SPECTROSCOPIC EVIDENCE FOR THE SYMMETRICAL LOCATION OF TYROSINE-D ANDTYROSINE-Z IN PHOTOSYSTEM-II

Saturation-recovery EPR spectroscopy has been used to probe the locati on of the redox-active tyrosines, Y-D (tyrosine 160 of the D2 polypept ide, cyanobacterial numbering) and Y-Z (tyrosine 161 of the D1 polypep tide), relative to the non-heme Fe(II) in Mn-depleted photosystem II ( PSII). Measurements have been made on PSII membranes isolated from spi nach and on PSII core complexes purified from the cyanobacterium Synec hocystis sp. PCC 6803. In the case of Synechocystis PSII, site-directe d mutagenesis of the Y-D residue to either phenylalanine (Y160F) or me thionine (Y160M) was done to eliminate the dark-stable Y-D(.) species and, thereby, allow direct spectroscopic observation of the Y-Z(.) EPR signal. The spin-lattice relaxation transients of both Y-D(.) and Y-Z (.) were non-single-exponential due to a dipolar interaction with one of the other paramagnetic species in PSII. Measurements on CN--treated , Mn-depleted cyanobacterial PSII, in which the non-heme Fe(II) was co nverted into its low-spin, diamagnetic state, proved that the non-heme Fe(II) was the sole spin-lattice relaxation enhancer for both the Y-D (.) and Y-Z(.) radicals, This justified the use of a dipolar model in order to fit the saturation-recovery EPR data, which were taken over t he temperature range 4-70 K. The dipolar rate constants extracted from the fits were identical in magnitude and had the same temperature dep endence for both Y-D(.) and YZ(.). The observation of identical dipola r interactions between Y-D(.) and Y-Z and the non-heme Fe(II) shows th at the distance from each tyrosine to the non-heme Fe(II) is the same. The calculated distance of 37 +/- 5 Angstrom between Y-D or Y-Z and t he non-heme Fe(II) agrees well with the distance predicted from the st ructure of the reaction center from purple bacteria. These results con stitute the first spectroscopic evidence for a symmetric location of t yrosines D and Z in PSII and are consistent with the existence of a C- 2 symmetry axis among the chromophores of PSII, as in the purple bacte rial reaction center.