Main Light-harvesting Complex Research Articles

Unraveling the electronic origin of a special feature in the triplet-minus-singlet spectra of carotenoids in natural photosystems.

The influence of carotenoid triplet states on the Qy electronic transitions of chlorophylls has been observed in experiments on light-harvesting complexes over the past three decades, but the interpretation of the resulting spectral feature in the triplet minus singlet (T-S) absorption spectra of photosystems is still debated, as the physical-chemical explanation of this feature has been elusive. Here, we resolve this debate, by explaining the T-S spectra of pigment complexes over the Qy-band spectral region through a comparative study of chlorophyll-carotenoid model dyads and larger pigment complexes from the main light harvesting complex of higher plants (LHCII). This goal is achieved by combining state-of-the-art time-dependent density functional theory with analysis of the relationship between electronic properties and nuclear structure, and by comparison to the experiment. We find that the special signature in the T-S spectra of both model and natural photosystems is determined by singlet-like triplet excitations that can be described as effective singlet excitations on chlorophylls influenced by a stable electronic triplet on the carotenoid. The comparison with earlier experiments on different light-harvesting complexes confirms our theoretical interpretation of the T-S spectra in the Qy spectral region. Our results indicate an important role for the chlorophyll-carotenoid electronic coupling, which is also responsible for the fast triplet-triplet energy transfer, suggesting a fast trapping of the triplet into the relaxed carotenoid structure. The gained understanding of the interplay between the electronic and nuclear structures is potentially informative for future studies of the mechanism of photoprotection by carotenoids.

Autofluorescence-based high-throughput isolation of nonbleaching Cyanidioschyzon merolae strains under nitrogen-depletion.

Photosynthetic organisms maintain optimum levels of photosynthetic pigments in response to environmental changes to adapt to the conditions. The identification of cyanobacteria strains that alleviate bleaching has revealed genes that regulate levels of phycobilisome, the main light-harvesting complex. In contrast, the mechanisms of pigment degradation in algae remain unclear, as no nonbleaching strains have previously been isolated. To address this issue, this study attempted to isolate nonbleaching strains of the unicellular red alga Cyanidioschyzon merolae after exposure to nitrogen (N)-depletion based on autofluorescence information. After four weeks under N-depletion, 13 cells from 500,000 cells with almost identical pre- and post-depletion chlorophyll a (Chl a) and/or phycocyanin autofluorescence intensities were identified. These nonbleaching candidate strains were sorted via a cell sorter, isolated on solid medium, and their post-N-depletion Chl a and phycocyanin levels were analyzed. Chl a levels of these nonbleaching candidate strains were lower at 1-4 weeks of N-depletion similar to the control strains, however, their phycocyanin levels were unchanged. Thus, we successfully isolated nonbleaching C. merolae strains in which phycocyanin was not degraded under N-depletion, via autofluorescence spectroscopy and cell sorting. This versatile method will help to elucidate the mechanisms regulating pigments in microalgae.

Photosynthetic Light Harvesting and Thylakoid Organization in a CRISPR/Cas9 Arabidopsis Thaliana LHCB1 Knockout Mutant.

Light absorbed by chlorophylls of Photosystems II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is trimeric LHCII. In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of LHCII. In absence of LHCB1, the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss, leading to a pale green phenotype and growth delay. The Photosystem II absorption cross-section was smaller, while the Photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring Photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by lower Photosystem I over Photosystem II reaction center ratio and by the dephosphorylation of LHCII and Photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knockout lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

Harvesting Far-Red Light with Plant Antenna Complexes Incorporating Chlorophyll d.

