A time-resolved FTIR difference study of the plastoquinone Q(A) and redox-active tyrosine Y(Z) interactions in photosystem II

In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the Y(Z)/(ox)Q(A)/^-/Y(Z)Q(A) vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 ± 2 °C), with a spectral resolution of 4 cm^-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of Y(Z)/(ox) and Q(A)/^- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the Y(Z)/(ox) decay, but does substantially slow down the Q(A)/^- decay. By making use of the difference in the decay kinetics using DCMU, the Q(A)/^- /Q(A) signals could be separated from the Y(Z)/(ox)/Y(Z) signals and a pure Q(A)/^-/Q(A) difference spectrum obtained. By comparison of the Y(Z)/(ox)Q(A)/^-/Y(Z)Q(A) difference spectrum with the pure Q(A)/^-/Q(A) difference spectrum, a large differential band at 1706/1699 cm^-1 could be identified and associated with Y(Z) oxidation. In contrast, an intense band at 1478 cm^-1, whose DCMU-sensitive decay follows the Q(A)/^- decay based on the chlorophyll a fluorescence measurements, was present in all of the time- resolved spectra. Since no significant reversible Chl⁺ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion Q(A)/^- [Berthomieu, C. Nabedryk, E. Mantele, W., and Breton, J. (1990) FEBS Lett. 269, 363-367].