Photosystem II is a unique enzyme which catalyzes light induced water oxidation. This process is driven by highly oxidizing ensemble of four Chl molecules, PD1, PD2, ChlD1 and ChlD2 called, P680. Excitation of one of the Chls in P680 leads to the primary charge separation, P680+Pheo-. Pheo- transfers electrons sequentially to the primary quinone acceptor QA and the secondary quinone acceptor QB. P680+ in turn extracts electrons from Mn4CaO5 cluster, a site for the water oxidation. There are two redox active tyrosines, TyrZ and TyrD, found in PSII. They are symmetrically located on the D1 and D2 central proteins. Only TyrZ acts as intermediate electron carrier between P680 and Mn4CaO5 cluster, while TyrD does not participate in the linear electron flow and stays oxidized under light conditions. Both tyrosines are involved in PCET. The reduced TyrD undergoes biphasic oxidation with the fast (msec-sec time range) and the slow (tens of seconds time range) kinetic phases. We assign these phases to two populations of PSII centers with proximal or distal water positions. We also suggest that the TyrD oxidation and stability is regulated by the new small lumenal protein subunit, PsbTn. The possible involvement of PsbTn protein in the proton translocation mechanism from TyrD is suggested. To assess the possible localization of primary cation in P680 the formation of the triplet state of P680 and the oxidation of TyrZ and TyrD were followed under visible and far-red light. We proposed that far-red light induces the cation formation on ChlD1. Transmembrane interaction between QB and TyrZ has been studied. The different oxidation yield of TyrZ, measured as a S1 split EPR signal was correlated to the conformational change of protein induced by the QB presence at the QB-site. The change is transferred via H-bonds to the corresponding His-residues via helix D of the D1 protein.

Photosystem II is unique! It remains the only enzyme that can oxidize water using light as energy input. Water oxidation in photosystem II is catalyzed by the CaMn4 cluster. The electrons extracted from the CaMn4 cluster are transferred to P680+ via the tyrosine residue D1-Tyr161 (YZ). Favorable oxidation of YZ is coupled to a proton transfer along a hydrogen bond to the nearby D1-His190 residue, resulting in the neutral radical YZ•. By illuminating photosystem II at cryogenic temperatures, YZ• can be trapped in a stable state. Magnetic interaction between this radical and the CaMn4 cluster gives rise to a split electron paramagnetic resonance (EPR) signal with characteristics that depend on the oxidation state (S state) of the cluster. The mechanism by which the split EPR signals are formed is different depending on the S state. In the S0 and S1 states, split signal induction proceeds via a P680+-centered mechanism, whereas in the S2 and S3 states, our results show that split induction stems from a Mn-centered mechanism. This S state-dependent pattern of split EPR signal induction can be correlated to the charge of the CaMn4 cluster in the S state in question and has prompted us to propose a general model for the induction mechanism across the different S states. At the heart of this model is the stability or otherwise of the YZ•–(D1-His190)+ pair during cryogenic illumination. The model is closely related to the sequence of electron and proton transfers from the cluster during the S cycle. Furthermore, the important hydrogen bond between YZ and D1-His190 has been investigated by following the split EPR signal formation in the different S states as a function of pH. All split EPR signals investigated decrease in intensity with a pKa of ~4-5. This pKa can be correlated to a titration event that disrupts the essential hydrogen bond, possibly by a direct protonation of D1-His190.  This has important consequences for the function of the CaMn4 cluster as this critical YZ–D1-His190 hydrogen bond steers a multitude of reactions at the cluster.

Proton coupled electron transfer reactions often involve tyrosine residues, because when oxidized, the phenolic side chain deprotonates. Tyrosine Z (YZ) is responsible for extracting electrons in a stepwise fashion from the oxygen evolving-complex in order to build enough potential to oxidize water. This process requires that each step YZ must deprotonate and reprotonate in order to maintain the high midpoint potential that is necessary to oxidize the oxygen-evolving complex, which makes YZ highly involved in proton coupled electron transfer reactions. In this thesis YZ has been studied within oxygen-evolving photosystem II utilizing electron paramagnetic resonance spectroscopy to monitor the tyrosyl radical that is formed upon light excitation. Kinetic analysis of YZ has shed light on the factors that are important for PSII to carry out water oxidation at the oxygen-evolving complex. Most notably the strong hydrogen-bonding network and the midpoint potential of YZ have been shown to be integral aspects of the water splitting reactions of PSII. By studying YZ within oxygen-evolving PSII, conclusions are readily applied to the native system.

