Spectroscopic and Enzymatic Characterization of the Active Site Dinuclear Metal Center of Calcineurin:  Implications for a Mechanistic Role

• Published in: Biochemistry (Easton) Vol. 36; no. 35; pp. 10727 - 10734
• Main Authors: Yu, Lian, Golbeck, John, Yao, Janet, Rusnak, Frank
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02-09-1997
• Subjects: Aniline Compounds; Animals; Binding Sites; Brain; Calcineurin; Calmodulin-Binding Proteins - chemistry; Calmodulin-Binding Proteins - metabolism; Calmodulin-Binding Proteins - physiology; Catalysis; Cattle; Dithionite - metabolism; Electron Spin Resonance Spectroscopy; Ferric Compounds - chemistry; Ferric Compounds - metabolism; Ferrous Compounds - metabolism; Hydrogen Peroxide - metabolism; Organophosphorus Compounds; Oxidation-Reduction; Phosphates - pharmacology; Phosphoprotein Phosphatases - chemistry; Phosphoprotein Phosphatases - metabolism; Phosphoprotein Phosphatases - physiology; Rats; Substrate Specificity; Titrimetry; Zinc - chemistry; Zinc - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi970519g

The active site of bovine brain calcineurin contains an Fe3+−Zn2+ dinuclear metal center. Replacement of Zn2+ with Fe2+ yields a mixed valence Fe3+−Fe2+ center that exhibits a characteristic EPR signal that can be used as a convenient spectroscopic probe of the active site. Addition of product phosphate to both the Fe3+−Fe2+ and Fe3+−Zn2+ forms of calcineurin led to perturbations of the respective EPR signals, indicating that phosphate affects the environment of the paramagnetic centers. Anaerobic titrations of the iron-substituted Fe3+−Fe2+ enzyme with dithionite resulted in a gradual loss of activity toward pNPP that paralleled the loss of intensity of the EPR signal of the mixed valence diiron center. During dithionite reduction, an EPR resonance with g ≈ 12 appeared. The intensity of this resonance increased when the spectrum was recorded in a parallel mode cavity and was therefore 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 valence oxidation state represents the catalytically competent form of the cluster. The dependence of the enzyme activity on the redox state of the cluster has implications for a mechanistic role.