title: Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase.
abstract: 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 Em 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.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Schwartz
authors.forename: E
authors.initials: E
authors.lastname: Yikilmaz
authors.forename: C K
authors.initials: CK
authors.lastname: Vance
authors.forename: S
authors.initials: S
authors.lastname: Vathyam
authors.forename: R L
authors.initials: RL
authors.lastname: Koder
authors.forename: A F
authors.initials: AF
authors.lastname: Miller
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005973:Glutamine; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase