X-ray Absorption Spectroscopy of the Iron Site in Escherichia coli Fe(III) Superoxide Dismutase

The local structure of the iron site in ferric superoxide dismutase from Escherichia coli has been characterized by X-ray absorption spectroscopy. In the resting state of the enzyme at pH 7.0, the iron is five-coordinate with an average metal-ligand bond length of 1.98 A. Binding of azide causes a reduction in the intensity of the bound state 1s-->3d transition and an increase of 0.08 A in average bond length. Both are indicative of an increase in the iron coordination number. Raising the pH from 7.0 to 10.5 causes a similar 0.08 A increase in the average bond length, again suggesting an increase in the iron coordination number. At intermediate pH (9.4), the average bond length is 2.03 A, consistent with an approximately 50:50 mixture of the limiting high and low pH forms. Similarly, the absorption edge structure varies continuously from pH 7 to 10.5. These spectra can be fit to a titration curve with a pKa of approximately 9.8. These data suggest that the pH-dependent transition, previously identified by UV-vis, EPR, and activity measurements, may be the conversion of the iron from five- to six-coordinate, presumably through coordination by hydroxide. The 1s-->3d transition for ferric superoxide dismutase at high pH is broader but not significantly less intense than that at pH 7. This suggests that the high pH form may be significantly distorted from octahedral symmetry. At pH 7, the ferric and ferric + azide samples undergo slow X-ray induced photoreduction.