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The Q y and B x excitation energy transfer (EET) in the minor light-harvesting complex CP29 (LHCII B4.1) antenna complex of Pisum sativum was characterized using a computational approach. We applied Förster resonance energy transfer (FRET) and the transition density cube (TDC) method to estimate the Coulombic coupling, based on a combination of classical molecular dynamics and quantum mechanics/molecular mechanics calculations. Employing TDC instead of FRET mostly affects the EET between chlorophylls (Chls) and carotenoids (Crts), as expected due to the Crts being spatially more challenging for FRET. Only between Chls, effects are found to be small (about only 0.1 EET efficiency change when introducing TDC instead of FRET). Effects of structural sampling were found to be small, illustrated by a small average standard deviation for the Q y state coupling elements (FRET/TDC: 0.97/0.94 cm -1 ). Due to the higher flexibility of the B x state, the corresponding deviations are larger (FRET/TDC between Chl-Chl pairs: 17.58/22.67 cm -1 , between Crt-Chl pairs: 62.58/31.63 cm -1 ). In summary, it was found for the Q band that the coupling between Chls varies only slightly depending on FRET or TDC, resulting in a minute effect on EET acceptor preference. In contrast, the coupling in the B band spectral region is found to be more affected. Here, the S 2 (1 B u ) states of the spatially challenging Crts may act as acceptors in addition to the B states of the Chls. Depending on FRET or TDC, several Chls show different Chl-to-Crt couplings. Interestingly, the EET between Chls or Crts in the B band is found to often outcompete the corresponding decay processes. The individual efficiencies for B band EET to Crts vary however strongly with the chosen coupling scheme (e.g., up to 0.29/0.99 FRET/TDC efficiency for the Chl a 604/neoxanthin pair). Thus, the choice of the coupling scheme must involve a consideration of the state of interest.

Microbial rhodopsin (also called retinal protein)-carotenoid conjugates represent a unique class of light-harvesting (LH) complexes, but their specific interactions and LH properties are not completely elucidated as only few rhodopsins are known to bind carotenoids. Here, we report a natural sodium-ion (Na + )-pumping Nonlabens (Donghaeana) dokdonensis rhodopsin (DDR2) binding with a carotenoid salinixanthin (Sal) to form a thermally stable rhodopsin-carotenoid complex. Different spectroscopic studies were employed to monitor the retinal-carotenoid interaction as well as the thermal stability of the protein, while size-exclusion chromatography (SEC) and homology modeling are performed to understand the protein oligomerization process. In analogy with that of another Na + -pumping protein Krokinobacter eikastus rhodopsin 2 (KR2), we propose that DDR2 (studied concentration range: 2 × 10 -6 to 4 × 10 -5 M) remains mainly as a pentamer at room temperature and neutral pH, while heating above 55 °C partially converted it into a thermally less stable oligomeric form of the protein. This process is affected by both the pH and concentration. At high concentrations (4 × 10 -5 to 2 × 10 -4 M), the protein adopts a pentamer form reflected in the excitonic circular dichroism (CD) spectrum. In the presence of Sal, the thermal stability of DDR2 is increased significantly, and the pigment is stable even at 85 °C. The results presented could have implications in designing stable rhodopsin-carotenoid antenna complexes.

Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP-FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle. Interacting with the compact OCP state, FRP completely prevents the possibility of OCP domain separation and formation of the signaling state capable of interacting with the antenna. The structural element that prevents FRP binding and formation of the complex is the short α-helix at the beginning of the N-terminal domain of OCP, which masks the primary site in the C-terminal domain of OCP. We determined the rate of opening of this site and show that it remains exposed long after the relaxation of the red OCP states. Observations of the OCP transitions on the ms time scale revealed that the relaxation of the orange photocycle intermediates is accompanied by an increase in the interaction of the carotenoid keto group with the hydrogen bond donor tyrosine-201. Our data refine the current model of photoinduced OCP transitions and the interaction of its intermediates with FRP.

We investigated the fluorescence kinetics of LH2 complexes from Marichromatium purpuratum , the cryo-EM structure of which has been recently elucidated with 2.4 Å resolution. The experiments have been carried out as a function of the excitation density by varying both the excitation fluence and the repetition rate of the laser excitation. Instead of the usual multiexponential fitting procedure, we applied the less common phasor formalism for evaluating the transients because this allows for a model-free analysis of the data without a priori knowledge about the number of processes that contribute to a particular decay. For the various excitation conditions, this analysis reproduces consistently three lifetime components with decay times below 100 ps, 500 ps, and 730 ps, which were associated with the quenched state, singlet-triplet annihilation, and fluorescence decay, respectively. Moreover, it reveals that the number of decay components that contribute to the transients depends on whether the excitation wavelength is in resonance with the B800 BChl a molecules or with the carotenoids. Based on the mutual arrangement of the chromophores in their binding pockets, this leads us to conclude that the energy transfer pathways within the LH2 complex of this species differ significantly from each other for exciting either the B800 BChl molecules or the carotenoids. Finally, we speculate whether the illumination with strong laser light converts the LH2 complexes studied here into a quenched conformation that might be related to the development of the non-photochemical quenching mechanism that occurs in higher plants.

The allosteric regulation of protein function proves important in many life-sustaining processes. In plant photosynthesis, LHCII, the major antenna complex of Photosystem II, employs a delicate switch between light harvesting and photoprotective modes. The switch is triggered by an enlarged pH gradient (ΔpH) across the thylakoid membranes. Using molecular simulations and quantum calculations, we show that ΔpH can tune the light-harvesting potential of the antenna via allosteric regulation of the excitonic coupling in chlorophyll-carotenoid pairs. To this end, we propose how the LHCII excited state lifetime is coupled to the environmental conditions. In line with experimental findings, our theoretical model provides crucial evidence toward the elucidation of the photoprotective switch of higher plants at an all-atom resolution.

Chlorophyll triplet excited states are byproducts of photosynthetic processes that can indirectly harm biological membranes by forming highly reactive oxygen species. A crucial photoprotective mechanism evolved by plants to counter this threat involves the triplet energy transfer from chlorophylls to carotenoid molecules, in which triplet states are not reactive. In the major light-harvesting complex of photosystem II (LHCII), the two central luteins play an important role in the mechanism, but it has been shown that carotenoid triplets are formed even when other carotenoids replace them in their binding sites. In this work, we have investigated carotenoid triplet formation in LHCII isolated from Arabidopsis thaliana npq1lut2 plants, in which violaxanthin replaces lutein. Although transient absorption spectroscopy showed altered singlet excited-state dynamics in the mutant LHCII without lutein, these antennae formed carotenoid triplets that were spectrally and dynamically identical to the wild-type protein. We conclude that lutein-binding sites in LHCII have conserved characteristics to ensure efficient triplet energy transfer to the carotenoid molecules that they accommodate, making the identity of the carotenoid trivial per se.

