Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide - Evidence for a dithiol-disulfide equilibrium and implications for redox regulation

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent 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 regulati on. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO ) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibi ted CaN with an IC50 of 3-8 mu m and the inactivation was reversed by 2,3-d imercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydro gen peroxide resulted in a concentration-dependent inactivation. Analysis o f the free thiol content performed on the H2O2 inactivated enzyme demonstra ted that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and w ith the dithiol oxidoreductase thioredoxin. We propose that a bridging of t wo closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same C ys residues causes the inactivation. Our data implicate a possible involvem ent of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was cons istent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation o f the enzyme no significant changes in the EPR spectrum were observed rulin g out that Fe2+ is present in the active enzyme and that the dinuclear meta l centre is the target for the oxidative inactivation of CaN.