Investigation of the differences in the local protein environments surrounding tyrosine radicals Y_Z^• and Y_D^• in photosystem II using wild-type and the D2-tyr160Phe mutant of synechocystis 6803

The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (Y_D) of the D2 polypeptide and Tyr161 (Y_Z) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680⁺. Spectroscopic characterization of YZ* has been hampered by the simultaneous presence of the much more stable Y_D^•, the short lifetime of Y_Z^•, and the difficulty in trapping the Y_Z^• radical at low temperature. We present here a method for obtaining an uncontaminated Y_Z^• radical, trapped by freezing under illumination of PSII core complexes isolated from Y_D-less mutants of Synechocystis 6803. Specific labeling with deuterium of the β-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the Y_D-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the Y_Z^• EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped Y_Z^• radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of Y_Z^•, this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ^•. We have compared the environments of Y_Z^• and Y_D^• by EPR, ¹H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of Y_Z^• and Y_D^• differ in line shape and line width. (2) Both Y_Z^• and Y_D^• exhibit D₂O-exchangeable ¹H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of Y_D^•, indicating the hydrogen bond to be well-defined. In the case of Y_Z^• it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) Y_D^• possesses three additional weak couplings that disappear in D₂O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 Å. (4) All of the ¹H couplings of Y_D^• are sharp, which is indicative of a well-ordered protein environment. (5) All of the ¹H couplings in the Y_Z^• spectrum are broad. The environment surrounding Y_Z^• appears to be more disordered and solvent-accessible.