We report quantum chemical calculations using multireference perturbation theory (MRPT) with the density matrix renormalization group (DMRG) plus photothermal deflection spectroscopy measurements to investigate the manifold of carotenoid excited states and establish their energies relative to the bright state (S 2 ) as a function of nuclear reorganization. We conclude that the primary photophysics and function of carotenoids are determined by interplay of only the bright (S 2 ) and lowest-energy dark (S 1 ) states. The lowest-lying dark state, far from being energetically distinguishable from the lowest-lying bright state along the entire excited-state nuclear reorganization pathway, is instead computed to be either the second or first excited state depending on what equilibrium geometry is considered. This result suggests that, rather than there being a dark intermediate excited state bridging a non-negligible energy gap from the lowest-lying dark state to the lowest-lying bright state, there is in fact no appreciable energy gap to bridge following photoexcitation. Instead, excited-state nuclear reorganization constitutes the bridge from S 2 to S 1 , in the sense that these two states attain energetic degeneracy along this pathway.

Solar light harvesting begins with electronic energy transfer in structurally complex light-harvesting antennae such as the peridinin chlorophyll- a protein from dinoflagellate algae. Peridinin chlorophyll- a protein is composed of a unique combination of chlorophylls sensitized by carotenoids in a 4:1 ratio, and ultrafast spectroscopic methods have previously been utilized in elucidating their energy-transfer pathways and timescales. However, due to overlapping signals from various chromophores and competing pathways and timescales, a consistent model of intraprotein electronic energy transfer has been elusive. Here, we used a broad-band two-dimensional electronic spectroscopy, which alleviates the spectral congestion by dispersing excitation and detection wavelengths. Interchromophoric couplings appeared as cross peaks in two-dimensional electronic spectra, and these spectral features were observed between the peridinin S 2 states and chlorophyll- a Q x and Q y states. In addition, the inherently high time and frequency resolutions of two-dimensional electronic spectroscopy enabled accurate determination of the ultrafast energy-transfer dynamics. Kinetic analysis near the peridinin S 1 excited-state absorption, which forms in 24 fs after optical excitation, reveals an ultrafast energy-transfer pathway from the peridinin S 2 state to the chlorophyll- a Q x state, a hitherto unconfirmed pathway critical for fast interchromophoric transfer. We propose a model of ultrafast peridinin chlorophyll- a protein photophysics that includes (1) a conical intersection between peridinin S 2 and S 1 states to explain both the ultrafast peridinin S 1 formation and the residual peridinin S 2 population for energy transfer to chlorophyll- a , and (2) computationally and experimentally derived peridinin S 2 site energies that support the observed ultrafast peridinin S 2 to chlorophyll-a Q x energy transfer.

Fucoxanthin chlorophyll (Chl) a/ c-binding proteins (FCPs) are unique light-harvesting antennas in diatoms. Recent time-resolved fluorescence analysis of photosystem I with FCP associated (PSI-FCPI) has mainly shown excitation energy transfer among Chls a from FCPI to PSI in tens of picoseconds. However, it remains unclear how each pigment, especially carotenoids and Chl c, in the FCPI is functionally related to the energy transfer in a femtosecond time range. Here, we reveal ultrafast excitation energy transfer mechanism in the PSI-FCPI preparations isolated from a diatom, Chaetoceros gracilis, by means of femtosecond time-resolved fluorescence spectroscopy with an upconversion system. Compared with the fluorescence lifetime components of PSI core-like complexes, the energy transfer of Chl c → Chl a in the FCPI was observed within hundreds of femtoseconds, and the energy in the FCPI was transferred to PSI in ∼2 ps. The comparative fluorescence analyses provide physical insights into the energy transfer machinery within FCPI and from FCPI to PSI.

We used elastic incoherent neutron scattering (EINS) to find out if structural changes accompanying local hydrogen bond rupture are also reflected in global dynamical response of the protein complex. Chromatophore membranes from LH2-only strains of the photosynthetic bacterium Rhodobacter sphaeroides, with spheroidenone or neurosporene as the major carotenoids, were subjected to high hydrostatic pressure at ambient temperature. Optical spectroscopy conducted at high pressure confirmed rupture of tertiary structure hydrogen bonds. In parallel, we used EINS to follow average motions of the hydrogen atoms in LH2, which reflect the flexibility of this complex. A decrease of the average atomic mean square displacements of hydrogen atoms was observed up to a pressure of 5 kbar in both carotenoid samples due to general stiffening of protein structures, while at higher pressures a slight increase of the displacements was detected in the neurosporene mutant LH2 sample only. These data show a correlation between the local pressure-induced breakage of H-bonds, observed in optical spectra, with the altered protein dynamics monitored by EINS. The slightly higher compressibility of the neurosporene mutant sample shows that even subtle alterations of carotenoids are manifested on a larger scale and emphasize a close connection between the local structure and global dynamics of this membrane protein complex.

In microbial rhodopsins (also called retinal proteins), the retinal chromophore is used for harvesting light. A carotenoid molecule has been reported to complement the retinal as light harvesting antenna in bacterial retinal proteins, although examples are scarce. In this paper, we present the formation of a novel antenna complex between thermophilic rhodopsin (TR) and the carotenoid salinixanthin (Sal). The complex formation and its structure were studied using UV-visible absorption as well as circular dichroism (CD) spectroscopies. Our studies indicate that the complex is formed in both the trimeric and monomeric forms of TR. CD spectroscopy suggests that excitonic coupling takes place between retinal and Sal. The binding of Sal with artificial TR pigments derived from synthetic retinal analogues further supports the contribution of the retinal chromophore to the CD spectrum. These studies further support the possibility of interaction between the 4-keto ring of the Sal and the retinal in TR-Sal complexes. Temperature-dependent CD spectra indicate that the positive band (ca. 482 nm) of the bisignate CD spectra of the studied complexes originates from the contribution of excitonic coupling and induced chirality of Sal in the protein binding site. The presence of a relatively smaller glycine residue in the vicinity of the retinal chromophore in TR is proposed to be crucial for binding with Sal. The results are expected to shed light on the mechanism of retinal-carotenoid interactions in other biological systems.

Energetics, protein dynamics, and electronic coupling are the key factors in controlling both electron and energy transfer in photosynthetic bacterial reaction centers (RCs). Here, we examine the rates and mechanistic pathways of the P + H A - radical-pair charge recombination, triplet state formation, and subsequent triplet energy transfer from the triplet state of the bacteriochlorophyll dimer (P) to the carotenoid in a series of mutant RCs (L131LH + M160LH (D1), L131LH + M197FH (D2), and L131LH + M160LH + M197FH (T1)) of Rhodobacter sphaeroides. In these mutants, the electronic structure of P is perturbed and the P/P + midpoint potential is systematically increased due to addition of hydrogen bonds between P and the introduced residues. High-resolution, broad-band, transient absorption spectroscopy on the femtosecond to microsecond timescale shows that the charge recombination rate increases and the triplet energy transfer rate decreases in these mutants relative to the wild type (WT). The increase of the charge recombination rate is correlated to the increase in the energy level of P + H A - and the increase in the P/P + midpoint potential. On the other hand, the decrease in rate of triplet energy transfer in the mutants can be explained in terms of a lower energy of 3 P and a shift in the electron spin density distribution in the bacteriochlorophylls of P. The triplet energy-transfer rate follows the order of WT > L131LH + M197FH > L131LH + M160LH > L131LH + M160LH + M197FH, both at room temperature and at 77 K. A pronounced temperature dependence of the rate is observed for all of the RC samples. The activation energy associated to this process is increased in the mutants relative to WT, consistent with a lower 3 P energy due to the addition of hydrogen bonds between P and the introduced residues.

