In‐Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy
| Autor: | Yury Kutin, Müge Kasanmascheff, Shari L. Meichsner |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Free Radicals
Stereochemistry 010402 general chemistry ribonucleotide reductase 01 natural sciences Catalysis Cofactor law.invention chemistry.chemical_compound Biosynthesis law In vivo Ribonucleotide Reductases Escherichia coli Biophysics | Hot Paper Electron paramagnetic resonance Research Articles chemistry.chemical_classification metalloenzymes biology 010405 organic chemistry Electron Spin Resonance Spectroscopy General Chemistry General Medicine 0104 chemical sciences Amino acid Enzyme Ribonucleotide reductase chemistry biology.protein Tyrosine tyrosyl radicals DNA unnatural amino acids Research Article EPR spectroscopy |
| Zdroj: | Angewandte Chemie (International Ed. in English) |
| ISSN: | 1521-3773 1433-7851 |
| Popis: | 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, F3Y122 ., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions. The in vivo structure of the redox‐active tyrosyl radical in ribonucleotide reductase that catalyses the biosynthesis of DNA building blocks was explored using advanced EPR spectroscopic techniques. Furthermore, the radical form of a tyrosine derivative within the cells was generated. Our results mark the initial steps towards gaining unique insights into the redox reactions involving tyrosyl radicals under physiological conditions. |
| Databáze: | OpenAIRE |
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