Core Of Photosystem Research Articles

Xanthophyll cycle pigment localization and dynamics during exposure to low temperatures and light stress in vinca major

The distribution of xanthophyll cycle pigments (violaxanthin plus antheraxanthin plus zeaxanthin [VAZ]) among photosynthetic pigment-protein complexes was examined in Vinca major before, during, and subsequent to a photoinhibitory treatment at low temperature. Four pigment-protein complexes were isolated: the core of photosystem (PS) II, the major light-harvesting complex (LHC) protein of PSII (LHCII), the minor light-harvesting proteins (CPs) of PSII (CP29, CP26, and CP24), and PSI with its LHC proteins (PSI-LHCI). In isolated thylakoids 80% of VAZ was bound to protein independently of the de-epoxidation state and was found in all complexes. Plants grown outside in natural sunlight had higher levels of VAZ (expressed per chlorophyll), compared with plants grown in low light in the laboratory, and the additional VAZ was mainly bound to the major LHCII complex, apparently in an acid-labile site. The extent of de-epoxidation of VAZ in high light and the rate of reconversion of Z plus A to V following 2.5 h of recovery were greatest in the free-pigment fraction and varied among the pigment-protein complexes. Photoinhibition caused increases in VAZ, particularly in low-light-acclimated leaves. The data suggest that the photoinhibitory treatment caused an enrichment in VAZ bound to the minor CPs caused by de novo synthesis of the pigments and/or a redistribution of VAZ from the major LHCII complex.

Spectroscopic characterization of pigment binding proteins in normal-grown and iron-stressed thermophilic cyanobacteria Synecococcus sp.

The results of low temperature absorption, fluorescence and hole burning spectroscopy of pigment binding proteins in normal-grown and iron-stressed thermophilic Synecococcus sp. are reported. These experiments revealed that the growing of Synecococcus sp. under the iron-limited condition affects spectral characteristics and excited energy transfer (EET) in pigment proteins. The comparison of low temperature absorption spectra of normal-grown and iron-stressed thermophilic cyanobacteria well documents major changes in composition of the antenna systems and in composition of the core of photosystem II. The absorption of membrane chlorophyll in particular, is blue-shifted from 679 nm in normal cells to 673 nm which is caused by absorption of chlorophyll binding protein CP 34 in stressed sample. The presence of CP 34 in the photosystem II (PS 1 1 Abbreviations: APC, allophycocyanin; CC, cylindrical core; Chl, chlorophyll; LT, low temperature; PBS, phycobilisomes; PSB, phonon sideband; PC, phycocyanin; PR, peripheral rods; PS, photosystem; RC, reaction center, ZPH, zero phonon hole. II) has also been seen in the low temperature fluorescence spectra where the increased luminescence at 685 nm has been observed. This implies the decreased efficiency of photosynthesis in iron-limited sample. We studied the energy transfer in PS II by the means of fluorescence and absorption hole-burning spectroscopy which enabled to study the influence of iron-stressed condition on energy transfer and pigment–protein interaction (Debye–Waller factor). The stressed cells exhibit the broadened spectral hole width at 682 and 685.5 nm.

Topography of the Photosystem I Core Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

PsaA and PsaB are homologous integral membrane proteins that form the heterodimeric core of photosystem I. Domain-specific antibodies were generated to examine the topography of PsaA and PsaB. The purified photosystem I complexes from the wild type strain of Synechocystis sp. PCC 6803 were treated with eight proteases to study the accessibility of cleavage sites in PsaA and PsaB. Proteolytic fragments were identified using the information from N-terminal amino acid sequencing, reactivity to antibodies, apparent mass, and specificity of proteases. The extramembrane loops of PsaA and PsaB differed in their accessibility to proteases, which indicated the folded structure of the loops or their shielding by the small subunits of photosystem I. NaI-treated and mutant photosystem I complexes were used to identify the extramembrane loops that were exposed in the absence of specific small subunits. The absence of PsaD exposed additional proteolytic sites in PsaB, whereas the absence of PsaE exposed sites in PsaA. These studies distinguish PsaA and PsaB in the structural model for photosystem I that has been proposed on the basis of x-ray diffraction studies (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., and Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973). Using osmotically shocked cells for protease treatments, the N terminus of PsaA was determined to be on the n side of the photosynthetic membranes. Based on these data and available published information, we propose a topological model for PsaA and PsaB.

