A Long-Lived Fe(III)-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mössbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods

Abstract

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mössbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.

Figure 1

Long-lived Int1 intermediate formed during the H200C-HPCA reaction with O₂ monitored by stopped-flow spectroscopy. H200C-HPCA (440 μM) was mixed with O₂ gas-saturated buffer (1.8 mM) in 200 mM MOPS, pH 7.5 at 4 °C. Spectra were recorded between 1.26 ms and 100 s with pathlengths of 2 mm. Spectra in bold are as follows: H200C-HPCA complex, red; Int1, blue; final ring-opened product species, purple. Inset: Reduced H200C HPCD (400 μM)-HPCA (200 μM) was mixed with O₂ gas-saturated buffer (1.8 mM) in 200 mM MOPS, pH 7.5 and monitored at 610 nm using a path length of 10 mm at 4 °C. Enzyme without substrate bound does not react with O₂.

Figure 2

Comparison of active sites of the WT HPCD and the H200C variant in complex with HPCA. Structure overlay of the WT HPCD (PDB 4GHG) and H200C (PDB 5BWH) structures. Atom color code: gray, carbon (HPCD); yellow, carbon (H200C); dark blue, nitrogen (HPCD); blue, nitrogen (H200C); dark red, oxygen (HPCD); red, oxygen (H200C); green, sulfur (H200C); bronze, iron (HPCD); purple, iron (H200C). Cartoons depict secondary structure elements for the H200C variant (gray) and HPCD (light blue). WatA-C represent crystallographically observed (not metal-coordinated) solvent in the active site.

Figure 3

(A) Zero-field, 4.2 K Mössbauer spectrum (black) of H200C-HPCA (simulation, red curve) and the minority species with ΔE_Q = 2.32 mm/s and δ = 1.20 mm/s (magenta curve). The spectra shown in panels A and B are raw data. Spectra shown in panels C and D were obtained by removing the high-spin ferric and high-spin ferrous impurities. (B, C) Spectra of the oxygenated intermediate, H200C-HPCA_Int1 = Int1, recorded at 4.2 K in parallel applied magnetic fields as indicated. The arrow in (B) points at the high-energy line of a doublet attributed to resting enzyme. (D) B = 0.2 T spectrum recorded at 10 K. The spectra in (C) and (D) contain a 6-line pattern (green curve) associated with the excited M_S = ±2 doublet of the S = 2 multiplet. The absorption of this feature increases with increasing temperature, showing that D > 0. The dominant contributions of the central feature, blue in (C), are from the M_S = 0 state and M_S = ± 1 doublet.

Figure 4

4.2 K Mössbauer spectra of Int1 recorded in variable, parallel applied magnetic fields of (A) 0.2 T, (B) 0.5 T, (C) 1.0 T, (D) 2.0 T, and (E) 3.0 T. The black hash-mark curves are the spectra that result after subtraction of simulations for the minority Fe^II (6%) and Fe^III (8%) contaminants from the raw data. The Fe^III contaminant was simulated with parameters given in the caption of Figure S4. Red lines are spin Hamiltonian simulations using the parameters listed in Table 1.

Figure 5

Parallel mode X-band EPR spectra of H200C-HPCA_Int1 recorded at 21 K. (A) ¹⁶O Int1, black curve. The red curve is an S = 2 SpinCount simulation based on eq 3 using D = +1.6 cm⁻¹, E/D = 0.055, g_z = 2.01, σ(E/D) = 0.005 and 0.57 mT packet line width. (B) Comparison of spectra of the ¹⁶O sample of (A) (black) with a sample of Int1 enriched with ~ 70% ¹⁷O₂ (purple curve). The purple curve has been scaled to match the peak amplitude of the g = 8.04 feature of the ¹⁶O sample. (C) Experimental spectrum of the ¹⁷O₂ enriched sample (black) with simulations for A_eff (¹⁷O) = 7 MHz (red), 17 MHz (blue), and 30 MHz (green). Conditions: 9.37 GHz frequency, 20 mW nonsaturating microwave power, 1 mT modulation, T = 21 K.

Figure 6

(Signal × T) vs T plot of Int1 obtained from analysis of the variable temperature parallel mode EPR signal at g = 8.04 shown in Figure S6.

Figure 7

Structural candidates for Fe^III complex Int1.

Figure 8

DFT models for Int1. Panel (A) shows Model I, and includes second- and third-sphere residues Y257, H248, N157, C200, W192, R243, R293, and three crystallographic waters (WatA-C) adjacent to the active site (PDB 5BWH). Panel (B) shows Model II for Int1; the residues R243, R293, and two of the crystallographic waters (WatA and WatB) have been removed. Panel (C) shows Model III in which the substrate acetic acid side chain has been truncated and is replaced by a methyl group. Hydrogen atoms have been omitted for clarity.

Figure 9

Spin density plot for the OOH conformation (Figure 7, right) of Model III. Regions of blue correspond to spin-up density, and regions of green correspond to spin-down density. Note that O_C1 and O_C2 (in the foreground) display both spin-up and spin-down densities. Hydrogens are not shown for clarity.

Figure 10

A_eff vs α calculated for O_C1 and O_C2 of the HPCA substrate, and O_proximal and proton carrying O_distal of the hydroperoxo ligand. The green dashed line indicates the experimentally determined upper limit for A_eff for the hydroperoxo oxygens. The plot was prepared using β = 70°. The DFT-calculated principal components of A(¹⁷O_proximal) are [−5.8, −16.2, −21.0] MHz. Angles α and β position z’ of the EFG in the x,y,z frame of the ZFS tensor. Figure S8 shows the direction of the unique axis relative to the DFT structure.

Figure 11

Diagram depicting the DFT-calculated transformation from a superoxo species to Int1 as described by transfer of a proton and an electron from the substrate to the superoxo ligand (proton-coupled electron transfer, PCET). The water molecule indicated is crystallographic WatC. Solid and dashed lines indicate the lowest spin septet and quintet states, respectively, for each conformation. Atoms highlighted in red are involved in hydrogen bonding interactions that affect the transfer. Energies on the left and right frame refer to the energies of the S = 3 states.

Figure 12

(A) Schematic depicting the crossing of the donor and acceptor levels. (B) Contour plot of the orbital of the transferring spin-down electron at the level crossing where the populations of this orbital at the donor and acceptor are equal.

Scheme 1

Proposed Oxygen Activation Pathway for the H200C-HPCD Variant That Involves Conversion of a Ferric Superoxo Intermediate to a Distal-Protonated Hydroperoxo, Semiquinone Radical Intermediate via Inner-Sphere PCET. ^a In the ES complex and superoxo intermediate the substrate is protonated at O_C1 (red superscript H).