Fig. 1: Structure of human wildtype MnSOD and protonation differences between oxidation states.

a The active sites of tetrameric MnSOD are found in positively charged cavities made by two subunits. Solvent and substrate must pass through gateway residues His30 and Tyr34 to interact with the catalytic Mn ion. The distance between Tyr34(O^H) and His30(N^δ1) is 5.4 Å. Blue dashed lines indicate hydrogen bonds. The inset indicates the chain identity derived crystallographically, where the asymmetric unit is composed of an AB dimer, and the CD dimer is generated through symmetry to form the native tetrameric assembly. b Room temperature neutron structures revealed changes in protonation among WAT1 and Gln143 and alterations in the hydrogen bond network of WAT1-Gln143-Trp123. Dotted lines indicate hydrogen bonds ≥ 1.8 Å while hashed lines indicate SSHBs that are hydrogen bonds < 1.8 Å. c A shared proton, indicated with rounded dots, was observed between His30 and Tyr166. This proton helps modulate the protonation state of N^δ1(His30) when the Mn ion changes oxidation states. d Tyr34 is deprotonated in Mn³⁺SOD and protonated in Mn²⁺SOD. There is no water within hydrogen bond distance on Tyr34 in Mn²⁺SOD. e Superposition of wildtype MnSOD (yellow, PDB ID 5VF9) and Tyr34Phe MnSOD (red, PDB ID 9BWR) active sites with a root-mean-square deviation of 0.07 Å among C^α atoms. Panel a was created from MnSOD X-ray structure (PDB ID 5VF9)⁷⁰, and panels (b–d) were created from the neutron structures of Mn³⁺SOD (PDB ID 7KKS) and Mn²⁺SOD (PDB ID 7KKW)¹³. All hydrogen positions were experimentally determined except for solvent molecules in panel a that were randomly generated to accentuate the solvent in the active site funnel. All distances are in Å.