¹⁸O Kinetic Isotope Effects in Non-Heme Iron Enzymes: Probing the Nature of Fe/O₂ Intermediates

Abstract

Reported here are the competitive 18 O/ 16 O kinetic isotope effects ( 18 O KIEs) on k cat /K m (O 2 ) for three non-heme iron enzymes that activate O 2 at an iron center coordinated by a 2-His-1-carboxylate facial triad: taurine dioxygenase (TauD), S-(2)-hydroxypropylphosphonic acid epoxidase (HppE), and 1-aminocyclopropyl-1-carboxylic acid oxidase (ACCO). The comparison of the measured 18 O KIEs with calculated 18 O equilibrium isotope effects ( 18 O EIEs) reveals an excellent correlation with the proposed mechanisms for these enzymes. 18 O KIEs of 1.0104 ± 0.0002 (TauD), 1.0120 ± 0.0002 (HppE), and 1.0215 ± 0.0005 (ACCO) suggest the formation in the rate limiting step of O 2 activation of an Fe III -alkylperoxo, Fe III -OOH, and Fe IV =O species, respectively. By probing only the steps from initial O 2 binding up to and including the first irreversible step of O 2 activation, the measured 18 O KIEs can be a valuable companion to pre-steady state kinetic analyses in studying the complex catalytic mechanisms of non-heme iron enzymes.The O 2 -activating, non-heme iron enzymes catalyze a wide range of oxygenation and oxidation reactions with important biological implications, such as DNA repair, hypoxic response, collagen biosynthesis, and histone demethylation. 1 Most of these enzymes contain a single iron center coordinated by two His and one Asp/Glu residues in a tridentate binding motif referred to as "2-His-1-carboxylate facial triad". Understanding the O 2 -activation process for these enzymes may provide key insights into the source of their divergent substrate specificity despite similarly coordinated active site metal centers. HppE is a reductase-dependent non-heme iron enzyme that catalyzes the epoxidation of S-HPP, the last step in the biosynthesis of the antibiotic fosfomycin. 9 The mechanism of HppE is not as well known as for TauD, formation of an Fe III -OOH species being proposed to involve either a hydrogen atom transfer (HAT) from S-HPP or proton-coupled electron transfer (PCET) from the reductant. 10 The measured 18 O KIE for HppE is 1.0120 ± 0.0002 at 25 °C, using FMN in the presence of NADH as the reductant (Figure 1

Electronic Structure Analysis of the Oxygen‐Activation Mechanism by FeII‐ and α‐Ketoglutarate (αKG)‐Dependent Dioxygenases

“…Our calculations revealed that the nucleophilic attack of the distal O atom of the O 2 adduct on the carbonyl group in αKG via a bicyclic transition state (TS1) is the rate-limiting step, which is between the addition of O 2 to the enzyme:Fe II /αKG/substrate complex and the generation of the Fe IV –oxo intermediate, as confirmed by recent experimental studies. [40] Decay of 7 TS1 affords a HS Fe III bound to an oxyl radical, whereas 5 TS1 evolves into a HS Fe II chelated by peroxosuccinate. According to the calculated energetics using DFT and ab initio methods, the septet reaction pathway might compete with the quintet channel.…”

Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study

“…Metal binding was examined by isothermal titration calorimetry [84]. 18 O-kinetic isotope effect measurements were used to identify the first irreversible reaction of O 2 activation, the step following Fe(III)-superoxo formation [85]. It also has proven useful to supplement these experimental results with computational approaches, such as quantum mechanics/molecular mechanics and density functional theory calculations [57, 86–93], and by comparison to biomimetic models [94, 95].…”

Enzymatic Chemistry of Cyclopropane, Epoxide, and Aziridine Biosynthesis

“…To study the reactive oxygen species more closely, the 18 O KIE on k cat / K m(O2) for the reaction with native substrate, ( S )-HPP, was measured to be 1.0120, the magnitude of which is consistent with the formation of an [Fe III -OOH] species in the rate-determining step. 219 Formation of the Fe III -OOH species could involve direct hydrogen atom abstraction from the substrate ( 150 → 153 ). It may also be generated via a rate-limiting proton coupled electron transfer to form a ferric peroxy intermediate (151) that is then used to oxidize substrate directly ( 151 → 154 ), 220 or to form an [Fe IV =O] species which oxidizes substrate ( 151 → 152 → 155 ).…”