EUROISMAR 2019

One of the main application fields of EPR in biological samples is the investigation of paramagnetic centers (like metal ions or amino acid radicals) in proteins. Electron-nuclear double resonance (ENDOR) allows obtaining information about naturally occurring, nuclear spins (¹H, ¹⁴N, …) in the immediate neighborhood of such electron spin centers. Interactions with remote nuclei (≥ 7 Å) are often poorly resolved owed to the weakness of the interaction. The strongest coupling is typically exerted by ¹H nuclei. However, their ubiquity in biological macromolecules leads to crowding of ¹H ENDOR spectra. ¹⁹F nuclei interact almost as strongly with electron spins as 1H does, and it is often possible to replace ¹H by ¹⁹F nuclei due to the bioisosterism of the two atoms. We introduce here ¹⁹F nuclei as spin labels for ENDOR at high EPR fields (3.4 T/94 GHz), where the ¹⁹F resonance frequency is reasonably (> 8 MHz) separated from all other nuclear resonances. The feasibility of this approach is shown at three stages. First, it is demonstrated that couplings between a nitroxide radical and ¹⁹F nuclear spin are detectable for distances up to 15 Å on small, synthetic models. For r ≳ 6 Å distances are found well consistent with the point-dipole model. In a second set of experiments on spin labelled RNA duplexes, data of comparable quality could be obtained. Finally, first applications of the method are shown on ¹⁹F labelled E. coli ribonucleotide reductase, providing geometrical information about the intermediate tyrosyl radical Y₃₅₆● in the long range radical transfer pathway of this enzyme. The results indicate that the method of 19F ENDOR could find application as a general tool in ESR spectroscopy for the intermediate distance range (~ 5 – 15 Å), as did PELDOR for the large distance range (~ 20 – 100 Å).