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 characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn2+, Fe3+ or Fe2+ 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 important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn2+, Fe3+ and 1H in the presence of Fe2+ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe2+SOD and Fe2+(Mn)SOD by 15N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe2+ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E(m) in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH- coordinated to the metal ion. (C) 2000 Elsevier Science S.A.
|Number of pages||10|
|Journal||Journal of Inorganic Biochemistry|
|State||Published - Jul 1 2000|
Bibliographical noteFunding Information:
Acknowledgment is made to the donors of The Petroleum Research Fund, administered by the A.C.S., for partial support of this research under ACS-PRF 33266-AC4,3. A.-F.M. is pleased to thank the N.S.F. for financial support (MCB9728793) and N.I.H. for generous financial aid in acquiring an NMR spectrometer (RR 06468). AFM also thanks the reviewers of the manuscript for exceptionally thoughtful reviews.
- Metal ion specificity
- Redox potential
- Superoxide dismutase
ASJC Scopus subject areas
- Inorganic Chemistry