EPR EVIDENCE THAT THE M+ RADICAL, WHICH IS OBSERVED IN 3 SITE-DIRECTED MUTANTS OF PHOTOSYSTEM-II, IS A TYROSINE RADICAL

Isotopic labeling of Synechocystis sp. PCC 6803 and EPR spectroscopy have been used to demonstrate that photosystem II contains two redox active tyrosines, D and Z (Barry, B. A. and Babcock, G. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7099-7103; Boerner, R. J. and Barry, B. A. (1993) J. Biol. Chem. 268, 17151-17154). Another organic radical, M+, has recently been observed in site-directed mutants in which a non-redox active amino acid is substituted at either the putative D or putative Z sites (Boerner, R. J., Bixby, K. A., Nguyen, A. P., Noren, G. H., Debus, R. J., and Barry, B. A. (1993) J. Biol. Chem. 268, 1817-1823; Noren, G. H., and Barry, B. A. (1992) Biochemistry 31, 3335-3342). Here, we use isotopic labeling to determine the chemical identity of M+. Upon incorporation of perdeuterated tyrosine into photosystem II, the M+ EPR signal narrows to approximately 12-13 G. Labeling with 3,5-deuterated tyrosine results in an isotropic doublet with splittings of 11 G. Our results show that M+ is a tyrosine radical with unique spectroscopic properties.