Electronic structure of neutral tryptophan radicals in ribonucleotide reductase studied by epr and endor spectroscopy

New transient protein-linked radicals have been analyzed by EPR and ENDOR spectroscopy in the radical enzyme ribonucteotide reductase of E coK, particularly in the mutant Y122F of the protein R2, during the reconstitution of the diiron center. Two different tryptophan radicals (Wa° and W0°) with life-times of several minutes are formed at room temperature. Wa° is freeze-trapped and investigated by EPR and ENDOR in normal and selectively deuterated proteins at 20K Two hyperfine couplings from the βmethylene protons, hyperfine tensors of two a-protons and the complete nitrogen hyperfine tensor are determined. Based on the absence of a large hyperfine coupling from the N-H proton, which could be expected for a cation radical, and on comparison of the experimental data with theoretical spin densities from density functional calculations, WV is assigned to an oxidized neutral tryptophan radical. A small anisotropic hyperfine coupling detected in selectively deuterated W." is tentatively assigned to a proton which is hydrogen bonded to the nitrogen of VW. A similar spin density distribution was obtained also for the second tryptophan radical. WV observed at room temperature by stopped-flow EPR is also assigned to an oxidized neutral radical. The site of Wa° and WtT in protein R2 has been determined from the comparison of the conformation of the βprotons (from EPR data) with X-ray structure data to W111 and W107, respectively, in close neighborhood to the iron center. For the first time, detailed hyperfine parameters are determined for protein-linked oxidized neutral tryptophan radicals. Using the same technique of iron reconstitution, in the mutant R2-Y122H, where the site of the catalytic essential stable tyrosyl radical has been replaced by a histidine, a new stable radical exhibiting a strong magnetic metal interaction is observed by EPR and ENDOR.