In‐Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy

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.