ELECTRONIC-STRUCTURE OF NEUTRAL TRYPTOPHAN RADICALS IN RIBONUCLEOTIDEREDUCTASE STUDIED BY EPR AND ENDOR SPECTROSCOPY

Two different tryptophan radicals (W-a(.) and W-b(.)) with lifetimes o f several minutes at room temperature are formed during the reconstitu tion of the diiron center in the Escherichia coli ribonucleotide reduc tase mutant protein R2 Y122F. Detailed hyperfine parameters are for th e first time determined for protein-linked oxidized neutral tryptophan radicals. Wa(.) is freeze-trapped and investigated by EPR and ENDOR i n protonated and selectively deuterated proteins at 20 K. Two hyperfin e couplings from the beta-methylene protons, hyperfine tensors of two alpha-protons, and the complete nitrogen hyperfine tensor are determin ed. Based on the absence of a large hyperfine coupling from the N-H pr oton, which would be expected for a cation radical, and on comparison of the experimental data with theoretical spin densities from density functional calculations, W-a(.) is assigned to an oxidized neutral try ptophan radical. A small anisotropic hyperfine coupling detected in se lectively deuterated Wa is tentatively assigned to a proton which is h ydrogen bonded to the nitrogen of W-a(.). A similar spin density distr ibution as for W-a(.) is obtained also for the second tryptophan radic al, W-b(.) observed by EPR at room temperature, which is also assigned to an oxidized neutral radical.