Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase (original) (raw)

We are addressing the puzzling metal ion specificity of Fe-and Mn-containing superoxide dismutases (SODs) [see C.K. Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461-467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location 21 31 21 characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn , Fe or Fe are bound, based on EPR and NMR spectroscopy. However, the mutant's Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD's activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be 21 31 1 important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn , Fe and H in the 21 presence of Fe distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion 21 21 15 activity. We have directly observed the side chain of the active site Gln in Fe SOD and Fe (Mn)SOD by N NMR. The very different 21 chemical shifts indicate that the active site Gln interacts differently with Fe in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density m 2 on the metal ion. It may do this by stabilizing OH coordinated to the metal ion.