Plants harvest photons for photosynthesis using light-harvesting complexes (LHCs)-an array of chlorophyll proteins that can reversibly switch from harvesting to energy-dissipation mode to prevent over-excitation and damage of the photosynthetic apparatus. In unicellular algae and lower plants this process requires the LHCSR proteins which senses over-acidification of the lumen trough protonatable residues exposed to the thylakoid lumen to activate quenching reactions. Further activation is provided by replacement of the violaxanthin ligand with its de-epoxidized product, zeaxanthin, also induced by excess light. We have produced the ppLHCSR1 protein from Physcomitrella patens by over-expression in tobacco and purified it in either its violaxanthin- or the zeaxanthin-binding form with the aim of analyzing their spectroscopic properties at either neutral or acidic pH. Using femtosecond spectroscopy, we demonstrated that the energy dissipation is achieved by two distinct quenching mechanism which are both activated by low pH. The first is present in both ppLHCSR1-Vio and ppLHCSR1-Zea and is characterized by 30-40ps time constant. The spectrum of the quenching product is reminiscent of a carotenoid radical cation, suggesting that the pH-induced quenching mechanism is likely electron transfer from the carotenoid to the excited Chl a. In addition, a second quenching channel populating the S state of carotenoid via energy transfer from Chl is found exclusively in the ppLHCSR1-Zea at pH5. These results provide proof of principle that more than one quenching mechanism may operate in the LHC superfamily and also help understanding the photoprotective role of LHCSR proteins and the evolution of LHC antennae.
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 (S) 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 S/ICT equilibration. On the basis of a two-state model representing the S and ICT states on the same S/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 S/ICT state as well as separate the S and ICT spectral signatures. The results are consistent with the concept that the S and ICT states are part of one potential energy surface.
We used ultrafast transient absorption spectroscopy to study excited-state dynamics of the keto-carotenoid fucoxanthin (Fx) and its two derivatives: 19'-butanoyloxyfucoxanthin (bFx) and 19'-hexanoyloxyfucoxanthin (hFx). These derivatives occur in some light-harvesting systems of photosynthetic microorganisms, and their presence is typically related to stress conditions. Even though the hexanoyl (butanoyl) moiety is not a part of the conjugated system of hFx (bFx), their absorption spectra in polar solvents exhibit more pronounced vibrational bands of the S state than for Fx. The effect of the nonconjugated acyloxy moiety is further observed in transient absorption spectra, which for Fx exhibit characteristic features of an intramolecular charge transfer (ICT) state in all polar solvents. For bFx and hFx, however, much weaker ICT features are detected in methanol, and the spectral markers of the ICT state disappear completely in polar, but aprotic acetonitrile. The presence of the acyloxy moiety also alters the lifetimes of the S/ICT state. For Fx, the lifetimes are 60, 30, and 20 ps in n-hexane, acetonitrile, and methanol, whereas for bFx and hFx, these lifetimes yield 60, 60, and 40 ps, respectively. Testing the S/ICT state lifetimes of hFx in other solvents revealed that some ICT features can be induced only in polar, protic solvents (methanol, ethanol, and ethylene glycol). Thus, bFx and hFx represent a rather rare example of a system in which a nonconjugated functional group significantly alters excited-state dynamics. By comparison with other carotenoids, we show that a keto group at the acyloxy tail, even though it is not in conjugation, affects the electron distribution along the conjugated backbone, resulting in the observed decrease of the ICT character of the S/ICT state of bFx and hFx.
Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)—abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.
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