# Structure-Function Studies of Ribonucleotide Reductase

> **NIH NIH R01** · HARVARD UNIVERSITY · 2021 · $82,263

## Abstract

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.

## Key facts

- **NIH application ID:** 10387984
- **Project number:** 3R01GM047274-29S1
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** DANIEL G. NOCERA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $82,263
- **Award type:** 3
- **Project period:** 1992-04-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10387984

## Citation

> US National Institutes of Health, RePORTER application 10387984, Structure-Function Studies of Ribonucleotide Reductase (3R01GM047274-29S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10387984. Licensed CC0.

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