| Abstract
| - The reaction center of photosystem II (PSII) of the oxygenicphotosynthetic electron transportchain contains two redox-active tyrosines, Tyr160 (YD) ofthe D2 polypeptide and Tyr161 (YZ) of the D1polypeptide, each of which may be oxidized by the primary electrondonor, P680+. Spectroscopiccharacterization of YZ• has been hampered bythe simultaneous presence of the much more stableYD•,the short lifetime of YZ•, and the difficultyin trapping the YZ• radical at lowtemperature. We presenthere a method for obtaining an uncontaminatedYZ• radical, trapped by freezing underillumination ofPSII core complexes isolated from YD-less mutants ofSynechocystis 6803. Specific labeling withdeuteriumof the β-methylene-3,3- or of the ring 3,5-protons of the PSIIreaction center tyrosines in the YD-lessD2-Tyr160Phe mutant results in a change in the hyperfine structure ofthe YZ• EPR signal, furtherconfirming that this signal indeed arises from tyrosine. Thetrapped YZ• radical is also stable forseveralmonths at liquid nitrogen temperature. Due to both the absence ofcontaminating paramagnetic speciesand the stability at low temperature of YZ•,this mutant core complex constitutes an excellentexperimentalsystem for the spectroscopic analysis of YZ•.We have compared the environments of YZ•and YD• byEPR, 1H ENDOR, and TRIPLE spectroscopies using both mutantand wild-type core complexes, with thefollowing observations: (1) the EPR spectra ofYZ• and YD• differin line shape and line width. (2) BothYZ• and YD• exhibitD2O-exchangeable 1H hyperfine coupling near 3MHz, consistent with the presenceof a hydrogen bond from a proton donor to the phenolic oxygen atom of aneutral tyrosyl radical. Thishyperfine coupling is sharp in the case ofYD•, indicating the hydrogen bond to bewell-defined. In thecase of YZ• it is broad, suggestive of adistribution of hydrogen-bonding distances. (3)YD• possessesthree additional weak couplings that disappear in D2O,arising from three or fewer protons (protein orsolvent) located within a shell between 4.5 and 8.5 Å. (4) All ofthe 1H couplings of YD• aresharp,which is indicative of a well-ordered protein environment. (5) Allof the 1H couplings in the YZ•spectrumare broad. The environment surroundingYZ• appears to be more disordered andsolvent-accessible.
|
