Saturation-recovery EPR spectroscopy has been used to probe the location of the redoxactive tyrosines, YD (tyrosine 160 of the D2 polypeptide, cyanobacterial numbering) and Yz (tyrosine 161 of the D1 polypeptide), relative to the non-heme Fe(II) in -depleted photosystem II (PSII). Measurements have been made on PSII membranes isolated from spinach and on PSII core complexes purified from the cyanobacterium Synechocystis sp. PCC 6803. In the case of Synechocystis PSII, sitedirected mutagenesis of the YD residue to either phenylalanine (Y160F) or methionine (Y160M) was done to eliminate the dark-stable YD.species and, thereby, allow direct spectroscopic observation of the Yz.EPR signal. The spin-lattice relaxation transients of both YD and Yz were non-single-exponential due to a dipolar interaction with one of the other paramagnetic species in PSII. Measurements on CN- treated, -depleted cyanobacterial PSII, in which the non-heme Fe(II) was converted into its low-spin, diamagnetic state, proved that the non-heme Fe(II) was the sole spin-lattice relaxation enhancer for both the YD and Yz radicals. This justified the use of a dipolar model in order to fit the saturation-recovery EPR data, which were taken over the temperature range 4-70 K. The dipolar rate constants extracted from the fits were identical in magnitude and had the same temperature dependence for both YD and Yz. The observation of identical dipolar interactions between YD' and Yz and the non-heme Fe(II) shows that the distance from each tyrosine to the non-heme Fe(II) is the same. The calculated distance of 37 ± 5 Å between YD or Yz and the non-heme Fe(II) agrees well with the distance predicted from the structure of the reaction center from purple bacteria. These results constitute the first spectroscopic evidence for a symmetric location of tyrosines D and Z in PSII and are consistent with the existence of a C2 symmetry axis among the chromophores of PSII, as in the purple bacterial reaction center.
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