The long-term goal of this proposal is to delineate the mechanisms by which amino-acid radicals participate in both productive and destructive redox reactions in living organisms. Controlled radical chemistry occurs in enzymes that use amino acids in catalytic and multistep electron-transfer reactions. Amino-acid radical enzymes are involved in a range of chemical transformations some of which are fundamental to aerobic life on Earth. It is of vital interest to delineate these chemical reactions in great detail for a numbe of reasons. These include, for example, developing anticancer drugs, understanding the interactions of non-steroidal anti- inflammatory drugs (NSAIDs) such as aspirin and ibuprofen with their protein targets, and laying the foundation for sustainable solar energy production. Importantly, there is also a sinister side to amino-acid radicals as these species are generated during oxidative stress conditions and can cause significant cellular damage. Despite the biochemical importance of amino-acid radicals, surprisingly little is known about their fundamental thermodynamic and kinetic properties. It is very challenging to study these species due to their highly oxidizing and reactive nature. As a result, no guide is currently available for comparing the formal reduction potentials of amino-acid radicals and correlating these values with the properties of the surrounding protein. Studies aimed at characterizing the proton-coupled electron transfer (PCET) reactions associated with tyrosine oxidation-reduction are currently of high interest but are largely conducted on small-molecule models free in solution. The correlation between the solution chemistry and the protein chemistry is not straightforward. Furthermore, it is well known that the protein matrix can modulate the lifetime of the amino- acid radical by many orders of magnitude but there is little information on how this occurs. We have developed a protein system that can provide novel and important information about these issues. The a3X constructs are well-structured proteins supporting reversible amino-acid oxidation-reduction and major radical stabilization. Protein reengineering, solution NMR spectroscopy, (very) high potential protein voltammetry, quantum chemical methods, time-resolved laser spectroscopy, and protein hydrogen exchange (HX) methods will be employed to characterize and refine the redox properties of the a3X proteins. This system will be developed along three connected paths. First, the a3X proteins will be used to generate a unique protein-based thermodynamic ladder for interpreting the thermodynamic and kinetic effects of mutations and chemical modifications of amino-acid radicals in natural systems. Second, this project seeks to complement and significantly extend prior solution studies by characterizing tyrosine/phenol-based proton-coupled electron transfer in a structured protein environment. Third, we will investigate how the dynamic properties of the protein ensemble may influence tyrosine radical formation, stabilization and decay.

Public Health Relevance

Protein radicals are involved in a range of both beneficial as well as harmful chemical reactions in living organisms. Little is known about these species since they are highly challenging to study in their natural protein environment and in model systems. We have developed a library of well-structured model proteins that enables studies of the fundamental chemical properties of amino-acid radicals.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079190-08
Application #
9212814
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2006-12-01
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
8
Fiscal Year
2017
Total Cost
$269,785
Indirect Cost
$98,785
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
O'Brien, Evan S; Lin, Danny W; Fuglestad, Brian et al. (2018) Improving yields of deuterated, methyl labeled protein by growing in H2O. J Biomol NMR 71:263-273
Glover, Starla D; Tyburski, Robin; Liang, Li et al. (2018) Pourbaix Diagram, Proton-Coupled Electron Transfer, and Decay Kinetics of a Protein Tryptophan Radical: Comparing the Redox Properties of W32• and Y32• Generated Inside the Structurally Characterized ?3W and ?3Y Proteins. J Am Chem Soc 140:185-192
Lee, Wankyu; Kasanmascheff, Müge; Huynh, Michael et al. (2018) Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm. Biochemistry 57:3402-3415
Ravichandran, Kanchana R; Zong, Allan B; Taguchi, Alexander T et al. (2017) Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer. J Am Chem Soc 139:2994-3004
Ravichandran, Kanchana R; Taguchi, Alexander T; Wei, Yifeng et al. (2016) A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes. J Am Chem Soc 138:13706-13716
Ravichandran, Kanchana R; Taguchi, Alexander T; Wei, Yifeng et al. (2016) A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes. J Am Chem Soc :
O'Brien, Evan S; Nucci, Nathaniel V; Fuglestad, Brian et al. (2015) Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN. J Biol Chem 290:30879-87
Glover, Starla D; Jorge, Christine; Liang, Li et al. (2014) Photochemical tyrosine oxidation in the structurally well-defined ?3Y protein: proton-coupled electron transfer and a long-lived tyrosine radical. J Am Chem Soc 136:14039-51
Ravichandran, Kanchana R; Liang, Li; Stubbe, JoAnne et al. (2013) Formal reduction potential of 3,5-difluorotyrosine in a structured protein: insight into multistep radical transfer. Biochemistry 52:8907-15
Tommos, Cecilia; Valentine, Kathleen G; Martínez-Rivera, Melissa C et al. (2013) Reversible phenol oxidation and reduction in the structurally well-defined 2-Mercaptophenol-??C protein. Biochemistry 52:1409-18

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