Oxidation of Cu, Zn-superoxide dismutase by the myeloperoxidase/hydrogen peroxide/chloride system: functional and structural effects.
This study investigated the functional and structural effects of bovine Cu, Zn-superoxide dismutase (Cu, Zn-SOD) oxidation by the myeloperoxidase (MPO)/hydrogen peroxide (H2O2)/chloride system and reagent hypochlorous acid (HOCl). Exposure to HOCl led to a fast inactivation accompanied by structural alterations. The residual SOD activity depended on the reactants concentration ratio and on the exposure time. The concomitant high consumption of HOCl indicated the presence of multiple targets on the protein. As assessed by SDS/PAGE, HOCl caused the dissociation of the protein into protomers at 16 kDa stable to both SDS and reducing conditions. Results from isoelectric focusing gels showed that exposure to HOCl induced the formation of modified protein derivatives, with a more acidic net electric charge than the parent molecule, consistent with the presence of additional ions observed in the electrospray ionization mass spectra. The reaction of protein with HOCl resulted in changes in protein conformation as assessed by the UV fluorescence and oxidation of the unique methionine and tyrosine, chlorination of several lysines with formation of chloramines. There was no significant formation of dityrosine and carbonyl groups. Exposure to high levels of HOCl resulted in complete enzyme inactivation, loss of additional lysine, histidine and arginine residues and coincident detection of weakly bound zinc and copper using 4-pyridylazaresorcinol. Collectively, the results suggest that the decrease of the dismutase activity is probably related to both dissociation into protomers and unfolding due to extensive oxidative modifications of amino acids.
Auchère
Françoise
F
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris 5, CNRS UMR 8601, 45 rue des Saints Pères 75270 Paris, France.
Capeillère-Blandin
Chantal
C
D000818:Animals; D002417:Cattle; D003300:Copper / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects* / Q000201:enzymology; D006834:Hydrazines / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006997:Hypochlorous Acid / Q000494:pharmacology*; D007525:Isoelectric Focusing; D008239:Lysine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D006834:Hydrazines; C446799:dinitrophenylhydrazine; D014443:Tyrosine; D006997:Hypochlorous Acid; D003300:Copper; D008715:Methionine; D006861:Hydrogen Peroxide; D009195:Peroxidase; D013482:Superoxide Dismutase; D015032:Zinc; D008239:Lysine