Indications for a dimeric organization of the antenna-depleted reaction center core of Photosystem II in thylakoids of intermittent light grown pea plants

Presently there is a debate and contradictory results on the supramolecular structure of Photosystem II (PS II) in the thylakoid membranes of cyanobacteria, green algae and higher plants that is related to the question whether PS II exists as dimer or monomer in the different membranes. PS II is composed of several subunits. The core complex, the smallest entity that is able to split water, consists of the subunits D1-D2, cyt b 559, CP43, CP47, and the 33 kDa protein. In vivo, this core complex is connected to peripheral light harvesting complexes: in cyanobacteria to the phycobilisomes and in green algae and higher plants to the Chl a/ b binding protein, the LHC II. In in vitro experiments the supramolecular structure of the core complexes has been found to be dimeric or monomeric depending on a variety of experimental factors.

Effect of 3-formylchromones substituted with 4-aminosalicylic acid and some other aniline derivatives on photosynthesis inhibition in spinach chloroplasts

The inhibitory effect was investigated of 16 different 3-formylchromone derivatives, the condensation products of 6-R1-3-formylchromone with 4-aminosalicylic acid and of the adducts of 6-R1-3-formylchromone withn-alcohols and aminosalicylic acids or some other derivatives of aniline, on the photochemical activity of spinach chloroplasts. The inhibitory activity of the compounds studied correlated with the lipophilicity of the R1 and R2 (alkoxy) substituents. Using fluorescence study it was found that the site of action of the studied effectors is photosystem (PS) 2. By EPR spectroscopy it was confirmed that the studied effectors interact with the intermediates Z+/Y+ which are situated in D 1 and D 2 proteins on the donor side of PS 2 which is reflected in a partial decrease in the photosynthetic electron flow through PS 2 to PS 1. It was found that the core of PS 2 is not damaged.

Stabilization of oxygen evolution and primary electron transport reactions in photosystem II against heat stress with glycinebetaine and sucrose

The protective action of co-solutes, such as sucrose and glycinebetaine, againts the thermal inactivation of photosystem II function was studied in untreated and Mn-depleted photosystem II preparations. It was shown that, in addition to the reactions that depend on the oxygen-evolving activity of the photosystem, those that implicate more intimately the reaction center itself are protected by high concentrations of osmolytes. However, the temperature required to inhibit oxygen evolution totally in the presence of osmolytes is lower than that required to eliminate reactions, such as P680 (primary electron donor in photosystem II) photo-oxidation and pheophytin photoreduction, which only involve charge separation and primary electron transport processes. The energy storage measured from the thermal dissipation yield during photoacoustic experiments and the yield of variable fluorescence are also protected to a significant degree (up to 30%) at temperatures at which oxygen evolution is totally inhibited. It is suggested that a cyclic electron transport reaction around photosystem II may be preserved under these conditions and may be responsible for the energy storage measured at relatively high temperatures. This interpretation is also supported by thermoluminescence data involving the recombination between reduced electron acceptors and oxidized electron donors at −30 and −55 °C. The data also imply that a high concentration of osmolyte allows the stabilization of the photosystem core complex together with the oxygen-evolving complex. The stabilization effect is understood in terms of the minimization of protein-water interactions as proposed by the theory of Arakawa and Timasheff ( Biophys. J., 47 (1985) 411–414).

Aggregation of Chlorophyll a′ in Aqueous Methanol

ABSTRACTAn aggregate of chlorophyll a′ (Chi a′, C132‐epimer of ChI a) formed in a methanovwater (40160, vol/vol) mixed solvent was examined by visible absorption, circular dichro‐ism (CD), fluorescence and resonance Raman spectrosco‐pies in relation to its possible involvement in the core of photosystem I reaction center. The Chl a′ aggregate exhibited a sharp, double‐peaked absorption spectrum (690 and 715 nm) accompanied by an intense, conservative CD signal. The fluorescence excitation polarization spectrum showed that the doublet results from the exciton splitting in a single aggregate species. Time‐dependent changes in the spectroscopic properties clearly point to a simple transformation process from one molecular species to another, though a minor component appears to coexist. This conclusion was supported also by the principal multicom‐ponent spectral estimation analysis of the transients of absorption spectra. The species formed at the initial stage is most probably a T‐shaped dimer or oligomer, which is then gradually converted into the final major product, presumably a stacked dimer. In both of these states, the Chl molecules are linked together via direct coordination of the C13’keto carbonyl oxygen onto the Mg atom of neighboring molecules, as suggested by almost identical resonance Raman spectra in ordinary and deuterated methanovwater mixed solvents. The stacked dimers probably further associate to form a colloidal state in this solvent system. Based on these results, a model for the Chl a′ aggregation is proposed.