"Laser flash spectroscopy of photosystem II" Photosystem II (PS II) of plants and cyanobacteria oxidizes water in a light-powered reaction. Thereby, this protein is the ultimate source of the atmospheric oxygen. The capacity to oxidize water is owed to two properties of PS II: (i) The midpoint potential of the oxidizing chlorophyll moiety is increased by 0.6 V compared to photosystem I or photochemical reaction centers of anoxygenic bacteria, and (ii) the energy requirements of the four steps needed for the tetravalent oxidation of water are adapted to the energy of red light quanta. This thesis deals with two particular aspects, namely: 1. The coupling of the electron transfer from tyrosine Z (YZ) to the primary donor (P680+) to proton transfer, and an inquiry on the role of a positive charge on YZox (plus base cluster) in increasing the oxidizing potential at the catalytic site. 2. The localization of the electron hole, P680+, among the excitonically coupled four inner chlorophyll a molecules, and an estimation of the midpoint potential differences between them. Electron-proton-coupling by YZ This study was carried out with PS II core complexes from spinach or pea with a deactivated (removed) manganese cluster. The reduction of P680+ was investigated as a function of pH by detecting the laser flash induced absorption changes with nanosecond resolution. Two kinetic components were found with different pH-dependence and activation energies. The alteration of kinetic parameters by H/D isotope substitutions or by addition of divalent cations implied two different types of YZ-oxidation: At acidic pH the electron transfer was coupled with proton transfer, whereas in the alkaline region it was more rapid and no longer controlled by proton transfer. The conversion between both mechanisms occured at pH 7.4. This value corresponds either to the apparent pK of YZ itself (i.e. of the hydroxy group of the phenol ring) or to the pK of an acid-base-cluster, which includes YZ. Independent measurements of pH-transients by following the absorption changes of hydrophilic proton indicators corroborated this notion. The data were interpreted as indicating that the phenolic proton of YZ was released into the medium at acidic, but not at alkaline pH. The electron transfer and proton release characteristics of intact, oxygen-evolving PS II resembled those in deactivated samples kept at alkaline pH. We concluded that the electron transfer from YZ to P680+ in the native system was not coupled with proton transfer into the bulk. This has shed doubt on a popular hypothesis on the role of YZ as 'hydrogen abstractor' from bound water. On the other hand, the energetic constraints of water oxidation could be eased by the positive upcharging during oxidation of YZox plus its base cluster. On the localization of the electron hole of P680+ Photooxidation of PS II oxidizes the set of four innermost chlorophyll a molecules giving rise to the only spectroscopically defined species P680+. The deconvolution of difference spectra into bands of pigments is ambiguous. By using photoselective excitation of antennae, i.e. chl a molecules with site specific energies at the long wavelength border of the mean Qy-band, and by polarized detection, it was possible to tag P680+QA-/P680QA and 3P680/P680 difference spectra with a further parameter, the (wavelength-dependent) anisotropy r. Results obtained at liquid nitrogen temperature (77 K) can be clearly interpreted in terms of two chl a monomer bands. The two main components of the P680+QA-/P680QA difference spectrum were marked by two distinct values of the anisotropy and could be interpreted in a straightforward manner: the bleaching of a band at 675 nm belonging to the charged species (chl a+) and an electrochromic blue-shift of a nearby chl a from 684 to 682 nm. The main bleaching band of the 3P680/P680 spectrum (at 77 K) can be apparently attributed to a third (or several) chl a component(s). The analysis of the P680+QA-/P680QA spectrum at cryogenic temperature is compatible with monomeric chl a bands. On the other hand, one could assume a system of excitonically coupled core pigments, as it was recently introduced in the literature on the basis of energy transfer studies ('multimer model'). However, in view of the clear indications for an electrochromic band shift and the location of the bleaching band, which absorbs in a wavelength region of monomeric chl a, one assumption of the 'multimer model' should be questioned. Presumably, the excitonic couplings are rather weak, in particular between each of the two central chl a-molecules (PA/PB) and its respective accessory chl a (BA/BB), because of (i) the distances and (ii) different site energies of the monomeric chromophores. At room temperature, the absorption difference and anisotropy spectra of P680+QA-/P680QA were clearly altered. The anisotropy data indicated that the changes could no longer exclusively be ascribed to thermal broadening of individual bands. The localization of the positive charge on one pigment, analogous to the situation at 77 K, was now unlikely. Hence, the midpoint potential differences between the inner four chlorophyll a molecules were small and were estimated as approximately 15 meV.