Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV=HO species

Abstract

Iron oxygenases generate elusive transient oxygen species to catalyze substrate oxygenation in a wide range of metabolic processes. Here we resolve the reaction sequence and structures of such intermediates for the archetypal non-heme Fe(II) and alpha-ketoglutarate-dependent dioxygenase TauD. Time-resolved Raman spectra of the initial species with (16)O(18)O oxygen unequivocally establish the Fe(IV) horizontal lineO structure. (1)H/(2)H substitution reveals direct interaction between the oxo group and the C1 proton of substrate taurine. Two new transient species were resolved following Fe(IV) horizontal lineO; one is assigned to the nu(FeO) mode of an Fe(III) horizontal line O(H) species, and a second is likely to arise from the vibration of a metal-coordinated oxygenated product, such as Fe(II) horizontal line O horizontal line C(1) or Fe(II) horizontal line OOCR. These results provide direct insight into the mechanism of substrate oxygenation and suggest an alternative to the hydroxyl radical rebinding paradigm.

Fig. 1.

Postulated mechanism of taurine oxygenation by TauD. States A, B, C, and F have been experimentally observed; state D is consistent with the chemistry of biomimetic model compounds; the radical rebinding mechanism of state G is inferred from studies of heme-containing oxygenases.

Fig. 2.

Resolution of transient oxygen species of TauD. (A) Raman spectra with ¹H- and ²H-taurine at -36 °C are shown as isotope differences (Gray), which reveal changes in oxygen vibrations as pairs of inverted bands (Fig. S2). Black traces show simulated spectra. Frequencies of a laser plasma line (▴) and a major ethylene glycol peak (♦) are indicated. Previously reported data for ¹H-taurine at 0.22 s (6) are shown for comparison. (B) Intensity profiles of individual F4, F3, and FX species in simulated difference spectra. The ∼800 cm^-1 shift was modeled as (i) separate but overlapped species, F4 and FX (Solid Line), or (ii) as F4 alone (Dashed Line).

Fig. 3.

Sensitivity of TauD intermediates to ¹H/²H substitution. Data are shown as ¹⁶O - ¹⁸O isotopic differences observed at 1.5 s (Gray) along with simulated spectra (Black). Individual contributions of F4 (Red), FX (Blue), and baselines (Dashed Line) are shown. Intensities of individual spectra in both regions were adjusted for clarity. Experimental conditions are the same as in Fig. 2.

Fig. 4.

Sensitivity of TauD intermediates to ¹⁶O¹⁸O substitution. Intensities of isotopic difference spectra (Gray) between symmetrically ( or ) and asymmetrically (¹⁶O¹⁸O) labeled derivatives were normalized by using an internal standard, and the simulated spectra (Black) were obtained by using the results in Fig. 2. Experimental conditions are the same as in Fig. 3 except for an increased spectral resolution.

Fig. 5.

Effect of metal binding on oxygen vibrations of secondary alcohol derivatives. Infrared absorption spectra of (a) isopropanol, (b) Na⁺ isopropoxide, (c) Zn²⁺ diisopropoxide, and (d) Zn²⁺ monoisopropoxide complexes are shown as ¹⁶O - ¹⁸O isotopic difference. Assignment of O─C─C symmetrical and asymmetrical stretching modes is shown by arrows. The ν_OCCsm region in traces b and c is magnified for clarity.

Fig. 6.

Possible mechanisms of taurine oxygenation by TauD. The structure of the active site following O─O bond cleavage (A) is the Fe^IV═O species. According to the standard pathway, species A oxidizes substrate to form the Fe─OH complex (B), which is followed by radical rebinding to form an alcohol (C) and decomposition into products (D). Alternative pathways involve formation of an alkoxide (C^′) upon concerted (B → C^′) or stepwise (B → B^′ → C^′ deprotonation via a transient Fe^III─O^- species. Atoms originating from O₂ are grayed; α-ketoglutarate (R₁) and taurine (R₂) are partially abbreviated.