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 absorpti on spectroscopy (XAS). The analysis of the data provides information o n the coordination number (six) and geometry (octahedral) of the iron( III) ion in the active site which seems to remain essentially unpertur bed with respect to the active iron(II)-containing enzyme. The first c oordination 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 hi stidine ligands is confirmed. The oxidized form is still able to bind o-substituted phenols, the adduct being characterized by an intense re d 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) c m(-1), typical of iron(III) phenolate complexes, confirms that this in hibitor binds to the metal centre.