Characterization of the Oxidized Form of Catechol 2,3-dioxygenase From Pseudomonas-putida Mt-2

The oxidized inactive extradiol cleaving catechol 2,3-dioxygenase from Pseudomonas putida mt-2 has been characterized through X-ray absorption spectroscopy (XAS). The analysis of the data provides information on the coordination number (six) and geometry (octahedral) of the iron(III) ion in the active site which seems to remain essentially unperturbed with respect to the active iron(II)-containing enzyme. The first coordination shell still appears to be composed by 2 nitrogen (at 0.207 nm) and 4 oxygen atoms (at 0.192 nm). The presence of at least two histidine ligands is confirmed. The oxidized form is still able to bind o-substituted phenols, the adduct being characterized by an intense red colour. The appearance in the electronic spectrum of the adduct of a charge transfer band at 538 nm with epsilon congruent to 2000 M(-1) cm(-1), typical of iron(III) phenolate complexes, confirms that this inhibitor binds to the metal centre.