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

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 (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.

SUBMITTER: Meichsner SL 

PROVIDER: S-EPMC8453577 | biostudies-literature |

REPOSITORIES: biostudies-literature