The Escherichia Coll ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y(122)center dot) in R2 generate a transient cysteinyl radical (C(439)center dot) in R1 through a pathway thought to involve amino acid radical intermediates [Y(122)center dot -> W-48 -> Y-356 within R2 to Y-731 -> Y-730 -> C-439 within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced Y-356 with a variety of fluorinated tyrosine analogues (2,3-F2Y, 3,5-F2Y, 2,3,5-F3Y, 2,3,6-F3Y, and F4Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivatives have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pK(a)S that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these FnY356-R2s are reported. The results suggest that the rate-determining step can be changed from a physical step to the radical propagation step by altering the reduction potential Of Y(356)center dot using these analogues. As the difference in potential of the FnY center dot relative to Y center dot becomes > 80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y356 is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO2Y356-R2, we assume that 2,3,5-F3Y356, 2,3,6-F3Y356, and F4Y356-R2s are all deprotonated at pH > 7.5. We show that they all efficiently initiate nucleotide reduction. If this assumption is correct, then a hydrogen-bonding pathway between W-48 and Y-356 of R2 and Y-731 of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.