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 ) cofactor for activity. The knowledge on the in vitro Y structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y . 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 are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y 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 Y , in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.