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Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

Photosynthetic electron transport is regulated by cyclic and pseudocyclic electron flow (CEF and PCEF) to maintain the balance between light availability and metabolic demands. CEF transfers electrons from photosystem I to the plastoquinone pool with two mechanisms, dependent either on PGR5/PGRL1 or on the type I NADH dehydrogenase-like (NDH) complex. PCEF uses electrons from photosystem I to reduce oxygen and in many groups of photosynthetic organisms, but remarkably not in angiosperms, it is catalyzed by flavodiiron proteins (FLVs). In this study, Physcomitrella patens plants depleted in PGRL1, NDH and FLVs in different combinations were generated and characterized, showing that all these mechanisms are active in this moss. Surprisingly, in contrast to flowering plants, Physcomitrella patens can cope with the simultaneous inactivation of PGR5- and NDH-dependent CEF but, when FLVs are also depleted, plants show strong growth reduction and photosynthetic activity is drastically reduced. The results demonstrate that mechanisms for modulation of photosynthetic electron transport have large functional overlap but are together indispensable to protect photosystem I from damage and they are an essential component for photosynthesis in any light regime., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Foundation.)

Although light is essential for photosynthesis, when in excess, it may damage the photosynthetic apparatus, leading to a phenomenon known as photoinhibition. Photoinhibition was thought as a light-induced damage to photosystem II; however, it is now clear that even photosystem I may become very vulnerable to light. One main characteristic of light induced damage to photosystem II (PSII) is the increased turnover of the reaction center protein, D1: when rate of degradation exceeds the rate of synthesis, loss of PSII activity is observed. With respect to photosystem I (PSI), an excess of electrons, instead of an excess of light, may be very dangerous. Plants possess a number of mechanisms able to prevent, or limit, such damages by safe thermal dissipation of light energy (non-photochemical quenching, NPQ), slowing-down of electron transfer through the intersystem transport chain (photosynthesis-control, PSC) in co-operation with the Proton Gradient Regulation (PGR) proteins, PGR5 and PGRL1, collectively called as short-term photoprotection mechanisms, and the redistribution of light between photosystems, called state transitions (responsible of fluorescence quenching at PSII, qT), is superimposed to these short term photoprotective mechanisms. In this manuscript we have generated a number of higher order mutants by crossing genotypes carrying defects in each of the short-term photoprotection mechanisms, with the final aim to obtain a direct comparison of their role and efficiency in photoprotection. We found that mutants carrying a defect in the ΔpH-dependent photosynthesis-control are characterized by photoinhibition of both photosystems, irrespectively of whether PSBS-dependent NPQ or state transitions defects were present or not in the same individual, demonstrating the primary role of PSC in photoprotection. Moreover, mutants with a limited capability to develop a strong PSBS-dependent NPQ, were characterized by a high turnover of the D1 protein and high values of Y(NO), which might reflect energy quenching processes occurring within the PSII reaction center.

The regulation of photosynthesis is crucial to efficiently support the assimilation of carbon dioxide and to prevent photodamage. One key regulatory mechanism is the pseudo-cyclic electron flow (PCEF) mediated by class-C flavodiiron proteins (FLVs). These enzymes use electrons coming from Photosystem I (PSI) to reduce oxygen to water, preventing over-reduction in the acceptor side of PSI. FLVs are widely distributed among organisms performing oxygenic photosynthesis and they have been shown to be fundamental in many different conditions such as fluctuating light, sulfur deprivation and plant submersion. Moreover, since FLVs reduce oxygen they can help controlling the redox status of the cell and maintaining the microoxic environment essential for processes such as nitrogen fixation in cyanobacteria. Despite these important roles identified in various species, the genes encoding for FLV proteins have been lost in angiosperms where their activity could have been at least partially compensated by a more efficient cyclic electron flow (CEF). The present work reviews the information emerged on FLV function, analyzing recent structural data that suggest FLV could be regulated through a conformational change., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photoprotection by mitigating overreduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo-cyclic electron flow mediated by the flavodiiron proteins FLV. In this work, we generated P. patens plants carrying both pgrl1 and flva knock-out mutations. A comparative analysis of the WT, pgrl1, flva, and pgrl1 flva lines suggests that cyclic and pseudo-cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, although both contribute to photosystem I protection from overreduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change whereas PGRL1 has a major role upon sustained strong illumination., (© 2018 John Wiley & Sons Ltd.)

