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

Mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide (O2●−) into hydrogen peroxide (H2O2) and molecular oxygen (O2), serving as a key defense against oxidative damage. Here, we structurally characterize the H2O2 exit-route in MnSOD, using a kinetically impaired Gln143Asn variant. In the wild-type enzyme, Gln143 participates in proton transfer reactions with the Mn3+-bound solvent (WAT1) to drive redox cycling of the metal for effective O2●− dismutation. Substitution with Asn disrupts the proton transfer, as Asn is too far away from WAT1, and stalls redox transitions in the Gln143Asn variant. This generates a slow-motion version of catalysis, that maximizes the likelihood of trapping and visualizing the H2O2 exit process with improved experimental control. Results reveal that Gln143Asn substitution introduces a cavity, which permits conformational flexibility of Tyr34, that would be sterically disallowed in the wild-type.