Tyrozine D oxidation and redox equilibrium in photosystem II

Tyrosine D (Tyr_D) is an auxiliary redox active tyrosine residue in photosystem II (PSII). The mechanism of Tyr_D 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_D. Tyr_D oxidation kinetics and competing recombination reactions were measured after a single saturating flash in the absence and presence of DCMU (inhibitor of the Q_B-site) in the pH range of 4.7-8.5. Two kinetic phases of Tyr_D 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_D 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 S₂Q_A^- recombination outcompetes the slow phase of Tyr_D oxidation by the S₂ state. Modelling of the whole complex of these electron transfer events associated with Tyr_D 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_D vicinity.