀^+⋅ reduction by Tyr_Z are slowed down from the hundreds of μs time range to the ms time range, whereas both the S₁Tyr_Z^• → S₂Tyr_Z and the S₃Tyr_Z^• → S₀Tyr_Z + O₂ transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr_Z phenol and its environment, likely the Tyr-O···H···Nϵ-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr_Z being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, His-190, are responsible for these changes.     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

What can we still learn from the electrochromic band-shifts in Photosystem II?

Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the Q_X absorption of Phe_D1 differs, a band-shift in the Q_X region of Phe_D2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of Tyr_D. In contrast, a band-shift due to the formation of either Q_A^•- or Tyr_Z^• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to Phe_D1 and at ~ 544 nm as expected with Q130 H-bonded to Phe_D1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O₂-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of P_D1 is blue shifted and in the PsbA3/T179H mutant. Upon Tyr_Z^•Q_A^•- formation the Soret band of P_D1 is red shifted and the Soret band of Chl_D1 is blue shifted. In contrast, only P_D1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to P_D1 is biphasic for all the S-state transitions except for S₁ to S₂, and shows that: i) the proton release in S₀ to S₁ occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S₂ to S₃, in T. elongatus, are significantly faster than often considered. The nature of S₂Tyr_Z^• is discussed in view of the models in the literature involving intermediate states in the S₂ to S₃ transition.     

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.