| Photosystem II (PSII) of oxygenic photosynthesis is continuously exposed to high oxygen concentrations, which in the presence of triplet excited-state chlorophyll (Chl) species leads to extremely harmful radical oxygen species. For this reason the flow of excited-state energy that arrives from the PSII antenna in the reaction center must be strictly regulated. A key component of the regulation of PSII is a process known as nonphotochemical quenching (NPQ); NPQ involves the dissipation of Chl singlet excited states into heat in the PSII antenna. By this process, the excited-state lifetime in the PSII antenna is decreased and the flow of excitations into the RC is diminished, thus allowing the plant to adapt to different light levels by preventing photoinduced damage. The antenna xanthophylls appear to play a major role in NPQ, as violaxanthin is enzymatically converted to zeaxanthin during the process. Plant fitness under stress conditions critically depends on their ability to protect themselves from photodamage, an issue that will become ever more relevant for agricultural production in a warming global climate and ensuing increased aridity. It is therefore imperative that we fully understand the physical and chemical mechanisms that lie at the basis of regulated light-harvesting in oxygenic photosynthesis. In this proposal we aim at uncovering the molecular basis that underlies NPQ phenomena in PSII. In all likelihood it is by rapidly induced conformational changes and/or exchanged cofactors that closely spaced pigments are transformed from long-lived energy transfer partners with minimal losses to energy sinks, where Chl excited states become strongly coupled to short-lived dissipative decay channels.The most likely molecular processes underlying NPQ are energy transfer processes from Chl to xanthophylls, or electron transfer from xanthophylls to Chl or the formation of charge transfer states between neighbouring Chls. Our approach to tackle this issue is two-fold: First, we will employ a series of covalently linked artificial light-harvesting systems consisting of phthalocyanine as Chl mimicks and carotenoids that have variable conjugation lengths and different functional substitutions on their backbone. By investigating these systems with ultrafast time-resolved spectroscopy, we will establish the factors that determine the rates and direction of energy flow between carotenoids and phthalocyanine as a function of these parameters. By systematically varying the midpoint potential of the phthalocyanine and the conjugation length of the carotenoid, the rate constant of electron transfer from carotenoids to Pc will be tested as a function of these parameters. These studies will form the basis on which to interpret the results of ultrafast experiments on quenched PSII preparations . Second, we will apply ultrafast time-resolved experiments on PSII in vivo in its quenched and unquenched states in plants and diatoms/brown algae. The experiments will be carried out on whole leaves and on chloroplasts of Arabidopsis and its mutants with deficient or enhanced NPQ response. Moreover, membranes of brown algae that are known for their highly developed NPQ response will be studied. These experiments will be corroborated by similar time-resolved spectroscopic experiments on quenched aggregates of the PSII antenna. By recording the data with sufficient signal-to-noise ratio, a low transient concentration of the quenching species will become apparent in the spectral evolution. By capturing the spectroscopic signatures of quenching states, we will be able to identify their molecular nature. Finally, artificial four-helix bundles with bound carotenoids and phthalocyanines will be investigated. These systems change conformation as a function of pH which brings phthalocyanine and carotenoid closer together, with singlet-excited state quenching as a result thereby mimicking NPQ in the PSII antenna. |
