Two-Dimensional ESEEM Spectroscopy of Nitrogen Hyperfine Couplings in Methemerythrin and Azidomethemerythrin

The application of a two-dimensional ESEEM spectroscopy, known as HYSCORE, to the study of the hyperfine couplings from the nitrogens of histidine and azide ligands in mixed-valent states of methemerythrin and azidomethemerythrin produced by low-temperature (77 K) γ-irradiation is described. The HYSCORE spectra contain groups of well-separated cross-peaks from directly coordinated and remote histidine nitrogens, and from nitrogens of the azide ligand. The observation of cross-features correlating double-quantum transitions allows direct estimation of the hyperfine couplings from all coordinated histidine nitrogens of semimethemerythrin which vary between 5 and 15 MHz as well as their changes upon azide binding. The results obtained show that two-dimensional spectroscopy provides significantly more information compared to one-dimensional techniques for the characterization of the paramagnetic centers involving large numbers of magnetically nonequivalent nuclei. This work forms the basis for the application of two-dimensional ESEEM in conjunction with low-temperature reduction for the detailed characterization of nitrogen coordination in diiron centers in proteins of unknown crystal structure.