Journal
JOURNAL OF INORGANIC BIOCHEMISTRY
Volume 80, Issue 3-4, Pages 247-256Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/S0162-0134(00)00086-6
Keywords
superoxide dismutase; redox potential; glutamine; metal ion specificity
Funding
- NCRR NIH HHS [RR 06468] Funding Source: Medline
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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. Sec. 120(3) (1998) 461-467]. Here, we test the significance to activity and active site integrity of the Gin side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gin 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 Gin 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 H-1 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 Gin in Fe2+SOD and Fe2+(Mn)SOD by N-15 NMR. The very different chemical shifts indicate that the active site Gin interacts differently with Fe2+ in the two proteins. Since a shorter distance from Gin to Fe and stronger interaction with Fe correlate with a lower E-m in Fe(Mn)SOD, Gin 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. All rights reserved.
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