Proton-coupled electron transfer (PCET) is a ubiquitous mechanistic motif in biology and mediates primary metabolic steps involving energy transduction, radical initiation and transport and the activation of substrates at cofactors. The control of highly reactive intermediates is achieved via coupling proton transfer (PT) and electron transfer (ET) processes. Management of radicals in biology is of particular relevance to human health, making a detailed understanding of PCET essential. Enzymes operating by PCET mechanisms are therapeutic targets with wide-ranging applications including chemotherapy, anti-retroviral drugs and anti-inflammatory agents. The proposed research program seeks to study biological PCET by employing a two-pronged multidisciplinary approach where studies of both natural and model systems synergistically combine to yield a detailed understanding of PCET. A major effort will be devoted to the role of PCET in amino acid radical initiation and transport over the 35 ? electron/proton coupled pathway in E. coli ribonucleotide reductase (RNR). RNR consists of two homodimeric subunits, ?2 and ?2, and is a key therapeutic target associated with a variety of diseases. The research plan relies on newly created biochemical and biophysical methods. Radicals will be generated on photoactive peptides or from non-natural amino acids, thereby bypassing the normal radical generation process of RNR. Fluorotyrosines will be site-specifically introduced to tune the thermo- dynamics and kinetics of ET and PT in radical transport within individual ?2 and ?2 subunits. The development of photo?2 is particularly impactful because it enables the study of the complete RNR enzyme, yielding a faithful platform for the study of radical transport across both subunits. Examination of radical transport in the fully assembled enzyme will permit the study of how anticancer/antiviral agents target disease by regulating radical transport in RNR. The research plan will be extended by utilizing triproline peptide scaffolds to model the PCET pathways of relevance to RNR. The modular nature of this platform permits key amino acid residues of to be systematically varied to gain mechanistic insight into how the electron and proton couple in RNR. The examination of how cofactor active sites are generated by PCET will be examined with hangman porphyrins, which are faithful structural and functional mimics of heme hydroperoxidase enzymes. The hangman models will provide a platform to photochemically generate Compounds I and II, allowing for the kinetics of their generation and reactivity to be defined. These systems will also allow for the electron and proton transfer distances in the PCET pathway to be independently controlled. The principles that emerge from these studies will be applied to explain the functions of a variety of enzymes and proteins that derive their activity from PCET including the origin of extremely large isotope effects in biology.

Public Health Relevance

Proton coupled electron transfer (PCET) underpins the primary metabolic steps of mitochondrial respiration and radical generation and transport. Many different diseases have been identified to originate from these processes including, but not limited to, type 2 diabetes, Parkinson's disease, atherosclerotic heart disease, stroke, Alzheimer's disease, cancer other diseases related to oxidative stress. Currently there are no cures for such diseases providing an imperative for understanding the PCET so that drugs may be designed to treat mitochondrial, metabolic and oxidative-stress induced cytopathies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047274-22
Application #
8530239
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1992-04-01
Project End
2016-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
22
Fiscal Year
2013
Total Cost
$406,352
Indirect Cost
$161,507
Name
Harvard University
Department
None
Type
Other Domestic Higher Education
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Pizano, Arturo A; Olshansky, Lisa; Holder, Patrick G et al. (2013) Modulation of Y356 photooxidation in E. coli class Ia ribonucleotide reductase by Y731 across the *2:*2 interface. J Am Chem Soc 135:13250-3
Holder, Patrick G; Pizano, Arturo A; Anderson, Bryce L et al. (2012) Deciphering radical transport in the large subunit of class I ribonucleotide reductase. J Am Chem Soc 134:1172-80
Pizano, Arturo A; Lutterman, Daniel A; Holder, Patrick G et al. (2012) Photo-ribonucleotide reductase ?2 by selective cysteine labeling with a radical phototrigger. Proc Natl Acad Sci U S A 109:39-43
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Reece, Steven Y; Lutterman, Daniel A; Seyedsayamdost, Mohammad R et al. (2009) Re(bpy)(CO)3CN as a probe of conformational flexibility in a photochemical ribonucleotide reductase. Biochemistry 48:5832-8
Petersen, Poul B; Roberts, Sean T; Ramasesha, Krupa et al. (2008) Ultrafast N-H vibrational dynamics of cyclic doubly hydrogen-bonded homo- and heterodimers. J Phys Chem B 112:13167-71
Yao, Lishan; Yan, Honggao; Cukier, Robert I (2007) A molecular dynamics study of the ligand release path in yeast cytosine deaminase. Biophys J 92:2301-10
Reece, Steven Y; Seyedsayamdost, Mohammad R; Stubbe, JoAnne et al. (2007) Direct observation of a transient tyrosine radical competent for initiating turnover in a photochemical ribonucleotide reductase. J Am Chem Soc 129:13828-30

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