X-ray absorption characterization of the bacterial superoxide dismutases and NMR characterization of substrate binding to phthalate dioxygenase.

Abstract

The Mn- and FeSODs are among the fastest enzymes known and have been shown to have nearly identical protein structures by crystallography. However, the Mn- and FeSODs appear to differ in their ability to bind anions and in their ability to function at high pH. X-ray absorption characterization of the structures of the metal sites in the Fe- and MnSODs from E. coli are reported. The metal in the Fe(III) enzyme is shown to increase in coordination number from 5 to 6 on addition of azide and fluoride, and also on increasing the pH. The latter is interpreted as binding of hyroxide, with an apparent pK of 9.6. In contrast, the Mn enzyme does not show a pH dependent change in coordination number for either the Mn(II) or the Mn(III) oxidation state. Azide causes a minor perturbation at the Mn(II) site, while causing an apparent increase in coordination for Mn(III)SOD. Examination of an iron substituted form of MnSOD, shows a coordination number of six at pH 8, which decreases to five below pH 6. Thus, the Fe in Fe(Mn)SOD also binds hydroxide, with a pK that is approximately 2 pH units lower. $\sp{19}$F NMR studies of fluorophthalates bound to metal substituted derivatives of phthalate dioxygenase (PDO) are reported. T $\sb1$s of 5 and 17 ms were measured for 3-F and 4-F phthalate, respectively, bound to Co(II)PDO. Using the Solomon relations $\rm (\tau\sb{c} = 1{-}10$ ps for the cobalt ion) gave Co to F distance estimates of 3.6-5.0 A for the 4-F and 5.0-6.2 A for the 3-F positions. The geometric model based on these results suggest that the metal ion lies in the plane that bisects the phthalate ring, and that it resides closer to the 4- position than to the 3- position. These data are fully consistent with a mechanism in which O$\sb2$ binds to the metal in a side-on fashion, although an end-on orientation may also be possible.