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We performed picosecond fluorescence measurements on well-defined Photosystem II (PSII) supercomplexes from Arabidopsis with largely varying antenna sizes. The average excited-state lifetime ranged from 109 ps for PSII core to 158 ps for the largest C(2)S(2)M(2) complex in 0.01% alpha-DM. Excitation energy transfer and trapping were investigated by coarse-grained modeling of the fluorescence kinetics. The results reveal a large drop in free energy upon charge separation (>700 cm(-1)) and a slow relaxation of the radical pair to an irreversible state (similar to 150 ps). Somewhat unexpectedly, we had to reduce the energy-transfer and charge-separation rates in complexes with decreasing size to obtain optimal fits. This strongly suggests that the antenna system is important for plant PSII integrity and functionality, which is supported by biochemical results. Furthermore, we used the coarse-grained model to investigate several aspects of PSII functioning. The excitation trapping time appears to be independent of the presence/absence of most of the individual contacts between light-harvesting complexes in PSII supercomplexes, demonstrating the robustness of the light-harvesting process. We conclude that the efficiency of the nonphotochemical quenching process is hardly dependent on the exact location of a quencher within the supercomplexes

This thesis describes fluorescence spectroscopy experiments on photosynthetic complexes that cover the primary photosynthetic processes, from the absorption of light by photosynthetic pigments to a charge separation (CS) in the reaction center (RC). Fluorescence spectroscopy is a useful tool in photosynthetic particles, because the latter are densely packed with fluorescence pigments like chlorophylls (Chl). The fluorescence of each pigment is affected by its environment and provide information about structure and dynamics of the photosynthetic complexes. In this thesis time-resolved fluorescence of Chl molecules is used for studying the ultrafast kinetics in membrane particles of photosystem II (PSII) (chapter 2, 3 and 4). In chapter 5 fluorescence lifetime imaging microscopy (FLIM) of is applied to study entire chloroplasts, either in the leaf or in isolated chloroplast form. The advantage of FLIM is that the interactions of the fluorescence pigments in both photosystems can be spatially resolved up to a resolution of 0.5 x 0.5 x 2 µm to indentify and quantify photosynthetic processes in their natural environment. Excitation energy transfer and charge separation in PSII membranes (chapter 2,3 and 4) In this thesis time-resolved fluorescence measurements of PSII containing membranes, the so called BBY particles, are performed in low-light conditions with open reaction centers. The BBY particles do not contain photosystem I (PSI) or stroma lamellae, but do support electron transfer and carry out oxygen evolution with high activity and are comparable with the grana in vivo. The fluorescence decay kinetics of the BBY particles are faster than observed in previous studies and also faster than observed for PSII in chloroplasts and thylakoid preparations. The average lifetime is 150 ps, which, together with previous annihilation experiments on light-harvesting complex II (LHCII) suggests that excitation migration from the antenna complexes contributes significantly to th

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F 0- and the F m-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 ¿m in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].

We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation