A Protein Environment-Modulated Energy Dissipation Channel in LHCII Antenna Complex.

The major light-harvesting complex of photosystem II (LHCII) is the main contributor to sunlight energy harvesting in plants. The flexible design of LHCII underlies a photoprotective mechanism whereby this complex switches to a dissipative state in response to high light stress, allowing the rapid dissipation of excess excitation energy (non-photochemical quenching, NPQ). In this work, we locked single LHCII trimers in a quenched conformation after immobilization of the complexes in polyacrylamide gels to impede protein interactions. A comparison of their pigment excited-state dynamics with quenched LHCII aggregates in buffer revealed the presence of a new spectral band at 515 nm arising after chlorophyll excitation. This is suggested to be the signature of a carotenoid excited state, linked to the quenching of chlorophyll singlet excited states. Our data highlight the marked sensitivity of pigment excited-state dynamics in LHCII to structural changes induced by the environment.

The robustness of the terminal emitter site in major LHCII complexes controls xanthophyll function during photoprotection.

Xanthophylls in light harvesting complexes perform a number of functions ranging from structural support to light-harvesting and photoprotection. In the major light harvesting complex of photosystem II in plants (LHCII), the innermost xanthophyll binding pockets are occupied by lutein molecules. The conservation of these sites within the LHC protein family suggests their importance in LHCII functionality. In the present work, we induced the photoprotective switch in LHCII isolated from the Arabidopsis mutant npq1lut2, where the lutein molecules are exchanged with violaxanthin. Despite the differences in the energetics of the pigments and the impairment of chlorophyll fluorescence quenching in vivo, we show that isolated complexes containing violaxanthin are still able to induce the quenching switch to a similar extent to wild type LHCII monomers. Moreover, the same spectroscopic changes take place, which suggest the involvement of the terminal emitter site (L1) in energy dissipation in both complexes. These results indicate the robust nature of the L1 xanthophyll binding domain in LHCII, where protein structural cues are the major determinant of the function of the bound carotenoid.

Spectroscopic Properties of Violaxanthin and Lutein Triplet States in LHCII are Independent of Carotenoid Composition.

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.

Energy transfer dynamics in a red-shifted violaxanthin-chlorophyll a light-harvesting complex.

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690 nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S2 and hot S1 states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.

Equilibration Dependence of Fucoxanthin S1 and ICT Signatures on Polarity, Proticity, and Temperature by Multipulse Femtosecond Absorption Spectroscopy.

To demonstrate the value of the multipulse method in revealing the nature of coupling between excited states and explore the environmental dependencies of lowest excited singlet state (S1) and intramolecular charge transfer (ICT) state equilibration, we performed ultrafast transient absorption pump-dump-probe and pump-repump-probe spectroscopies on fucoxanthin in various solvent conditions. The effects of polarity, proticity, and temperature were tested in solvents methanol at 293 and 190 K, acetonitrile, and isopropanol. We show that manipulation of the kinetic traces can produce one trace reflecting the equilibration kinetics of the states, which reveals that lower polarity, proticity, and temperature delay S1/ICT equilibration. On the basis of a two-state model representing the S1 and ICT states on the same S1/ICT potential energy surface, we were able to show that the kinetics are strictly dependent on the initial relative populations of the states as well as the decay of the ICT state to the ground state. Informed by global analysis, a systematic method for target analysis based on this model allowed us to quantify the population transfer rates throughout the life of the S1/ICT state as well as separate the S1 and ICT spectral signatures. The results are consistent with the concept that the S1 and ICT states are part of one potential energy surface.

Efficient light-harvesting using non-carbonyl carotenoids: Energy transfer dynamics in the VCP complex from Nannochloropsis oceanica.

Violaxanthin-chlorophyll a protein (VCP) from Nannochloropsis oceanica is a Chl a-only member of the LHC family of light-harvesting proteins. VCP binds carotenoids violaxanthin (Vio), vaucheriaxanthin (Vau), and vaucheriaxanthin-ester (Vau-ester). Here we report on energy transfer pathways in the VCP complex. The overall carotenoid-to-Chla energy transfer has efficiency over 90%. Based on their energy transfer properties, the carotenoids in VCP can be divided into two groups; blue carotenoids with the lowest energy absorption band around 480nm and red carotenoids with absorption extended up to 530nm. Both carotenoid groups transfer energy efficiently from their S2 states, reaching efficiencies of ~70% (blue) and ~60% (red). The S1 pathway, however, is efficient only for the red carotenoid pool for which two S1 routes characterized by 0.33 and 2.4ps time constants were identified. For the blue carotenoids the S1-mediated pathway is represented only by a minor route likely involving a hot S1 state. The relaxed S1 state of blue carotenoids decays to the ground state within 21ps. Presence of a fraction of non-transferring red carotenoids with the S1 lifetime of 13ps indicates some specific carotenoid-protein interaction that must shorten the intrinsic S1 lifetime of Vio and/or Vau whose S1 lifetimes in methanol are 26 and 29ps, respectively. The VCP complex from N. oceanica is the first example of a light-harvesting complex binding only non-carbonyl carotenoids with carotenoid-to-chlorophyll energy transfer efficiency over 90%.

