# Proton Coupled Electron Transfer Mechanism of Ribonucleotide Reductase

> **NIH NIH R01** · HARVARD UNIVERSITY · 2020 · $407,250

## Abstract

Abstract
Proton-coupled electron transfer (PCET) is a ubiquitous mechanism in biology, serving as the basis for
mediating steps involving biosynthesis of both primary and secondary metabolites, radical generation and
transport, and the activation of substrates at cofactors. The control of highly reactive radical intermediates is
achieved via 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
(RNR), which performs reversible long-range charge-transfer that spans 35 Å and two subunits (α and β) upon
applications including chemotherapy, anti-retroviral drugs and anti-inflammatory agents. The proposed
research program seeks to define PCET at a detailed mechanistic level by focusing on ribonucleotide reductase
every turnover. This process occurs via a pathway of redox-active amino acids, rendering RNR a paradigm for
the study of PCET in biology. An interdisciplinary approach integrates a suite of experimental methods
encompassing biochemistry, transient spectroscopy, synthesis and electrochemistry to target three specific
aims. Owing to the sensitivity of the coupling between the proton and electron, we seek to define how
conformational gating targets the PCET pathway. We will concentrate on tyrosine dyads and triads of the PCET
pathway and introduce canonical and non-native point mutations to address the effects of driving force,
electrostatic local environment and hydrogen bonding interactions involving these tyrosine clusters. In tandem
with these biochemical inquiries, studies of cofacially aligned tyrosine model dyads will be investigated to
direct link between allostery and radical transport. A second specific aim targets PCET across the α | β protein
define how the energetics of radical generation are affected by stacking and hydrogen bonding. These studies
will uncover how conformational gating controls RNR activity at an atomistic level and thus will establish a
interface with the goal of identifying critical residues that mediate proton transfer attendant to radical
transport. The subunit interface of RNR is a critical nexus of the PCET pathway and provides an access point
for therapeutics designed to affect enzymatic function. The third specific aim seeks to understand the interplay
between allosteric activation in α2 and reduction of the Y122• cofactor, 35 Å away in β2. To address this directly,
we will embark on an investigation of the kinetics and thermodynamics of PCET through β by employing new
photoβ2s. We will extend our studies to a RNR class featuring a (Mn Fe ) state to initiate forward PCET
 IV IV
through the enzyme by photoinitiating the active state for radical generation. Together, the principles that
emerge from addressing these specific aims will be applied to explain the functions of a variety of enzymes and
proteins that derive their activity from PCET.

## Key facts

- **NIH application ID:** 9849298
- **Project number:** 5R01GM047274-28
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** DANIEL G. NOCERA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $407,250
- **Award type:** 5
- **Project period:** 1992-04-01 → 2021-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9849298

## Citation

> US National Institutes of Health, RePORTER application 9849298, Proton Coupled Electron Transfer Mechanism of Ribonucleotide Reductase (5R01GM047274-28). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9849298. Licensed CC0.

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