D. Koulougliotis et al., SPECTROSCOPIC EVIDENCE FOR THE SYMMETRICAL LOCATION OF TYROSINE-D ANDTYROSINE-Z IN PHOTOSYSTEM-II, Biochemistry, 34(9), 1995, pp. 2850-2856
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.