Redox control of calcineurin by targeting the binuclear Fe(2+)-Zn(2+) center at the enzyme active site.
The interaction of protein serine/threonine phosphatase calcineurin (CaN) with superoxide and hydrogen peroxide was investigated. Superoxide specifically inhibited phosphatase activity of CaN toward RII (DLDVPIPGRFDRRVSVAAE) phosphopeptide in tissue and cell homogenates as well as the activity of the enzyme purified under reducing conditions. Hydrogen peroxide was an effective inhibitor of CaN at concentrations several orders of magnitude higher than superoxide. Inhibition by superoxide was calcium/calmodulin-dependent. Nitric oxide (NO) antagonized superoxide action on CaN. We provide kinetic and spectroscopic evidence that native, catalytically active CaN has a Fe(2+)-Zn(2+) binuclear center in its active site that is oxidized to Fe(3+)-Zn(2+) by superoxide and hydrogen peroxide. This oxidation is accompanied by a gain of manganese dependence of enzyme activity. CaN isolated by a conventional purification procedure was found in the oxidized, ferric enzyme form, and it became increasingly dependent on divalent cations. These results point to a complex redox regulation of CaN phosphatase activity by superoxide, which is modified by calcium, NO, and superoxide dismutase.
Namgaladze
Dmitry
D
Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Universität Konstanz, D-78457 Konstanz, Germany.
Hofer
H Werner
HW
Ullrich
Volker
V
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D019703:Calcineurin / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019271:Hypoxanthine / Q000494:pharmacology; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010748:Phosphopeptides / Q000737:chemistry; D011487:Protein Conformation; D013481:Superoxides / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
D010748:Phosphopeptides; D013481:Superoxides; D019271:Hypoxanthine; D006861:Hydrogen Peroxide; D007501:Iron; D014969:Xanthine Oxidase; D019703:Calcineurin; D015032:Zinc