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

Abstract

Reported here are the competitive ¹⁸O/¹⁶O kinetic isotope effects (¹⁸O KIEs) on k_cat/K_m(O₂) for three non-heme iron enzymes that activate O₂ 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 ¹⁸O KIEs with calculated ¹⁸O equilibrium isotope effects (¹⁸O EIEs) reveals an excellent correlation with the proposed mechanisms for these enzymes. ¹⁸O 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 O₂ activation of an Fe^III-alkylperoxo, Fe^III-OOH, and Fe^IV=O species, respectively. By probing only the steps from initial O₂ binding up to and including the first irreversible step of O₂ activation, the measured ¹⁸O KIEs can be a valuable companion to pre-steady state kinetic analyses in studying the complex catalytic mechanisms of non-heme iron enzymes.

The O₂-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 O₂-activation process for these enzymes may provide key insights into the source of their divergent substrate specificity despite similarly coordinated active site metal centers.¹

Several recent studies have employed the measurement of competitive ¹⁸O/¹⁶O kinetic isotope effects (¹⁸O KIEs) on k_cat/K_m(O₂) for O₂-activating metalloenzymes, in order to probe the early steps involved in O₂ activation and to reveal the nature of the metal/O₂ intermediate formed in the rate determining step (RDS) of k_cat/K_m(O₂).^2,3 Reported here are the ¹⁸O KIEs for three non-heme iron enzymes that activate O₂ at an iron center coordinated by a 2-His-1-carboxylate facial triad: taurine dioxygenase (TauD), S-(2)-hydroxypropylphosphonic acid (S-HPP) epoxidase (HppE), and 1-aminocyclopropyl-1-carboxylic acid oxidase (ACCO). These ¹⁸O KIE measurements allow, for the first time, a direct comparison of the O₂ activation process by non-heme iron enzymes employing different substrates and coreductants.¹

TauD is an α-ketoglutarate (αKG)-dependent non-heme iron enzyme that catalyzes the hydroxylation of taurine in bacteria.⁴ Its mechanism has been extensively investigated and several Fe/O₂ intermediates have been characterized, including a high-valent Fe^IV=O species.^5–7 The RDS of k_cat/K_m(O₂) in TauD is proposed to be the attack of an Fe^III-OO^•− species on αKG to form a cyclic alkylperoxo intermediate, which then undergoes oxidative decarboxylation to form the Fe^IV=O species. The measured ¹⁸O KIE for TauD is 1.0102 ± 0.0002 at 30 °C (Figure 1).⁸

Figure 1.

Isotope fractionation plots for TauD (●), HppE (■), and ACCO (◆). The fits for obtaining ¹⁸O KIEs are shown in solid, dashed, and dotted lines, respectively.⁸ Conditions: TauD: 0.2 μM TauD, 0.4–0.6 mM O₂, 2 mM taurine, 2 mM αKG, 0.2 mM ascorbate, 50 mM BisTris pH 6.2, 30 °C; HppE: 10μM HppE, 0.4–0.6 mM O₂, 1 mM S-HPP, 11 μM FMN, 1.5 mM NADH, 20 mM Tris-HCl pH 7.5, 25 °C; ACCO: 5 μM ACCO, 0.3–0.5 mM O₂, 3 mM ACC, 20 mM ascorbate, 20 mM NaHCO₃, 100 mM NaCl, 100 mM MOPS pH 7.2, 25 °C.

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 ¹⁸O KIE for HppE is 1.0120 ± 0.0002 at 25 °C, using FMN in the presence of NADH as the reductant (Figure 1).⁸ This value is similar to the one obtained for TauD, suggesting a similar oxidation state for the Fe/O₂ intermediate formed in the RDS.

ACCO is an ascorbate-dependent non-heme iron enzyme that catalyzes the last step in ethylene biosynthesis, an important plant hormone.¹¹ Recent steady-state kinetic studies of ACCO suggest that substrate oxidation occurs after the RDS of O₂ activation, which most probably involves the formation of an Fe^IV=O species.¹² The ¹⁸O KIE for ACCO is 1.0215 ± 0.0005 at 25 °C (Figure 1), one of the largest values measured for O₂-activating metalloenzymes.¹³

Competitive ¹⁸O KIEs on k_cat/K_m(O₂) reflect changes in the oxygen bond order that occur in all steps from initial O₂ binding up to and including the first irreversible step.¹⁹ To help interpret the measured ¹⁸O KIEs, calculated ¹⁸O equilibrium isotope effects (¹⁸O EIEs) can be obtained from vibrational frequencies of reactants and products,¹⁴ following the formalism developed by Bigeleisen and Mayer.²⁰ These calculated ¹⁸O EIEs can be used as upper limits for the measured ¹⁸O KIEs (assuming a negligible isotope effect contribution from the reaction coordinate frequency),²¹ allowing a direct comparison between model reactions and experimental values. Using known frequencies for Fe^III-OO^•−,¹⁵ Fe^III-OOH,¹ and Fe^IV=O species,⁷ the relevant Fe/O₂ intermediates for the studied enzymes,^1,22 we calculated ¹⁸O EIEs of 1.0080, 1.0172, and 1.0287, respectively (Table 1).⁸ In addition, ¹⁸O EIEs of 1.0187 and 1.0143 were calculated for an Fe-alkylperoxo species, Fe^III-OO^tBu,^16,17 and an Fe-peroxocarbonate species,¹⁸ respectively, the latter being similar to a proposed intermediate in TauD.⁶

Table 1.