Chlorophyll (Chl) triplet states generated in photosynthetic light-harvesting complexes (LHCs) can be quenched by carotenoids to prevent the formation of reactive singlet oxygen. Although this quenching occurs with an efficiency close to 100% at physiological temperatures, the Chl triplets are often observed at low temperatures. This might be due to the intrinsic temperature dependence of the Dexter mechanism of excitation energy transfer, which governs triplet quenching, or by temperature-induced conformational changes. Here, we report about the temperature dependence of Chl triplet quenching in two LHCs. We show that both the effects contribute significantly. In LHC II of higher plants, the core Chls are quenched with a high efficiency independent of temperature. A different subpopulation of Chls, which increases with lowering temperature, is not quenched at all. This is probably caused by the conformational changes which detach these Chls from the energy-transfer chain. In a membrane-intrinsic LHC of dinoflagellates, similarly two subpopulations of Chls were observed. In addition, another part of Chl triplets is quenched by carotenoids with a rate which decreases with temperature. This allowed us to study the temperature dependence of Dexter energy transfer. Finally, a part of Chls was quenched by triplet-triplet annihilation, a phenomenon which was not observed for LHCs before.

To explore the photoprotection role of multicompositional carotenoid (Car) in photosynthetic purple bacteria, we investigated, by means of triplet excitation profile (TEP) combined with steady-state optical spectroscopies, the core light-harvesting complex-reaction center of a mutant strain of Rhodobacter sphaeroides (m-LH1-RC) at room temperature. TEP spectra revealed that spheroidene and derivative (Spe) preferentially protect bacteriochlorophylls (BChls) of relatively lower site energy by quenching the triplet excitation ( 3 BChl*); however, spirilloxanthin (Spx) does so irrespective to the site energy of BChls. Triplet excitation results showed the triplet excitation energy-transfer (EET) reaction in a timescale of ∼0.5 μs from Spe and derivatives as a major component (∼85%) to Spx as a minor component (∼8%), suggesting the coexistence of different kinds of Cars in the individual LH1 complex. The nonequivalent quenching potency and the triplet EET reaction between Cars constitute the cooperative photoprotection by multicompositional Cars in bacterial photosynthesis.

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower pK a 3.5 and another at the higher pK a 8.4, that affect the pigment's formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of the binding site by the retinal chromophore induces protein conformational alterations which in turn affect the carotenoid conformation, which precedes the formation of the retinal-protein covalent bond. Our examination of synthetic retinal analogues in which the ring structure was modified revealed that, in the absence of sal, the retinal ring structure affects the rate of pigment formation and that the intact structure is needed for efficient pigment formation. However, the presence of sal abolishes this effect, and all-trans retinal and its modified ring analogues bind at a similar rate.

We present a direct comparison of two-photon spectra of various carotenoid-tetrapyrrole dyads and phthalocyanines (Pc) as well as chlorophylls (Chl) in the spectral range between 950 and 1360 nm, corresponding to one-photon spectra between 475 and 680 nm. For carotenoids (Car) with 8, 9, or 10 conjugated double bonds, the two-photon absorption cross section of states below the optical allowed carotenoid S 2 is at least about 3-10 times higher than that of Pc or chlorophyll a and b at 550/1100 nm. A quantitative comparison of spectra from Pc with and without carotenoids of eight and nine conjugated double bonds confirms energy transfer from optically forbidden carotenoid states to Pc in these dyads. When considering that less than 100% efficient energy transfer reduces the two-photon contribution of the carotenoids in the spectra, the actual Car two-photon cross sections relative to Chl/Pc are even higher than a factor of 3-10. In addition, strong spectroscopic two-photon signatures at energies below the optical allowed carotenoid S 2 state support the presence of additional optical forbidden carotenoid states such as S*, S x , or, alternatively, contributions from higher vibronic or hot S 1 states dominating two-photon spectra or energy transfer from the carotenoids. The onset of these states is shifted about 1500-3500 cm -1 to lower energies in comparison to the S 2 states. Our data provides evidence that two-photon excitation of the carotenoid S*, S x , or hot S 1 states results in energy transfer to tetrapyrroles or chlorophylls similar to that observed with the Car S 1 two-photon excitation.

The present study investigates the structure of lycopene aggregates both in vitro and in vivo using ultraviolet-visible (UV-vis) and Raman spectroscopies. The electronic absorption bands of the J- and H-aggregates in vitro shift to lower and higher energies, respectively, compared to that of the lycopene monomer. Along with these results, the frequencies of the ν 1 Raman bands were shifted to lower and higher frequencies, respectively. By plotting the frequencies of the ν 1 Raman band against the S 0 → S 2 transition energy, a linear relationship between the data set with different aggregation conformations can be obtained. Therefore, the band positions depending on the different conformations can be explained based on the idea that the effective conjugated C═C chain lengths within lycopene molecules are different due to the environmental effect (site-shift effect) caused by the aggregation conformation. Applying this knowledge to the in vivo measurement of a tomato fruit sample, the relationship between the aggregation conformation of lycopene and the spectral patterns observed in the UV-vis as well as Raman spectra in different parts of tomato fruits was discussed in detail. The results showed that the concentration of lycopene (particularly that of the J-aggregate) specifically increased, whereas that of chlorophyll decreased, with ripening. Furthermore, Raman imaging indicated that lycopene with different aggregate conformations was distributed inhomogeneously, even within one sample. The layer formation in tomato tissues with high concentrations of J- and H-aggregates was successfully visualized. In this manner, the presence of lycopene distributions with different aggregate conformations was unveiled in vivo.

This spectroscopic study investigates the origin of the transient feature of the S* excited state of carotenoids bound in LH1 complexes from purple bacteria. The studies were performed on two RC-LH1 complexes from Rba. sphaeroides strains that bound carotenoids with different carbon-carbon double bond conjugation N, neurosporene (N = 9) and spirilloxanthin (N = 13). The S* transient spectral feature, originally associated with an elusive and optically silent excited state of spirilloxanthin in the LH1 complex, may be successfully explained and mimicked without involving any unknown electronic state. The spectral and temporal characteristics of the S* feature suggest that it is associated with triplet-triplet annihilation of carotenoid triplets formed after direct excitation of the molecule via a singlet fission mechanism. Depending on pigment homogeneity and carotenoid assembly in the LH1 complex, the spectro-temporal component associated with triplet-triplet annihilation may simply resolve a pure T-S spectrum of a carotenoid. In some cases (like spirilloxanthin), the T-S feature will also be accompanied by a carotenoid Stark spectrum and/or residual transient absorption of minor carotenoid species bound into LH1 antenna complex.

