High-resolution X-ray structure of Gln143Asn manganese superoxide dismutase captures multiple hydrogen peroxide-binding sites.

Abstract

Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide into hydrogen peroxide and molecular oxygen, serving as a key defence against oxidative damage. Despite extensive studies, the full structural characterization of H₂O₂-binding sites in MnSOD remains largely unexplored. Previous H₂O₂-soaked MnSOD structures have identified two distinct H₂O₂-binding sites: one directly ligated to the catalytic manganese (LIG position) and another at the active-site gateway (PEO position) between the second-shell residues Tyr34 and His30. In this study, a kinetically impaired Gln143Asn MnSOD variant is used to trap and explore additional H₂O₂-binding sites beyond the second-shell solvent gate. In the wild-type enzyme, Gln143 mediates proton transfers with the manganese-bound solvent (WAT1) to drive redox cycling of the metal, which is necessary for effective superoxide dismutation. Substitution with Asn stalls catalysis because the increased distance from WAT1 disrupts critical proton-coupled electron-transfer (PCET) events, and the redox cycling of the active-site metal is impaired. This, in turn, stalls the electrostatic cycling of positive charge on the enzyme surface and enhances the likelihood of trapping transient H₂O₂-bound states in this variant. The results reveal several H₂O₂ molecules leading up to the active site, in addition to the canonical LIG and PEO positions.