Hydrogen Bonding of Redox-Active Tyrosine Z of Photosystem II Probed by FTIR Difference Spectroscopy

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Abstract
The TyrZ•/TyrZ FTIR difference spectrum is reported for the first time in Mn-depleted photosystem II (PS II)-enriched membranes of spinach, in PS II core complexes of Synechocystis sp. PCC 6803 WT, and in the mutant lacking TyrD (D2-Tyr160Phe). In Synechocystis, the ν7‘a(CO) and δ(COH) infrared modes of TyrZ are proposed to account at 1279 and 1255 cm-1. The frequency of these modes indicate that TyrZ is protonated at pH 6 and involved in a strong hydrogen bond to the side chain of a histidine, probably D1-His190. A positive signal at 1512 cm-1 is assigned to the ν(CO) mode of TyrZ• on the basis of the 27 cm-1 downshift observed upon 13C-Tyr labeling at the Tyr ring C4 carbon. A second IR signal, at 1532 cm-1, is tentatively assigned to the ν8a(CC) mode of TyrZ•. The frequency of the ν(CO) mode of TyrZ• at 1512 cm-1 is comparable to that observed at 1513 cm-1 for the Tyr• obtained by UV photochemistry of tyrosinate in solution, while it is higher than that of TyrD• in WT PS II at 1503 cm-1 and that of non-hydrogen-bonded TyrD• in the D2-His189Gln mutant at 1497 cm-1 [Hienerwadel, R., Boussac, A., Breton, J., Diner, B. A., and Berthomieu, C. (1997) Biochemistry36, 14712−14723]. This latter work and the present FTIR study suggest that hydrogen bonding induces an upshift of the ν(CO) IR mode of tyrosyl radicals and that TyrZ• forms (a) stronger hydrogen bond(s) than TyrD• in WT PS II. Alternatively, the frequency difference between TyrZ• and TyrD• ν(CO) modes could be explained by a more localized positive charge near the tyrosyl radical oxygen of TyrD• than TyrZ•. The TyrZ•/TyrZ spectrum obtained in Mn-depleted PS II membranes of spinach shows large similarities with the S3‘/S2‘ spectrum characteristic of radical formation in Mn-containing but Ca2+-depleted PS II, in support of the assignment using ESEEM of TyrZ• as being responsible for the split EPR signal observed upon illumination in these conditions [Tang, X.-S., Randall, D. W., Force, D. A., Diner, B. A., and Britt, R. D. (1996) J. Am. Chem. Soc.118, 7638−7639]. The peak at 1514 cm-1 is assigned to the ν(CO) mode of TyrZ• in these preparations, which indicates that Mn depletion only very slightly perturbs the immediate environment of TyrZ• phenoxyl.