Vibrational frequencies (cm⁻¹) of Fe/O₂ species, the corresponding calculated ¹⁸O EIEs, and experimental ¹⁸O E/KIEs.

molecule	Frequency (cm−1)			18O EIE (calc)a	18O EIE (expt)b	18O KIE (expt)c
	mode	ν16-16	ν18-16
FeIII-OO•−	Fe-Od	555	526	1.0080	1.0054 (Mb)	NDe
	O-Od	1136	1100f
FeIII-OOH	Fe-Og	621	599	1.0172	1.0113 (Hr)	1.0120 (HppE)
	O-Og	844	820f
	O-Hh	3539	3527
	O-O-Hh	1205	1199
FeIII-OOtBu	Fe-Oi	637	612	1.0187	ND	ND
	O-Oj	860	829f
	O-tBuj	746	738
	Fe-Ok	547	524	1.0143	ND	1.0102 (TauD)
	O-Ok	884	863f
	O-Ck	965	946
	O-C-Ok	728	710
FeIV=O	Fe-Ol	821	787	1.0287	ND	1.0215 (ACCO)

^aThe ¹⁸O EIEs were calculated using the Bigeleisen-Mayer equation and known frequencies (Ref ⁸).

^bMeasured ¹⁸O EIEs for O₂-binding proteins myoglobin (Mb) and hemerythrin (Hr) (Ref ¹⁴).

^cThis work.

^dRef ¹⁵.

^eNot determined.

^fν_18-16 was calculated as follows: ν_18-16 = (ν_16-16 ν _18-18)^½ (Ref ⁸).

^gRef ¹.

^hRef ¹⁴.

ⁱRef ¹⁶.

^jRef ¹⁷.

^kRef ¹⁸.

^lRef ⁷.

The comparison of the measured ¹⁸O KIEs with calculated ¹⁸O EIEs reveals an excellent correlation with the proposed mechanisms for these enzymes (Scheme 1). In TauD, the formation of the cyclic alkylperoxo intermediate in the rate determining step is supported by the measured ¹⁸O KIE of 1.0102 ± 0.0002, higher than the calculated ¹⁸O EIE for formation of an Fe^III-OO^•− species (1.0080) and the measured ¹⁸O EIE for Mb (1.0054 ± 0.0006, Table 1),¹⁴ but lower than the calculated ¹⁸O EIE for an Fe-peroxocarbonate species (1.0143). The ¹⁸O KIE of 1.0120 ± 0.0002 for HppE is similar to the measured ¹⁸O EIE of 1.0113 ± 0.0005 for Hr¹⁴ and lower than the calculated ¹⁸O EIE of 1.0172 for an Fe^III-OOH species (Table 1), suggesting a rate determining formation of an Fe^III-OOH species.²³ The relatively small measured ¹⁸O KIEs in TauD and HppE could have been diminished vs. calculated ¹⁸O EIEs due to the presence of a partially rate limiting O₂ binding step. This appears not to be the case for HppE, where preliminary data support a ¹⁸O/¹⁶O discrimination free from kinetic complexity.²³ For TauD, a kinetically significant O₂ binding step cannot be ruled out,⁶ yet the excellent agreement between the measured ¹⁸O KIE and the calculated ¹⁸O EIE (Table 1) strongly suggests a rate limiting formation of the cyclic alkylperoxo intermediate. Thus, in both TauD and HppE the observed ¹⁸O KIEs are concluded to be mechanistic in origin.

Scheme 1.

Proposed mechanisms of O₂ activation for TauD, HppE, and ACCO (RDS = proposed rate determining step of k_cat/K_m(O₂), R-H = taurine, Asc = ascorbate).

For ACCO, the large ¹⁸O KIE of 1.0215 ± 0.0005 (lower than only one calculated ¹⁸O EIE of 1.0287, Table 1) implies a significant change in the oxygen bond order and points toward Fe^IV=O species formation as the RDS of O₂ activation (Scheme 1).¹³ In ACCO, the oxidation of the Fe^III-O₂^•− species to Fe^III-OOH by ascorbate is proposed to be reversible, similar to the reversible O₂ binding observed in hemerythrin.¹⁴ Interestingly, the initial inner sphere reduction of O₂ and formation of an Fe-OO^•− species does not appear to be rate limiting for any of these enzymes, in accordance with a recently proposed reversible O₂ binding as a requisite for the specific reactivity of non-heme iron enzymes.²⁴ This is in contrast to a rate limiting and irreversible outer sphere activation of O₂ by glucose oxidase.^2,3

The studies reported herein reveal the ability of ¹⁸O KIE measurements to dissect the complex catalytic mechanisms of non-heme iron enzymes. By probing only the steps from O₂ binding to the first irreversible step of O₂ activation, these measurements offer a valuable companion to pre-steady state kinetic analyses, which investigate the nature of the intermediate immediately preceding the RDS. In the present case, measured ¹⁸O KIEs for three non-heme iron enzymes that use different reductants in the course of O₂ activation have been directly related to each enzyme’s distinct chemical mechanism.