The reaction center of photosystem II (PSII) contains two redox-active tyrosines, Tyrd and Tyrz, which are Tyr160 and Tyr161 of the D2 and Dl proteins, respectively. We have introduced five site-directed mutations in the D2 protein in the vicinity of Tyrd to analyze the consequences of the mutations on spectral and functional properties of Tyrdox. Characterization of three mutants, P161A and P161L (Pro161 changed to A1a and Leu, respectively) and Q164L (Gln164 mutated to Leu), is emphasized. Of these three mutants, only P161L is an obligate photoheterotroph; it is capable of oxygen evolution, but is photoinactivated rapidly. The D2 protein of this mutant migrates slower on a SDS-polyacrylamide gel. The EPR spectrum of Tyrdox is modified in the three mutants. The EPR spectra of Tyrdox in wild type and the mutants were characterized in detail by comparison of EPR spectra of thylakoids from cells grown in the presence and absence of tyrosine that was deuterated in specific positions. The experimentally obtained EPR spectra of wild type, P161 A, and Q164L could be simulated satisfactorily using current theoretical models. The angle between one of the hydrogens on the β-methylene carbon and the 2pz orbital at C1 of the tyrosine ring was found to change slightly but significantly as a function of the mutations (52° in wild type, 50° in P161A, and 48° in Q164L). The overall electronic structure of Tyrdox is quite unaffected; only minor redistribution of the unpaired electron spin is observed between the wild type and the mutated systems. In all three strains, the spin density is in the range from 0.34-0.38 at carbon atom C1, −0.08 at C2 and C6, and 0.26-0.29 at C4 and the oxygen. In the P161L mutant a dark-stable, wide radical is observed with an intermediate g value (g = 2.0042). This EPR spectrum is also sensitive to tyrosine deuteration. However, the EPR spectrum is not easily understood in terms of a single Tyr radical and could not be simulated adequately. Possible reasons for this are discussed. Nonetheless, the results presented here imply the sensitivity of EPR spectroscopy to monitor changes in geometry and spin distribution in radicals as a function of alterations in their immediate protein environment.
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