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

摘要

Tyrosine (Y_D) in the D2 reaction centre polypeptide of photosystem II(PSII) is redox-active and, under illumination, froms 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 n PSII was probed by EPR in conjunction with carefully designed site specific isotope labelling, A comprehensive series of different selectively ~2H-, ~(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 bound length of 1.5 A. 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.