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A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes
[Image: see text] Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y(122)•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino a...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2016
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224885/ https://www.ncbi.nlm.nih.gov/pubmed/28068088 http://dx.doi.org/10.1021/jacs.6b08200 |
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author | Ravichandran, Kanchana R. Taguchi, Alexander T. Wei, Yifeng Tommos, Cecilia Nocera, Daniel G. Stubbe, JoAnne |
author_facet | Ravichandran, Kanchana R. Taguchi, Alexander T. Wei, Yifeng Tommos, Cecilia Nocera, Daniel G. Stubbe, JoAnne |
author_sort | Ravichandran, Kanchana R. |
collection | PubMed |
description | [Image: see text] Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y(122)•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y(122) ⇆ [W(48)] ⇆ Y(356) in β2 to Y(731) ⇆ Y(730) ⇆ C(439) in α2). Equilibration of Y(356)•, Y(731)•, and Y(730)• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y(122)• and Y(356)• has not been detected. Our recent report of Y(356)• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y(122) (F(3)Y(122)•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F(3)Y(122)• and Y(356)•. Incubation of F(3)Y(122)•-β2, Y(731)F-α2 (or Y(730)F-α2), CDP, and ATP at different temperatures (2–37 °C) provides ΔE°′(F(3)Y(122)•–Y(356)•) of 20 ± 10 mV at 25 °C. The pH dependence of the F(3)Y(122)• ⇆ Y(356)• interconversion (pH 6.8–8.0) reveals that the proton from Y(356) is in rapid exchange with solvent, in contrast to the proton from Y(122). Insertion of 3,5-difluorotyrosine (F(2)Y) at Y(356) and rapid freeze-quench EPR analysis of its reaction with Y(731)F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F(2)Y(356)• generation by the native Y(122)•. F(n)Y-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y(122) and C(439) is ∼200 meV uphill. |
format | Online Article Text |
id | pubmed-5224885 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-52248852017-01-11 A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes Ravichandran, Kanchana R. Taguchi, Alexander T. Wei, Yifeng Tommos, Cecilia Nocera, Daniel G. Stubbe, JoAnne J Am Chem Soc [Image: see text] Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y(122)•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y(122) ⇆ [W(48)] ⇆ Y(356) in β2 to Y(731) ⇆ Y(730) ⇆ C(439) in α2). Equilibration of Y(356)•, Y(731)•, and Y(730)• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y(122)• and Y(356)• has not been detected. Our recent report of Y(356)• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y(122) (F(3)Y(122)•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F(3)Y(122)• and Y(356)•. Incubation of F(3)Y(122)•-β2, Y(731)F-α2 (or Y(730)F-α2), CDP, and ATP at different temperatures (2–37 °C) provides ΔE°′(F(3)Y(122)•–Y(356)•) of 20 ± 10 mV at 25 °C. The pH dependence of the F(3)Y(122)• ⇆ Y(356)• interconversion (pH 6.8–8.0) reveals that the proton from Y(356) is in rapid exchange with solvent, in contrast to the proton from Y(122). Insertion of 3,5-difluorotyrosine (F(2)Y) at Y(356) and rapid freeze-quench EPR analysis of its reaction with Y(731)F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F(2)Y(356)• generation by the native Y(122)•. F(n)Y-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y(122) and C(439) is ∼200 meV uphill. American Chemical Society 2016-09-20 2016-10-19 /pmc/articles/PMC5224885/ /pubmed/28068088 http://dx.doi.org/10.1021/jacs.6b08200 Text en Copyright © 2016 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Ravichandran, Kanchana R. Taguchi, Alexander T. Wei, Yifeng Tommos, Cecilia Nocera, Daniel G. Stubbe, JoAnne A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes |
title | A >200
meV Uphill Thermodynamic Landscape for Radical
Transport in Escherichia coli Ribonucleotide Reductase
Determined Using Fluorotyrosine-Substituted Enzymes |
title_full | A >200
meV Uphill Thermodynamic Landscape for Radical
Transport in Escherichia coli Ribonucleotide Reductase
Determined Using Fluorotyrosine-Substituted Enzymes |
title_fullStr | A >200
meV Uphill Thermodynamic Landscape for Radical
Transport in Escherichia coli Ribonucleotide Reductase
Determined Using Fluorotyrosine-Substituted Enzymes |
title_full_unstemmed | A >200
meV Uphill Thermodynamic Landscape for Radical
Transport in Escherichia coli Ribonucleotide Reductase
Determined Using Fluorotyrosine-Substituted Enzymes |
title_short | A >200
meV Uphill Thermodynamic Landscape for Radical
Transport in Escherichia coli Ribonucleotide Reductase
Determined Using Fluorotyrosine-Substituted Enzymes |
title_sort | >200
mev uphill thermodynamic landscape for radical
transport in escherichia coli ribonucleotide reductase
determined using fluorotyrosine-substituted enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224885/ https://www.ncbi.nlm.nih.gov/pubmed/28068088 http://dx.doi.org/10.1021/jacs.6b08200 |
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