title: Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase.
abstract: We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)SOD] and Fe-SOD to elucidate the basis for the inactivity of Fe-sub-(Mn)SOD, despite its apparent similarity to Fe-SOD. The active site of (reduced) Fe2+-sub-(Mn)SOD is qualitatively similar to that of native Fe2+-SOD, indicating similar active site structures and coordination environments for Fe2+. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe2+-sub-(Mn)SOD's retention of ability to reduce O2*- [Vance and Miller (1998) J. Am. Chem. Soc. 120(3), 461-467]. The active site of (oxidized) Fe3+-sub-(Mn)SOD differs from that of Fe3+-SOD with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe3+. Although Fe3+-sub-(Mn)SOD binds the small anions N3- and F-, the KD for N3- is tighter than that of Fe3+-SOD, suggesting that the (Mn)SOD protein favors anion binding more than does the (Fe)SOD protein. The EPR spectral consequences of binding F- are reminiscent of those observed upon binding the first F- to Fe3+-SOD, but the EPR spectrum obtained upon binding N3- is different, consistent with crystallographic observation of a different binding mode for N3- in Thermus thermophilus Mn-SOD than Fe-SOD [Lah, M., et al. (1995) Biochemistry 34, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N3- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe3+-sub-(Mn)SOD and the differences in the Fe3+ coordination indicated by the EPR spectra are consistent with Fe3+-sub-(Mn)SOD's inability to oxidize O2*- and suggest that its low E degrees is due to perturbation of the oxidized state.
authors.affiliation: Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: C K
authors.initials: CK
authors.lastname: Vance
authors.forename: A F
authors.initials: AF
authors.lastname: Miller
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005296:Ferrous Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D017186:Titrimetry
chemical_list: D005296:Ferrous Compounds; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase