SPIN-DENSITY DISTRIBUTION, CONFORMATION, AND HYDROGEN-BONDING OF THE REDOX-ACTIVE TYROSINE Y-Z IN PHOTOSYSTEM-II FROM MULTIPLE ELECTRON MAGNETIC-RESONANCE SPECTROSCOPIES - IMPLICATIONS FOR PHOTOSYNTHETIC OXYGEN EVOLUTION

The oxidized form of the redox-active Y-Z tyrosyl residue involved in photosynthetic oxygen evolution has been generated and trapped in Mn-d epleted Photosystem II core complexes from a D2-Y160F mutant strain of Synechocystis 6803. This system eliminates interference from P-700+ a nd Y-D(.) and allowed characterization of Y-Z(.) by using a combinatio n of specific H-2-labeling and electron magnetic-resonance techniques that included CW-EPR, frequency-modulated and transient detected ENDOR , and H-2-ESEEM. Using these complementary techniques, we have carried out a detailed evaluation of the hyperfine structure of Y-Z(.) and ob tained the dipolar interactions to weakly coupled nuclei, the strongly anisotropic tensors of the ring-hydrogens, and the more isotropic int eractions to the beta-methylene site. No (H2O)-H-2 exchangeable featur es could be detected by CW-ENDOR, implying that Y-Z is not involved in a well-ordered hydrogen bond in its radical form. From the hyperfine coupling tensors the spin-density distribution of Y-Z(.) was derived w ith the following values: C-1 (0.37), C-2,C-6 (-0 07), C-3,C-5 (0.26), and C-4-O (0.25). These values are similar to those reported for othe r tyrosyl radicals, both hydrogen bonded and non-hydrogen bonded. We c onclude that tyrosyl radicals are not tuned to specific function by la rge-scale modulations of their spin density through hydrogen-bonding e ffects. ENDOR and H-2-ESEEM spectra representing the hyperfine interac tions of the beta-methylene site provided evidence of rotational mobil ity about the tyrosyl C-1-C-beta bond. A quantitative analysis of the H-2-ESEEM data provided a distribution in theta of about 14 degrees. T he observation of mobility in the Y-Z site, and the lack of a well-ord ered hydrogen bond are inconsistent with the conventional view of Y-Z as a pure electron-transfer component in Photosystem II. Rather, we su ggest a hydrogen-atom transfer function for Y-Z in water oxidation. Wi thin this model, the (Mn)(4)/Y-Z center forms the Oxygen-Evolving Comp lex of Photosystem II where the (Mn)(4) cluster binds substrate water and delocalizes oxidizing equivalents and Y-Z acts by abstracting hydr ogens from substrate water in either a concerted or sequential fashion .