Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro

Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.     

The C-terminal Extension Peptide of Non-photoconvertible Water-Soluble Chlorophyll-Binding Proteins (Class II WSCPs) Affects Their Solubility and Stability: Comparative Analyses of the Biochemical and Chlorophyll-Binding Properties of Recombinant Brassica, Raphanus and Lepidium WSCPs with or Without Their C-terminal Extension Peptides

Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.     

Are tyrosine residues involved in the photoconversion of the water‐soluble chlorophyll‐binding protein of Chenopodium album?

Non‐photosynthetic and hydrophilic chlorophyll (Chl) proteins, called water‐soluble Chl‐binding proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non‐photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin‐like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant proteins (Y13A, Y14A, Y87A, Y134A, Y13‐14A, Y13‐87A, Y13‐134A, Y14‐87A, Y14‐134A, Y87‐134A, Y13‐14‐87A, Y13‐14‐134A, Y13‐87‐134A, Y14‐87‐134A and Y13‐14‐87‐134A) using site‐directed mutagenesis. Amazingly, all the mutant proteins retained not only chlorophyll‐binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13‐14‐87‐134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion.     

Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein

Two kinds of water-soluble chlorophyll (Chl) proteins (WSCPs) have been found, e.g., a WSCP from Chenopodium, Atriplex, Polygonum, and Amaranthus species (class I) and that from Brassica, Raphanus, and Lepidium species (class II). Classes I and II WSCPs differ mainly in their photoconvertiblity. Class I WSCPs show a light-induced absorption change, whereas Class II WSCPs do not. The molecular and functional properties of Class I WSCP are largely uncertain. On the other hand, recent studies on the adaptation of plants to osmotic stress revealed the participation of drought-stress induced proteins with molecular masses of 20-22 kDa possessing a sequence similarity with class II WSCPs. This mini review focuses on the molecular signature of class II WSCPs. The physiological function of class II WSCPs has not been clarified either, but, their water-solubility, low Chl content, and stress-inducibility suggested little contribution to photosynthesis. Several molecular properties predicting its physiological role are as follows. The WSCP tetramer, may have only one or no Chl molecules in each subunit. All WSCPs possess a motif for Künitz-type proteinase inhibitor family in their sequence. WSCP is induced by drought- and heat-stresses suggesting its protective role during stress conditions. Monomeric recombinant apo-WSCP is able to remove Chls from the thylakoid membrane in aqueous solution and form into a tetramer. Brassica-WSCP contains a signal sequence targeted to endoplasmic reticulum. The highly conserved, C-terminal region is missing in the mature WSCP. Possible functions of class II WSCPs in plant tissues are discussed.     

Three-Step Photoconversion of Only Three Subunits of the Water-Soluble Chlorophyll-Binding Protein Tetramer from Chenopodium album

Water-soluble chlorophyll (Chl)-binding proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A₇₄₂/A₆₆₈ ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.     

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium

Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.     

Synthetic Studies on C-Nucleosides

Chenopodium album has a non-photosynthetic chlorophyll protein known as the water-soluble chlorophyll (Chl)-binding protein (WSCP). The C. album WSCP (CaWSCP) is able to photoconvert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton. Reducing reagents such as β-mercaptoethanol or dithiothreitol inhibit photoconversion, indicating that S–S bridge(s) in CaWSCP are quite important for it. Recently, we found that the mature region of CaWSCP contains five cysteine residues; Cys2, Cys30, Cys48, Cys63, and Cys144. To identify which cysteine residues are involved in the photoconversion, we generated five mutants (C2S, C30S, C48S, C63S, and C144S) by site-directed mutagenesis. Interestingly, C48S, C63S, and C144S mutants showed the same Chl-binding activity and photoconvertibility as those of the recombinant wild-type CaWSCP-His, while the C2S and C30S mutants completely lost Chl-binding activity. Our findings indicated that the S–S bridge between Cys2 and Cys30 in each CaWSCP subunit is essential for Chl-binding activity.     