A protein kinase in the core of photosystem II.

In green plants, several intrinsic protein components of the photosystem II (PS II) complexes are subject to reversible phosphorylation on threonine residues. Evidence from mutant and inhibitor studies indicates that multiple kinases are involved. The protein kinases appear to be membrane-bound and redox-regulated, with activity requiring reducing conditions. We report the identification of a protein kinase activity which copurifies with a core complex of PS II and is capable of phosphorylating the photosystem proteins and associated light-harvesting complex. The enzyme is a distinct and novel protein whose close proximity to the photosystem reaction center is confirmed by its rapid inactivation under strong red light irradiation in the presence of oxygen.

Organization of Photosystem I Polypeptides (Identification of PsaB Domains That May Interact with PsaD)

PsaA and PsaB are homologous integral membrane-proteins that form the heterodimeric core of photosystem i (PSI). We used subunit-deficient PSI complexes from the mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 to examine interactions between PsaB and other PSI subunits. Incubation of the wild-type PSI with thermolysin yielded 22-kD C-terminal fragments of PsaB that were resistant to further proteolysis. Modification of the wild-type PSI with N-hydroxysuccinimidobiotin and subsequent cleavage by thermolysin showed that the lysyl residues in the 22-kD C-terminal domain were inaccessible to modification by N-hydroxysuccinimidobiotin. The absence of PsaE, PsaF, PsaI, PsaJ, or PsaL facilitated accumulation of 22-kD C-terminal fragments of PsaB but did not alter their resistance to further proteolysis. When the PsaD-less PSI was treated with thermolysin, the 22-kD C-terminal fragments of PsaB were rapidly cleaved, with concomitant accumulation of a 16-kD fragment and then a 3.4-kD one. We mapped the N termini of these fragments by N-terminal amino acid sequencing and the C termini from their positive reaction with an antibody against the C-terminal peptide of PsaB. The cleavage sites were proposed to be in the extramembrane loops on the cytoplasmic side. Western blot analyses showed resistance of PsaC and PsaI to proteolysis prior to cleavage of the 22-kD fragments. Therefore, we propose that PsaD shields two extramembrane loops of PsaB and protects the C-terminal domain of PsaB from in vitro proteolysis.

Factors influencing PS II particle array formation in Arabidopsis thaliana chloroplasts and the relationship of such arrays to the thermostability of PS II

Chloroplasts of the thermal-stable fatty acid desaturase mutants JB67 and LK3 of Arabidopsis thaliana are characterised by the presence of regular arrays of freeze-fracture particles associated with the core and light-harvesting antennae of Photosystem II (Tsvetkova et al. (1994) Biochim. Biophys. Acta 1192, 263–271). Similar arrays were found to be induced in the membranes of chloroplasts isolated from wild-type plants by resuspending the chloroplasts in media containing Tricine and/or high concentrations of compatible co-solutes such as sorbitol. The thermal stability of their chloroplasts was also increased under such conditions. The increased tendency to form PS II particle arrays, and the enhanced thermal stability of PS II, in chloroplasts isolated from the mutants and wild-type chloroplasts suspended in different media, appear to be reflections of the increased stability of protein-protein interactions between and within PS II units, respectively. The role of lipids in determining the formation of freeze-fracture particle arrays in the mutants is discussed in terms of the observed changes in lipid composition and their possible role in the control of lipid/protein synthesis.

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.

Evolution of heliobacteria: Implications for photosynthetic reaction center complexes

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein shows rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

Function and organization of photosystem I in a cyanobacterial mutant strain that lacks PsaF and PsaJ subunits.

