A multifrequency HYSCORE study of weakly coupled nuclei in frozen solutions of high-spin aquometmyoglobin

In this work, we show the extreme power of multifrequency HYSCORE (hyperfine sublevel correlation spectroscopy) techniques to unravel the hyperfine interactions of the electron spin with the remote nuclei in the heme site of high-spin ferric heme proteins. Horse heart aquo-metmyoglobin was used as a model system to demonstrate the power of these techniques. Experimental evidence was collected and assigned to protons of the proximal histidine ligand, to the mesoprotons of the heme ligand, and to two different protons of the distal water ligand. The latter difference relates to the stabilization of the water ligand by the E7His residue. Furthermore, HYSCORE signals of the remote N(delta) of the proximal (F8) and N(epsilon) of the distal (E7) histidine were detected. Finally, correlation peaks from the lesser-abundant (13)C nuclei of the heme ligand could be detected. These novel results allow dissection of the hyperfine couplings into individual contributions and calculation of the spin density in the pi and sigma orbitals, thus completing earlier electron paramagnetic resonance and liquid-state NMR data.