Different Response of Carbonyl Carotenoids to Solvent Proticity Helps To Estimate Structure of the Unknown Carotenoid from Chromera velia.

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.

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.

Excited-state dynamics of monomeric and aggregated carotenoid 8'-apo-ß-carotenal.

Excited-state properties of monomeric and aggregated carbonyl carotenoid 8'-apo-ß-carotenal were studied by means of femtosecond transient absorption spectroscopy. For monomers, the polarity-dependent behavior characteristic of carotenoids with conjugated carbonyl group was observed. In n-hexane the S(1) lifetime is 25 ps, but it is shortened to 8 ps in methanol. This shortening is accompanied by the appearance of new spectral bands in transient absorption spectrum. On the basis of analysis of the transient absorption spectra of monomeric 8'-apo-ß-carotenal in n-hexane and methanol, we propose that the polarity-induced spectral bands are due to the S(1)(A(g)(-))-S(3)(A(g)(+)) transition, which is enhanced upon breaking the symmetry of the molecule. This symmetry breaking is caused by the conjugated carbonyl group; it is much stronger in polar solvents where the S(1) state gains significant charge-transfer character. Upon addition of water to methanol solution of 8'-apo-ß-carotenal we observed formation of aggregates characterized by either blue-shifted (H-aggregate) or red-shifted (J-aggregate) absorption spectrum. Both aggregate types exhibit excited-state dynamics significantly different from those of monomeric 8'-apo-ß-carotenal. The lifetime of the relaxed S(1) state is 20 and 40 ps for the H- and J-aggregates, respectively. In contrast to monomers, aggregation promotes formation of triplet state, most likely by homofission occurring between tightly packed molecules within the aggregate.

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.

The analysis of PS II photochemical activity using single and multi-turnover excitations.

Paper describes chlorophyll a fluorescence measurements in algal cells, and intact plant leaves and isolated chloroplasts. It focuses on amplitude and 10 µs-resolved kinetics of variable fluorescence responses upon excitation with fluorescence-saturating pulses (SP) and with 25 µs saturating single turnover flashes (STF) which are exposed before, during and after a 100 s actinic illumination (AL) of low and high intensity. In addition to the amply documented suppression of the maximal variable fluorescence from F(m) to F(m)('), the relative proportion of the distinguished O-J-, J-I- and I-P-phases of an SP-induced response is shown to be distinctly different in dark- and light-adapted leaves. The O-J-phase in the 0.01-1 ms time range is much less sensitive to light adaptation than the other phases in the 1-200 ms range. In algae and chloroplasts, the amplitude F(m)(STF) of the STF-induced response is hardly affected by a shift from the dark- to the light-activated steady state. The results support the hypothesis that the maximal variable fluorescence F(m) induced by a multiple-turnover, fluorescence-saturating pulse (SP), is associated with the release of photochemical and photoelectrochemical quenching. It is argued that the OJIPMT- or Kautsky induction curve of variable chlorophyll fluorescence in the 0-100 s time range is the reflection of the release of photochemical quenching supplemented with a temporary Photosystem I (PSI)-dependent photoelectric stimulation and transient release of photoelectrochemical quenching of radiative energy loss in the Photosystem II (PSII) antennas, rather than solely of a decrease in PSII photochemical activity as is usually concluded.

Excited-state properties of the 16kDa red carotenoid protein from Arthrospira maxima.

We have studied spectroscopic properties of the 16kDa red carotenoid protein (RCP), which is closely related to the orange carotenoid protein (OCP) from cyanobacteria. Both proteins bind the same chromophore, the carotenoid 3'-hydroxyechinenone (hECN), and the major difference between the two proteins is lack of the C-terminal domain in the RCP; this results in exposure of part of the carotenoid. The excited-state lifetime of hECN in the RCP is 5.5ps, which is markedly longer than in OCP (3.3ps) but close to 6ps obtained for hECN in organic solvent. This confirms that the binding of hECN to the C-terminal domain in the OCP changes conformation of hECN, thereby altering its excited-state properties. Hydrogen bonds between the C-terminal domain and the carotenoid are also absent in the RCP. This allows the conformation of hECN in the RCP to be similar to that in solution, which results in comparable excited-state properties of hECN in solution. The red-shift of the RCP absorption spectrum is most likely due to aggregation of RCP induced by hydrophobic nature of hECN that, when exposed to buffer, stimulates formation of assemblies minimizing contact of hECN with water. We suggest that the loss of the C-terminal domain renders the protein amphipathic, containing both hydrophobic (the exposed part of hECN) and hydrophilic (N-terminal domain) regions, and may help the RCP to interact with lipid membranes; exposed hECN can penetrate into the hydrophobic environment of the lipid membrane, possibly to provide additional photoprotection.

