Electron spin echo modulation spectra were recorded for a blue copper protein rusticyanin. The protein is amenable to molecular biology methods and can be prepared as a stable form lacking one of the two histidine ligands to copper (H85A). The w.t. and H85A forms of the protein were examined in depth to fully characterize the ESEEM spectroscopic properties of imidazole nitrogens in the copper ligand field. The use of timing variables (tau selection) in the three pulse experiment as a means to identify peaks has been established. A graphical method of identifying peaks and quantifying superhyperfine parameters from experimental data has also been demonstrated. The electron spin echo spectra suggest that ~m=1 transitions of both spin manifolds may be observed by a judicious choice of tau in a three pulse experiment. This greatly improves the accuracy of the simulation process and removes ambiguity in misidentifying peaks by an improper association. A graphical representation of the peaks allows one to predict the condition of """"""""exact cancellation"""""""" and thereby remove ambiguities in quantifying the superhyperfine parameters. As regards the w.t. and H85A forms of the protein rusticyanin, the absence of the second histidine ligand puts more spin density onto the remaining histidine ligand to copper.
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