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, F3Y122., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.

GdIII‐19F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance

“…[20] In-cell ENDOR measurements have previously been reported for strongly coupled nuclei located less than 1 nm away from the electron spin. [17] This includes 1 H ENDOR of a stable tyrosyl radical in E. coli ribonucleotide reductase [22] and 31 P and 1 H ENDOR of low molecular mass Mn II complexes in the cell. [23,24] Here, we demonstrate the feasibility of conducting incell Gd III -19 F Mims ENDOR for distance measurements in proteins, expanding the accessible distance range for EPR techniques to the native cellular environment.…”

“…In‐cell ENDOR measurements have previously been reported for strongly coupled nuclei located less than 1 nm away from the electron spin [17] . This includes 1 H ENDOR of a stable tyrosyl radical in E. coli ribonucleotide reductase [22] and 31 P and 1 H ENDOR of low molecular mass Mn II complexes in the cell [23, 24] …”

Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy

“…Electron spin resonance (ESR) spectroscopy, in particular, pulsed electron‐electron double resonance (PELDOR or DEER) spectroscopy has emerged as a powerful tool to study protein complexes, even in the cellular environments. [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ] PELDOR data reveals the ensemble conformational heterogeneity and in favorable cases can resolve the thermodynamic and kinetic aspects with spatiotemporal resolution. [ 22 , 23 , 24 , 25 , 26 , 27 ] The nitroxide‐based methane thiosulfonate spin label (MTSL) is the most preferred tag for proteins.…”

Dance with spins: site-directed spin labeling coupled to electron paramagnetic resonance spectroscopy directly inside cells

“…32,33 Nowadays, it represents an established technique for studying Reactive Oxygen Species ROS (spin-trapping approach), tyrosyl radicals and metals complexes in cellular and animal models. [34][35][36] On the other hand, protocols to exploit the advantages of paramagnetic labels for the study of diamagnetic biomolecules in cells are still under development. SDLS-EPR is particularly interesting for intracellular studies because of the negligible background signal.…”