Hyperoxidation of PRX-IIE was assayed as described above using the FOX assay with 400 µM H2O2 as substrate and increasing CuOOH concentrations. Furthermore, the oxidation state was investigated by electrospray ionization coupled with mass spectrometry (ESI-MS; Esquire 3000, Bruker Daltonics, Bremen, Germany). 10–20 µM of prereduced protein in 100 mM Tris-HCl, pH 8.0, was incubated with 5 mM DTT and different CuOOH concentrations or 0.5 mM DTT and increasing H2O2 concentrations for 1 h at room temperature (RT). Excess low molecular weight reagents were removed by acetone precipitation and proteins were resuspended in H2O. Dilutions were prepared in 30% EtOH, 0.1% formic acid (FA) and the mixture was introduced into the ESI-MS. Instrumental settings: Capillary voltage = 4.000 V. Nebulizer gas pressure = 15 psi. Drying gas flow = 4.0 L/min. Drying gas temperature = 300 °C. Mass-to-charge (m/z) values: 650-1200. Mass spectra were deconvoluted using the software provided by the manufacturer (DataAnalysis, Bruker Daltonics, Bremen, Germany).

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