Electron spin-echo modulation spectroscopic study of the type I copper center associated with stellacyanin from Rhusvernicifera Examination of the graphical analysis of ESEEM spectra for two dissimilar weakly coupled 14N nuclei

Abstract

The type I copper protein stellacyanin was examined by electron spin-echo modulation (ESEEM) spectroscopy in order to test the multi-frequency approach of analyzing weak I = 1 hyperfine interactions when two slightly dissimilar contributions to the echo modulation interferogram are present. Two distinct contributions to the stellacyanin spectrum are resolved by the multi-frequency acquisition of ESEEM spectra, and the graphical approach to their deconvolution is described. The stellacyanin ESEEM spectrum can be completely explained in terms of two imino nitrogen contributions assigned to the histidine side chain ligands to copper whose ring orientations are perpendicular to one another (minimally 60°). The spin Hamiltonian parameters assigned to these two nitrogen atoms are: e²Qq = 1.45 MHz, η = 0.9, A = 2.0 MHz; and e²Qq = 1.50 MHz, η = 0.9, A = 1.5 MHz. Analysis of the double quantum transition lineshape indicates that the nuclear hyperfine interaction tensor is very nearly isotropic. There is no evidence in the data reported here for the detection of an amino nitrogen interaction that would arise from the predicted ligand GlnN_ε.