The First Direct Characterization of a High-Valent Iron Intermediate in the Reaction of an α-Ketoglutarate-Dependent Dioxygenase: A High-Spin Fe(IV) Complex in Taurine/α-Ketoglutarate Dioxygenase (TauD) from Escherichia coli

Abstract

The Fe(II)- and α-ketoglutarate(αKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O₂ to a five-coordinate, (His)₂(Asp)-facially coordinated Fe(II) center to which αKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O₂ on the ketone carbonyl of αKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)O^2-) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)O^2- complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O₂ has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature γ-radiolysis of the trapped intermediate) by Mössbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its Mössbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related αKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O₂-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)O^2- species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.