PH-DEPENDENT CHARGE EQUILIBRIA BETWEEN TYROSINE-D AND THE S-STATES IN PHOTOSYSTEM .2. ESTIMATION OF RELATIVE MIDPOINT REDOX POTENTIALS

The effect of protonation events on the charge equilibrium between tyrosine-D and the water-oxidizing complex in photosystem II has been studied by time-resolved measurements of the EPR signal II(slow) at room temperature. The flash-induced oxidation of Y(D) by the water-oxidizing complex in the S2 state is a monophasic process above pH 6.5 and biphasic at lower pHs, showing a slow and a fast phase. The half-time of the slow phase increases from about 1 s at pH 8.0 to about 20 s at pH 5.0, whereas the half-time of the fast phase is pH independent (0.4-1 s). The dark reduction of Y(D)+ was followed by measuring the decay of signal II(slow) at room temperature. Y(D)+ decays in a biphasic way on the tens of minutes to hours time scale. The minutes phase is due to the electron transfer to Y(D)+ from the S0 state of the water-oxidizing complex. The half-time of this process increases from about 5 min at pH 8.0 to 40 min at pH 4.5. The hours phase of Y(D)+ has a constant half-time of about 500 min between pH 4.7 and 7.2, which abruptly decreases above pH 7.2 and below pH 4.7. This phase reflects the reduction of Y(D)+ either from the medium or by an unidentified redox component of PSII in those centers that are in the S1 state. The titration curve of the half-times for the oxidation of Y(D) reveals a proton binding with a pK around 7.3-7.5 that retards the electron transfer from Y(D) to the water-oxidizing complex. We propose that this monoprotic event reflects the protonation of an amino acid residue, probably histidine-190 on the D2 protein, to which Y(D) is hydrogen bonded. The titration curves for the oxidation of Y(D) and for the reduction of Y(D)+ show a second proton binding with pK almost-equal-to 5.8-6.0 that accelerates the electron transfer from Y(D) to the water-oxidizing complex and retards the process in the opposite direction. This protonation most probably affects the water-oxidizing complex. From the measured kinetic parameters, the lowest limits for the equilibrium constants between the S0Y(D)+ and the S1Y(D) as well as between the S1Y(D)+ and S2Y(D) states were estimated to be 5 and 750-1000, respectively. These equilibrium constants permitted us to derive a relative redox potential scheme showing that the S1/S0 redox couple of the water-oxidizing complex is at least 40 mV more negative than the Y(D)+/Y(D) couple and the latter is at least 170 mV more negative than the S2/S1 redox couple of the water-oxidizing complex. Together with available data in the literature, these estimations place the Y(D)+/Y(D) couple at approximately 720-760 mV.