Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates

Abstract

Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD−HPCA and H200N−HPCA, respectively) with O(2). A blue intermediate forms within 20 ms of mixing of O(2) with H200N−HPCA (H200N(Int1)(HPCA)). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin Fe(III) (S = 5/2) antiferromagnetically coupled to a radical (S(R) = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200N(Int1)(HPCA) decays over the next 2 s, possibly through an Fe(II) intermediate (H200N(Int2)(HPCA)), to yield the product and the resting Fe(II) enzyme. Reaction of FeHPCD−HPCA with O(2) results in rapid formation of a colorless Fe(II) intermediate (FeHPCD(Int1)(HPCA)). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCD(Int1)(HPCA) suggests it is an intermediate occurring after O(2) activation and attack. The similar Mössbauer parameters for FeHPCD(Int1)(HPCA) and H200N(Int2)(HPCA) suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O(2) via the iron does occur, leading to aromatic ring cleavage.

Figure 1

H200N-HPCA + O₂ reaction monitored by stopped-flow. Panel A shows diode array spectra recorded between 3 ms and 2 s after mixing stoichiometric 640 μM (sites) H200N-HPCA anaerobic complex with O₂-saturated buffer (~ 1.8 mM) (1:1) at 4 °C in 200 mM MOPS pH 7.5 (2 mm pathlength). The thick line spectra are for the specific times shown. Thin line cyan, 3 – 32 ms; gray, 32 ms – 2 s. The inset shows spectrum that results from subtracting the spectrum at 3 ms from that at 32 ms (cyan) and the spectrum of HPCA quinone produced by treatment of HPCA with mushroom tyrosinase (blue, dashed). Panel B shows the time course of the same reaction as in Panel A, but in 200 mM MES buffer pH 5.5. Inset: Comparison of the spectra of the 610 nm feature formed at pH 5.5, 7.5, and 9.0.

Figure 2

Time dependent parallel mode EPR spectra from the H200N-HPCA + O₂ reaction. Parallel mode EPR spectra in the g = 8 region of RFQ samples frozen between 0 (top, unreacted) and 2 s after mixing 1.65 mM anaerobic H200N-HPCA complex with O₂-saturated buffer (1:1) at 4 °C in 200 mM MOPS buffer, pH 7.5 are shown. EPR conditions: Frequency 9.35 GHz; microwave power, 50.4 mW; modulation amplitude, 1 mT, and temperature 50 K. Inset: Time course of the g = 8.2 signal intensity (•). The solid line is the fit to the optically monitored time course at 610 nm from Figure S1.

Figure 3

Parallel-mode EPR spectra (colored) and simulations (black) for H200N-HPCA frozen 40 ms after reaction with O₂ at temperatures of (A) 10 K and (B) 2 K. Prior to mixing with O₂-saturated buffer: 1.5 mM H200N-HPCA, 200 mM MOPS buffer pH 7.5. Simulation parameters: A: S₁ = 5/2, S_R=1/2, J = +25 cm^-1, D₁ = 1 cm^-1, E/D₁ = 0.12, g_1z = 2.015, g_Rz = 2.00. B: S = 2, D = -4 cm^-1, E/D = 0.15, g_z = 2.00. EPR conditions: microwaves, 20 mW (A), 0.2 mW (B) at 9.29 GHz; modulation, 1.0 mT. The intensity of B has been reduced by a factor of 2. The inset shows a plot of signal times temperature for the g = 8.2 feature and a theoretical fit to this intensity.