Carotenoids in energy transfer and quenching processes in Pcb and Pcb-PS I complexes from Prochlorothrix hollandica.

Chlorophyll (Chl) a/b-binding proteins from Prochlorothrix hollandica known as Pcb antennae were studied by femtosecond transient absorption technique to identify energy transfer rates and pathways in Pcb and Pcb-PS I complexes. Carotenoids transfer energy to Chl with low efficiency of approximately 25% in Pcb complexes. Interestingly, analysis of transient absorption spectra identified a pathway from the hot S(1) state of zeaxanthin and/or beta-carotene as the major energy transfer channel between carotenoids and chlorophylls in Pcb whereas the S(2) state contributes only marginally to energy transfer. Due to energetic reasons, no energy transfer is possible via the relaxed S(1) state of carotenoids. The low overall energy transfer efficiency of carotenoids recognizes chlorophylls as the main light-harvesting pigments. Besides Chl a, presence of Chl b, which transfers energy to Chl a with nearly 100% efficiency, significantly broadens the spectral range accessible for light-harvesting and improves cross section of Pcb complexes. The major role of carotenoids in Pcb is photoprotection.

Photochemical and photoelectrochemical quenching of chlorophyll fluorescence in photosystem II.

This paper deals with kinetics and properties of variable fluorescence in leaves and thylakoids upon excitation with low intensity multi-turnover actinic light pulses corresponding with an excitation rate of about 10 Hz. These show a relatively small and amply documented rise in the sub-s time range towards the plateau level F(pl) followed by a delayed and S-shaped rise towards a steady state level F(m) which is between three and four fold the initial dark fluorescence level F(o). Properties of this retarded slow rise are i) rate of dark recovery is (1-6 s)(-1), ii) suppression by low concentration of protonophores, iii) responsiveness to complementary single turnover flash excitation with transient amplitude towards a level F(m) which is between five and six fold the initial dark fluorescence level F(o) and iv) in harmony with and quantitatively interpretable in terms of a release of photoelectrochemical quenching controlled by the trans-thylakoid proton pump powered by the light-driven Q cycle. Data show evidence for a sizeable fluorescence increase upon release of (photo) electrochemical quenching, defined as qPE. Release of qPE occurs independent of photochemical quenching defined here as qPP even under conditions at which qPP = 1. The term photochemical quenching, hitherto symbolized by qP, will require a new definition, because it incorporates in its present form a sizeable photoelectrochemical component. The same is likely to be true for definition and use of qN as an indicator of non photochemical quenching.

On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains.

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q(A) of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F (o) towards a maximum F (m) (STF) when Q(A) becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20-30% increase when the second partner is applied within 20-100 micros after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2-0.4 and 2-5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1-4 kHz) shows (i) in the first 3-4 flashes a transient overshoot 20-30% above the F (m) (STF) = 3*F (o) level reached in the 1st flash with a partial decline towards a dip D in the next 2-3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F (m) = 5*F (o) in the 3-60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S(0) fraction with Q(B)-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q(B)-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: accumulation and function of QB-nonreducing centers.

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in Fm relative to Fo, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of QB-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of QB-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.

Evidence for localisation of accumulated chlorophyll cation on the D1-accessory chlorophyll in the reaction centre of photosystem II.

Absorption and circular dichroism spectra of Photosystem II (PS II) reaction centres (RC) were studied and compared with spectra calculated on the basis of point-dipole approximation. Chlorophyll cation was accumulated during a light treatment of PS II RC in the presence of artificial electron acceptor silicomolybdate. Light-induced difference spectra and their calculated counterparts revealed the location of accumulated cation at the accessory chlorophyll of the D1 protein subunit.

Excitonic interactions in the reaction centre of photosystem II studied by using circular dichroism.

Changes in excitonic interactions of photosystem II (PSII) reaction centre (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor (pheophytin (Pheo)) were analysed using circular dichroism (CD). The CD spectrum of PSII RC shows positive bands at 417, 435 and 681 and negative bands at 447 and 664 nm. Oxidation of the primary donor by illuminating the sample in the presence of silicomolybdate resulted in nearly symmetric decrease of CD amplitudes at 664 and 684 nm. In the Soret region, the maximum bleaching of CD signal was detected at 449 and 440 nm. Accumulation of reduced Pheo in the presence of dithionite brought about much lower changes in CD amplitudes than P680 oxidation. In this case, only a small asymmetric bleaching at 680 and 668 nm in the red region and a bleaching at 445, 435 and 416 nm in the Soret region has been detected. Therefore, we suppose that the contribution of the Pheo of the primary acceptor to the total CD signal of RC is negligible. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was strongly temperature-dependent. The reversible CD bleaching was completely inhibited below 200 K, although the reduced Pheo was accumulated even at a temperature of 77 K. Since the temperature does not influence the excitonic interaction, the temperature dependence of the CD changes upon Pheo reduction does not support the model of Pheo excitonically interacting with the other chlorophylls (Chl) of the RC. We propose that Pheo should not be considered as a part of a multimer model.