Resolving spectral overlap in ENDOR by chirp echo Fourier transform detection

Abstract

Electron nuclear double resonance (ENDOR) spectroscopy is a powerful technique for probing the structure and function of paramagnetic centers via measuring the magnetic interactions of unpaired electrons with nearby nuclear spins. For systems with multiple magnetic nuclei, commonly encountered in transition metal complexes in catalysis or metalloproteins, ENDOR spectra often become very crowded due to the broad, anisotropic hyperfine (HF) and nuclear quadrupole (NQ) interactions in disordered systems. In this work, we substitute the Hahn echo in Davies ENDOR by a chirp echo of the Kunz–Böhlen–Bodenhausen scheme to resolve spectral overlap in a second dimension. Fourier transformation of the chirp echo directly yields an additional EPR dimension without increasing measurement time and reveals correlations between nuclear and electron transitions, thereby resolving spectral overlap, shown here for ¹H, ^14/15N and ⁶³Cu in ENDOR spectra of the copper protein ScoI. Different influences of interactions along the two dimensions and the possibility for selective excitation to address specific spectral components are exploited to disentangle the small copper NQ-coupling from the large, anisotropic HF-coupling. Simple, efficient frequency-domain simulations reproduce the experimental 2D Chirp Echo Epr SpectroscopY (CHEESY) ENDOR spectra and provide a basis to extract spin Hamiltonian parameters. Limitations and benefits of CHEESY ENDOR are discussed in comparison to established ENDOR techniques, 2D Mims ENDOR and HYEND, which reveals a competitive signal-to-noise ratio for CHEESY ENDOR due to the inherent FT advantage and RF-chirp compatibility to enhance sensitivity. These features expand the scope and feasibility of ENDOR investigations to a wider range of applications.