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 fla sh-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) vibrati onal difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), wit h 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 kinetic s of Y-Z(OX) and Q(A)(-) were independently monitored by EPR and chlor ophyll a fluorescence, respectively, under the same experimental condi tions, These measurements confirmed that the addition of DCMU to Tris- washed PSII samples does not significantly affect the Y-Z(OX) decay, b ut does substantially slow down the QA(-) 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 spec trum, a large differential band at 1706/1699 cm(-1) could be identifie d and associated with Yz oxidation. In contrast, an intense band at 14 78 cm(-1), whose DCMU-sensitive decay follows the QA(-) decay based on the chlorophyll a fluorescence measurements, was present in all of th e time-resolved spectra. Since no significant reversible Chl(+) radica ls could be detected by the EPR measurements under our experimental co nditions, we confirm that this band most likely arises only from the s emiquinone anion QA(-).