Escherichia coli class Ia ribonucleotide reductase (RNR) catalyzes the formation of all four deoxynucleoside 5′‐diphosphates from their corresponding nucleoside 5′‐diphosphates. Class Ia RNRs are composed of two homodimeric subunits, α2 and β2, that form the active complex. A stable tyrosyl radical (Y122•) in the β2 subunit generates a transient cysteine radical (C439•) located 35 Å away in the α2 subunit by a mechanism that is proposed to involve the concerted movement of protons and electrons (proton‐coupled electron transfer or PCET) through several aromatic amino acid residues (Y122 ‐ [W48] ‐ Y356 in β2 to Y731 ‐ Y730 ‐ C439 in α2). 2,3,5‐trifluorotyrosine (2,3,5‐F3Y) has been site‐specifically inserted at Y122 using a polyspecific aminoacyl tRNA synthetase to modulate the driving force of radical propagation. The diferric‐2,3,5‐F3Y• radical cofactor self assembles in vitro producing 0.8‐1.0 F3Y•/β2. The reaction of Y122(2,3,5)F3Y‐β2, α2, substrate and effector generates 0.3‐0.5 equiv. of a putative Y356• that is kinetically and chemically competent in nucleotide reduction. At 5°C, complete re‐oxidation of 2,3,5‐F3Y by Y356• is recorded and represents our first observation of the disappearance and reappearance of the stable radical at position 122. Altering the driving force for radical propagation by simply 10 mV partially lifts the protein conformational gate that governs radical transport allowing mechanistic insight into the details of PCET.