Ribonucleotide reductases (RNRs) catalyze the conversion of all four ribonucleotides to deoxyribonucleotides and are essential for DNA synthesis in all organisms. The active form of E. coli Ia RNR is composed of two homodimers that form the active α2β2 complex. Catalysis is initiated by long-range radical translocation over a ∼32 Å proton-coupled electron transfer (PCET) pathway involving Y356β and Y731α at the interface. Resolving the PCET pathway at the α/β interface has been a long-standing challenge due to the lack of structural data. Herein, molecular dynamics simulations based on a recently solved cryogenic-electron microscopy structure of an active α2β2 complex are performed to examine the structure and fluctuations of interfacial water, as well as the hydrogen-bonding interactions and conformational motions of interfacial residues along the PCET pathway. Our free energy simulations reveal that Y731 is able to sample both a flipped-out conformation, where it points toward the interface to facilitate interfacial PCET with Y356, and a stacked conformation with Y730 to enable collinear PCET with this residue. Y356 and Y731 exhibit hydrogen-bonding interactions with interfacial water molecules and, in some conformations, share a bridging water molecule, suggesting that the primary proton acceptor for PCET from Y356 and from Y731 is interfacial water. The conformational flexibility of Y731 and the hydrogen-bonding interactions of both Y731 and Y356 with interfacial water and hydrogen-bonded water chains appear critical for effective radical translocation along the PCET pathway. These simulations are consistent with biochemical and spectroscopic data and provide previously unattainable atomic-level insights into the fundamental mechanism of RNR.