Stereoelectronic Properties of Metalloenzymes. 5. Identification and Assignment of Ligand Hyperfine Splittings in the Electron Spin Resonance Spectrum of Galactose Oxidase

Time-averaged electron spin resonance spectra for native galactose oxidase suggested two nitrogen-liganded atoms based on the partial resolution of superhyperfine structure in the parallel region of the spectrum. Electron spin resonance data obtained for 63Cu-galactose oxidase as well as the 19F×-enzyme complex and imidazole-enzyme complex indicate that not only are there two nitrogen atoms present in the native protein but that these arise from liganded imidazole (histidine) nitrogen atoms. Interpretation of these spectra is dependent on the observation of superhyperfine structure in the parallel, perpendicular, and “overshoot” regions of all the spectra. A detailed discussion and an explanation for the interpretations are presented.