# Understanding Protein Radicals

> **NIH NIH R01** · TEXAS A&M AGRILIFE RESEARCH · 2021 · $303,000

## Abstract

Oxidoreductases use metallocofactors, organic molecules and four types of amino acids (tyrosine, tryptophan,
cysteine and glycine) to perform electron transfer (ET) and proton-coupled electron transfer (PCET) reactions.
The four amino acids serve as one-electron redox mediators in biocatalytic and multistep electron/hole transfer
(radical transfer) processes some of which are essential to life on earth. There is also a sinister side to amino-
acid ET/PCET since these reactions can be induced at oxidative stress conditions and cause significant cellular
damage. It is extremely challenging to experimentally resolve the thermodynamic and kinetic redox properties
of a single amino-acid residue. The highly oxidizing and reactive nature of amino-acid radicals increases the
barrier for experimental characterization even further. Consequently, key information to support the
understanding of one of nature's essential redox tools is missing. This research project is based on a family of
well-structured model proteins with the unique capability of providing both reversible reduction potentials
(E°'s) and detailed PCET kinetic/mechanistic information on tyrosine (Y) and tryptophan (W) oxidation-
reduction reactions. The so-called a3X proteins are based on a three-helix bundle (a3) with an aromatic residue
at interior position 32 (X32). The a3X system will be developed along three connected paths to gain novel
information on the fundamental factors that govern Y/W multistep electron/hole transfers. (1) DE°'s and
solution NMR structures obtained on the current a3X family will be analyzed using density functional theory
(DFT) and quantum mechanics/molecular mechanics (QM/MM) methods. The goal is to obtain a robust
computational protocol for quantitatively predicting DE°'s of Y and W residues. The computational protocol
will be used to identify W analogs with significantly shifted potentials relative to the potential of native W.
Selected analogs will be inserted at site 32 in a3W and their E°'s measured by protein film voltammetry.
Iterative prediction/test cycles will refine and validate the computational protocol. (2) a3 proteins containing
one (a3Y & a3W) and two (a3YX & a3WX, X = Y, W & analogs) redox-active aromatic residues and a tethered
ruthenium (Ru) photosensitizer will be made. These proteins will be used to: (a) Determine the rate of radical
formation in Ru-a3Y and Ru-a3W as a function of the PCET driving force to determine the reorganization
energies associated with the Y32·/Y32 and W32·/W32 redox pairs. This is a fundamental parameter (l) in ET and
PCET rate constant expressions and thus far unknown for protein Y·/Y and W·/W redox pairs. (b) Generate a
comprehensive description of Y/W multistep electron/hole transfers based on rigorously determined structural
(electron donor/acceptor & proton donor/acceptor spatial organization), thermodynamic (DE°' & DpKa), and
kinetic (kPCET & l values) parameters. (3) Advanced NMR and EPR methods will be used t...

## Key facts

- **NIH application ID:** 10209779
- **Project number:** 2R01GM079190-11A1
- **Recipient organization:** TEXAS A&M AGRILIFE RESEARCH
- **Principal Investigator:** Cecilia Tommos
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $303,000
- **Award type:** 2
- **Project period:** 2008-08-04 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10209779

## Citation

> US National Institutes of Health, RePORTER application 10209779, Understanding Protein Radicals (2R01GM079190-11A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10209779. Licensed CC0.

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