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: alpha 2 and beta 2. alpha 2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. beta 2 contains the stable diiron tyrosyl radical (Y-122 center dot) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over > 35 angstrom between the Y-122 center dot in beta 2 and C-439 in the active site of alpha 2. A docking model of alpha 2 beta 2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y-730 and Y-731 in alpha 2. In this study the function of residues Y-730 and Y-731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha 2 and Y731NH2Y-alpha 2 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 beta 2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730 center dot or NH2Y731 center dot in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha 2s make deoxynucleotides. These results show that the NH2Y center dot can oxidize C-439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha 2. The observed NH2Y center dot may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.