Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase

Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese supero xide dismutase. Biochemical and spectroscopic characterization of site-dire cted mutants has allowed functional characterization of these residues in t he wild-type (wt) enzyme. X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures. T he Y34A mutant, as well as the previously reported Y34F mutant, retained es sentially the full superoxide dismutase activity of the wild-type enzyme, a nd the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the subst rate access funnel. Mutation of the outer sphere solvent pocket residue Q14 6 has more dramatic effects. The Q146E mutant is isolated as an apoprotein lacking dismutase activity, Q146L and Q146H mutants retain only 5- 10% of t he dismutase activity of the wild-type enzyme. The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the s uperoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophil um, which is active in both Mn and Fe forms. Interestingly, the iron-substi tuted Q146H protein also exhibits low dismutase activity, which increases a t lower pH. Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.