Probing the electronic structure of tyrosine radical YD in photosystem II by EPR spectroscopy using site specific isotope labelling in Spirodela oligorrhiza

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Tyrosine (Y D ) in the D2 reaction centre polypeptide of photosystem II (PSII) is redox-active and, under illumination, forms a dark-stable radical Y D . The origin of its stability and the functional role of Y D are not well understood. For understanding the electronic structure and reactivity of Y D , it is crucial to unambiguosly establish its hyperfine structure. There is considerable variation in the hyperfine data of Y D and their interpretation in literature. In the present study, the hyperfine structure of tyrosine radical Y D in PSII was probed by EPR in conjunction with carefully designed site specific isotope labelling. A comprehensive series of different selectively 2 H-, 13 C- or 17 O-labeled tyrosine were synthesized and incorporated in Spirodela oligorrhiza with more than 95% enrichment. The 13 C- and 17 O-hyperfine interactions were obtained from spectral simulations. From the anisotropy of the hyperfine interactions the spin densities at all phenoxyl ring positions were precisely obtained. Comparison of the absolute differences in individual spin densities between Y D and neutral tyrosine radical in vitro with those of computationally calculated spin densities yield excellent agreement for a well ordered hydrogen bond between Y D and the surrounding protein matrix with a bond length of 1.5 Å. Enantioselective labeling confirms that the β-methylene hydrogens of Y D in S. oligorrhiza are oriented in a highly constrained specific position making Y D strongly immobilized, thereby ensuring a firm hydrogen bond of the phenoxyl oxygen to the protein matrix.