Abstract Proton-coupled electron transfer (PCET) is a ubiquitous mechanism in biology, serving as the basis for mediating steps involving biosynthesis of metabolites, radical generation and transport, and the activation of substrates at cofactors. The control of highly reactive radical intermediates is achieved by coupling proton and electron transfer processes. Management of radicals in biology is of particular relevance to human health, as enzymes operating by PCET are therapeutic targets with wide-ranging applications including chemotherapy, anti-retroviral and anti-bacterial drugs and anti-inflammatory agents. Of the enzymes that operate by PCET, ribonucleotide reductases (RNRs) are exceptional in their biological function and are paramount to health, as the enzymes produce the DNA building blocks for life. The central role of RNRs in nucleic acid metabolism has made the human RNR the target of five clinically used therapeutics that shut down the PCET pathway and, consequently, nucleotide reduction. The class Ia RNR is the exemplar of biological PCET; its function originates from a reversible long-range radical transport pathway that spans 35 Å and two subunits (α and β) upon every turnover. An interdisciplinary approach integrates a suite of experimental methods encompassing biochemistry, steady-state and transient biophysical spectroscopies, synthesis, and electrochemistry to target three specific aims. Specific Aim 1 seeks to address the role of PCET in nucleotide reduction, both in the substrate activation phase involving the conserved radical at the “top face” of the active site, as well as in the radical substrate reduction phase at the “bottom face” of the active site. Work will be advanced by (i) leveraging newly developed selenocysteine incorporation methodologies to alter proton inventories and electron affinities, (ii) examining rate constants of individual steps using model compounds, and (iii) structurally capturing forward radical transport leading into the active site. As the coupling between the proton and electron along the radical transport pathway is the target of conformational gating by the enzyme, Specific Aim 2, is designed to identify amino acid networks that govern allosteric PCET regulation between the α and β subunits, and to rigorously define the structural dynamics at the interface that modulate RNR activity. This work is guided by new structural insights afforded from cryo-EM studies, which allow both the nature of subunit interactions and the networks of amino acids that connect the catalytic, specificity, and activity sites of the intact enzyme to be identified. The structural and temporal visualization of subunit dynamics that come from these studies will inform on the design of new small molecule therapeutics targeting the subunit interface. Specific Aim 3 will utilize biochemical and molecular biology innovations to elucidate initial events of radical transfer within the β- subunit with a focus on a critical...