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Orientations of carboxylate groups coupled to the Mn cluster in the photosynthetic oxygen-evolving center as studied by polarized ATR-FTIR spectroscopy.

It is essential to clarify the structures and interactions of amino acids surrounding the Mn cluster in photosystem II (PSII) to understand the molecular mechanism of photosynthetic oxygen evolution. In this study, polarized attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was applied for the first time to PSII to investigate the orientation of carboxylate groups coupled to the oxygen-evolving Mn cluster. PSII membranes from spinach were oriented on the surface of a silicon ATR crystal, and flash-induced polarized ATR-FTIR difference spectra for the S(1) --> S(2) transition (S(2)/S(1) spectra) were obtained. The distribution of membrane orientations by mosaic spread was estimated from the semiquinone CO peak in polarized Q(A)(-)/Q(A) difference spectra recorded using the same oriented sample by buffer exchange. The orientations of carboxylate groups coupled to the Mn cluster were estimated from the dichroic ratios of the symmetric COO(-) bands in the polarized S(2)/S(1) ATR-FTIR spectra. We found that most of the carboxylate groups perturbed during the S(1) --> S(2) transition, due to direct ligation to the Mn cluster or though a hydrogen bond network, have orientations in a relatively narrow angle range of 34-48 degrees with respect to the membrane normal. Implications of the obtained orientations and the changes upon formation of S(2) are discussed on the basis of the information from previous FTIR studies and the X-ray structures. The results in this study show that polarized ATR-FTIR difference spectroscopy is a fruitful method for investigating the orientations and their reaction-induced changes in redox cofactors and coupled amino acid side chains in photosynthetic proteins.