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Nannochloropsis is an eukaryotic alga of the phylum Heterokonta, originating from a secondary endosymbiotic event. In this work, we investigated how the photosynthetic apparatus responds to growth in different light regimes in Nannochloropsis gaditana. We found that intense illumination induces the decrease of both photosystem I and II contents and their respective antenna sizes. Cells grown in high light showed a larger capacity for electron transport, with enhanced cyclic electron transport around photosystem I, contributing to photoprotection from excess illumination. Even when exposed to excess light intensities for several days, N. gaditana cells did not activate constitutive responses such as nonphotochemical quenching and the xanthophyll cycle. These photoprotection mechanisms in N. gaditana thus play a role in acclimation to fast changes in illumination within a time range of minutes, while regulation of the electron flow capacity represents a long-term response to prolonged exposure to excess light.

Nannochloropsis gaditana belongs to Eustigmatophyceae, a class of eukaryotic algae resulting from a secondary endosymbiotic event. Species of this class have been poorly characterized thus far but are now raising increasing interest in the scientific community because of their possible application in biofuel production. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of only chlorophyll a, with violaxanthin and vaucheriaxanthin esters as the most abundant carotenoids. In this study, the photosynthetic apparatus of this species was analyzed by purifying the thylakoids and isolating the different pigment-binding complexes upon mild solubilization. The results from the biochemical and spectroscopic characterization showed that the photosystem II antenna is loosely bound to the reaction center, whereas the association is stronger in photosystem I, with the antenna-reaction center super-complexes surviving purification. Such a supramolecular organization was found to be conserved in photosystem I from several other photosynthetic eukaryotes, even though these taxa are evolutionarily distant. A hypothesis on the possible selective advantage of different associations of the antenna complexes of photosystems I and II is discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II., Results: In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex., Conclusion: We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed.

Time-resolved fluorescence measurements were performed on isolated core and intact Photosystem I (PS I) particles and stroma membranes from Arabidopsis thaliana to characterize the type of energy-trapping kinetics in higher plant PS I. Target analysis confirms the previously proposed "charge recombination" model. No bottleneck in the energy flow from the bulk antenna compartments to the reaction center has been found. For both particles a trap-limited kinetics is realized, with an apparent charge separation lifetime of approximately 6 ps. No red chlorophylls (Chls) are found in the PS I-core complex from A. thaliana. Rather, the observed red-shifted fluorescence (700-710 nm range) originates from the reaction center. In contrast, two red Chl compartments, located in the peripheral light-harvesting complexes, are resolved in the intact PS I particles (decay lifetimes 33 and 95 ps, respectively). These two red states have been attributed to the two red states found in Lhca 3 and Lhca 4, respectively. The influence of the red Chls on the slowing of the overall trapping kinetics in the intact PS I complex is estimated to be approximately four times larger than the effect of the bulk antenna enlargement.

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to "normal" chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm(-1)), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red-most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705 nm and originates from a broad absorption band peaking at 690 nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.

In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while beta-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The "red" lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima: one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The "blue"-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T-S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2-Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale.

In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I.

Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.

Photosystem II of higher plants is a multisubunit transmembrane complex composed of a core moiety and an extensive peripheral antenna system. The number of antenna polypeptides per core complex is modulated following environmental conditions in order to optimize photosynthetic performance. In this study, we used a barley (Hordeum vulgare) mutant, viridis zb63, which lacks photosystem I, to mimic extreme and chronic overexcitation of photosystem II. The mutation was shown to reduce the photosystem II antenna to a minimal size of about 100 chlorophylls per photosystem II reaction centre, which was not further reducible. The minimal photosystem II unit was analysed by biochemical methods and by electron microscopy, and found to consist of a dimeric photosystem II reaction centre core surrounded by monomeric Lhcb4 (chlorophyll protein 29), Lhcb5 (chlorophyll protein 26) and trimeric light-harvesting complex II antenna proteins. This minimal photosystem II unit forms arrays in vivo, possibly to increase the efficiency of energy distribution and provide photoprotection. In wild-type plants, an additional antenna protein, chlorophyll protein 24 (Lhcb6), which is not expressed in viridis zb63, is proposed to associate to this minimal unit and stabilize larger antenna systems when needed. The analysis of the mutant also revealed the presence of two distinct signalling pathways activated by excess light absorbed by photosystem II: one, dependent on the redox state of the electron transport chain, is involved in the regulation of antenna size, and the second, more directly linked to the level of photoinhibitory stress perceived by the cell, participates in regulating carotenoid biosynthesis.

