Mechanism for Catechol Ring Cleavage by Non-Heme Iron Intradiol Dioxygenases:  A Hybrid DFT Study

The mechanism of the catalytic reaction of protocatechuate 3,4-dioxygenase (3,4-PCD), a representative intradiol dioxygenase, was studied with the hybrid density functional method B3LYP. First, a smaller model involving only the iron first-shell ligands (His460, His462, and Tyr408) and the substrates (catechol and dioxygen) was used to probe various a priori plausible reaction mechanisms. Then, an extended model involving also the most important second-shell groups (Arg457, Gln477, and Tyr479) was used for the refinement of the preselected mechanisms. The computational results suggest that the chemical reactions constituting the catalytic cycle of intradiol dioxygenases involve:  (1) binding of the substrate as a dianion, in agreement with experimental suggestions, (2) binding of dioxygen to the metal aided by an electron transfer from the substrate to O₂, (3) formation of a bridging peroxo intermediate and its conformational change, which opens the coordination site trans to His462, (4) binding of a neutral XOH ligand (H₂O or Tyr447) at the open site, (5) proton transfer from XOH to the neighboring peroxo ligand yielding the hydroperoxo intermediate, (6) a Criegee rearrangement leading to the anhydride intermediate, and (7) hydrolysis of the anhydride to the final acyclic product. One of the most important results obtained is that the Criegee mechanism requires an in-plane orientation of the four atoms (two oxygen and two carbon atoms) mainly involved in the reaction. This orientation yields a good overlap between the two σ orbitals involved, C−C σ and O−O σ*, allowing an efficient electron flow between them. Another interesting result is that under some conditions, a homolytic O−O bond cleavage might compete with the Criegee rearrangement. The role of the second-shell residues and the substituent effects are also discussed.