Photosystem I functions as a light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic membranes of cyanobacteria and chloroplasts. A mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 that contains a deletion of the psaF gene and a transcriptionally inactive psaJ gene has assembled photosystem I complexes that lack PsaF, a lumenal protein and PsaJ, a 4-kDa hydrophobic protein. The cells of the mutant and wild type strains have similar rates of photosynthetic electron transfer and P700+ rereduction under linear and cyclic electron transfer conditions. Analysis of flash-induced absorption transients at 700 nm demonstrate that the absence of PsaF in purified mutant photosystem I did not affect the rate of P700 rereduction by cytochrome c553. Therefore, PsaF is not essential for docking of cytochrome c553. We also studied the organization of the proteins of mutant and wild type photosystem I by comparing their accessibility to digestion by thermolysin or to removal by 1 M NaI. The PsaA-PsaB subunits were more easily degraded by thermolysin in the mutant photosystem I. Thermolysin cleavage of PsaB yielded two major fragments that were immunoreactive with an antibody raised against the C terminus of PsaB. The N termini of these PsaB peptides mapped at Ile482 and Ile498 residues, thus identifying a surface-exposed domain of the core of photosystem I. The PsaE subunit could be removed by 1 M NaI and was rapidly digested by thermolysin in the mutant but not in the wild type photosystem I. Therefore, PsaF and PsaJ subunits of photosystem I have dispensable accessory roles in the function and organization of the complex.

Isolation and characterization of three membranebound chlorophyll-protein complexes from four dinoflagellate species

Employing discontinuous sucrose density gradient centrifugation of n -dodecyl β-d-maltoside-solubilized thylakoid membranes, three chlorophyll (Chl)-protein complexes containing Chl a , Chl c 2 and peridinin in different proportions, were isolated from the dinoflagellates Symbiodinium microadriaticum, S. kawagutii, S. pilosum and Heterocapsa pygmaea . In S. microadriaticum , the first complex, containing 13% of the total cellular Chl a , and minor quantities of Chl c 2 and peridinin, is associated with polypeptides with apparent molecular mass ( M r ) of 8-9 kDa, and demonstrated inefficient energy transfer from the accessory pigments to Chl a . The second complex contains Chl a , Chl c 2 and peridinin in a molar ratio of 1:1:2, associated with two apoproteins of M r 19-20 kDa, and comprises 45%, 75% and 70%, respectively, of the cellular Chl a , Chl c 2 and peridinin. The efficient energy transfer from Chl c 2 and peridinin to Chl a in this complex is supportive of a light-harvesting function. This Chl a - c 2 - peridin-protein complex represents the major light-harvesting complex in dinoflagellates. The third complex obtained contains 12% of the cellular Chl a , and appears to be the core of photosystem I, associated with a light-harvesting complex. This complex is spectroscopically similar to analogous preparations from different taxonomic groups, but demonstrates a unique apoprotein composition. Antibodies against the water-soluble peridinin-Chl a -protein (sPCP) light-harvesting complexes failed to cross-react with any of the thylakoid-associated complexes, as did antibodies against Chl a - c -fucoxanthin apoprotein (from diatoms). Antibodies against the P 700 apoprotein of plants did not cross-react with the photosystem I complex. Similar results were observed in the other dinoflagellates.

Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves

Monocotyledonous leaves subjected to osmotica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recently produced antibody probes, the effects on the stabilization of thylakoid membranes of applying either difluoromethylarginine (DFMA), a specific inhibitor of putrescine synthesis via arginine decarboxylase, or the polyamines Spd, Spm, or diaminopropane (Dap) to osmotically shocked oat leaves (Avena sativa L.) have been investigated. High protein levels were maintained in thylakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; as indicated by the level of large subunits). Applications of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine applications and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discussed.

The D1 protein of the photosystem II reaction‐centre complex accumulates in the absence of D2: analysis of a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking cytochrome d559

The reaction center core of photosystem II, a multiprotein membrane bound complex, is composed of a heterodimer of two proteins, D1 and D2. A random mutagenesis technique was used to isolate a photosystem II deficient mutant, CP6t16, of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Nucleotide sequence analysis showed that the primary lesion in CP6t16 is an ochre mutation introducing a translational stop codon in the psbE gene, encoding the alpha-subunit of cytochrome b559, an integral component of the PSII complex. Analysis of the protein composition of CP6t16 thylakoid membranes isolated in the presence of serine protease inhibitors revealed that, in the absence of cytochrome b559, the D2 protein is also absent. However, the D1 protein is stably incorporated in these membranes, suggesting that the synthesis and integration of D1 are independent of those of D2 and cytochrome b559.

