In-Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy.
Shari L MeichsnerYury KutinMüge KasanmascheffPublished in: Angewandte Chemie (International ed. in English) (2021)
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 (Y122 . ) cofactor for activity. The knowledge on the in vitro Y122 . structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y122 . . 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 Y122 . are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y122 . 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, F3 Y122 . , in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.
Keyphrases
- induced apoptosis
- cell cycle arrest
- escherichia coli
- healthcare
- amino acid
- endothelial cells
- single molecule
- endoplasmic reticulum stress
- cell death
- single cell
- stem cells
- oxidative stress
- mesenchymal stem cells
- energy transfer
- cell proliferation
- molecular dynamics simulations
- induced pluripotent stem cells
- social media
- iron deficiency
- electron transfer