Electron spin echo studies of the copper complexes of conalbumin

Electron spin echo envelope spectroscopy was used to probe the two metal binding sites of Cu(II)-conalbumin. The echo envelope spectrum of Cu(II)-conalbumin-oxalate, with metal ion at either one or both of the binding sites, contains lines arising from the interaction of the electron spin of Cu(II) with bound imidazole, demonstrating histidine ligation to the metal ion. The 13C superhyperfine interaction of bound [13C]oxalate, obtained from the ratio of the electron spin echo envelopes of Cu(II)-conalbumin-[13C]oxalate to that of Cu(II)-conalbumin-[12C]oxalate, is about twice the free precession frequency and indicates a contact interaction between 13C and Cu(II). This study indicates that oxalate is directly coordinated to the metal ion. Over the pH range 7.0 to 10.0, where Cu(II)-conalbumin binds carbonate as an associated anion, the echo envelope spectrum indicates that at least one imidazole ligand is coordinated to Cu(II). Below pH 6.0 and above pH 11.0, imidazole coordination is not observed.