Spectroscopic and enzymatic characterization of the active site dinuclear metal center of calcineurin: Implications for a mechanistic role

Lian Yu, John Golbeck, Janet Yao, Frank Rusnak

Research output: Contribution to journalArticle

54 Scopus citations

Abstract

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 paremagnetic 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.

Original languageEnglish (US)
Pages (from-to)10727-10734
Number of pages8
JournalBiochemistry
Volume36
Issue number35
DOIs
StatePublished - Sep 2 1997

All Science Journal Classification (ASJC) codes

  • Biochemistry

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