Amino acid substitution at the dimeric interface of human manganese superoxide dismutase.

The side chains of His30 and Tyr166 from adjacent subunits in the homotetramer human manganese superoxide dismutase (Mn-SOD) form a hydrogen bond across the dimer interface and participate in a hydrogen-bonded network that extends to the active site. Compared with wild-type Mn-SOD, the site-specific mutants H30N, Y166F, and the corresponding double mutant showed 10-fold decreases in steady-state constants for catalysis measured by pulse radiolysis. The observation of no additional effect upon the second mutation is an example of cooperatively interacting residues. A similar effect was observed in the thermal stability of these enzymes; the double mutant did not reduce the major unfolding transition to an extent greater than either single mutant. The crystal structures of these site-specific mutants each have unique conformational changes, but each has lost the hydrogen bond across the dimer interface, which results in a decrease in catalysis. These same mutations caused an enhancement of the dissociation of the product-inhibited complex. That is, His30 and Tyr166 in wild-type Mn-SOD act to prolong the lifetime of the inhibited complex. This would have a selective advantage in blocking a cellular overproduction of toxic H2O2.