G-VALUES AS A PROBE OF THE LOCAL PROTEIN ENVIRONMENT - HIGH-FIELD EPR OF TYROSYL RADICALS IN RIBONUCLEOTIDE REDUCTASE AND PHOTOSYSTEM-II

The stable tyrosyl radicals in E. coli ribonucleotide reductase (RNR) and photosystem II (PSII) were studied by high-field 245 GHz 8.7T EPR. The relaxation properties of Tyr-D degrees, the stable radical in PSII, are different depending on the spin-state of the manganese-cluster within the protein. Microwave power studies indicated that at high fields and low temperatures the RNR radical relaxes comparably to Tyr-D degrees when the Mn-cluster is in the paramagnetic state. This verified the existence of a weak exchange coupling between the tyrosyl radical and the nearby binuclear iron site in RNR. A detailed study of the g-values of the two radicals was carried out, and these g-values were compared to those obtained from an existing 9 GHz single-crystal EPR study of a model tyrosyl radical. The g-values of the radicals were calculated using the unrestricted Hartree-Fock MNDO molecular orbital method with PM3 parameterization and the theory of g-values of radicals developed by Stone. The spin-orbit coupling contribution to the g-values were evaluated using a modified version of Roothaan's theory of electronic excitation. The local environment of the radicals were incorporated into these calculations by inclusion of point charges and hydrogen bonds. The method described here is independent of adjustable parameters except those inherent to the PM3-MNDO method. The calculated g-values were in good agreement with those measured experimentally. The differences in g-tensors of the tyrosyl radical in RNR, PSII, and the model system are explained in terms of stabilization of the nonbonding orbitals of the tyrosyl oxygen by electrostatic and hydrogen-bonding interactions. The g-value calculations also indicate that Tyr-D degrees is less strongly hydrogen-bonded than in the model system. By incorporating existing ENDOR data into our analysis, we have been able to localize the hydrogen-bond donor to Tyr-DO relative to the phenyl-ring: a hydrogen bond distance of similar to 1.7 Angstrom, bond angle of similar to 140 degrees, and dihedral angle of similar to 35 degrees. This approach which combines high-field EPR measurements of g-values with molecular orbital calculations is generally applicable to organic radicals.