Tyrosyl radicals (Y center dot s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F(n)Ys, n = 1-4) are promising tools for studying the mechanism of Y center dot formation and reactivity, as their pK(a) values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F(3)Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F(n)Ys while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F(n)Ys site-specifically into the two subunits (alpha 2 and beta 2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y center dot s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F(n)Y(s), and the resulting proteins are isolated in good yields. F(n)Ys incorporated at position 122 of beta 2, the site of a stable Y center dot in wild-type RNR, generate long-lived F(n)Y center dot s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y(122)(2,3,5)F(3)Y-beta 2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y center dot can accumulate. The distinct EPR properties of the 2,3,5-F(3)Y center dot facilitate spectral subtractions that make detection and identification of new Y center dot s straightforward.