Seemingly redundant parallel pathways for electron transfer (ET), composed of identical sets of cofactors, are a cornerstone feature of photosynthetic reaction centers (RCs) involved in light-energy conversion. In native bacterial RCs, both A and B branches house one bacteriochlorophyll (BChl) and one bacteriopheophytin (BPh), but the A branch is used exclusively. Described herein are the results obtained for two Rhodobacter capsulatus RCs with an unnaturally high degree of cofactor asymmetry, two BPh on the RC's B side and two BChl on the A side. These pigment changes derive, respectively, from the His(M180)Leu mutation [a BPh (Φ B ) replaces the B-side BChl (B B )], and the Leu(M212)His mutation [a BChl (β A ) replaces the A-side BPh (H A )]. Additionally, Tyr(M208)Phe was employed to disfavor ET to the A branch; in one mutant, Val(M131)Glu creates a hydrogen bond to H B to enhance ET to H B . In both Φ B mutants, the decay kinetics of the excited primary ET donor (P*) resolve three populations with lifetimes of ∼9 ps (50-60%), ∼40 ps (10-20%), and ∼200 ps (20-30%), with P + Φ B - formed predominantly from the 9 ps fraction. The 50-60% yield of P + Φ B - is the highest yet observed for a Φ B -containing RC. The results provide insight into factors needed for efficient multistep ET.

Ultrafast photochemistry of pharaonis halorhodopsin (p-HR) in the intact membrane of Natronomonas pharaonis has been studied by photoselective femtosecond pump-hyperspectral probe spectroscopy with high time resolution. Two variants of this sample were studied, one with wild-type retinal prosthetic groups and another after shifting the retinal absorption deep into the blue range by reducing the Schiff base linkage, and the results were compared to a previous study on detergent-solubilized p-HR. This comparison shows that retinal photoisomerization dynamics is identical in the membrane and in the solubilized sample. Selective photoexcitation of bacterioruberin, which is associated with the protein in the native membrane, in wild-type and reduced samples, demonstrates conclusively that unlike the carotenoids associated with some bacterial retinal proteins the carrotenoid in p-HR does not act as a light-harvesting antenna.

Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation-bacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.

Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

Carotenoid-to-bacteriochlorophyll energy transfer has been widely investigated in bacteriochlorophyll (BChl) a-containing light harvesting complexes. Blastochloris viridis utilizes BChl b, whose absorption spectrum is more red-shifted than that of BChl a. This has implications on the efficiency and pathways of carotenoid-to-BChl energy transfer in this organism. The carotenoids that comprise the light-harvesting reaction center core complex (LH1-RC) of B. viridis are 1,2-dihydroneurosporene and 1,2-dihydrolycopene, which are derivatives of carotenoids found in the light harvesting complexes of several BChl a-containing purple photosynthetic bacteria. Steady-state and ultrafast time-resolved optical spectroscopic measurements were performed on the LH1-RC complex of B. viridis at room and cryogenic temperatures. The overall efficiency of carotenoid-to-bacteriochlorophyll energy transfer obtained from steady-state absorption and fluorescence measurements were determined to be ∼27% and ∼36% for 1,2-dihydroneurosporene and 1,2-dihydrolycopene, respectively. These results were combined with global fitting and target analyses of the transient absorption data to elucidate the energetic pathways by which the carotenoids decay and transfer excitation energy to BChl b. 1,2-Dihydrolycopene transfers energy to BChl b via the S2 → Qx channel with kET2 = (500 fs)(-1) while 1,2-dihydroneurosporene transfers energy via S1→ Qy (kET1 = (84 ps)(-1)) and S2 → Qx (kET2 = (2.2 ps)(-1)) channels.

Femtosecond heterodyne transient grating spectroscopy was employed to investigate the nonradiative decay pathway from the S2 (1(1)Bu(+)) state to the S1 (2(1)Ag(-)) state of peridinin in methanol solution. Just as previously observed by this laboratory for β-carotene in benzonitrile, the real (absorption) and imaginary (dispersion) components of the transient grating signal obtained with Fourier transform spectral interferometry from peridinin exhibit ultrafast responses indicating that S2 state decays in 12 fs to produce an intermediate state, Sx. The excited state absorption spectrum from the Sx state of peridinin, however, is found to be markedly blue-shifted from that of β-carotene because it makes a substantial contribution to the signal observed with 40 fs, 520 nm pulses. The results of a global target analysis and numerical simulations using nonlinear response functions and the multimode Brownian oscillator model support the assignment of Sx to a displaced conformation of the S2 state rather than to a vibrationally excited (or hot) S1 state. The Sx state in peridinin is assigned to a structure with a distorted conjugated polyene backbone moving past an activation-energy barrier between planar and twisted structures on the S2 potential surface. The lengthened lifetime of the Sx state of peridinin in methanol, 900 ± 100 fs, much longer than that typically observed for carotenoids lacking carbonyl substituents, ∼150 fs, can be attributed to the slowing of torsional motions by solvent friction. In peridinin, the system-bath coupling is significantly enhanced over that in β-carotene solution most likely due to the intrinsic intramolecular charge transfer character it derives from the electron withdrawing nature of the carbonyl substituent. An important additional implication is that the Sx state, and the distorted structures reached subsequently along the torsional gradient on the S2 potential surface, may serve as the principal excitation energy transfer donors to chlorophyll a in the peridinin-chlorophyll a protein from dinoflagellates.

This paper presents a spectroscopic investigation of deoxyperidinin, a synthetic peridinin analogue in which the carbonyl functional group in peridinin was replaced by a nonconjugated methylene group. Steady-state and ultrafast time-resolved absorption and fluorescence spectroscopic experiments are carried out on deoxyperidinin in n-hexane and acetonitrile at room temperature and in 2-methyltetrahydrofuran at 77 K. The spectra of deoxyperidinin have higher vibronic resolution compared to those of peridinin. The higher resolution is due to a substantial reduction in both molecular conformational disorder and inhomogeneous broadening of the spectra of deoxyperidinin compared to peridinin. Features in the steady-state absorption spectrum of deoxyperidinin that are not evident in the spectrum of peridinin are unambiguously assigned to the forbidden S0 (1(1)Ag(-)) → S1 (2(1)Ag(-)) absorption transition. The characteristics of both the steady-state and time-resolved spectra are interpreted using EOM-CCSD, SAC-CI, and MNDO-PSDCI quantum computational formalisms that provided a theoretical framework for understanding the photophysical properties of the molecules.

