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.
Highlights
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 avoid the production of harmful singlet oxygen
It was found that in LHCII more than 90% of Chl triplets are at room temperature efficiently quenched primarily by two lutein molecules even avoiding the formation of singlet oxygen.[14,15]
During the last step the sample was purified via fast protein liquid chromatography (FPLC) in order to reduce the content of monomeric LHCIIs and free pigments and frozen only once
Summary
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 avoid the production of harmful singlet oxygen. The extensive studies of the excitation energy transfer within LHCII show very fast Chl b to Chl a relaxation, occurring on a timescale of several ps.[4,16,17,18,19] Due to the much slower total singlet excited state decay of LHCII (lifetime of isolated LHCII E 3.5 ns) and an inter-system crossing rate of B10 nsÀ1, mainly Chl a triplet states are formed.[14,20] From the crystal structure it can be seen that all Chls a are in close proximity with either one of two central luteins or neoxanthin.[7,21] This spatial arrangement of the pigment molecules leads to efficient quenching of the Chl triplet states.[22] The fourth Car, either violaxanthin or zeaxanthin depending on the stress conditions of plants or algae before protein purification, is located at the periphery of the protein backbone and was shown not to contribute to triplet quenching.[23,24]
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