Frequency and time domain ¹⁹F ENDOR spectroscopy: role of nuclear dipolar couplings to determine distance distributions

¹⁹F electron-nuclear double resonance (ENDOR) spectroscopy is emerging as a method of choice to determine molecular distances in biomolecules in the angstrom to nanometer range. However, line broadening mechanisms in ¹⁹F ENDOR spectra can obscure the detected spin-dipolar coupling that encodes the distance information, thus limiting the resolution and accessible distance range. So far, the origin of these mechanisms has not been understood. Here, we employ a combined approach of rational molecular design, frequency and time domain ENDOR methods as well as quantum mechanical spin dynamics simulations to analyze these mechanisms. We present the first application of Fourier transform ENDOR to remove power broadening and measure <i>T</i>_2n of the ¹⁹F nucleus. We identify nuclear dipolar couplings between the fluorine and protons up to 14 kHz as a major source of spectral broadening. When removing these interactions by H/D exchange, an unprecedented spectral width of 9 kHz was observed suggesting that, generally, the accessible distance range can be extended. In a spin labeled RNA duplex we were able to predict the spectral ENDOR line width, which in turn enabled us to extract a distance distribution. This study represents a first step towards a quantitative determination of distance distributions in biomolecules from ¹⁹F ENDOR.