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.

PsbS-dependent and -independent mechanisms regulate carotenoid-chlorophyll energy coupling in grana thylakoids.

In higher plants, PsbS is known to play a key role in the regulation of photosynthetic light harvesting. However, the molecular mechanism and role of electronic carotenoid-chlorophyll (Chl) interactions for the downregulation of excess excitation (nonphotochemical energy quenching, NPQ) are still poorly understood. Here, we explored carotenoid â†’ Chl energy transfer in isolated grana thylakoid membranes from mutants either deficient in or overexpressing PsbS. Since it was suggested that PsbS regulates the supramolecular protein network to control NPQ, we varied this network by diluting the grana protein densities. Our results indicate that different electronic quenching mechanisms are operative in grana thylakoids: a PsbS-dependent mechanism and a membrane protein density-dependent mechanism that is also operative in the absence of PsbS.

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.

The back and forth of energy transfer between carotenoids and chlorophylls and its role in the regulation of light harvesting.

Many aspects in the regulation of photosynthetic light-harvesting of plants are still quite poorly understood. For example, it is still a matter of debate which physical mechanism(s) results in the regulation and dissipation of excess energy in high light. Many researchers agree that electronic interactions between chlorophylls (Chl) and certain states of carotenoids are involved in these mechanisms. However, in particular, the role of the first excited state of carotenoids (Car S1) is not easily revealed, because of its optical forbidden character. The use of two-photon excitation is an elegant approach to address directly this state and to investigate the energy transfer in the direction Car S1 â†’ Chl. Meanwhile, it has been applied to a large variety of systems starting from simple carotenoid-tetrapyrrole model compounds up to entire plants. Here, we present a systematic summary of the observations obtained by two-photon excitation about Car S1 â†’ Chl energy transfer in systems with increasing complexity and the correlation to fluorescence quenching. We compare these observations directly with the energy transfer in the opposite direction, Chl â†’ Car S1, for the same systems as obtained in pump-probe studies. We discuss what surprising aspects of this comparison led us to the suggestion that quenching excitonic Car-Chl interactions could contribute to the regulation of light harvesting, and how this suggestion can be connected to other models proposed.

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.

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.