Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide: Evidence for a dithiol–disulfide equilibrium and implications for redox regulation
2000
Bogumil, Ralf | Namgaladze, Dmitry | Schaarschmidt, Daniel | Schmachtel, Tilman | Hellstern, Simon | Mutzel, Rupert | Ullrich, Volker
Calcineurin (CaN) is a Ca2+âand calmodulin (CaM)âdependent serine/threonine phosphatase containing a dinuclear Fe–Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN Aâsubunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3–8âµm and the inactivation was reversed by 2,3âdimercaptoâ1âpropane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentrationâdependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4âdithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN Aâsubunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+–Zn2+ dinuclear centre. Upon H2O2âmediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.
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