The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 mus to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 mus (at 20 degrees C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 degrees C, pH 6.4). In the second part of the study, the temperature dependence (-2.7 to 27.5 degrees C) of the rate constant of dioxygen formation (600/s at 20 degrees C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q B site in the D1 subunit and thus block the electron transfer from Q A to Q B . Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å . The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q B site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q A , which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334 -342). This discovery is discussed in the context of proton transfer to the lumen.The process of photosynthesis converts solar energy into biochemically amenable energy. A distinction is made between oxygenic and anoxygenic photosynthesis. In the latter sulfur compounds, hydrogen gas or organic materials serve as electron source. In contrast, oxygenic photosynthesis in higher plants, algae, and cyanobacteria uses water as an electron source and generates molecular oxygen, thereby maintaining the level of oxygen in the atmosphere. Water oxidation takes place at the large homodimeric protein-cofactor complex photosystem II (PSII), 7 a light-driven water:plastoquinone oxidoreductase harbored in the thylakoid membrane (1-3). The structure of the PSII core complex (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus is known from x-ray crystallographic studies at a current resolution of 2.9 Å (4, 5). The photochemical reaction center (RC) in PSII is of type II and structurally related to the RC of purple bacteria (pbRC) (6), which perform anoxygenic photosynthesis. The PSII-RC contains four chlorophyll a (Chla) molecules (P D1 , P D2 , Chl D1 , and Chl D2 ), two pheophytins (Pheo D1 and Pheo D2 ), and two plastoquinones (PQ) (Q A and Q B ) with a nonheme iron in between. These cofactors are embedded in a heterodimeric protein matrix formed by subunits D1 and D2 (systematic names: PsbA and PsbD, respectively) and are arranged in two pseudo-C2 symmetric branches with respect to a 2-fold rotation axis, which crosses the non-heme iron and is oriented normal to the membrane plane.Photons from sunlight are collected by antenna proteins of PSII, and the excitation energy is transferred to the RC, where it gives rise to the formation of the radical pair P D1 ⅐ϩ Pheo D1 ⅐Ϫ and subsequent electron transfer to the fixed single-electron transmitter Q A . The electron hole at P D1 ⅐ϩ is able to abstract electrons via the redox-active tyrosine Y Z (Tyr-161A) from the heteronuclear Mn 4 Ca cluster located at the lumenal (d...

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The D1 protein (PsbA) of photosystem II (PSII) from Thermosynechococcus elongatus is encoded by a psbA gene family that is typical of cyanobacteria. Although the transcription of these three genes has been studied previously (Kós, P. B., Deák, Z., Cheregi, O., and Vass, I. (2008) Biochim. Biophys. Acta 1777, 74 -83), the protein quantification had not been possible due to the high sequence identity between the three PsbA copies. The successful establishment of a method to quantify the PsbA proteins on the basis of reverse phase-LC-electrospray mass ionization-MS/MS (RP-LC-ESI-MS/MS) enables an accurate comparison of transcript and protein level for the first time ever. Upon high light incubation, about 70% PsbA3 could be detected, which closely corresponds to the transcript level. It was impossible to detect any PsbA2 under all tested conditions. The construction of knock-out mutants enabled for the first time a detailed characterization of both whole cells and also isolated PSII complexes. PSII complexes of the ⌬psbA1/psbA2 mutant contained only copy PsbA3, whereas only PsbA1 could be detected in PSII complexes from the ⌬psbA3 mutant. In whole cells as well as in isolated complexes, a shift of the free energy between the redox pairs in the PsbA3 complexes in comparison with PsbA1 could be detected by thermoluminescence and delayed fluorescence measurements. This change is assigned to a shift of the redox potential of pheophytin toward more positive values. Coincidentally, no differences in the Q A -Q B electron transfer could be observed in flash-induced fluorescence decay or prompt fluorescence measurements. In conclusion, PsbA3 complexes yield a better protection against photoinhibition due to a higher probability of the harmless dissipation of excess energy.

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