Increasing the absorption cross section of plants by introducing far-red absorbing chlorophylls (Chls) has been proposed as a strategy to boost crop yields. To make this strategy effective, these Chls should bind to the photosynthetic complexes without altering their functional architecture. To investigate if plant-specific antenna complexes can provide the protein scaffold to accommodate these Chls, we have reconstituted the main light-harvesting complex (LHC) of plants LHCII in vitro and in silico, with Chl d. The results demonstrate that LHCII can bind Chl d in a number of binding sites, shifting the maximum absorption ∼25 nm toward the red with respect to the wild-type complex (LHCII with Chl a and b) while maintaining the native LHC architecture. Ultrafast spectroscopic measurements show that the complex is functional in light harvesting and excitation energy transfer. Overall, we here demonstrate that it is possible to obtain plant LHCs with enhanced far-red absorption and intact functional properties.

Superradiance of bacteriochlorophyll c aggregates in chlorosomes of green photosynthetic bacteria

Chlorosomes are the main light-harvesting complexes of green photosynthetic bacteria that are adapted to a phototrophic life at low-light conditions. They contain a large number of bacteriochlorophyll c, d, or e molecules organized in self-assembling aggregates. Tight packing of the pigments results in strong excitonic interactions between the monomers, which leads to a redshift of the absorption spectra and excitation delocalization. Due to the large amount of disorder present in chlorosomes, the extent of delocalization is limited and further decreases in time after excitation. In this work we address the question whether the excitonic interactions between the bacteriochlorophyll c molecules are strong enough to maintain some extent of delocalization even after exciton relaxation. That would manifest itself by collective spontaneous emission, so-called superradiance. We show that despite a very low fluorescence quantum yield and short excited state lifetime, both caused by the aggregation, chlorosomes indeed exhibit superradiance. The emission occurs from states delocalized over at least two molecules. In other words, the dipole strength of the emissive states is larger than for a bacteriochlorophyll c monomer. This represents an important functional mechanism increasing the probability of excitation energy transfer that is vital at low-light conditions. Similar behaviour was observed also in one type of artificial aggregates, and this may be beneficial for their potential use in artificial photosynthesis.

Two-photon absorption and excitation spectroscopy of carotenoids, chlorophylls and pigment-protein complexes.

In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden ("dark") and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than ∼600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.

Preparation of high-purity R-phycoerythrin and R-phycocyanin from Pyropia yezoensis in membrane chromatography

R-phycoerythrin and R-phycocyanin are the main light-harvesting pigment-protein complexes in Pyropia yezoensis. They have bright colors and a high degree of fluorescence, and are now widely used in biomedicine, food, and cosmetic industries. Therefore, membrane chromatography was used to isolate and purify phycobiliprotein from P. yezoensis. Algal cells were disrupted by repeated freeze-thaw cycles. Then ammonium sulfate was added into the crude phycobiliprotein extract, so that the saturation of ammonium sulfate in the solution reached 20%, 30%, and 40% in sequence. At 30% and 40% ammonium sulfate saturation, precipitates of R-phycoerythrin and R-phycocyanin mixture in different proportions were obtained. The precipitates were ultra-filtrated to remove the salt, and then, the retentate was taken for membrane chromatographic treatment. In the membrane chromatographic treatment, salt-sensitive PVDF (polyvinyl difluoride) membranes were used for the follow-up experiments. By adjusting the concentration of ammonium sulfate, the membrane system changed the binding strength of various proteins, and then separated different types of phycobiliproteins. The final products of R-phycoerythrin (Aλmax/A280 purity 4.25, yield 45%) and R-phycocyanin (two purity grades: purity 5.8, yield 14.70% and purity 4.18, yield 39.89%) were obtained and then characterized by spectrophotometric and fluorometric measurements and SDS-PAGE. However, the large-scale production of R-phycocyanin and R-phycoerythrin remains a bottleneck as the column chromatography is difficult to be applied for industrial production. This method avoids the problem of easy clogging of column chromatography and can be useful for mass-production of R-phycoerythrin and R-phycocyanin in the future based on the laboratory results.

The role of far-red spectral states in the energy regulation of phycobilisomes

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.

How reduced excitonic coupling enhances light harvesting in the main photosynthetic antennae of diatoms

Strong excitonic interactions are a key design strategy in photosynthetic light harvesting, expanding the spectral cross-section for light absorption and creating considerably faster and more robust excitation energy transfer. These molecular excitons are a direct result of exceptionally densely packed pigments in photosynthetic proteins. The main light-harvesting complexes of diatoms, known as fucoxanthin-chlorophyll proteins (FCPs), are an exception, displaying surprisingly weak excitonic coupling between their chlorophyll (Chl) a's, despite a high pigment density. Here, we show, using single-molecule spectroscopy, that the FCP complexes of Cyclotella meneghiniana switch frequently into stable, strongly emissive states shifted 4-10 nm toward the red. A few percent of isolated FCPa complexes and ∼20% of isolated FCPb complexes, on average, were observed to populate these previously unobserved states, percentages that agree with the steady-state fluorescence spectra of FCP ensembles. Thus, the complexes use their enhanced sensitivity to static disorder to increase their light-harvesting capability in a number of ways. A disordered exciton model based on the structure of the main plant light-harvesting complex explains the red-shifted emission by strong localization of the excitation energy on a single Chl a pigment in the terminal emitter domain due to very specific pigment orientations. We suggest that the specific construction of FCP gives the complex a unique strategy to ensure that its light-harvesting function remains robust in the fluctuating protein environment despite limited excitonic interactions.

Apoprotein heterogeneity increases spectral disorder and a step-wise modification of the B850 fluorescence peak position

It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric ‘ring’ of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 ‘rings’ are present in “low-light” adapted cells and that an unknown chaperon process creates multiple sub-types of ‘rings’ with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.

Engineering a pH-Regulated Switch in the Major Light-Harvesting Complex of Plants (LHCII): Proof of Principle.

Under excess light, photosynthetic organisms employ feedback mechanisms to avoid photodamage. Photoprotection is triggered by acidification of the lumen of the photosynthetic membrane following saturation of the metabolic activity. A low pH triggers thermal dissipation of excess absorbed energy by the light-harvesting complexes (LHCs). LHCs are not able to sense pH variations, and their switch to a dissipative mode depends on stress-related proteins and allosteric cofactors. In green algae the trigger is the pigment-protein complex LHCSR3. Its C-terminus is responsible for a pH-driven conformational change from a light-harvesting to a quenched state. Here, we show that by replacing the C-terminus of the main LHC of plants with that of LHCSR3, it is possible to regulate its excited-state lifetime solely via protonation, demonstrating that the protein template of LHCs can be modified to activate reversible quenching mechanisms independent of external cofactors and triggers.

LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells

To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.

Investigating the excited electronic states of carotenoids in the main plant lightharvesting complex (LHCII) via femtosecond pump-probe spectroscopy

No abstract available.

In vitro stereospecific hydration activities of the 3-vinyl group of chlorophyll derivatives by BchF and BchV enzymes involved in bacteriochlorophyll c biosynthesis of green sulfur bacteria.

The photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum produces bacteriochlorophyll (BChl) c pigments bearing a chiral 1-hydroxyethyl group at the 3-position, which self-aggregate to construct main light-harvesting antenna complexes, chlorosomes. The secondary alcoholic hydroxy group is requisite for chlorosomal aggregation and biosynthesized by hydrating the 3-vinyl group of their precursors. Using recombinant proteins of Cba. tepidum BchF and BchV, we examined in vitro enzymatic hydration of some 3-vinyl-chlorophyll derivatives. Both the enzymes catalyzed stereoselective hydration of zinc 3-vinyl-8-ethyl-12-methyl-bacteriopheophorbide c or d to the zinc 31 R-bacteriopheophorbide c or d homolog, respectively, with a slight amount of the 31 S-epimric species. A similar R-stereoselectivity was observed in the BchF-hydration of zinc 3-vinyl-8-ethyl- and propyl-12-ethyl-bacteriopheophorbides c, while their BchV-hydration gave a relatively larger amount of the 31 S-epimers. The in vitro stereoselective hydration confirmed the in vivo production of the S-epimeric species by BchV. The enzymatic hydration for the above 8-propylated substrate proceeded more slowly than that for the 8-ethylated, and the 8-isobutylated substrate was no longer hydrated. Based on these results, biosynthetic pathways of BChl c homologs and epimers are proposed.

