Anion binding to the four-copper form of bovine erythrocyte superoxide dismutase: Mechanistic implications.

The binding of thiocyanate, azide, and other anions to superoxide dismutase (SOD) containing copper in both the copper and zinc sites of the native enzyme (Cu2Cu2SOD) has been studied. Electron spin resonance spectroscopy was used to show that binding of SCN- to Cu2Cu2SOD breaks the imidazolate bridge between the two copper centers. Thiocyanate displaces the bridging histidine from the Cu2+ ion in the copper site and also replaces the aspartic acid ligand from the Cu2+ ion in zinc site. This conclusion is supported by studies with Ag2Cu2SOD, where silver is in the native copper site and copper is in the native zinc site. At low concentrations, N3- ion displaces axially coordinated water from Cu2Cu2SOD. At higher concentrations, N3- also breaks the imidazolate bridge. Parallel behavior was observed for SCN- and N3- binding to a model compound for Cu2Cu2SOD, showing that the difference between the two anions is a consequence of their copper binding properties and is not due to secondary interactions with the protein active site. The SCN- complex of Cu2Cu2SOD is catalytically active, indicating that the intact imidazolate bridge is not essential to the mechanism of superoxide dismutase action. Phosphate ion breaks the histidine bridge in Cu2Cu2SOD, whereas cyanate, formate, and fluoride ions do not. At about pH 11, hydroxide ion promotes the irreversible formation of a copper complex of deprotonated peptide nitrogen atoms for Cu2Cu2SOD but not for Cu2Zn2SOD in the same pH range.