2-DIMENSIONAL ESEEM SPECTROSCOPY OF NITROGEN HYPERFINE COUPLINGS IN METHEMERYTHRIN AND AZIDOMETHEMERYTHRIN

The application of a two-dimensional ESEEM spectroscopy, known as HYSC ORE, to the study of the hyperfine couplings from the nitrogens of his tidine and azide ligands in mixed-valent states of methemerythrin and azidomethemerythrin produced by low-temperature (77 K) gamma-irradiati on 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-featu res correlating double-quantum transitions allows direct estimation of the hyperfine couplings from all coordinated histidine nitrogens of s emimethemerythrin which vary between 5 and 15 MHz as well as their cha nges upon azide binding. The results obtained show that two-dimensiona l spectroscopy provides significantly more information compared to one -dimensional techniques for;the characterization of the paramagnetic c enters involving large numbers of magnetically nonequivalent nuclei. T his work forms the basis for the application of two-dimensional ESEEM in conjunction with low-temperature reduction for the detailed charact erization of nitrogen coordination in diiron centers in proteins of un known crystal structure.