In order to estimate the possible structure of the unknown carbonyl carotenoid related to isofucoxanthin from Chromera velia denoted as isofucoxanthin-like carotenoid (Ifx-l), we employed steady-state and ultrafast time-resolved spectroscopic techniques to investigate spectroscopic properties of Ifx-l in various solvents. The results were compared with those measured for related carotenoids with known structure: fucoxanthin (Fx) and isofucoxanthin (Ifx). The experimental data were complemented by quantum chemistry calculations and molecular modeling. The data show that Ifx-l must have longer effective conjugation length than Ifx. Yet, the magnitude of polarity-dependent changes in Ifx-l is larger than for Ifx, suggesting significant differences in structure of these two carotenoids. The most interesting spectroscopic feature of Ifx-l is its response to solvent proticity. The transient absorption data show that (1) the magnitude of the ICT-like band of Ifx-l in acetonitrile is larger than in methanol and (2) the S1/ICT lifetime of Ifx-l in acetonitrile, 4 ps, is markedly shorter than in methanol (10 ps). This is opposite behavior than for Fx and Ifx whose S1/ICT lifetimes are always shorter in protic solvent methanol (20 and 13 ps) than in aprotic acetonitrile (30 and 17 ps). Comparison with other carbonyl carotenoids reported earlier showed that proticity response of Ifx-l is consistent with presence of a conjugated lactone ring. Combining the experimental data and quantum chemistry calculations, we estimated a possible structure of Ifx-l.

ECD, ROA, and VCD were used to characterize astaxanthin conformers that differ in their arrangements of the β-ionone ring in respect to the chain. We obtained ECD spectra experimentally, and the ECD, ROA, and VCD spectra of both individual conformers and conformation-averaged mixtures were predicted using quantum-chemical calculations at the CAM-B3LYP level of theory using the PCM solvation model. The chiroptical methods employed (particularly ECD and ROA) were considerably more sensitive to conformational changes of astaxanthin compared to "mono-signed" conventional Raman spectroscopy. Strikingly, conformers that are the same optical isomers (e.g., of 3S,3'S-astxanthin), while geometrically nearly mirror images, exhibited sign-inversed ECD and ROA spectra. The conformational sensitivity of these chiroptical methods makes them a promising tool in the study of carotenoids in the natural environment (for instance, in de novo algal or yeast astaxanthin sources).

Ultrafast transient electronic absorption, one- and two- dimensional electronic-vibrational spectroscopies were used to study the nonradiative relaxation dynamics of β-apo-8'-carotenal (bapo), a model aldehyde containing carotenoid, in cyclohexane and acetonitrile solutions. 2D electronic-vibrational (2DEV) spectroscopy allows for a direct correlation between the intrinsically coupled electronic and vibrational degrees of freedom, which are thought to play an important role in driving relaxation of bapo from the bright S2 and lower-lying dark S1 state. Line shapes of features in the 2DEV spectra allow us to make more definitive assignments of excited state vibrations of bapo in acetonitrile. Anisotropy studies definitively demonstrate that the excited state dynamics of bapo do not involve a trans-cis isomerization, counter to prior hypotheses. For specific vibrational modes, the electronic and vibrational line shapes remain correlated beyond the decay of the S2 excited state, indicating that the transfer of molecules to the S1 state is impulsive and involves a conical intersection in the vertical Franck-Condon region.

Lycopene biosynthesis in photosynthetic organisms controls the metabolic flux of reaction center carotenoids like β-carotene and lutein through the geometric four-step isomerization of 7,9,9',7'-tetra-cis-lycopene (prolycopene) to its all-trans form. In plants and cyanobacteria, a redox-controlled flavoenzyme carotenoid isomerase catalyzes the prolycopene isomerization although its functional loss inside the chloroplast can be rescued by light. In order to address the chloroplast-specificity and efficiency of the light-induced isomerization reaction, we need to critically understand the excited state dynamics of prolycopene and the nature of electronic states that lead to the isomerization. Using broadband femtosecond transient absorption spectroscopy, we observe ∼610 fs rise of the long-lived triplet state from the photoexcited S2 with a quantum yield of ∼0.19. The triplet state eventually triggers the first C═C bond isomerization at the symmetric 9 or 9' position on the tetra-cis backbone to yield the tri-cis product with 15% quantum efficiency. However, direct sensitization of the photoreactive triplet state via meso-tetraphenyl porphyrin sensitizer under steady state illumination leads to an efficient production of all-trans-lycopene with 58% quantum yield. Our work implies that chlorophyll-enriched chloroplasts should form an optimized photoreaction vessel for prolycopene isomerization, and synthetic utilization of such cis-carotenoids can lead to efficient triplet harvesting photon-conversion devices.

Bixin, a carotenoid found in annatto (Bixa orellana), is unique among natural carotenoids by being water-soluble. We stabilized free radicals from bixin on the surface of silica-alumina (Si-Al) and characterized them by pulsed electron-nuclear double resonance (ENDOR). DFT calculations of unpaired electron spin distribution for various bixin radicals predict the EPR hyperfine couplings. Least-square fitting of experimental ENDOR spectra by spectra calculated from DFT hyperfine couplings characterized the radicals trapped on Si-Al. DFT predicts that the trans bixin radical cation is more stable than the cis bixin radical cation by 1.26 kcal/mol. This small energy difference is consistent with the 26% trans and 23% cis radical cations in the ENDOR spectrum. The remainder of the ENDOR spectrum is due to several neutral radicals formed by loss of a H(+) ion from the 9, 9', 13, or 13' methyl group, a common occurrence in all water-insoluble carotenoids previously studied. Although carboxyl groups of bixin strongly affect its solubility relative to other natural carotenoids, they do not alter properties of its free radicals based on DFT calculations and EPR measurements which remain similar to typical water-insoluble carotenoids.

Excitation energy transfer (EET) in peridinin-chlorophyll-protein (PCP) complexes is dominated by the S1 → Qy pathway, but the high efficiencies cannot be solely explained by this one pathway. We postulate that EET from peridinin S2 excitons may also be important. We use complete active space configuration interaction calculations and the transition density cube method to calculate Coulombic couplings between peridinin and chlorophyll a in the PCP complex of the dinoflagellate Amphidinium carterae and compare monomeric and dimeric delocalized peridinin S2 excited states. Our calculations show that the S2 → Qy EET pathway from peridinin to chlorophyll a is the dominant energy transfer pathway from the S2 excited state in PCP, with several values in the sub-picosecond range. This result suggests that the S2 → Qy EET pathway may be responsible for the reported chlorophyll a bleaching signature seen in experiment at around 200 fs, and not the S2 → Qx pathway as previously suggested.

The fate of triplet excited states in the Fenna-Matthew-Olson (FMO) pigment-protein complex is studied by means of time-resolved nanosecond spectroscopy and exciton model simulations. Experiments reveal microsecond triplet excited-state energy transfer between the bacteriochlorophyll (BChl) pigments, but show no evidence of triplet energy transfer to molecular oxygen, which is known to produce highly reactive singlet oxygen and is the leading cause of photo damage in photosynthetic proteins. The FMO complex is exceptionally photo stable despite the fact it contains no carotenoids, which could effectively quench triplet excited states of (bacterio)chlorophylls and are usually found within pigment-protein complexes. It is inferred that the triplet excitation is transferred to the lowest energy pigment, BChl 3, within the FMO complex, whose triplet state energy is shifted by pigment-protein interactions below that of the singlet oxygen excitation. Thus, the energy transfer to molecular oxygen is blocked and the FMO does not need carotenoids for photo protection.

