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

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 decades of research, the product exit pathway in MnSOD remains understudied, due to the enzyme’s rapid, diffusion-limited catalysis. Here, we structurally characterize the H ₂ O ₂ exit route in MnSOD, using a kinetically impaired Gln143Asn variant. In the wild-type enzyme, Gln143 participates in proton transfer reactions with the Mn ³⁺ -bound solvent (WAT1) to drive redox cycling of the metal for effective O ₂ ^●− 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 H ₂ O ₂ exit process with improved experimental control. Results reveal that the Gln143Asn substitution introduces a cavity, which permits conformational flexibility of Tyr34, that would be sterically disallowed in the wild-type. This flexibility of Tyr34 narrows the gateway between the second-shell residues Tyr34 and His30 and restricts diffusion of the Mn ²⁺ -bound peroxide out of the metal’s primary coordination sphere. Also, peroxide occupies a secondary binding site between Tyr34 and His30. The combination of Tyr34 flexibility and the enzyme’s inability to redox cycle causes a “traffic jam” of peroxide at the mouth of the blocked gateway. Overall, our findings provide novel structural insight into the mechanism of product trafficking in MnSOD and underscore the utility of rational mutagenesis in accessing elusive mechanistic states.