Fluorinated tyrosines (FnY's, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y's, a stable Y radical (Y center dot) in the beta subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in beta and 731 and 730 in alpha) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y-122-beta and the X-ray structures of each resulting beta with a diferric cluster are reported and compared with wt-beta 2 crystallized under the same conditions. The essential diferric-FnY center dot cofactor is self-assembled from apo FnY-Chi 2, Fe2+, and O-2 to produce similar to 1Y center dot/beta 2 and similar to 3 Fe3+/beta 2. The FnY center dot are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-beta 2. Each FnY center dot-beta 2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the F-19 nucleus provide unique signatures of each FnY- that are readily distinguishable from unlabeled Y center dot's. The variability of the abiotic FnY pK(a)'s (6.4 to 7.8) and reduction potentials (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y center dot's in catalysis and to investigate the importance and identity of hopping Y.'s within redox active proteins proposed to protect them from uncoupled radical chemistry.