Characterization of red-shifted phycobilisomes isolated from the chlorophyll f-containing cyanobacterium Halomicronema hongdechloris

Phycobilisomes are the main light-harvesting protein complexes in cyanobacteria and some algae. It is commonly accepted that these complexes only absorb green and orange light, complementing chlorophyll absorbance. Here, we present a new phycobilisome derived complex that consists only of allophycocyanin core subunits, having red-shifted absorption peaks of 653 and 712nm. These red-shifted phycobiliprotein complexes were isolated from the chlorophyll f-containing cyanobacterium, Halomicronema hongdechloris, grown under monochromatic 730nm-wavelength (far-red) light. The 3D model obtained from single particle analysis reveals a double disk assembly of 120–145Å with two α/β allophycocyanin trimers fitting into the two separated disks. They are significantly smaller than typical phycobilisomes formed from allophycocyanin subunits and core-membrane linker proteins, which fit well with a reduced distance between thylakoid membranes observed from cells grown under far-red light. Spectral analysis of the dissociated and denatured phycobiliprotein complexes grown under both these light conditions shows that the same bilin chromophore, phycocyanobilin, is exclusively used. Our findings show that red-shifted phycobilisomes are required for assisting efficient far-red light harvesting. Their discovery provides new insights into the molecular mechanisms of light harvesting under extreme conditions for photosynthesis, as well as the strategies involved in flexible chromatic acclimation to diverse light conditions.

The specific localizations of phosphorylated Lhcb1 and Lhcb2 isoforms reveal the role of Lhcb2 in the formation of the PSI-LHCII supercomplex in Arabidopsis during state transitions

State transitions are an important photosynthetic short-term response that maintains the excitation balance between photosystems I (PSI) and II (PSII). In plants, when PSII is preferentially excited, LHCII, the main heterotrimeric light harvesting complex of PSII, is phosphorylated by the STN7 kinase, detaches from PSII and moves to PSI to equilibrate the relative absorption of the two photosystems (State II). When PSI is preferentially excited LHCII is dephosphorylated by the PPH1 (TAP38) phosphatase, and returns to PSII (State I). Phosphorylation of LHCII that remain bound to PSII has also been observed. Although the kinetics of LHCII phosphorylation are well known from a qualitative standpoint, the absolute phosphorylation levels of LHCII (and its isoforms) bound to PSI and PSII have been little studied. In this work we thoroughly investigated the phosphorylation level of the Lhcb1 and Lhcb2 isoforms that compose LHCII in PSI-LHCII and PSII-LHCII supercomplexes purified from WT and state transition mutants of Arabidopsis thaliana. We found that, at most, 40% of the monomers that make up PSI-bound LHCII trimers are phosphorylated. Phosphorylation was much lower in PSII-bound LHCII trimers reaching only 15–20%. Dephosphorylation assays using a recombinant PPH1 phosphatase allowed us to investigate the role of the two isoforms during state transitions. Our results strongly suggest that a single phosphorylated Lhcb2 is sufficient for the formation of the PSI-LHCII supercomplex. These results are a step towards a refined model of the state transition phenomenon and a better understanding of the short-term response to changes in light conditions in plants.

Highly efficient energy transfer from a carbonyl carotenoid to chlorophyll a in the main light harvesting complex of Chromera velia