Light-harvesting complex I (LHCI), which serves as a peripheral antenna for photosystem I (PSI) in green plants, consists mainly of four polypeptides, Lhca1-4. We report room temperature emission properties of individual reconstituted monomeric Lhca proteins (Lhca1, -2, -3, and -4) and dimeric Lhca1/4, performed by steady-state and time-resolved fluorescence techniques. The emission quantum yields of the samples are approximately 0.12, 0.085, 0.081, 0.041, and 0.063 for Lhca1, -2, -3, -4, and the -1/4 dimer, respectively, which is considerably lower than the value of 0.22 found for light-harvesting complex II (LHCII), the main peripheral antenna complex of photosystem II in green plants. The decay components of LHCI proteins can be divided in two categories: Lhca1 and Lhca3 have decay times of 1.1-1.6 ns and 3.3-3.6 ns, and Lhca2 and Lhca4 have decay times of 0.7-0.9 ns and 3.1-3.2 ns. These categories seem to correlate with the pigment composition of the samples. All decay times are faster than that observed previously for LHCII. When the absolute emission yields and the lifetimes of the Lhca samples are combined, the overall emission properties of the individual Lhca proteins are expressed in terms of their emitting dipole moment strength. In the samples without extreme red states, that is, Lhca1 and Lhca2, the emitting dipole moment has a value close to unity (relative to monomeric chlorophyll in acetone), which is similar to that for LHCII, whereas, in the samples with the red-most state (F-730), that is, Lhca3, -4, and the -1/4 dimer, the emitting dipole moment has a value less than unity (0.6-0.8), which can be explained by mixing the red-most (exciton) state with a dark charge-transfer state, as suggested in previous PSI red pigment studies. In addition, we find a lifetime component of approximately 50-150 ps in all red-pigment-containing samples, which cannot be due to "slow" energy transfer, but is instead assigned to an unrelaxed state of the pigment-protein, which, on this time-scale, is converted into the final emitting state.

We report on the association of the antenna system to the reaction center in Photosystem I. Biochemical analysis of mutants depleted in antenna polypeptides showed that the binding of the antenna moiety is strongly cooperative. The minimal building block for the antenna system was shown to be a dimer. Specific protein-protein interactions play an important role in antenna association, and the gap pigments, bound at the interface between core and antenna, are proposed to mediate these interactions Gap pigments have been characterized by comparing the spectra of the Photosystem I to those of the isolated antenna and core components. CD spectroscopy showed that they are involved in pigment-pigment interactions, supporting their relevance in energy transfer from antenna to the reaction center. Moreover, gap pigments contribute to the red-shifted emission forms of Photosystem I antenna. When compared with Photosystem II, the association of peripheral antenna complexes in PSI appears to be more stable, but far less flexible and functional implications are discussed.

In this study, triplet quenching, the major photoprotection mechanism in antenna proteins, has been studied in the light-harvesting complex of photosystem I (LHC-I). The ability of carotenoids bound to LHC-I subunit Lhca4, which is characterized by the presence of the red-most absorption components at wavelength >700 nm, to protect the system through quenching of the chlorophyll triplet states, has been probed, by analyzing the induction of carotenoid triplet formation. We have investigated this process at low temperature, when the funneling of the excitation toward the low-lying excited states of the Chls is stronger, by means of optically detected magnetic resonance (ODMR), which is well-suited for investigation of triplet states in photosynthetic systems. The high selectivity and sensitivity of the technique has made it possible to point out the presence of specific interactions between carotenoids forming the triplet states and specific chlorophylls characterized by red-shifted absorption, by detection of the microwave-induced Triplet minus Singlet (T-S) spectra. The effect of the red forms on the efficiency of triplet quenching was specifically probed by using the Asn47His mutant, in which the red forms have been selectively abolished (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Lack of the red forms yields into a reduced efficiency of the triplet quenching in LHC-I thus suggesting that the "red Chls" play a role in enhancing triplet quenching in LHC-I and, possibly, in the whole photosystem I.

