Hydrogen peroxide induce modifications of human extracellular superoxide dismutase that results in enzyme inhibition

Superoxide dismutase (EC-SOD) controls the level of superoxide in the extracellular space by catalyzing the dismutation of superoxide into hydrogen peroxide and molecular oxygen. In addition, the enzyme reacts with hydrogen peroxide in a peroxidase reaction which is known to disrupt enzymatic activity. Here, we show that the peroxidase reaction supports a site-specific bond cleavage. Analyses by peptide mapping and mass spectrometry shows that oxidation of Pro112 supports the cleavage of the Pro112–His113 peptide bond. Substitution of Ala for Pro112 did not inhibit fragmentation, indicating that the oxidative fragmentation at this position is dictated by spatial organization and not by side-chain specificity. The major part of EC-SOD inhibited by the peroxidase reaction was not fragmented but found to encompass oxidations of histidine residues involved in the coordination of copper (His98 and His163). These oxidations are likely to support the dissociation of copper from the active site and thus loss of enzymatic activity. Homologous modifications have also been described for the intracellular isozyme, Cu/Zn-SOD, reflecting the almost identical structures of the active site within these enzymes. We speculate that the inactivation of EC-SOD by peroxidase activity plays a role in regulating SOD activity in vivo, as even low levels of superoxide will allow for the peroxidase reaction to occur.

Oxidative modifications of human EC-SOD induced by hydrogen peroxide.Figure optionsDownload as PowerPoint slideHighlights
► Hydrogen peroxide inhibits EC-SOD by oxidation of amino acid residues in spatial proximity to the active site copper.
► The coordination sphere of copper is disrupted by the oxidation of His98 or His163.
► Protein fragmentation is mediated by cleavage of the Pro112-His113 peptide bond.
► Fragmentation is induced by hydrogen abstraction on the carbon of Pro112.