A perfluorinated aromatic link was used as a synthon in the design of bis-azo dyes.

Tyrosine D (Tyr) is an auxiliary redox active tyrosine residue in photosystem II (PSII). The mechanism of Tyr oxidation was investigated by EPR spectroscopy, flash-induced fluorescence decay and thermoluminescence measurements in PSII enriched membranes from spinach. PSII membranes were chemically treated with 3mM ascorbate and 1mM diaminodurene and subsequent washing, leading to the complete reduction of Tyr. Tyr oxidation kinetics and competing recombination reactions were measured after a single saturating flash in the absence and presence of DCMU (inhibitor of the Q-site) in the pH range of 4.7-8.5. Two kinetic phases of Tyr oxidation were observed by the time resolved EPR spectroscopy - the fast phase (msec-sec time range) and the pH dependent slow phase (tens of seconds time range). In the presence of DCMU, Tyr oxidation kinetics was monophasic in the entire pH range, i.e. only the fast kinetics was observed. The results obtained from the fluorescence and thermoluminescence analysis show that when forward electron transport is blocked in the presence of DCMU, the SQ recombination outcompetes the slow phase of Tyr oxidation by the S state. Modelling of the whole complex of these electron transfer events associated with Tyr oxidation fitted very well with our experimental data. Based on these data, structural information and theoretical considerations we confirm our assignment of the fast and slow oxidation kinetics to two populations of PSII centers with different water positions (proximal and distal) in the Tyr vicinity.

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Charge separation is a key component of the reactions cascade of photosynthesis, by which solar energy is converted to chemical energy. From this photochemical reaction, two radicals of opposite charge are formed, a highly reducing anion and a highly oxidising cation. We have previously proposed that the cation after far-red light excitation is located on a component different from PD1, which is the location of the primary electron hole after visible light excitation. Here, we attempt to provide further insight into the location of the primary charge separation upon far-red light excitation of PS II, using the EPR signal of the spin polarized 3P680 as a probe. We demonstrate that, under far-red light illumination, the spin polarized 3P680 is not formed, despite the primary charge separation still occurring at these conditions. We propose that this is because under far-red light excitation, the primary electron hole is localized on ChlD1, rather than on PD1. The fact that identical samples have demonstrated charge separation upon both far-red and visible light excitation supports our hypothesis that two pathways for primary charge separation exist in parallel in PS II reaction centres. These pathways are excited and activated dependent of the wavelength applied.

The tyrosine residue D2-Tyr160 (Tyr) in photosystem II (PSII) can be oxidized through charge equilibrium with the oxygen evolving complex in PSII. The kinetics of the electron transfer from Tyr has been followed using time-resolved EPR spectroscopy after triggering the oxidation of pre-reduced Tyr by a short laser flash. After its oxidation Tyr is observed as a neutral radical (Tyr) indicating that the oxidation is coupled to a deprotonation event. The redox state of Tyr was reported to be determined by the two water positions identified in the crystal structure of PSII [Saito et al. (2013) Proc. Natl. Acad. Sci. USA 110, 7690]. To assess the mechanism of the proton coupled electron transfer of Tyr the oxidation kinetics has been followed in the presence of deuterated buffers, thereby resolving the kinetic isotope effect (KIE) of Tyr oxidation at different H/D concentrations. Two kinetic phases of Tyr oxidation - the fast phase (msec-sec time range) and the slow phase (tens of seconds time range) were resolved as was previously reported [Vass and Styring (1991) Biochemistry 30, 830]. In the presence of deuterated buffers the kinetics was significantly slower compared to normal buffers. Furthermore, although the kinetics were faster at both high pH and pD values the observed KIE was found to be similar (~2.4) over the whole pL range investigated. We assign the fast and slow oxidation phases to two populations of PSII centers with different water positions, proximal and distal respectively, and discuss possible deprotonation events in the vicinity of Tyr.