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 alpha-ketoglutarate(alphaKG)-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 O(2) to a five-coordinate, (His)(2)(Asp)-facially coordinated Fe(II) center to which alphaKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O(2) on the ketone carbonyl of alphaKG 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 O(2) has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/alpha-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 gamma-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 alphaKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O(2)-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.

Mechanism of Aromatic Amino Acid Hydroxylation

“…Similar species have been proposed as the active oxygen intermediates in several other non-heme iron monooxygenases (77). Very recently, an intermediate with spectroscopic properties consistent with high spin Fe (IV)O was detected in the reaction of the non-heme iron enzyme taurine/α-ketoglutarate dioxygenase (78); while the mechanisms of the α-ketoglutarate dependent hydroxylases differ in detail from that of the pterin dependent enzymes (79), in both, the iron is bound by a two histidineone carboxylate triad (80). Biomimetic studies have established the ability of Fe(IV) O species to catalyze aromatic hydroxylation, sulfoxidation, and epoxidation reactions (81)(82)(83)(84).…”

SyrB2 in syringomycin E biosynthesis is a nonheme Fe II α-ketoglutarate- and O 2 -dependent halogenase

“…The formation of the Fe IV AO ferryl species is likely to be a common intermediate in both the oxygenase and halogenase mechanism. The high-valent oxoiron species can break unactivated C-H bonds in substrates homolytically to produce the substrate radical and an Fe III -OH as donor of an OH⅐ by radical rebound to complete hydroxylation (24,25,28). We anticipate that there will be a chloride ion bound to the Fe IV intermediate of the SyrB2 active site.…”

Role of Substrate Positioning in the Catalytic Reaction of 4-Hydroxyphenylpyruvate Dioxygenase—A QM/MM Study

“…Notably, the Fe(IV)=O species has been trapped and characterized by spectroscopic methods for taurine 2-oxoglutarate dioxygenase and two other α-ketoacid dependent enzymes. [24][25][26] With respect to the hydroxylation reactions, for HPPD it was proposed that electrophilic attack on the aromatic ring of HPA leads to a high-spin Fe(III)/ring radical σ-complex. 8,9,14 The subsequent migration of the acetic acid side chain was found to proceed in two steps:…”