ABSTRACT
Deuteration at selected positions on the phenol ring and at the beta-methylene carbon for the YD.tyrosine radical in Photosystem II in the cyanobacterium Synechocystis 6803 was achieved by growing the organism under conditions in which it is a functional aromatic amino acid auxotroph (Barry, B. A., and Babcock, G. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7099-7103). A series of model tyrosine radicals, also deuterated at specific sites on the aromatic ring and the methylene group, was generated by UV irradiation of frozen solutions. The EPR spectra of the specifically deuterated in vivo radicals confirm that YD.is a tyrosine; nevertheless its spectra differ from those of the tyrosine models. By comparing the EPR spectra of the specifically deuterated radicals with those of the fully protonated, the hyperfine couplings of the various protons of both YD.and the model compound radicals were determined. For both species, the unpaired electron spin density distribution is essentially identical and follows an odd-alternant pattern with high rho values at the carbons ortho and para to the tyrosine phenol oxygen; the meta positions have low spin densities. The differences in EPR spectral characteristics for the two radicals are rationalized as arising from variations in the conformation of the beta-methylene group with respect to the phenol head group. Considering these EPR results and those reported for other model and naturally occurring tyrosine radicals, we conclude that this situation is general; there is little deviation in this class of compounds from the odd-alternant spin density distribution; variations in EPR lineshapes arise primarily from variations in beta-methylene orientation. The conformation of the -CH2- group in biologically active tyrosine radicals deviates from that observed in the models and may be functionally significant. Because the EPR spectrum of YZ., the second redox active tyrosine radical in Photosystem II, is identical to that of YD., we conclude that the two radicals are in similar protein environments, a conclusion that is supported by the protein sequences in the vicinity of the two radicals.
