X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism.

The extradiol-cleaving catechol 2,3-dioxygenase (2,3-CTD) isolated from Pseudomonas putida mt-2 and its catechol and ternary E.S.NO complexes are characterized by X-ray absorption spectroscopy (XAS). The intensities of the 1s-->3d transitions in the pre-edge spectra of the uncomplexed enzyme and its substrate complex show that the Fe(II) center is five-coordinate in both complexes, in agreement with earlier magnetic circular dichroism studies [Mabrouk, P. A., Orville, A. M., Lipscomb, J. D., & Solomon, E. I. (1991) J. Am. Chem. Soc. 113, 4053-4061]. Analysis of the EXAFS region of uncomplexed 2,3-CTD shows five N/O ligand atoms 2.09 A from the active site Fe(II). In the 2,3-CTD.catechol complex, one N/O atom is located at 1.93 A and four N/O type ligands are at 2.10 A. By comparison with FeII-(6TLA)(DBCH), the first well-characterized mononuclear Fe(II).catechol model complex, the 1.93 A scatterer is proposed to be the oxygen from the deprotonated hydroxyl group of the coordinated catecholate monoanion. Nitric oxide binds to the Fe(II) center in the enzyme.catechol complex without displacing the existing ligands, resulting in the formation of a six-coordinate complex, as indicated by the addition of a new N/O type scatterer at 1.74 A. Bond valence sum (BVS) analysis of the bond lengths derived from the EXAFS fits gives values that correspond to the iron oxidation states established for these complexes, thus lending credence to the coordination environment deduced for the iron center in those complexes. The present study provides the first evidence for a monoanionic substrate binding mode in an extradiol dioxygenase, which is distinct from the dianionic binding mode proposed for intradiol dioxygenases. We speculate that this difference in binding mode may have important ramifications for the site of aromatic ring cleavage in the subsequent oxygen insertion reactions.