The red-most fluorescence emission of photosystem I (733 nm at 4 K) is associated with the Lhca4 subunit of the antenna complex. It has been proposed that this unique spectral feature originates from the low energy absorption band of an excitonic interaction involving chlorophyll A5 and a second chlorophyll a molecule, probably B5 (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Because of the short distances between chromophores in Lhc proteins, the possibility that other pigments are involved in the red-shifted spectral forms could not be ruled out. In this study, we have analyzed the pigment-pigment interactions between nearest neighboring chromophores in Lhca4. This was done by deleting individual chlorophyll binding sites by mutagenesis, and analyzing the changes in the spectroscopic properties of recombinant proteins refolded in vitro. The red-shifted (733 nm) fluorescence peak, the major target of this analysis, was lost upon mutations affecting sites A4, A5, and B5 and was modified by mutating site B6. In agreement with the shorter distance between chlorophylls A5 and B5 (7.9 A) versus A4 and A5 (12.2 A) in Lhca4 (Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635), we conclude that the low energy spectral form originates from an interaction involving pigments in sites A5 and B5. Mutation at site B6, although inducing a 15-nm blue-shift of the emission peak, maintains the red-shifted emission. This implies that chromophores responsible for the interaction are conserved and suggests a modification in the pigment organization. Besides the A5-B5 pair, evidence for additional pigment-pigment interactions between chlorophylls in sites B3-A3 and B6-A6 was obtained. However, these features do not affect the red-most spectral form responsible for the 733-nm fluorescence emission band.

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.

EET in reconstituted Lhca4, a peripheral light-harvesting complex from Photosystem I of Arabidopsis thaliana, containing 10 chlorophylls and 2 carotenoids, was studied at room temperature by femtosecond transient absorption spectroscopy. Two spectral forms of Lut were observed in the sites L1 and L2, characterized by significantly different interactions with nearby chlorophyll a molecules. A favorable interpretation of these differences is that the efficiency of EET to Chls is about two times lower from the "blue" Lut in the site L1 than from the "red" Lut in the site L2 due to fast IC in the former case. A major part of the energy absorbed by the "red" Lut, approximately 60%-70%, is transferred to Chls on a sub-100-fs timescale from the state S(2) but, in addition, minor EET from the hot S(1) state within 400-500 fs is also observed. EET from the S(1) state to chlorophylls occurs also within 2-3 ps and is ascribed to Vio and/or "blue" Lut. EET from Chl b to Chl a is biphasic and characterized by time constants of approximately 300 fs and 3.0 ps. These rates are ascribed to EET from Chl b spectral forms absorbing at approximately 644 nm and approximately 650 nm, respectively. About 25% of the excited Chls a decays very fast-within approximately 15 ps. This decay is proposed to be related to the presence of the interacting Chls A5 and B5 located next to the carotenoid in the site L2 and may imply some photoprotective role for Lhca4 in the photosystem I super-complex.

We report on the results obtained by measuring the stoichiometry of antenna polypeptides in Photosystem I (PSI) from Arabidopsis thaliana. This analysis was performed by quantification of Coomassie blue binding to individual LHCI polypeptides, fractionation by SDS/PAGE, and by the use of recombinant light harvesting complex of Photosystem I (Lhca) holoproteins as a standard reference. Our results show that a single copy of each Lhca1-4 polypeptide is present in Photosystem I. This is in agreement with the recent structural data on PSI-LHCI complex [Ben Shem, A., Frolow, F. and Nelson, N. (2003) Nature, 426, 630-635]. The discrepancy from earlier estimations based on pigment binding and yielding two copies of each LHCI polypeptide per PSI, is explained by the presence of 'gap' and 'linker' chlorophylls bound at the interface between PSI core and LHCI. We showed that these chlorophylls are lost when LHCI is detached from the PSI core moiety by detergent treatment and that gap and linker chlorophylls are both Chl a and Chl b. Carotenoid molecules are also found at this interface between LHCI and PSI core. Similar experiments, performed on PSII supercomplexes, showed that dissociation into individual pigment-proteins did not produce a significant loss of pigments, suggesting that gap and linker chlorophylls are a peculiar feature of Photosystem I.

Photosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes.

Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at approximately 700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.