Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y₁₂₂•) within the β2 subunit to a cysteine residue (C₄₃₉) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y₁₂₂ → W₄₈ → Y₃₅₆ in β2, across the subunit interface to Y₇₃₁ → Y₇₃₀ → C₄₃₉ in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y₁₂₂• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH₂Y•). In the current study [¹⁵N]- and [¹⁴N]-NH₂Y₇₃₀• have been generated in H₂O and D₂O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH ₂Y₇₃₀•. The g tensor (g_x = 2.0052, g _y = 2.0042, g_z = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH₂ moiety are consistent with an intramolecular hydrogen bond within NH₂Y ₇₃₀•. This analysis is an essential first step in using the detailed structure of NH₂Y₇₃₀• to formulate a model for a PCET mechanism within α2 and for use of NH₂Y in other systems where transient Y•s participate in catalysis.