Mapping the Exit Route of Hydrogen Peroxide From the Manganese Superoxide Dismutase (MnSOD) Active Site.

Abstract

Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide (O [bullet]-) into hydrogen peroxide (H O ) and molecular oxygen (O ), serving as a key defense 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 Mn (LIG position) and another at the active site gateway (PEO position) between 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 Mn-bound solvent (WAT1) to drive redox cycling of the metal, necessary for effective O [bullet]- 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. Results reveal several H2O2 molecules leading up to the active site, in addition to the canonical LIG and PEO positions. SynopsisA high-resolution X-ray structure of a Gln143Asn variant of manganese superoxide dismutase reveals multiple hydrogen peroxide binding sites beyond the canonical LIG and PEO binding positions within the active site. These findings expand the known landscape of product peroxide-bound states in MnSOD.