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. & 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 degrees 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.