Oxidoreductases use metallocofactors, organic molecules, and four amino acids (Tyr, Trp, Cys, and Gly) to perform electron transfer (ET) and proton-coupled electron transfer (PCET) reactions. These four amino acids serve as one-electron (radical) redox mediators in biocatalytic and multistep electron/hole 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. Consequently, key information to support the understanding of one of nature’s essential redox tools is missing. Our research focuses on developing a family of well-structured model proteins with the unique capability of providing detailed mechanistic information on Tyr (Y) and Trp (W) oxidation-reduction reactions.

The α₃X model protein system

We have made a family of well-structured model proteins specifically designed to study Y and W radical formation, transfer, and decay. This model system provides us with the rare opportunity to study amino acid radical reactions under fully reversible conditions inside a structurally well-determined protein environment. The α₃X proteins were developed by combining protein design with site-specific incorporation of unnatural amino acids and detailed structural studies. We now aim to expand the α₃X system by constructing well-structured proteins containing two redox-active residues (α₃YX and α₃WX, X = Y, W & Y/W analogs). These proteins will be explored to examine fundamental ET/PCET properties associated with Y/W-based radical transfer.

C. Tommos, K. G. Valentine, M. C. Martínez-Rivera, L. Liang, V. R. Moorman, Reversible phenol oxidation and reduction in the structurally well-defined 2-mercaptophenol-α₃C protein. Biochemistry 52, 1409–1418 (2013).

K. R. Ravichandran, A. B. Zong, A. T. Taguchi, D. G. Nocera, J. Stubbe, C. Tommos, Formal reduction potentials of difluorotyrosine and trifluorotyrosine protein residues: Defining the thermodynamics of multistep radical transfer. J. Am. Chem. Soc. 139, 2994–3004 (2017).

Tools to study Y/W oxidation-reduction

Mechanistic studies of the α₃X proteins (and the α₃XX proteins under construction) require a range of biophysically oriented approaches. These include obtaining essential thermodynamic information using a (very) high potential protein film voltammetry approach developed by the Tommos lab. In collaborative work, we use laser spectroscopy and theoretical methods to determine X₃₂ radical formation and decay mechanisms. Through collaborative efforts, we also use advanced nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopic methods to connect the redox properties of X₃₂ with the structural and dynamical properties of the α₃ protein scaffold. Additionally, we will label our proteins with ruthenium (Ru) complexes to study the PCET kinetics of light-triggered radical formation (in Ru-α₃Y and Ru-α₃W) and radical transfer (in Ru-α₃YX and Ru-α₃WX). The overall goal is to obtain a comprehensive and predictive understanding of Y and W radicals in biological ET/PCET processes.

B. W. Berry, M. C. Martínez-Rivera, C. Tommos, Reversible voltammograms and a pourbaix diagram for a protein tyrosine radical. Proc. Natl. Acad. Sci. U.S.A. 109, 9739–9743 (2012).

A. Nilsen-Moe, C. R. Reinhardt, S. D. Glover, L. Liang, S. Hammes-Schiffer, L. Hammarström, C. Tommos, Proton-coupled electron transfer from tyrosine in the interior of a de novo protein: Mechanisms and primary proton acceptor. J. Am. Chem. Soc. 142, 11550–11559 (2020).