Intermediates in Non-Heme Iron Intradiol Dioxygenase Catalysis

Protocatechuate 3,4-dioxygenase (3,4-PCD) sequentially binds 3,4-(OH)₂-benzoate (PCA) and O₂ before catalyzing ring fissure. Structural and spectroscopic studies show that the 3,4-PCD active site Fe³⁺ has a trigonal bipyramidal ligand coordination with axial Tyr and His and equatorial His, Tyr, and OH^- ligands. It is proposed from structural and kinetic data that PCA binds progressively: i) to the Fe³⁺ via the C4-O^- displacing the OH^-, and ii) deeper in the site, yielding octahedral coordination geometry. Subsequently, PCA rotates so that: i) the C3-O^- becomes an equatorial ligand, ii) the C4-O^- displaces the axial Tyr to form an Fe³⁺-PCA chelate, and iii) rotation of the displaced Tyr opens an O₂ binding site next to PCA from which electrophilic attack on the dianionic PCA can occur. The importance of the axial Tyr ligand to catalysis is indicated by the fact that the turnover number of the Y447H mutant of 3,4-PCD is 600-fold lower than wild type. In accord with the proposed mechanism, kinetic measurements of reaction cycle intermediates showed that the decreased rate is due to slow substrate binding.