Site-specific insertion of 3-aminotyrosine into subunit α2 of E. coli ribonucleotide reductase: Direct evidence for involvement of Y₇₃₀ and Y₇₃₁ in radical propagation

E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: α2 and β2. α2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. β2 contains the stable diiron tyrosyl radical (Y₁₂₂·) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 Å between the Y₁₂₂· in β2 and C₄₃₉ in the active site of α2. A docking model of α2β2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y₇₃₀ and Y₇₃₁ in α2. In this study the function of residues Y₇₃₀ and Y₇₃₁ is investigated by their site-specific replacement with 3-aminotyrosine (NH₂Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y₇₃₀NH ₂Y-α2 and Y₇₃₁NH₂Y-α2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of β2, CDP, and ATP. The results reveal formation of an NH₂Y radical (NH₂Y₇₃₀· or NH₂Y ₇₃₁· ) in a kinetically competent fashion. Activity assays demonstrate that both NH₂Y-α2s make deoxynucleotides. These results show that the NH₂Y· can oxidize C₄₃₉ suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within α2. The observed NH₂Y· may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.