Kinetic and structural effects of interfacial interruption and protein nitration in human manganese superoxide dismutase

This dissertation investigated interfacial attributes of human manganese superoxide dismutase (MnSOD), a homotetramer with two structurally unique interfaces. A conserved dimeric interface is formed from the junction of two subunits and the tetrameric interface is formed from the interaction of two dimer pairs. Using 19F NMR, observation of chemical shift and linewidth changes for fluorine-labeled tyrosines in the dimeric and tetrameric interfaces of human MnSOD (Tyr169 and Tyr45 respectively) indicated greater rigidity in the dimeric interface compared to the tetrameric interface. Replacement of the dimeric, interfacial residue Glu162 with aspartate or alanine did not significantly alter the crystal structure of human MnSOD, though an interaction with a histidine ligand of the active site manganese was truncated. This interaction was mediated in E162D by an intervening solvent molecule. Catalytic activity for E162D and E162A was 5-20% that of wild-type enzyme and differential scanning calorimetry indicated a role for E162 in thermal stability. Nitration of Tyr34, a dimeric, interfacial residue involved in a hydrogen-bond network emanating from the active-site manganese, abolishes activity in human MnSOD. In vitro nitration of human MnSOD yielded 74% nitration of Tyr34 with moderate nitration of other aromatic residues. A 2.4 A structure of nitrated MnSOD aligned well with wild type though an NO 2 group covalently linked to Tyr34 is observed. The structure of nitro-MnSOD indicates that alteration of the hydrogen-bond network as well as steric blockade and electrostatic repulsion of substrate all account for the loss in catalytic activity associated with nitration. Taken together, these findings provide a role for the tetrameric interface in stability and the dimeric interface in catalysis. In addition, this dissertation provides a structural explanation for abolished catalytic activity associated with nitration of Tyr34 in MnSOD. Future therapeutic studies on this enzyme will involve the study of residues that form the dimeric interface of MnSOD.