Electron Magnetic Resonance of the Tyrosyl Radical in Ribonucleotide Reductase from Escherichia coli

Abstract

The spin density distribution of the Y122 tyrosyl radical in the R2 subunit of ribonucleotide reductase from Escherichia coli has been determined. Incorporation of isotopically labeled tyrosine into the protein has allowed us to measure the 17O hyperfine coupling by using EPR, giving a direct measure of the tyrosine phenol oxygen spin density, 0.29 ± 0.02. The hyperfine tensors of six protons of the radical have been determined by using ENDOR. Magnetic field selection allows a determination of the orientation of the hyperfine tensors relative to the g tensor. Electron−nuclear−nuclear triple resonance has been applied to establish the relative signs of three hyperfine couplings. These measurements give a more precise and more accurate picture of the spin density distribution in a protein tyrosyl radical than has been available previously. The 17O hyperfine splitting in tyrosyl radicals in aqueous glasses has also been measured. The differences in hyperfine couplings indicate that addition of a hydrogen bon...