We report on energy transfer pathways in the main light-harvesting complex of photosynthetic relative of apicomplexan parasites, Chromera velia. This complex, denoted CLH, belongs to the family of FCP proteins and contains chlorophyll (Chl) a, violaxanthin, and the so far unidentified carbonyl carotenoid related to isofucoxanthin. The overall carotenoid-to-Chl-a energy transfer exhibits efficiency over 90% which is the largest among the FCP-like proteins studied so far. Three spectroscopically different isofucoxanthin-like molecules were identified in CLH, each having slightly different energy transfer efficiency that increases from isofucoxanthin-like molecules absorbing in the blue part of the spectrum to those absorbing in the reddest part of spectrum. Part of the energy transfer from carotenoids proceeds via the ultrafast S2 channel of both the violaxanthin and isofucoxanthin-like carotenoid, but major energy transfer pathway proceeds via the S1/ICT state of the isofucoxanthin-like carotenoid. Two S1/ICT-mediated channels characterized by time constants of ~0.5 and ~4ps were found. For the isofucoxanthin-like carotenoid excited at 480nm the slower channel dominates, while those excited at 540nm employs predominantly the fast 0.5ps channel. Comparing these data with the excited-state properties of the isofucoxanthin-like carotenoid in solution we conclude that, contrary to other members of the FCP family employing carbonyl carotenoids, CLH complex suppresses the charge transfer character of the S1/ICT state of the isofucoxanthin-like carotenoid to achieve the high carotenoid-to-Chl-a energy transfer efficiency.

Membrane crystals of plant light-harvesting complex II disassemble reversibly in light.

Using the mass-measuring capability of scanning transmission electron microscopy, we demonstrate that membrane crystals of the main light-harvesting complex of plants possess the ability to undergo light-induced dark-reversible disassociations, independently of the photochemical apparatus. This is the first direct visualization of light-driven reversible reorganizations in an isolated photosynthetic antenna. These reorganizations, identified earlier by circular dichroism (CD), can be accounted for by a biological thermo-optic transition: structural changes are induced by fast heat transients and thermal instabilities near the dissipation, and self-association of the complexes in the lipid matrix. A comparable process in native membranes is indicated by earlier findings of essentially identical kinetics, and intensity and temperature dependences of the ΔCD in granal thylakoids.

Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii

State transitions represent a photoacclimation process that regulates the light-driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between photosystem I (PSI) and II (PSII) by shuttling LHCII, the main light-harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas reinhardtii in which it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This situation is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C.reinhardtii in states 1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state 2 is able to bind two LHCII trimers that contain all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This structure is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI-bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type III and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2.

Role of Carotenoids in Light-Harvesting Processes in an Antenna Protein from the Chromophyte Xanthonema debile

Chromophytes are an important group of microorganisms that contribute significantly to the carbon cycle on Earth. Their photosynthetic capacity depends on efficiency of the light-harvesting system that differs in pigment composition from that of green plants and other groups of algae. Here we employ femtosecond transient absorption spectroscopy to study energy transfer pathways in the main light-harvesting complex of Xanthonema debile, denoted XLH, which contains four carotenoids--diadinoxanthin, heteroxanthin, diatoxanthin, and vaucheriaxanthin--and Chl-a. Overall carotenoid-to-chlorophyll energy transfer efficiency is about 60%, but energy transfer pathways are excitation wavelength dependent. Energy transfer from the carotenoid S(2) state is active after excitation at both 490 nm (maximum of carotenoid absorption) and 510 nm (red edge of carotenoid absorption), but this channel is significantly more efficient after 510 nm excitation. Concerning the energy transfer pathway from the S(1) state, XLH contains two groups of carotenoids: those that have the S(1) route active (~25%) and those having the S(1) pathway silent. For a fraction of carotenoids that transfer energy via the S(1) channel, energy transfer is observed after both excitation wavelengths, though energy transfer times are different, yielding 3.4 ps (490 nm excitation) and 1.5 ps (510 nm excitation). This corresponds to efficiencies of the S(1) channel of ~85% that is rather unusual for a donor-acceptor pair consisting of a noncarbonyl carotenoid and Chl-a. Moreover, major carotenoids in XLH, diadinoxanthin and diatoxanthin, have their S(1) energies in solution lower than the energy of the acceptor state, Q(y) state of Chl-a. Thus, binding of these carotenoids to XLH must tune their S(1) energy to allow for efficient energy transfer. Besides the light-harvesting function, carotenoids in XLH also have photoprotective role; they quench Chl-a triplets via triplet-triplet energy transfer from Chl-a to carotenoid.