Spectroscopic measurement of a long-predicted active site pK in iron- superoxide dismutase from Escherichia coli

The accepted mechanism of Fe-containing superoxide dismutase (Fe-SOD) activity and inhibition by anions implies the existence of a group with a pK of 8.6-9.0 in the active site of reduced Fe-SOD [Bull, C. and Fee, J. A. (1985) J. Am. Chem. Soc. 107, 3295-3304]. We have performed pH titrations of reduced Fe-SOD by NMR spectroscopy and observe a pK of 8.5 at 30 °C which is the only pK affecting the active site between pH 5.5 and 10.5. Thus, we present the first spectroscopic evidence of the predicted pK. Although the pK is associated with chemical shift changes for almost all of the resonances of the active site, resonance line widths and the T₁ of a ligand proton are not significantly affected by the pK, indicating that there is no significant conformational change and only relatively minor effects on the electronic spin properties of Fe²⁺. The changes in chemical shift are probably caused by changes in hydrogen bonding to a ligand and attendant subtle perturbation of the Fe²⁺ paramagnetism upon loss of the proton with the pK of 8.5. The pK is also associated with a dramatic restriction of the exchange of at least one ligand proton. Thus, active site accessibility to solvent and OH^- decreases by more than 2 orders of magnitude upon loss of the proton with the pK of 8.5. Since OH^- is a competitive inhibitor of Fe-SOD, and thus a substrate analog, this dramatic and unusual decrease in accessibility to OH^- is consistent with the increase in the K(M) for O₂·^- that is associated with a pK near 9.