Singlet–triplet annihilation in single LHCII complexes

Abstract

In light harvesting complex II (LHCII) of higher plants and green algae, carotenoids (Cars) have an important function to quench chlorophyll (Chl) triplet states and therefore avoid the production of harmful singlet oxygen. The resulting Car triplet states lead to a non-linear self-quenching mechanism called singlet-triplet (S-T) annihilation that strongly depends on the excitation density. In this work we investigated the fluorescence decay kinetics of single immobilized LHCIIs at room temperature and found a two-exponential decay with a slow (3.5 ns) and a fast (35 ps) component. The relative amplitude fraction of the fast component increases with increasing excitation intensity, and the resulting decrease in the fluorescence quantum yield suggests annihilation effects. Modulation of the excitation pattern by means of an acousto-optic modulator (AOM) furthermore allowed us to resolve the time-dependent accumulation and decay rate (∼7 μs) of the quenching species. Inspired by singlet-singlet (S-S) annihilation studies, we developed a stochastic model and then successfully applied it to describe and explain all the experimentally observed steady-state and time-dependent kinetics. That allowed us to distinctively identify the quenching mechanism as S-T annihilation. Quantitative fitting resulted in a conclusive set of parameters validating our interpretation of the experimental results. The obtained stochastic model can be generalized to describe S-T annihilation in small molecular aggregates where the equilibration time of excitations is much faster than the annihilation-free singlet excited state lifetime.

Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy

“…2C). The obtained mean value of the lifetime was similar to the mean fluorescence lifetime in an ensemble of solubilized complexes [19] and to that of the single immobilized LHCII subject to the <50 W/cm 2 excitation power, when any non-linear annihilation effects are absent [20].…”

Time-resolved fluorescence measurements on leaves: principles and recent developments

“…The data gave the first indication that spectral separation of PSII from PSI kinetics was possible in time-resolved fluorescence measurements on intact leaves. However, since the measurement was performed with the laser excitation on the same spot of the leaf, it was impossible to achieve the F o state (the state with minimal fluorescence intensity, corresponding to fully oxidised PSII), and S–S/S–T annihilation, which can also occur at moderate laser powers (Gruber et al 2015), was not taken into account. More recently, fluorescence decay traces were measured on leaves during non-photochemical induction and relaxation (Sylak-Glassman et al 2014, 2016).…”

Wavelength-Dependent Optical Response of Single Photosynthetic Antenna Complexes from Siphonous Green Alga Codium fragile

“…A technique that clearly avoids ensemble averaging is optical single-molecule spectroscopy. Owing to the sensitivity of the chromophores to changes in their local electrostatic environment, this can be employed to map out conformational changes of the protein matrix as changes of the spectral parameters of the embedded chromophores. ,− Previously, single-molecule techniques have been applied successfully to LHCII. ,− Upon excitation around 630 nm, spectral fluctuations and in particular a temporal redshift of the emission became observable. The authors distinguished between a “moderate redshift” if the emission peaked around 690–700 nm and a “far redshift” if the emission peak was even further to the red. , It was suggested that the moderately red-shifted emission is associated with the formation of a mixed exciton-charge transfer (CT) state that accompanies the transition from the light-harvesting mode to the photoprotective mode. ,− Fluorescence lifetimes, unfortunately, were not reported for these features.…”