The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S2 of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

Two carotenoids with aryl rings were studied by femtosecond transient absorption spectroscopy and theoretical computational methods, and the results were compared with those obtained from their nonaryl counterpart, β-carotene. Although isorenieratene has more conjugated C═C bonds than β-carotene, its effective conjugation length, Neff, is shorter than of β-carotene. This is evidenced by a longer S1 lifetime and higher S1 energy of isorenieratene compared to the values for β-carotene. On the other hand, although isorenieratene and renierapurpurin have the same π-electron conjugated chain structure, Neff is different for these two carotenoids. The S1 lifetime of renierapurpurin is shorter than that of isorenieratene, indicating a longer Neff for renierapurpurin. This conclusion is also consistent with a lower S1 energy of renierapurpurin compared to those of the other carotenoids. Density functional theory (DFT) was used to calculate equilibrium geometries of ground and excited states of all studied carotenoids. The terminal ring torsion in the ground state of isorenieratene (41°) is very close to that of β-carotene (45°), but equilibration of the bond lengths within the aryl rings indicates that the each aryl ring forms its own conjugated system. This results in partial detachment of the aryl rings from the overall conjugation making Neff of isorenieratene shorter than that of β-carotene. The different position of the methyl group at the aryl ring of renierapurpurin diminishes the aryl ring torsion to ∼20°. This planarization results in a longer Neff than that of isorenieratene, rationalizing the observed differences in spectroscopic properties.

In this work we analyzed the infrared and visible transient absorption spectra of all-trans-β-apo-8'-carotenal in several solvents, differing in both polarity and polarizability at different excitation wavelengths. We correlate the solvent dependence of the kinetics and the band shape changes in the infrared with that of the excited state absorption bands in the visible, and we show that the information obtained in the two spectral regions is complementary. All the collected time-resolved data can be interpreted in the frame of a recently proposed relaxation scheme, according to which the major contributor to the intramolecular charge transfer (ICT) state is the bright 1Bu(+) state, which, in polar solvents, is dynamically stabilized through molecular distortions and solvent relaxation. A careful investigation of the solvent effects on the visible and infrared excited state bands demonstrates that both solvent polarity and polarizability have to be considered in order to rationalize the excited state relaxation of trans-8'-apo-β-carotenal and clarify the role and the nature of the ICT state in this molecule. The experimental observations reported in this work can be interpreted by considering that at the Franck-Condon geometry the wave functions of the S1 and S2 excited states have a mixed ionic/covalent character. The degree of mixing depends on solvent polarity, but it can be dynamically modified by the effect of polarizability. Finally, the effect of different excitation wavelengths on the kinetics and spectral dynamics can be interpreted in terms of photoselection of a subpopulation of partially distorted molecules.

Free radical scavenging properties of Lycopene (LYC), Phytofluene (PF), and Phytoene (PE) are studied experimentally, considering the electron transfer mechanism and using the ABTS(•+) decolouration assay. Density Functional Theory is used to obtain the theoretical UV-visible spectra of ABTS (neutral and cation) and to determine the free radical scavenger capacity in terms of the electron donor mechanism and the deactivation of singlet oxygen. The experimental data agreed very well with the theoretical results. Considering both mechanisms, LYC, PF, and PE can be considered good free radical scavengers, with LYC turning out to be the best and PE the worst antiradical. However, the triplets that formed following the deactivation of singlet oxygen presented similar capacity for donating electrons. In the case of triplets, the antiradical capacity of LYC, PF, and PE is similar in terms of the electron donor mechanism. Although the results indicated that PF and PE are not as effective antiradicals as LYC, which is to be expected due to the number of conjugated double bonds, they do present a higher antioxidant capacity than expected considering the small number of conjugated double bonds.

The excited state dynamics of carbonyl carotenoids is very complex because of the coupling of single- and doubly excited states and the possible involvement of intramolecular charge-transfer (ICT) states. In this contribution we employ ultrafast infrared spectroscopy and theoretical computations to investigate the relaxation dynamics of trans-8'-apo-β-carotenal occurring on the picosecond time scale, after excitation in the S2 state. In a (slightly) polar solvent like chloroform, one-dimensional (T1D-IR) and two-dimensional (T2D-IR) transient infrared spectroscopy reveal spectral components with characteristic frequencies and lifetimes that are not observed in nonpolar solvents (cyclohexane). Combining experimental evidence with an analysis of CASPT2//CASSCF ground and excited state minima and energy profiles, complemented with TDDFT calculations in gas phase and in solvent, we propose a photochemical decay mechanism for this system where only the bright single-excited 1Bu(+) and the dark double-excited 2Ag(-) states are involved. Specifically, the initially populated 1Bu(+) relaxes toward 2Ag(-) in 200 fs. In a nonpolar solvent 2Ag(-) decays to the ground state (GS) in 25 ps. In polar solvents, distortions along twisting modes of the chain promote a repopulation of the 1Bu(+) state which then quickly relaxes to the GS (18 ps in chloroform). The 1Bu(+) state has a high electric dipole and is the main contributor to the charge-transfer state involved in the dynamics in polar solvents. The 2Ag(-) → 1Bu(+) population transfer is evidenced by a cross peak on the T2D-IR map revealing that the motions along the same stretching of the conjugated chain on the 2Ag(-) and 1Bu(+) states are coupled.

We modeled excitation energy transfer (EET) in the peridinin-chlorophyll a-protein (PCP) complex of dinoflagellate Amphidinium carterae to determine which pathways contribute dominantly to the high efficiency of this process. We used complete active space configuration interaction (CAS-CI) to calculate electronic structure properties of the peridinin (PID) and chlorophyll a (CLA) pigments in PCP and the transition density cube (TDC) method to calculate Coulombic couplings between energy transfer donors and acceptors. Our calculations show that the S1 → Qy EET pathway from peridinin to chlorophyll a is the dominant energy transfer pathway in PCP, with two sets of interactions-between PID612 and CLA601 and between PID622 and CLA602-contributing most strongly. EET lifetimes for these two interactions were calculated to be 2.66 and 2.90, with quantum efficiencies of 85.75 and 84.65%, respectively. The calculated Coulombic couplings for EET between two peridinin molecules in the strongly allowed S2 excited states are extremely large and suggest excitonic coupling between pairs of peridinin S2 states. This methodology is also broadly applicable to the study of EET in other photosynthetic complexes and/or organic photovoltaics, where both single and double excitations are present and donor and acceptor molecules are tightly packed.

