High-field ENDOR and the sign of the hyperfine coupling

Abstract

Two different approaches for assigning electron nuclear double resonance (ENDOR) signals to their respectiveM s manifolds by a controlled generation of asymmetric ENDOR spectra, are described and applied to a number of systems. This assignment then allows a straightforward determination of the sign of the hyperfine coupling. Both approaches rely on a high thermal polarization that is easily achieved at high fields and low temperatures. For high-spin systems, such asS = 5/2 the assignment is afforded by the selective inversion of the | −3/2〉 → | −1/2〉 electron paramagnetic resonance (EPR) transition which is highly populated as compared to its symmetric counterpart, the |1/2〉 → |3/2〉 EPR transition, and therefore is easily identified. ForS = 1/2 the determination of the sign of the hyperfine coupling becomes possible when the cross- and nuclear-spin relaxation rates are much slower than the electron-spin relaxation rate and variable mixing time pulse ENDOR is used to measure the spectrum. Under these conditions the signals of theM s = 1/2 (α) manifold become negative when the mixing time is on order of the electron-spin relaxation time, whereas those of theM s =−1/2 (β) manifold remain positive. Under partial saturation of the nuclear transitions and short mixing time the opposite behavior is observed. Pulse W-band1H ENDOR experiments demonstrating these approaches were applied and the signs of the hyperfine couplings of the water ligands in Mn(H2O) 6 2+ , the Hα and Hβ histidine protons in the Cu(histidine)2 complex, the imidazole protons in Cu(imidazole) 4 2+ and the cysteine β-protons in nitrous oxide reductase were determined.