Fourier Transform Infrared Difference Study of Tyrosineo Oxidation and Plastoquinone Q_A Reduction in Pfaotosystem II

摘要

Two redox active tyrosines are present in the homologous poiypeptides Dl and D2 of photo-system II (PS II). Tyrz (Dl-161) is involved in the electron transfer reactions resulting in oxygen evolution, while Tyr_D (D2-160) usually forms a dark-stable radical. In Mn -depleted PS II, Tyr_D~(centre dot) can be slowly reduced by exogenous reductants. Charge separation then results in the oxidation of Tyr_D and Tyrz and the reduction of the primary electron acceptor Q_A. The semiquinone Q_A~- can be reoxidized by oxidants like ferricyanide. In the present work, experimental conditions leading to the generation of pure Q_A~-/Q_A or Tyr_D~(centre dot)/Tyr_D FTIR difference spectra have been optimized. Therefore, single-turnover flashes or short illuminationswere performed on PS II samples in the presence of exogenous reductants or oxidants. The Q_A~- and Tyr_D~(centre dot) radicals were generated with high yield and with a lifetime of several seconds or minutes allowing averaging of FTIR difference spectrawith high signal to noise ratio. Both Q_A~- formation and contributions at the electron donor side of PS II were monitored by EPR spectroscopy. In PS II samples at pH 6 in the presence of PMS, NH_2OH, and DCMU, EPR measurements show that Q_A~- is formedwith high yield upon a l s illumination at 10 °C, while no radical from the electron donor side of PS II is detected. Therefore the Q_A~-/Q_A FTIR spectrum obtained in these conditions shows only vibrational changes due to Q_A reduction in PS II. In contrast, a similar spectrum was recently interpreted in terms of dominant contributions from Chl~+/Chl signals [MacDonald, G. M., Steenhuis, I. J., & Barry, B. A. (1995) J. Biol. Chem. 270, 8420-8428], although the contribution from the electron acceptorQA was not quantified. In particular, it is shown here that the large positive signal at 1478 cm~(-1) is due to the Q_A~- state and not to a Chl~+ mode. This band is not downshifted upon ~(15)N- labeling of spinach PS II membranes within the ±1 cm~(-1)accuracy of the method and is therefore tentatively assigned to the v(C***O) mode of the plastosemiquinone Q_A~-. Also unchanged upon ~(15)N-labeling, signals at 1644 and/or 1630 cm~(-1) are possible candidates for the v(C=O) mode(s) of neutral Q_A in PSII. The Tyr_D~(centre dot)/Tyr_D FTIR spectrum is recorded at 4 °C on Tris-washed PS II membranes from spinach at pH 6 in the presence of phosphate, formate, and ferricyanide. EPR experiments performed on these samples show that almost all Tyr_D~(centre dot) is formed upon a l s illumination at 4 °C and that Tyr_D~(centre dot) is then reduced within 12 min in the dark. No contributions from Tyr_Z~(centre dot) or Q_A~- are detected 2 s after illumination. It is thus possible to optimize experimental conditions to record the FTIR difference spectrum only due to Tyr_D photooxidation in PS II-enriched membranes of spinach. The Tyr_D~(centre dot)/Tyr_D FTIR spectrum is compared to a cresol~(centre dot)/cresol FTIR difference spectrum obtained by UV irradiation at 10 K of cresol at pH 8. The spectral analogies observed between the in vivo and in vitro spectra recorded either in H_2O or in D_2O suggest that IR modes of Tyr_D contribute at 1513 and 1252 cm~(-1). These frequencies are characteristic of a protonated tyrosine. A positive signal is observed at 1506 cm~(-1) for cresol~(centre dot) and at 1504 cm~(-1) for the Tyr_D~(centre dot) state. This suggests contribution of the Tyr_D~(centre dot) side chain at 1504 cm~(-1). A band at 1473 cm~(-1) was previously assigned to the v(CO) mode of Tyr_D~(centre dot) [MacDonald, G. M., Bixby, K. A., & Barry, B. A. (1993) Proc, Natl Acad. Sci. U.S.A. 90, 11024-11028]. In contrast, no positive signal is observed at 1473 cm~(-1) in the Tyr_D~(centre dot)/Tyr_D FTIR. difference spectrum presented here. The Tyr_D~(centre dot)/Tyr_D spectrum also shows vibrational changes from peptide groups and amino acid side chains which are modified upon Tyr_D~(centre dot) forma