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 biochemical methods with the transient spectroscopy afforded by the requested instrumentation 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 interfacing the TRIR instrumentation with existing laser instrumentation to define kinetics of key intermediates associated with individual steps of RNR active site chemistry and with model compounds that faithfully capture the RNR active site chemistry. The TRIR technique will also be used the follow amide I and II stretches of key residues in amino acid networks that govern allosteric PCET regulation between the α and β subunits. 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. The TRIR will also aid in studies of Specific Aim 3 that utilize biochemical and molecular biology innovations to elucidate initial events of radical transfer within the β-subunit, with a focus on a critical tryptophan within the PCET pathway.