Modified EPR Spectra of the Tyrosine_D Radical in Photosystem II in Site-Directed Mutants of Synechocystis sp. PCC 6803: Identification of Side Chains in the Immediate Vicinity of Tyrosine_D on the D2 Protein

The oxidizing side of photosystem II contains two redox-active tyrosyl side chains, Tyrz and Tyr_D, and a cluster of Mn atoms involved in water oxidation. The structural environment of these components is unknown, and with computer-assisted modeling we have created a three-dimensional model for the structures around Tyr_z and Tyr_D [Svensson et al. (1990) EMBO J. 9, 2051–2059]. Both tyrosines are proposed to form hydrogen bonds to nearby histidine residues (for Synechocystis 6803, these are His 190 on the D1 and His 189 on the D2 proteins). We have tested this proposal by electron paramagnetic resonance (EPR) spectroscopy of Tyr_D^ox in mutants of the cyanobacterium Synechocystis 6803 carrying site-directed mutations in the D2 protein. In two mutants, where His 189 of the D2 protein is changed to either Tyr or Leu, the normal EPR spectrum from Tyr_D^ox is replaced by narrow, structureless radical signals with g-values similar to that of Tyr_D^ox (g ≈ 2.0050). The new radicals copurify with photosystem II, are dark-stable, destabilized by elevated pH, and light-inducible, and originate from radicals formed by oxidation. These properties are similar to those of normal Tyr_D^ox, and we assign the new spectra to Tyr_D^ox in an altered environment induced by the point mutation in His 189. In a third mutant, where G1n 164 of the D2 protein was mutated to Leu, we also observed a modified EPR spectrum from Tyr_D^ox. This is also consistent with the model in which this residue is found in the immediate vicinity of Tyr_D^ox. Thus the results provide experimental evidence supporting essential aspects of the structural model.