Escherichia coli ribonucleotide reductase is an alpha 2 beta 2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in alpha 2 by a stable diferric tyrosyl radical (Y(122)center dot) cofactor in beta 2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 A through a specific pathway of residues: Y(122)center dot -> W(48)-> Y(356) in beta 2 to Y(731) -> Y(730) -> C(439) in alpha 2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of beta. The resulting protein, Y(336)NH(2)Y-beta 2, and the previously generated proteins Y(731)NH(2)Y-alpha 2 and Y(730)NH(2)Y-alpha 2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (5), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y center dot) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH2Y center dot formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH2Y center dot formation, while the k(cat) of similar to 0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y center dot of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-alpha 2 and Y(731)NH(2)Y-alpha 2 have been solved and indicate minimal structural changes relative to wt-alpha 2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.