A novel role of water-soluble chlorophyll proteins in the transitory storage of chorophyllide

All chlorophyll (Chl)-binding proteins involved in photosynthesis of higher plants are hydrophobic membrane proteins integrated into the thylakoids. However, a different category of Chl-binding proteins, the so-called water-soluble Chl proteins (WSCPs), was found in members of the Brassicaceae, Polygonaceae, Chenopodiaceae, and Amaranthaceae families. WSCPs from different plant species bind Chl a and Chl b in different ratios. Some members of the WSCP family are induced after drought and heat stress as well as leaf detachment. It has been proposed that this group of proteins might have a physiological function in the Chl degradation pathway. We demonstrate here that a protein that shared sequence homology to WSCPs accumulated in etiolated barley (Hordeum vulgare) seedlings exposed to light for 2 h. The novel 22-kD protein was attached to the outer envelope of barley etiochloroplasts, and import of the 27-kD precursor was light dependent and induced after feeding the isolated plastids the tetrapyrrole precursor 5-aminolevulinic acid. HPLC analyses and spectroscopic pigment measurements of acetone-extracted pigments showed that the 22-kD protein is complexed with chlorophyllide. We propose a novel role of WSCPs as pigment carriers operating during light-induced chloroplast development.     

Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700-900 μmol photons m⁻² s⁻¹. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.     

Water soluble chlorophyll binding protein of higher plants: a most suitable model system for basic analyses of pigment-pigment and pigment-protein interactions in chlorophyll protein complexes

This short review paper describes spectroscopic studies on pigment-pigment and pigment-protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl-protein complexes.Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets.Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments.The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.     

Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera)

A water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera), hereafter termed BoWSCP, is categorized into the Class II WSCPs (non-photoconvertible WSCPs). Previous studies on BoWSCP have focused mainly on its biochemical characterization. In this study, we cloned the cDNA encoding BoWSCP. Sequence analysis revealed that the BoWSCP gene was composed of a single exon corresponding to 654 bp of an open reading frame encoding 218 amino acid residues, including 19 residues of a deduced signal peptide targeted to the endoplasmic reticulum (ER). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of native BoWSCP revealed that the molecular mass of the subunit was 19,008.523 Da, corresponding to a mature protein of 178 amino acids, indicating the removal of 21 residues in the C-terminal region. Functional BoWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (BoWSCP-His). When BoWSCP-His was mixed with thylakoid membranes in aqueous solution, BoWSCP-His was able to remove Chls from the thylakoid membranes. The absorption spectrum of the reconstituted BoWSCP-His was identical to that of the native BoWSCP. Chl binding analyses of BoWSCP-His revealed that the BoWSCP-His bound both Chl a and Chl b with almost the same affinity in 40% methanol solution, although the native BoWSCP had a higher content of Chl a. To reveal the intracellular localization of BoWSCP, we constructed a transgenic plant expressing the fluorescent protein fused with the N-terminal deduced signal peptide of BoWSCP. The fluorescence emitted from the chimeric protein was detected in the ER body, an ER-derived compartment observed only in Brassicaceae plants.     

Triplet-triplet energy transfer in Peridinin-Chlorophyll a-protein reconstituted with Chl a and Chl d as revealed by optically detected magnetic resonance and pulse EPR: Comparison with the native PCP complex from Amphidinium carterae

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at ∼ 620 and ∼ 640 nm in the Triplet-minus-Singlet (T − S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.     

Replacement of chlorophyll with di-vinyl chlorophyll in the antenna and reaction center complexes of the cyanobacterium Synechocystis sp. PCC 6803: Characterization of spectral and photochemical properties

Chlorophyll (Chl) a in a cyanobacterium Synechocystis sp. PCC 6803 was replaced with di-vinyl (DV)-Chl a by knock-out of the specific gene (slr1923), responsible for the reduction of a 8-vinyl group, and optical and photochemical properties of purified photosystem (PS) II complexes (DV-PS II) were investigated. We observed differences in the peak wavelengths of absorption and fluorescence spectra; however, replacement of Chl a with DV-Chl a had limited effects. On the contrary, photochemical reactions were highly sensitive to high-light treatments in the mutant. Specifically, DV-Chl a was rapidly bleached under high-light conditions, and we detected significant dissociation of complexes and degradation of D1 proteins (PsbA). By comparing the SDS-PAGE patterns observed in this study to those observed in spinach chloroplasts, this degradation is assigned to the acceptor-side photoinhibition. The delayed fluorescence in the nanosecond time region at 77 K was suppressed in DV-PS II, possibly increasing triplet formation of Chl molecules. Our findings provide insight into the evolutionary processes of cyanobacteria. The effects of pigment replacement on the optimization of reactions are discussed.     

Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting antenna proteins from two-helix and four-helix ancestors

The nuclear-encoded Chl a/b and Chl a/c antenna proteins of photosynthetic eukaryotes are part of an extended family of proteins that also includes the early light-induced proteins (ELIPs) and the 22 kDa intrinsic protein of PS II (encoded by psbS gene). All members of this family have three transmembrane helices except for the psbS protein, which has four. The amino acid sequences of these proteins are compared and related to the three-dimensional structure of pea LHC II Type I (Kühlbrandt and Wang, Nature 350: 130–134, 1991). The similarity of psbS to the three-helix members of the family suggests that the latter arose from a four-helix ancestor that lost its C-terminal helix by deletion. Strong internal similarity between the two halves of the psbS protein suggests that it in turn arose as the result of the duplication of a gene encoding a two-helix protein. Since psbS is reported to be present in at least one cyanobacterium, the ancestral four-helix protein may have been present prior to the endosymbiotic event or events that gave rise to the photosynthetic eukaryotes. The Chl a/b and Chl a/c antenna proteins, and the immunologically-related proteins in the rhodophytes may have had a common ancestor which was present in the early photosynthetic eukaryotes, and predated their division into rhodophyte, chromophyte and chlorophyte lineages. The LHC I-LHC II divergence probably occurred before the separation of higher plants from chlorophyte algae and euglenophytes, and the different Types of LHC I and LHC II proteins arose prior to the separation of angiosperms and gymnosperms.Abbreviations CAB Chl a/b-binding - ELIP early light-induced protein - FCP fucoxanthin-Chl a/c protein - PCR polymerase chain reaction - TMH trans-membrane helix     

Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O₂/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O₂/mg Chl/h in the absence and presence of CaCl₂, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

Recombinant water-soluble chlorophyll protein from Brassica oleracea var. Botrys binds various chlorophyll derivatives

A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var. Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli. The protein has been refolded in vitro to study its pigment binding behavior. Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein [Satoh, H., Nakayama, K., Okada, M. (1998) J. Biol. Chem. 273, 30568-30575]. WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding. WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein. We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes. If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited. We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl. This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives.     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (∼ 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A₁ instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.     

In vitro reassociation of phycobiliproteins and membranes to form functional membrane-bound phycobilisomes

A membrane-bound phycobilisome complex has been isolated from the cyanobacterium Fremyella diplosiphon grown in green light, thus containing phycoerythrin in addition to phycocyanin and allophycocyanin. The complex was dissociated by lowering the salt concentration. In the mixture obtained, no energy transfer from phycoerythrin to chlorophyll (Chl) a was observed. Reassociation of the phycobiliproteins and membrane mixture was carried out by a gradual increase of the salt concentration. The complex obtained after reassociation was characterized by polypeptide composition, absorbance and fluorescence emission spectra and electron microscopy. These analyses revealed similar composition and structure for the original and reconstituted membrane-bound phycobilisomes. Fluorescence emission spectra and measurements of Photosystem II activity demonstrated energy transfer from phycoerythrin to Chl a (Photosystem II) in the reconstituted complex. Reassociation of mixtures with varying phycoerythrin / Chl ratio showed that the phycobiliprotein concentration was critical in the reassociation process. Measurements of the amount of phycobilisomes reassociated with the photosynthetic membrane did not show saturation of binding when increasing the phycobiliprotein concentration. The ratio phycoerythrin / Chl a in the native complex was 7:1 (mg / mg). When the phycobiliprotein concentration was increased during the reassociation process, a ratio of 13–15 mg phycoerythrin / mg Chl a could be obtained. Under these conditions, only part of the phycobilisomes attached to the thylakoids was able to transfer energy to Photosystem II.