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

摘要

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(center dot)) cofactor for activity. The knowledge on the in vitro Y-122(center dot) structure and its radical distribution within the beta 2 subunit has accumulated over the years; yet little information exists on the in vivo Y-122(center dot). 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(center dot) are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y-122(center dot) per beta 2 distribution, supporting the key role of Y-center dot concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F3Y122 center dot, in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.