Differential Synthesis of Photosystem Cores and Light-Harvesting Antenna during Proplastid to Chloroplast Development in Spirodela oligorrhiza

Proplastids and etioplasts are common starting points for monitoring chloroplast development in higher plants. Although proplastids are the primary precursor of chloroplasts, most proplastid to chloroplast systems are cumbersome to study temporally. Conversely, the etioplast to chloroplast transition is initiated by light and is readily examined as a function of time. Etioplasts, however, are found mostly in plants germinated in the dark and are not an obligatory step in chloroplast development. We have chosen to study chloroplast ontogeny in Spirodela oligorrhiza (Kurtz) Hegelm (a C(3)-monocot) because of its unique ability to grow indefinitely in the dark. Ultrastructural, physiological, and molecular evidence is presented in support of a temporal, light-triggered proplastid to chloroplast transition in Spirodela. The dark-grown plants are devoid of chlorophyll, and upon illumination synchronously green over a 3- to 5-day period. Synthesis of chloroplast proteins involved in photosynthesis is coincident with thylakoid assembly, chlorophyll accumulation, and appearance of CO(2) fixation activity. Interestingly, the developmental sequence in Spirodela was slow enough to reveal that biosynthesis of the D1 photosystem II reaction center protein precedes biosynthesis of the major light-harvesting antenna proteins. This, coupled with the high chlorophyll a/b ratio observed early in development, indicated that reaction center assembly occurred prior to accumulation of the light-harvesting complexes. Thus, with Spirodela one can study proplastid to chloroplast conversions temporally in higher plants and follow the process on a time scale that enables a detailed dissection of plastid maturation processes.

Presence of two chlorophyll a′ molecules at the core of photosystem I

Presence of two chlorophyll a′ molecules at the core of photosystem I

Presence of two chlorophyll a′ molecules at the core of photosystem I

The pigment composition in cells and thylakoid membranes of a cyanobacterium, and in leaves and intact or P700-enriched chloroplasts of highest plants has been reinvestigated by solvent extraction/HPLC. In acetone extracts, the amount of chlorophyll (Chl) a ′ relative to Chlf a ′ on P700 was neatly twice that in chloroform extracts: the latter gave a result in line with the stochrophyl of Chl a ′ P700 · 1 found by chlorofrom extraction (Kobayashi, M. et al. (1998) Biochim. Biophys. Acta 936, S1 S90. By two step extraction last with chloroform then with acetone, at roughly equal amount of Chl a ′ was extracted in each step, while more than 90% of Chl a ′ was extracted already in the first step. For chloroplasts heavily enriched in P700, the residue of the last extraction exhibited a Chl a ′ molar ratio outreaching the equilibrium value (0.33) as verified by the second extraction HPLC. These results evidence that two Chl a ′ molecules exist at the Photosystem (PS) I core, and just one of them is of a site to which only such a hydropidia molecules as rectone is accessible. A preliminary discussion is given on the possible role of the two Chl a ′ molecules in PS I.

Presence of two chlorophyll a′ molecules at the core of photosystem I

The pigment composition in cells and thylakoid membranes of a cyanobacterium, and in leaves and intact or P700-enriched chloroplasts of highest plants has been reinvestigated by solvent extraction/HPLC. In acetone extracts, the amount of chlorophyll (Chl) a′ relative to Chlf a′ on P700 was neatly twice that in chloroform extracts: the latter gave a result in line with the stochrophyl of Chl a′ P700 · 1 found by chlorofrom extraction (Kobayashi, M. et al. (1998) Biochim. Biophys. Acta 936, S1 S90. By two step extraction last with chloroform then with acetone, at roughly equal amount of Chl a′ was extracted in each step, while more than 90% of Chl a′ was extracted already in the first step. For chloroplasts heavily enriched in P700, the residue of the last extraction exhibited a Chl a′ molar ratio outreaching the equilibrium value (0.33) as verified by the second extraction HPLC. These results evidence that two Chl a′ molecules exist at the Photosystem (PS) I core, and just one of them is of a site to which only such a hydropidia molecules as rectone is accessible. A preliminary discussion is given on the possible role of the two Chl a′ molecules in PS I.