Fourier transform infrared difference study of tyrosineD oxidation and plastoquinone QA reduction in photosystem II.
Two redox active tyrosines are present in the homologous polypeptides D1 and D2 of photo-system II (PS II). TyrZ (D1-161) is involved in the electron transfer reactions resulting in oxygen evolution, while TyrD (D2-160) usually forms a dark-stable radical. In Mn-depleted PS II, TyrD. can be slowly reduced by exogenous reductants. Charge separation then results in the oxidation of TyrD and TyrZ and the reduction of the primary electron acceptor QA. The semiquinone QA- can be reoxidized by oxidants like ferricyanide. In the present work, experimental conditions leading to the generation of pure QA-/QA or TyrD./TyrD FTIR difference spectra have been optimized. Therefore, single-turnover flashes or short illuminations were performed on PS II samples in the presence of exogenous reductants or oxidants. The QA- and TyrD. radicals were generated with high yield and with a lifetime of several seconds or minutes allowing averaging of FTIR difference spectra with high signal to noise ratio. Both QA- 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, NH2OH, and DCMU, EPR measurements show that QA- is formed with high yield upon a 1 s illumination at 10 degrees C, while no radical from the electron donor side of PS II is detected. Therefore the QA-/QA FTIR spectrum obtained in these conditions shows only vibrational changes due to QA 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, J. J., & Barry, B. A. (1995) J. Biol. Chem. 270, 8420-8428], although the contribution from the electron acceptor QA was not quantified. In particular, it is shown here that the large positive signal at 1478 cm-1 is due to the QA- state and not to a Chl+ mode. This band is not downshifted upon 15N-labeling of spinach PS II membranes within the +/- 1 cm-1 accuracy of the method and is therefore tentatively assigned to the v(C[symbol: see text]O) mode of the plastosemiquinone QA-. Also unchanged upon 15N-labeling, signals at 1644 and/or 1630 cm-1 are possible candidates for the v(C = O) mode(s) of neutral QA in PS II. The TyrD./TyrD FTIR spectrum is recorded at 4 degrees 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 TyrD. is formed upon a 1 s illumination at 4 degrees C and that TyrD. is then reduced within 12 min in the dark. No contributions from TyrZ. or QA- are detected 2 s after illumination. It is thus possible to optimize experimental conditions to record the FTIR difference spectrum only due to TyrD photooxidation in PS II-enriched membranes of spinach. The TyrD./TyrD FTIR spectrum is compared to a cresol./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 H2O or in D2O suggest that IR modes of TyrD 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. and at 1504 cm-1 for the TyrD. state. This suggests contribution of the TyrD. side chain at 1504 cm-1. A band at 1473 cm-1 was previously assigned to the v(CO) mode of TyrD. [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 TyrD./TyrD FTIR difference spectrum presented here. The TyrD./TyrD spectrum also shows vibrational changes from peptide groups and amino acid side chains which are modified upon TyrD. formation. Proton release at the PS II protein surface upon TyrD. formation is deduced from differential signals at the v(PO) modes of phosphate.
Hienerwadel
R
R
Section de Bioénergétique, CEA-Saclay, Gif-sur-Yvette, France.
Boussac
A
A
Breton
J
J
Berthomieu
C
C
D004578:Electron Spin Resonance Spectroscopy; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D045332:Photosystem II Protein Complex; D010971:Plastoquinone; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D014443:Tyrosine; D010971:Plastoquinone