Characterization of the arene-oxidizing intermediate in ToMOH as a diiron(III) species

Abstract

We report the generation and characterization of a diiron(III) intermediate formed during reaction with dioxygen of the reduced hydroxylase component of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1. The decay rate of this species is accelerated upon mixing with phenol, a substrate for this system. Under steady-state conditions, hydrogen peroxide was generated in the absence of substrate. The oxidized hydroxylase also decomposed hydrogen peroxide to liberate dioxygen in the absence of reducing equivalents. This activity suggests that dioxygen activation may be reversible. The linear free energy relationship determined from hydroxylation of para-substituted phenols under steady-state turnover has a negative slope. A value of rho < 0 is consistent with electrophilic attack by the oxidizing intermediate on the aromatic substrates. The results from these steady and pre-steady-state experiments provide compelling evidence that the diiron(III) intermediate is the active oxidant in the toluene/o-xylene monooxygenase system and is a peroxodiiron(III) transient, despite differences between its optical and Mössbauer spectroscopic parameters and those of other peroxodiiron(III) centers.

Figure 1

Mössbauer spectra of freeze-quenched samples from the reaction of reduced ToMOH:2ToMOD mixtures with O₂. The samples were frozen before mixing (A) and 0.14 s (B), 2 s (C), and 32 s (D) after mixing. The spectra (vertical bars) are collected at 4.2 K in a 50 mT field applied parallel to the γ-ray beam. The red and orange lines are simulated spectra of the diiron(III) intermediate and diiron(III) product, respectively. The spectrum of the diiron(III) intermediate (red) was modeled with a single quadrupole doublet with δ = 0.55 mm/s, ΔE_Q = 0.67 mm/s and line width = 0.27 mm/s. The spectrum of the diiron(III) product (orange) was simulated as a superposition of two equal intensity quadrupole doublets with parameters δ = 0.52 mm/s, ΔE_Q = 0.73 mm/s and line width = 0.32 for doublet 1 and δ = 0.52 mm/s, ΔE_Q = 0.97 mm/s and line width = 0.28 mm/s for doublet 2. They are plotted at the following absorption intensities: red, 44%, 41% and 13% in B, C, and D; orange, 41% in D. Unreacted diiron(II) protein accounts for ~ 56% of total Fe absorption at maximal accumulation of the intermediate (B). These parameters and intensities are the results of a global analysis of the entire set of the Mössbauer spectra including those that are not depicted here.

Figure 2

Overlay of Mössbauer spectra of the diiron(III) intermediate (red) and product (orange). The spectrum for the intermediate (red vertical bars) was prepared by removing the 56% diiron(II) contribution from the raw spectrum of the 0.14 s freeze-quenched sample (Figure 1B), and the product spectrum (orange vertical bars) was the spectrum of as-purified oxidized ToMOH. For comparison the two spectra are scaled to matching intensities. The solid lines are theoretical spectra simulated with parameters of the Fe species reported in the caption of Figure 1.

Figure 3

Speciation plot for reaction of diiron(II) ToMOH:2ToMOD with dioxygen. Reduced diiron(II) protein ( ) reacts with dioxygen at ~ 26 s⁻¹ to form a diiron(III) transient species ( ). Only ~45% of diiron(II) clusters give rise to this intermediate. The diiron(III) transient subsequently decays to the oxidized resting state ( ) at ~ 0.045 s⁻¹. The unreacted diiron(II) centers decay slowly at 0.02 s⁻¹ to generate several ill-defined ferric species, the production of which is not depicted. Solid lines are one- or two-exponential fits to the data.

Figure 4

Mössbauer spectra of double-mixing RFQ samples for reaction of the diiron(III) intermediate with buffer containing phenol. Reduced protein was allowed to react with O₂ for 0.17 s to generate the intermediate before mixing with phenol (A). After mixing with phenol, the reaction mixture was freeze-quenched at 0.14 s (B), 0.67 s (C), 5 s (D) and 100 s (E). The spectra (vertical bars) are collected at 4.2 K in a 50 mT applied field parallel to the γ-ray beam. The green, red and orange lines are simulated spectra, respectively, of the unreacted diiron(II) protein, diiron(III) intermediate and diiron(III) product. The spectra of the diiron(III) intermediate in the absence of phenol (red in A) and diiron(III) product (orange) were simulated using the parameters reported in the caption of Figure 1. The spectrum of the diiron(III) intermediate was perturbed slightly by the presence of phenol (see the following Figure 6) and was simulated with the altered parameters reported in text (red lines in B-E). The spectrum of the unreacted diiron(II) was modeled as a superposition of two quadrupole doublets with an intensity ratio of 10 to 1, and the following parameters: δ= 1.32 mm/s, ΔE_Q = 3.06 mm/s for the intense doublet, and δ = 1.39 mm/s and ΔE_Q = 2.13 mm/s for the minor doublet. The simulated spectra are plotted at the following absorption intensities: green, 61%, 60%, 58%, 57% and 29%; red, 39%, 30%, 14%, 0% and 0%; orange, 0%, 12%, 28%, 43% and 71% in A, B, C, D and E, respectively. The black lines overlaid with the spectra are composite spectra including all the Fe species mentioned above.

Figure 5

Speciation plot for reaction of the diiron(III) intermediate with phenol. The intermediate ( ) decays at 2 s⁻¹ to give rise to the early formation phase of diiron(III) product ( ). Diiron(II) clusters ( ) that do not traverse the intermediate oxidize more slowly, with a rate constant of ~0.01 s⁻¹ to product. Solid lines represent one- or two-exponential fits to the data.

Figure 6

Overlay of Mössbauer spectra of the diiron(III) intermediate in the absence (red) and presence (black) of phenol. These spectra were prepared by removing the unreacted diiron(II) contributions from the spectrum shown in Figure 4A and spectrum of a sample freeze-quenched at 70 ms after mixing with phenol. Addition of phenol results in smaller values for δ and ΔE_Q as compared to the intermediate generated in the absence of substrate (see text for parameters). Absorption lines also broaden in the presence of phenol by 0.03 and 0.06 mm/s, for low- and high-energy lines, respectively.

Figure 7

Optical spectrum of a sample following an RFQ double-mixing Mössbauer experiment. The broad absorption band is centered at ~ 580 nm.

Figure 8

Concentration of hydrogen peroxide evolved under steady-state conditions with () and without (•) phenol. Hydrogen peroxide is formed slowly as NADH is consumed by the enzyme system. The addition of substrate inhibits peroxide formation. Data were fit with hyperbolic functions.

Figure 9

Change in the concentration of hydrogen peroxide with time after mixing with ToMOH–ToMOD mixtures. Hydrogen peroxide is rapidly consumed and evolved dioxygen in a manner independent of the presence of substrate. A peroxide shunt pathway was not accessed because catechol was not observed in assay solutions that contained phenol.

Figure 10

Hammett plots for oxidation of p-substituted phenols by ToMO. The values of ρ were −1.3 and −1.7 for σ_m (■) and σ⁺ ( ) respectively. A negative reaction constant is indicative of electrophilic reactions. The goodness of fit as measured by R² for either σ value is 0.81.

Scheme 1

Dioxygen Activation at Iron-Heme and CBDI Centers

Scheme 2

Proposed Pathways for Substrate Hydroxylation

Scheme 3

Reaction of ToMOH_ox with Hydrogen Peroxide

Scheme 4

Substrate Hydroxylation and Dioxygen Activation by ToMOH