Oxidatively induced Cu for Mn exchange in protein phosphatase 1γ: A new method for active site analysis

Protein phosphatase 1 gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn2+ in the active site when expressed in Escherichia coli in a buffer containing MnCl2. Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1 gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155. (c) 2009 Elsevier Ltd. All rights reserved.