Carotenoid dark state to chlorophyll energy transfer in isolated light-harvesting complexes CP24 and CP29.

We present a comparison of the energy transfer between carotenoid dark states and chlorophylls for the minor complexes CP24 and CP29. To elucidate the potential involvement of certain carotenoid-chlorophyll coupling sites in fluorescence quenching of distinct complexes, varying carotenoid compositions and mutants lacking chlorophylls at specific binding sites were examined. Energy transfers between carotenoid dark states and chlorophylls were compared using the coupling parameter, [Formula: see text], which is calculated from the chlorophyll fluorescence observed after preferential carotenoid two-photon excitation. In CP24, artificial reconstitution with zeaxanthin leads to a significant reduction in the chlorophyll fluorescence quantum yield, [Formula: see text], and a considerable increase in [Formula: see text]. Similar effects of zeaxanthin were also observed in certain samples of CP29. In CP29, also the replacement of violaxanthin by the sole presence of lutein results in a significant quenching and increased [Formula: see text]. In contrast, the replacement of violaxanthin by lutein in CP24 is not significantly increasing [Formula: see text]. In general, these findings provide evidence that modification of the electronic coupling between carotenoid dark states and chlorophylls by changing carotenoids at distinct sites can significantly influence the quenching of these minor proteins, particularly when zeaxanthin or lutein is used. The absence of Chl612 in CP24 and of Chl612 or Chl603 in CP29 has a considerably smaller effect on [Formula: see text] and [Formula: see text] than the influence of some carotenoids reported above. However, in CP29 our results indicate slightly dequenching and decreased [Formula: see text] when these chlorophylls are absent. This might indicate that both, Chl612 and Chl603 are involved in carotenoid-dependent quenching in isolated CP29.

Carotenoid-chlorophyll coupling and fluorescence quenching in aggregated minor PSII proteins CP24 and CP29.

It is known that aggregation of isolated light-harvesting complex II (LHCII) in solution results in high fluorescence quenching, reduced chlorophyll fluorescence lifetime, and increased electronic coupling of carotenoid (Car) S1 and chlorophyll (Chl) Qy states, as determined by two-photon studies. It has been suggested that this behavior of aggregated LHCII mimics aspects of non-photochemical quenching processes of higher plants and algae. However, several studies proposed that the minor photosystem II proteins CP24 and CP29 also play a significant role in regulation of photosynthesis. Therefore, we use a simple protocol that allows gradual aggregation also of CP24 and CP29. Similarly, as observed for LHCII, aggregation of CP24 and CP29 also leads to increasing fluorescence quenching and increasing electronic Car S1-Chl Qy coupling. Furthermore, a direct comparison of the three proteins revealed a significant higher electronic coupling in the two minor proteins already in the absence of any aggregation. These differences become even more prominent upon aggregation. A red-shift of the Qy absorption band known from LHCII aggregation was also observed for CP29 but not for CP24. We discuss possible implications of these results for the role of CP24 and CP29 as potential valves for excess excitation energy in the regulation of photosynthetic light harvesting.

Direct interaction of the major light-harvesting complex II and PsbS in nonphotochemical quenching.

The photosystem II (PSII) subunit S (PsbS) plays a key role in nonphotochemical quenching, a photoprotective mechanism for dissipation of excess excitation energy in plants. The precise function of PsbS in nonphotochemical quenching is unknown. By reconstituting PsbS together with the major light-harvesting complex of PSII (LHC-II) and the xanthophyll zeaxanthin (Zea) into proteoliposomes, we have tested the individual contributions of PSII complexes and Zea to chlorophyll (Chl) fluorescence quenching in a membrane environment. We demonstrate that PsbS is stable in the absence of pigments in vitro. Significant Chl fluorescence quenching of reconstituted LHC-II was observed in the presence of PsbS and Zea, although neither Zea nor PsbS alone was sufficient to induce the same quenching. Coreconstitution with PsbS resulted in the formation of LHC-II/PsbS heterodimers, indicating their direct interaction in the lipid bilayer. Two-photon excitation measurements on liposomes containing LHC-II, PsbS, and Zea showed an increase of electronic interactions between carotenoid S1 and Chl states, Φ(Coupling)(CarS1-Chl), that correlated directly with Chl fluorescence quenching. These findings are in agreement with a carotenoid-dependent Chl fluorescence quenching by direct interactions of LHCs of PSII with PsbS monomers.

Carotenoid-chlorophyll coupling and fluorescence quenching correlate with protein packing density in grana-thylakoids.

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.

On the role of excitonic interactions in carotenoid-phthalocyanine dyads and implications for photosynthetic regulation.

In two recent studies, energy transfer was reported in certain phthalocyanine-carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S(1)) and phthalocyanines (Pcs) in the direction Pc â†’ Car S(1) (Kloz et al., J Am Chem Soc 133:7007-7015, 2011) as well as in the direction Car S(1) â†’ Pc (Liao et al., J Phys Chem A 115:4082-4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S(1) is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole-carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.

Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II.

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

Two-photon study on the electronic interactions between the first excited singlet states in carotenoid-tetrapyrrole dyads.

Electronic interactions between the first excited states (S(1)) of carotenoids (Car) of different conjugation lengths (8-11 double bonds) and phthalocyanines (Pc) in different Car-Pc dyad molecules were investigated by two-photon spectroscopy and compared with Car S(1)-chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective two-photon excitation of the Car S(1) state allowed sensitive monitoring of the flow of energy between Car S(1) and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S(1) and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked Car-Pc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S(1) → Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc → Car S(1) energy transfer. In phenylamino-linked Car-Pc dyads in toluene extremely large electronic interactions between the Car S(1) state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S(1)-Pc Q(y) interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.

Correlation of Car S(1) → Chl with Chl → Car S(1) energy transfer supports the excitonic model in quenched light harvesting complex II.

Recently, excitonic carotenoid-chlorophyll interactions have been proposed as a simple but effective model for the down-regulation of photosynthesis in plants. The model was proposed on the basis of quenching-correlated electronic carotenoid-chlorophyll interactions (Car S(1) → Chl) determined by Car S(1) two-photon excitation and red-shifted absorption bands. However, if excitonic interactions are indeed responsible for this effect, a simultaneous correlation of quenching with increased energy transfer in the opposite direction, Chl Q(y) → Car S(1), should be observed. Here we present a systematic study on the correlation of Car S(1) → Chl and Chl → Car S(1) energy transfer with the occurrence of red-shifted bands and quenching in isolated LHCII. We found a direct correlation between all four phenomena, supporting our conclusion that excitonic Car S(1)-Chl interactions provide low-lying states serving as energy traps and dissipative valves for excess excitation energy.

On the regulation of photosynthesis by excitonic interactions between carotenoids and chlorophylls.

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S(1), shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S(1)) and chlorophyll (Chl) states, phi(Coupling)(Car S(1)-Chl), correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and phi(Coupling)(Car S(1)-Chl) with the intensity of red-shifted bands in the Chl Q(y) and carotenoid absorption was also observed. These results suggest quenching excitonic Car S(1)-Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent down- and up-regulation of the photosynthetic activity and phi(Coupling)(Car S(1)-Chl) in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S(1)-Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, phi(Coupling)(Car S(1)-Chl) depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S(1)-Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoid-chlorophyll states serve as traps and dissipation valves for excess excitation energy.