SPECTROSCOPIC AND ENZYMATIC CHARACTERIZATION OF THE ACTIVE-SITE DINUCLEAR METAL CENTER OF CALCINEURIN - IMPLICATIONS FOR A MECHANISTIC ROLE

The active site of bovine brain calcineurin contains an Fe3+-Zn2+ dinu clear metal center. Replacement of Zn2+ with Fe2+ yields a mixed valen ce Fe3+-Fe2+ center that exhibits a characteristic EPR signal that can be used as a convenient spectroscopic probe of the active site. Addit ion of product phosphate to both the Fe3+-Fe2+ and Fe3+-Zn2+ forms of calcineurin led to perturbations of the respective EPR signals, indica ting that phosphate affects the environment of the paramagnetic center s, Anaerobic titrations of the iron-substituted Fe3+-Fe2+ enzyme with dithionite resulted in a gradual loss of activity toward pNPP that par alleled the loss of intensity of the EPR signal of the mixed valence d iiron center, During dithionite reduction, an EPR resonance with g app roximate to 12 appeared, The intensity of this resonance increased whe n the spectrum was recorded in a parallel mode cavity and was therefor e attributed to a paramagnetic center with integer spin. Oxidation of the Fe3+-Fe2+ cluster to the diferric state by hydrogen peroxide also led to a loss of activity. These results indicate that the mixed valen ce oxidation state represents the catalytically competent form of the cluster, The dependence of the enzyme activity on the redox state of t he cluster has implications for a mechanistic role.