Structure and reaction mechanism of catechol 2,3-dioxygenase (metapyrocatechase)

Catechol 2,3-dioxygenases catalyzes the extradiol ring-cleavage of catechol derivatives. The enzyme from Pseudomonas putida mt-2 (metapyrocatechase, MPC) is a homotetramer and prefers small monocyclic substrates. We have structurally characterized MPC at a 2.8-Angstrom resolution in the presence of 10% acetone. The subunit comprises the N- and C-terminal domains of a similar structure. The active site is located in a funnel-shaped space of the C-terminal domain. Within this space, an iron atom is directly ligated by three protein residues (His 153, His214, and Glu265), and an acetone molecule binds the Fe atom. The geometry at the Fe site is a distorted tetrahedral. C2/C3-substituted phenols were deprotonated when they bound to holo-MPC. The absorption spectra of the MPC complexes with o-nitrophenol, 2-hydroxybenzaldehyde, and m-nitrophenol showed a new broad absorption around 650 nm. Resonance Raman spectra of the o-nitrophenol-MPC complex excited at 632.8 nm showed the band assigned to the symmetric stretching of the nitro group of o-nitrophenolate. The conserved active-site residues, His199 and Tyr255, may play important roles in the deprotonation of the ligands and the formation of the new absorption band. We propose how the C2/C3-substituted phenolates bind to the Fe(II) center. (C) 2002 Elsevier Science B.V. All rights reserved.