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[摘要]：In this work, we investigated the oxidative modification of histidine residues induced by peroxidase and thiol oxidase activities of bovine copper-zinc superoxide dismutase (Cu-ZnSOD) using NMR and pulse EPR spectroscopy. ID NMR and 2D-NOESY were used to determine the oxidative damage at the Zn(II) and CLI(II) active sites as well as at distant histidines. Results indicate that during treatment of SOD with hydrogen peroxide (H2O2) or cysteine in the absence of bicarbonate anion (HCO3-), both exchangeable and nonexchangeable protons were affected. Both His-44 and His-46 in the Cu(II) active site were oxidized based on the disappearance of NOESY cross-peaks between CH and NH resonances of the imidazole rings. In the Zn(I I) site, only His-69, which is closer to His-44, was oxidatively modified. However, addition of HCO3- protected the active site His residues. Instead, resonances assigned to the His-41 residue, 11 angstrom away from the Cu(II) site, were completely abolished during both HCO3--stimulated peroxidase activity and thiol oxidase activity in the presence of HCO3-. Additionally, ESEEM/HYSCORE and ENDOR studies of SOD treated with peroxide/Cys in the absence of HCO3- revealed that hyperfine couplings to the distal and directly coordinated nitrogens of the His-44 and His-46 ligands at the Cu(II) active site were modified. In the presence of HCO3-, these modifications were absent. HCO3--mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities. |
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