The orange carotenoid protein (OCP) is a 35 kDa water-soluble protein involved in a photoprotective mechanism of the photosynthetic apparatus of cyanobacteria. The OCP protein contains a single molecule of the carotenoid 3'-hydroxyechinenone (3'-hECN). We have performed transient absorption studies at 77 K in the visible and near-infrared spectral ranges on 3'-hECN in solvent glass and in both inactive (orange) and active (red) forms of OCP. In the OCP the cryogenic temperature prohibited the protein from spontaneous conversion between activity stages and allowed us to study well-defined spectral forms of the protein. The studies show that each form of the OCP consists of two protein subpopulations having different photophysical properties of the bound 3'-hECN. At 77 K the inactive OCP reveals two lifetimes of the first excited state of 3'-hECN of 5.2 and 11 ps while in the active form of OCP these are 3.2 and 7.1 ps. We have also determined the energy of the first excited singlet state of 3'-hECN in long-lived subpopulations of both OCP forms at 77 K. This is 13,750 cm(-1) in the inactive OCP and 12,300 cm(-1) in the active OCP. Shortening of the lifetime and decrease of the energy of the first excited singlet state of 3'-hECN confirm the lengthening of the effective conjugation of the carotenoid upon the inactive-to-active conversion of OCP.

In the present paper, we provide an extended study of the vibrational signature of a butenolide carotenoid, peridinin, in various solvents by combining resonance Raman spectroscopy (RRS) with theoretical calculations. The presence of a Fermi resonance due to coupling between the lactonic C═O stretching and the overtone of the wagging of the C-H in the lactonic ring provides a spectroscopic way of differentiating between peridinins lying in different environments. This is a significant achievement, given that simultaneous presence of several peridinins (each with a peculiar photophysical role) in different environments occurs in the most important peridinin containing proteins, the peridinin-chlorophyll proteins (PCPs) and the Chl a-c2-peridinin binding proteins. In RRS, small modifications of solvent polarity can give rise to large differences in the intensity and splitting between the two bands, resulting from the Fermi resonance. By changing the polarity, we can tune the frequency of stretching of the C═O and, while the C-H wagging frequency is almost always constant in different solvents, move the system from a perfect resonance condition to off-resonance ones. We have corroborated our spectroscopic findings with a quasi-classical dynamical model of two coupled oscillators, and DFT calculations on peridinin in different solvents; we have also used calculations to complete the peridinin vibrational mode assignments in the 800-1600 cm(-1) region of RRS spectra, corresponding to polyene chain motion. Finally, the presence of Fermi resonance has been used to reinterpret previous vibrational spectroscopic experiments in PCPs.

Carotenoids play an essential role in photoprotection, interacting with other pigments to safely dissipate excess absorbed energy as heat. In cyanobacteria, the short time scale photoprotective mechanisms involve the photoactive orange carotenoid protein (OCP), which binds a single carbonyl carotenoid. Blue-green light induces the photoswitching of OCP from its ground state form (OCPO) to a metastable photoproduct (OCPR). OCPR can bind to the phycobilisome antenna and induce fluorescence quenching. The photoswitching is accompanied by structural and functional changes at the level of the protein and of the bound carotenoid. Here, we use broadband two-dimensional electronic spectroscopy to study the differences in excited state dynamics of the carotenoid in the two forms of OCP. Our results provide insight into the origin of the pronounced vibrational lineshape and oscillatory dynamics observed in linear absorption and 2D electronic spectroscopy of OCPO and the large inhomogeneous broadening in OCPR, with consequences for the chemical function of the two forms.

We present a quantum-mechanical investigation of the photophysics of a specific carotenoid, peridinin, which is present in light-harvesting complexes. The fundamental role played by the geometry in determining the position and character of its low-lying singlet electronic states is investigated using a multireference DFT approach in combination with a continuum solvation model. The main photophysical properties of peridinin appear to be governed by the lowest two singlet excited states, as no evidence points to an intermediate S* state and the energies of the upper excited states are too high to allow their population with excitation in the visible range. These two excited states (S1, 2(1)A(g)(-) and S2, 1(1)B(u)(+)) are highly connected through the conjugation path here characterized by the value of the bond length alternation (BLA). The S1 and S2 states present distinct natures for small BLA values, whereas for larger ones they become more similar in terms of both brightness and dipolar character and their energies become closer. The geometrical issue is thus of fundamental importance for a correct interpretation of the spectroscopic signatures of peridinin.

Bacteriochlorophyll a with Ni(2+) replacing the central Mg(2+) ion was used as an ultrafast excitation energy dissipation center in reconstituted bacterial LH1 complexes. B870, a carotenoid-less LH1 complex, and B880, an LH1 complex containing spheroidene, were obtained via reconstitution from the subunits isolated from chromatophores of Rhodospirillum rubrum . Ni-substituted bacteriochlorophyll a added to the reconstitution mixture partially substituted the native pigment in both forms of LH1. The excited-state dynamics of the reconstituted LH1 complexes were probed by femtosecond pump-probe transient absorption spectroscopy in the visible and near-infrared spectral region. Spheroidene-binding B880 containing no excitation dissipation centers displayed complex dynamics in the time range of 0.1-10 ps, reflecting internal conversion and intersystem crossing in the carotenoid, exciton relaxation in BChl complement, and energy transfer from carotenoid to the latter. In B870, some aggregation-induced excitation energy quenching was present. The binding of Ni-BChl a to both B870 and B880 resulted in strong quenching of the excited states with main deexcitation lifetime of ca. 2 ps. The LH1 excited-state lifetime could be modeled with an intrinsic decay time constant in Ni-substituted bacteriochlorophyll a of 160 fs. The presence of carotenoid in LH1 did not influence the kinetics of energy trapping by Ni-BChl unless the carotenoid was directly excited, in which case the kinetics was limited by a slower carotenoid S1 to bacteriochlorophyll energy transfer.

The spectroscopic properties of the peridinin-chlorophyll a-protein (PCP) from the coral symbiotic dinoflagellate Symbiodinium have been characterized by application of various ultrafast optical spectroscopies including femto- and nanosecond time-resolved absorption and picosecond time-resolved fluorescence (TRF) at 77 K. Excited state properties of peridinin and Chl a and their intramolecular interaction characteristics have been obtained from global fitting analysis and directed kinetic modeling of the data sets and compared to their counterparts known for the PCP from Amphidinium carterae. The lifetimes of the excited state of peridinin show close agreement with those known for the counterpart PCP, demonstrating that molecular interactions have the same characteristics in both complexes. More variances have been recorded for the excited state properties of Chl a including elongation of both the intramolecular energy transfer time between Chl's within the pair in the protein monomer and the excited state lifetime of the long wavelength form of Chl a (terminal acceptor). Kinetic modeling of formation of the peridinin triplet state has shown that the PCP is protected from potential photodamage due to an extremely fast peridinin triplet state formation of kTT = (14.4 ± 2.3) × 10(9) s(-1) ((70 ± 12)(-1) (ps)(-1)) that guarantees instantaneous depletion of Chl a triplets and prevents formation of harmful singlet oxygen ((1)ΔgO2).

