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In‐Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy
Author(s) -
Meichsner Shari L.,
Kutin Yury,
Kasanmascheff Müge
Publication year - 2021
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.202102914
Subject(s) - ribonucleotide reductase , electron paramagnetic resonance , cofactor , chemistry , in vivo , in vitro , biochemistry , enzyme , protein subunit , ribonucleotide , stereochemistry , biophysics , biology , nuclear magnetic resonance , nucleotide , gene , physics , microbiology and biotechnology
Abstract The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di‐iron tyrosyl radical (Y 122 . ) cofactor for activity. The knowledge on the in vitro Y 122 . structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y 122 . . Here, we characterize this essential radical in whole cells. Multi‐frequency EPR and electron‐nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y 122 . are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y 122 . per β2 distribution, supporting the key role of Y . concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F 3 Y 122 . , in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.