High-Resolution X-ray Structure of Gln143Asn Manganese Superoxide Dismutase Captures Multiple Hydrogen Peroxide...

Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide ( ${O_2}^{•-}$ ) into hydrogen peroxide ( $H_2{O}_2$ ) and molecular oxygen ( $O_2$ ), serving as a key defense against oxidative damage. Despite extensive studies, the full structural characterization of $H_2{O}_2$ -binding sites in MnSOD remains largely unexplored. Previous $H_2{O}_2$ -soaked MnSOD structures have identified two distinct $H_2{O}_2$ -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 $H_2{O}_2$ -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 ${O_2}^{•-}$ 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 $H_2{O}_2$ -bound states in this variant. Results reveal several $H_2{O}_2$ molecules leading up to the active site, in addition to the canonical LIG and PEO positions.