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 and two subunits (? and ?) 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 ? | ? 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 ?2 and reduction of the Y122? cofactor, 35 away in ?2. To address this directly, we will embark on an investigation of the kinetics and thermodynamics of PCET through ? by employing new photo?2s. 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.

Public Health Relevance

Proton-coupled electron transfer (PCET) represents a fundamental mechanism for biological control over the formation and utilization of highly oxidizing intermediates. This control is often imparted through macromolecular changes targeting the precise management of proton movement during electron flux. Sustaining many primary and secondary metabolic processes, PCET is intrinsically linked to the management of reactive oxygen species in vivo and diseased states such as Type 2 diabetes, Parkinson's disease, atherosclerotic heart disease, stroke, Alzheimer's disease and cancer, all of which can be traced back to disruption of PCET processes at the molecular-level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047274-28
Application #
9849298
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1992-04-01
Project End
2021-01-31
Budget Start
2020-02-01
Budget End
2021-01-31
Support Year
28
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
University-Wide
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Guo, Junling; Suástegui, Miguel; Sakimoto, Kelsey K et al. (2018) Light-driven fine chemical production in yeast biohybrids. Science 362:813-816
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; Minnihan, Ellen C; Lin, Qinghui et al. (2017) Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase. Biochemistry 56:856-868
Greene, Brandon L; Taguchi, Alexander T; Stubbe, JoAnne et al. (2017) Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase. J Am Chem Soc 139:16657-16665
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
Olshansky, Lisa; Greene, Brandon L; Finkbeiner, Chelsea et al. (2016) Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase. Biochemistry 55:3234-40
Olshansky, Lisa; Stubbe, JoAnne; Nocera, Daniel G (2016) Charge-Transfer Dynamics at the ?/? Subunit Interface of a Photochemical Ribonucleotide Reductase. J Am Chem Soc 138:1196-205
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

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