Figure 4

4.2 K zero field Mössbauer spectra of H200N-HPCA complexes. (A) H200N, 0.9 mM at pH 7.5 in 200 mM MOPS buffer. The colored line is a simulation. (B) Stoichiometric (sites) H200N-HPCA complex, 1.54 mM, at pH 7.5 in 200 mM MOPS buffer. Simulations of the doublets of the two enzyme-substrate complexes, H200N_ES1^HPCA (green) and H200N_ES2^HPCA (blue), are depicted by the colored lines. The black line represents the sum of the two species. (C-E) Reaction of 1.48 mM H200N-HPCA with O₂-saturated buffer (1:1). For the RFQ samples quenched at (C) 20 ms and (D) 400 ms, we have outlined in red the spectrum of the intermediate, H200N_Int1^HPCA. (E) Sample frozen (not by RFQ) at 3 min representing the end of the reaction. The colored line is a simulation of the spectrum. (F) B = 0 spectrum of the RFQ sample of Figure 5 prepared in a different experiment under the same conditions.

Figure 5

4.2 K Mössbauer spectra of H200N_Int1^HPCA recorded in parallel applied magnetic fields of (A) 4.0 T, and (B) 8.0 T. The central features of the spectra have unresolved contributions from the “splashed” ferrous contaminant. The red lines, drawn to represent 70 % of total Fe, are WMOSS spectral simulations based on eq 1 using the parameters listed in Table 1. This is the sample from Figure 4F.

Figure 6

(A) EPR spectra of isotopically enriched H200N_Int1^HPCA prepared with (red) natural abundance isotopes, (blue) 70% enriched ¹⁷O_2, (green) HPCA enriched to 68% with ¹⁷O at the C3 OH functional group. Samples were prepared as described in Figure 2. Conditions before mixing: 1.65 mM H200N-HPCA complex, saturated O₂-containing buffer, 200 mM MOPS pH 7.5 and 4 °C. EPR conditions: Frequency 9.29 GHz; microwave power, 20 mW; modulation, 1.0 mT, temperature, 10 K. The simulations of natural abundance (B) and 70% enriched ¹⁷O₂ (C) samples are for S = 2, D = 1.3 cm^-1, E/D = 0.12, with A_z^O,c of 0 (B) or 17 MHz (C).

Figure 7

FeHPCD-HPCA + O₂ reaction monitored by stopped-flow. Diode array spectra recorded between 4 ms and 2 s after mixing 100 μM (based on active sites) stoichiometric, anaerobic FeHPCD-HPCA complex with O₂ saturated buffer (~1.8 mM) at 4 °C in 200 mM MOPS pH 7.5 (2 mm pathlength). Inset: Reaction monitored by single wave stopped-flow spectroscopy at 380 nm. In the time range shown, the data (black line) can be fit to a sum of two exponential terms (red line) with the reciprocal relaxation times shown.

Figure 8

Mössbauer spectra from the FeHPCD-HPCA + O₂ reaction recorded at 4.2K for B = 0. (A): FeHPCD, (B): The anaerobic FeHPCD-HPCA complex, (C): FeHPCD-HPCA + O₂ at 15 ms after mixing with O₂, (D): FeHPCD-HPCA + O₂ at 65 ms after mixing with O₂, (E): 5 min after mixing. Conditions before mixing: ~ 1.8 mM enzyme-substrate complex, 200 mM MOPS, pH 7.5, and 4 °C all reactions. For (A) the sample concentration was ~ 0.9 mM.

Scheme 1. Proposed Reaction Mechanism for Extradiol Dioxygenases

For the studies described here, R is either –CH₂COO^- (HPCA) or -NO₂ (4NC). In the case of 4NC, the substrate is fully deprotonated, so the proton required for catalysis must derive from solvent.

Scheme 2

Kinetic Model from Stopped-flow Studies of the H200N-HPCA + O₂ Reaction.

Scheme 3. Intermediates from the FeHPCD and H200N Turnover Cycles

The intermediates shown are those proposed for the FeHPCD reaction with HPCA as the substrate. The colored boxes identify trapped intermediates that either correlate exactly with or are similar to these intermediates. The boxes with solid borders identify intermediates from solution RFQ studies reported here and in a previous study, while those with dashed borders are from crystal structures reported previously.