Light-harvesting complexes ensure necessary flow of excitation energy into photosynthetic reaction centers. In the present work, transient absorption measurements were performed on LH1-RC complexes isolated from two aerobic anoxygenic phototrophs (AAPs), Roseobacter sp. COL2P containing the carotenoid spheroidenone, and Erythrobacter sp. NAP1 which contains the carotenoids zeaxanthin and bacteriorubixanthinal. We show that the spectroscopic data from the LH1-RC complex of Roseobacter sp. COL2P are very similar to those previously reported for Rhodobacter sphaeroides, including the transient absorption spectrum originating from the intramolecular charge-transfer (ICT) state of spheroidenone. Although the ICT state is also populated in LH1-RC complexes of Erythrobacter sp. NAP1, its appearance is probably related to the polarity of the bacteriorubixanthinal environment rather than to the specific configuration of the carotenoid, which we hypothesize is responsible for populating the ICT state of spheroidenone in LH1-RC of Roseobacter sp. COL2P. The population of the ICT state enables efficient S1/ICT-to-bacteriochlorophyll (BChl) energy transfer which would otherwise be largely inhibited for spheroidenone and bacteriorubixanthinal due to their low energy S1 states. In addition, the triplet states of these carotenoids appear well-tuned for efficient quenching of singlet oxygen or BChl-a triplets, which is of vital importance for oxygen-dependent organisms such as AAPs.

The regulation of light-harvesting in photosynthesis under conditions of varying solar light irradiation is essential for the survival and fitness of plants and algae. It has been proposed that rearrangements of protein distribution in the stacked grana region of thylakoid membranes connected to changes in the electronic pigment-interaction play a key role for this regulation. In particular, carotenoid-chlorophyll interactions seem to be crucial for the down-regulation of photosynthetic light-harvesting. So far, it has been difficult to determine the influence of the dense protein packing found in native photosynthetic membrane on these interactions. We investigated the changes of the electronic couplings between carotenoids and chlorophylls and the quenching in grana thylakoids of varying protein packing density by two-photon spectroscopy, conventional chlorophyll fluorometry, low-temperature fluorescence spectroscopy, and electron micrographs of freeze-fracture membranes. We observed an increasing carotenoid-chlorophyll coupling and fluorescence quenching with increasing packing density. Simultaneously, the antennas size and excitonic connectivity of Photosystem II increased with increasing quenching and carotenoid-chlorophyll coupling whereas isolated, decoupled LHCII trimers decreased. Two distinct quenching data regimes could be identified that show up at different protein packing densities. In the regime corresponding to higher protein packing densities, quenching is strongly correlated to carotenoid-chlorophyll interactions whereas in the second regime, a weak correlation is apparent with low protein packing densities. Native membranes are in the strong-coupling data regime. Consequently, PSII and LHCII in grana membranes of plants are already quenched by protein crowding. We concluded that this ensures efficient electronic connection of all pigment-protein complexes for intermolecular energy transfer to the reaction centers and allows simultaneously sensitive regulation of light harvesting by only small changes in the protein packaging.

In oxygenic photosynthetic organisms, chlorophyll triplets are harmful excited states readily reacting with molecular oxygen to yield the reactive oxygen species (ROS) singlet oxygen. Carotenoids have a photoprotective role in photosynthetic membranes by preventing photoxidative damage through quenching of chlorophyll singlets and triplets. In this work we used mutation analysis to investigate the architecture of chlorophyll triplet quenching sites within Lhcb5, a monomeric antenna protein of Photosystem II. The carotenoid and chlorophyll triplet formation as well as the production of ROS molecules were studied in a family of recombinant Lhcb5 proteins either with WT sequence, mutated into individual chlorophyll binding residues or refolded in vitro to bind different xanthophyll complements. We observed a site-specific effect in the efficiency of chlorophyll-carotenoid triplet-triplet energy transfer. Thus chlorophyll (Chl) 602 and 603 appear to be particularly important for triplet-triplet energy transfer to the xanthophyll bound into site L2. Surprisingly, mutation on Chl 612, the chlorophyll with the lower energy associated and in close contact with lutein in site L1, had no effect on quenching chlorophyll triplet excited states. Finally, we present evidence for an indirect role of neoxanthin in chlorophyll triplet quenching and show that quenching of both singlet and triplet states is necessary for minimizing singlet oxygen formation.

Predicting the complete electronic structure of carotenoid molecules remains an extremely complex problem, particularly in anisotropic media such as proteins. In this paper, we address the electronic properties of nine relatively simple carotenoids by the combined use of electronic absorption and resonance Raman spectroscopies. Linear carotenoids exhibit an excellent correlation between (i) the inverse of their conjugation chain length N, (ii) the energy of their S0 → S2 electronic transition, and (iii) the position of their ν1 Raman band (corresponding to the stretching mode of their conjugated C═C bonds). For cyclic carotenoids such as β-carotene, this correlation is also observed between the latter two parameters (S0 → S2 energy and ν1 frequency), whereas their "nominal" conjugation length N does not follow the same relationship. We conclude that β-carotene and cyclic carotenoids in general exhibit a shorter effective conjugation length than that expected from their chemical structure. In addition, the effect of solvent polarizability on these molecular parameters was investigated for four of the carotenoids used in this study. We demonstrate that resonance Raman spectroscopy can discriminate between the different effects underlying shifts in the S0 → S2 transition of carotenoid molecules.

Excited state dynamics in LH2 complexes of two purple bacterial species were studied by broad-band two-dimensional electronic spectroscopy. The optical response was measured in the 500-600 nm spectral region on the 0-400 fs time scale. Global target analysis of two-dimensional (2D) transient spectra revealed the main energy transfer pathways between carotenoid S2, 1Bu(-) and S1 states and bacteriochlorophyll Qx state. Global analysis ascertained the evolutionary and vibration-associated spectra, which also indicated the presence of a higher-lying vibrational level in the carotenoid S1 state. The estimation of the spectral overlap between the 1Bu(-) state and the Qx state indicated a significant contribution of the 1Bu(-) state to the overall S2-to-Qx excitation energy transfer.

The orange carotenoid protein (OCP) is a crucial player in the process of nonphotochemical quenching in a large number of cyanobacteria. This water-soluble protein binds one pigment only, the keto carotenoid 3'-hydroxyechinenone, and needs to be photoactivated by strong (blue-green) light in order to induce energy dissipation within or from the phycobilisome, the main light harvesting system of these organisms. We performed transient-absorption spectroscopy on OCP samples frozen in the inactive and active forms at 77 K. By making use of target analysis we determined the excited state properties of the active form. Our results show that OCP photoactivation modifies the carotenoid excited state energy landscape. More specifically the photoactivated OCP is characterized by one state with predominantly ICT character (ICT/S1) and a lifetime of 2.3 ps, and another state with mainly S1 character (S1/ICT) with a lifetime of 7.6 ps. We also show that the kinetic model is fully consistent with the RT data obtained earlier (Berera et al., J. Phys. Chem. B 2012, 116, 2568-2574). We propose that this ICT/S1 state acts as the quencher in the OCP mediated nonphotochemical quenching.