Reaction centers from the YL167 mutant of Rhodobacter sphaeroides, containing a highly oxidizing bacteriochlorophyll dimer and a tyrosine residue substituted at Phe L167, were compared to reaction centers from the YM mutant, with a tyrosine at M164, and a quadruple mutant containing a highly oxidizing dimer but no nearby tyrosine residue. Distinctive features in the light-induced optical and EPR spectra showed that the oxidized bacteriochlorophyll dimer was reduced by Tyr L167 in the YL167 mutant, resulting in a tyrosyl radical, as has been found for Tyr M164 in the YM mutant. In the YL167 mutant, the net proton uptake after formation of the tyrosyl radical and the reduced primary quinone ranged from +0.1 to +0.3 H+/reaction center between pH 6 and pH 10, with a dependence that is similar to the quadruple mutant but different than the large proton release observed in the YM mutant. In the light-induced absorption spectrum in the 700-1000 nm region, the YL167 mutant exhibited unique changes that can be assigned as arising primarily from an approximately 30 nm blue shift of the dimer absorption band. The optical signals in the YL167 mutant were pH dependent, with a pKa value of approximately 8.7, indicating that the tyrosyl radical is stabilized at high pH. The results are modeled by assuming that the phenolic proton of Tyr L167 is trapped in the protein after oxidation of the tyrosine, resulting in electrostatic interactions with the tetrapyrroles and nearby residues.
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