STRUCTURE OF THE Y-D TYROSINE RADICAL IN PHOTOSYSTEM-II AS REVEALED BY H-2 ELECTRON-SPIN ECHO ENVELOPE MODULATION (ESEEM) SPECTROSCOPIC ANALYSIS OF HYDROGEN HYPERFINE INTERACTIONS

Electron spin echo envelope modulation (ESEEM) spectroscopic techniques have been used to characterize strong hydrogen hyperfine interactions in the Y-D tyrosine radical in randomly oriented photosystem II reaction center protein isolated from Synechocystis 6803. Strains of Synechocystis rendered auxotrophic for aromatic amino acids were used to incorporate H-2-label at the 3,5-ring or methylene bridge positions of Y-D* specifically. Stimulated echo envelope modulation was generated by conventional and pulse-swapping three-pulse sequences. Envelope-divided spectra of 3,5-H-2-/fully-protonated-Y-D* obtained for tau values from 143 to 1388 ns (9.132 GHz, 0.3265 T) display distinctive tau-suppression effects on the fundamental and double-quantum hyperfine frequencies. Simulations of the spectra yielded magnitudes and signs of the principal components of the total hyperfine tenser, [-3.0, -3.9, -1.1] MHz, and unpaired p(pi) spin density at the 3,5-positions of 0.25 was estimated from the isotropic coupling. Multifrequency studies of the methylene-H-2-labeled Y-D* displayed two pairs of hyperfine couplings that are assigned to two magnetically inequivalent H-2 nuclei. Simulations of the methylene-H-2 spectra give the following tenser values (axial dipolar tenser symmetry and signs assumed): H-2(beta,2), [A perpendicular to = 0.8, A parallel to = 2.2] MHz; H-2(beta,1), [A perpendicular to = 3.1, A parallel to = 4.5] MHz. Computations based on the standard description of beta-hyperfine coupling, constrained by the relative spectral amplitudes and the two isotropic coupling constants, revealed dihedral angles between the planes of the ring-C-1-C-beta-H-2(beta) atoms and phenol ring of 52 degrees and 68 degrees and an unpaired p pi spin density at C-1 of 0.37. There is no detectable static or dynamic dispersion in the dihedral angles from 4.2 to 77 K. The conformation of the methylene bridge is therefore fixed discretely by the protein. An independent estimation of the C-1 spin density of 0.36 was obtained from the common dipolar coupling strength (0.47 MHz) of H-beta,H-1 and H-beta,2 and the C-1-to-H-beta distance in L-tyrosine crystals. This allows estimation of static values for the H-2 isotropic beta-hyperfine coupling coefficients, B-0 and B-2, of <\0.1\ and 25.7 MHz, respectively. Comparison of the spin density distributions and dihedral angles among Y-D* and model tyrosine neutral radicals in solution, crystalline, and glassy environments reveals factors contributing to the tyrosine radical energetics and redox potential of the couple and suggests an electronic mechanism for enhancement of directional selectivity in electron transfers mediated by tyrosine and other organic cofactor radicals.