The oxidation produced by hydrogen peroxide on Ca-ATP-G-actin

We report here that in vitro exposure of monomeric actin to hydrogen peroxi de leads to a conversion of 6 of the 16 methionine residues to methionine s ulfoxide residues. Although the initial effect of H2O2 on actin is the oxid ation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, a nd Met355 are the other sites of the oxidative modification. Met44 and Met4 7 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medi um and are found in all four subdomains of actin. The conformations of subd omain 1, a region critical for the functional binding of different actin-bi nding proteins, and subdomain 2, which plays important roles in the polymer ization process and stabilization of the actin filament, are changed upon o xidation. The conformational changes are deduced from the increased exposur e of hydrophobic residues, which correlates with methionine sulfoxide forma tion. from the perturbations in tryptophan fluorescence, and from the decre ased susceptibility to limited proteolysis of oxidized actin.