Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ-lessons from the first crystal structure and kinetic studies

Abstract

Heme biosynthesis in Gram-positive bacteria follows a recently described coproporphyrin-dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme-HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV-vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five-coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H-bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H₂ O₂ molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (k_cat /K_M = 1.8 × 10² m^-1 ·s^-1 at pH 7.0) and occurs in a stepwise manner with a three-propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two-step reaction mechanism. Furthermore, the structure of coproheme-HemQ is compared to that of the phylogenetically related heme b-containing chlorite dismutases.

Database: Structural data are available in the PDB under the accession number 5LOQ.

Keywords: Gram-positive pathogens; HemQ; coproheme; heme b; heme biosynthesis.

Figure 1

Structure of coproheme‐LmHemQ. (A) Overlay of pentameric apo‐LmHemQ (4WWS, pale colors) and with pentameric coproheme‐HemLmQ (5LOQ, saturated colors) (B) Overlay of apo‐LmHemQ (4WWS: chainA) shown as a blue cartoon with all five subunits of coproheme‐LmHemQ (chain A: green, chain B: pink, chain C: cyan, chain D: yellow, chain E: red). R133 and R179 for apo‐LmHemQ are shown as blue sticks and for coproheme‐LmHemQ subunits as lines in the respective colors. Coprohemes are depicted in light gray. (C) Overlay as in (B) of the helix on the proximal side of the coproheme, where the proximal histidine (H174) and R179 are located.

Figure 2

Active site architecture of coproheme‐LmHemQ. (A) Distal residues and proximal histidine (H174) in all five subunits are depicted as green, pink, cyan, yellow, and red sticks. Residues of apo‐LmHemQ structure (4WWS) are presented in blue. Coproheme of all subunits is depicted as gray lines. (B) Distal and proximal residues of coproheme in chain A are represented as green sticks within the cartoon representation of the secondary structure. Electron density map (2mFo‐DFc) contoured at 1 σ is depicted as blue mesh.

Figure 3

Coproheme conformation and environment. Overlay of coproheme moieties bound to LmHemQ from all five subunits (top left), and H‐bonding interactions of coproheme propionates at positions 2 and 4 with the protein matrix in the respective subunits, residues within 3 Å of a propionate oxygen atom are depicted as sticks. The coordination of the proximal H174 to the coproheme iron is also shown.

Figure 4

Structural environment of p6 and p7. (A) Sequence alignment of LmHemQ and chlorite dismutases with available crystal structures. Residues with atoms within 3 Å of the propionate oxygen atoms of p6 are highlighted in green, of p7 in orange, and if both propionates are within the threshold it is highlighted in blue. The alignment was generated previously 9. Stick presentation of residues within 3 Å of p6 (B) and p7 (C) of chain A of coproheme‐LmHemQ.

Figure 5

UV‐vis and resonance Raman spectra of ferric and ferrous forms of coproheme‐LmHemQ and heme b‐LmHemQ in 50 mm phosphate buffer, pH 7.0. (A) UV‐vis and second derivative spectra of ferric coproheme‐LmHemQ and heme b‐HemQ. Heme b‐LmHemQ was formed by using a 5 : 1 stoichiometric excess of H₂O₂ to coproheme‐LmHemQ. (B) Resonance Raman spectra of ferric coproheme‐LmHemQ and heme b‐LmHemQ in the low frequency region showing the propionyl and vinyl bending modes. The inset shows the vinyl stretching modes region in polarized light. Experimental conditions: excitation wavelength 406.7 nm, laser power at the sample 5 mW; average of six spectra with 60‐min integration time (coproheme‐LmHemQ), average of four spectra with 40‐min integration time (heme b‐LmHemQ). (C) UV‐vis spectra of ferrous coproheme‐LmHemQ and heme b‐HemQ (D) Resonance Raman spectra of ferrous coproheme‐LmHemQ and heme b‐LmHemQ in the low frequency region showing the ν(Fe‐Im) stretching mode together with the propionyl and vinyl bending modes. Experimental conditions: excitation wavelength 441.6 nm, laser power at the sample 10 mW; average of six spectra (coproheme‐LmHemQ) and of three spectra (hemeb‐LmHemQ) with 30‐min integration time.

Figure 6

Enzymatic activity of coproheme‐LmHemQ mediated by H₂O₂. (A) Spectral conversion of 5 μm coproheme‐LmHemQ mediated by addition of 50 μm H₂O₂. The initial coproheme‐LmHemQ spectrum is depicted in black, the resulting final heme b‐LmHemQ spectrum in red. Selected spectra during conversion are shown in gray. The inset depicts time traces, extracted at 370 and 410 nm. (B) Kinetics of the H₂O₂‐mediated conversion of coproheme‐LmHemQ in 50 mm phosphate buffer, pH 7.0, are presented as Hanes plot.

Figure 7

Reaction of H₂O₂‐mediated coproheme conversion to heme b followed by electronic circular dichroism. (A) Spectral conversion of 18 μm coproheme‐LmHemQ mediated by stepwise titration with H₂O₂ (0–36 μm). Spectrum of coproheme‐LmHemQ is depicted as bold black line (0 μm H₂O₂), final spectrum as bold red line (36 μm H₂O₂). Bold green line, bold cyan line, bold pink line represent addition of 6, 18, and 27 μm H₂O₂, respectively. Thin lines in the respective color are at H₂O₂ concentrations lower than the one represented by a bold line (green: 3 μm; cyan: 9 μm, 15 μm; pink: 21 μm, 24 μm; red: 30 μm, 33 μm H₂O₂). The inset depicts the normalized changes in ellipticity at 388 (black) and 410 nm (orange) after each titration step versus the H₂O₂/coproheme‐LmHemQ ratio. (B, left panel) Time traces (black lines) of conversion of 30 μm coproheme‐LmHemQ to heme b‐LmHemQ followed by the change of ellipticity at 395 nm. Single exponential fits are depicted in red (first phase) and green (second phase). The time of H₂O₂ addition is indicated as gray line, spectra were shifted for clarity. (B, right panel) Plots of k _obs1 (red) and k _obs2 (green) versus H₂O₂ concentration with linear fits. Conditions: 50 mm phosphate buffer, pH 7.0.

Figure 8

Stoichiometry of the enzymatic activity of coproheme‐LmHemQ mediated by H₂O₂. (A) UV‐vis absorption spectra recorded following the stepwise titration of 18 μm coproheme‐LmHemQ with H₂O₂ (0–36 μm). (B) Plot of normalized absorbance changes at 375 (black), 395 (orange), and 410 nm (cyan) after each titration step versus the H₂O₂/coproheme‐LmHemQ ratio (including sigmoidal fits). (C) HPLC profiles and mass spectrometric analysis of samples from the titration experiment described in (A); coproheme (708.19 Da, black), monovinyl, monopropionate deuteroheme (662.18 Da, green), heme b (616.18 Da, red). (D) Relative amounts of the three porphyrin species (area under curve of HPLC profiles) at each H₂O₂/coproheme‐LmHemQ ratio. (E) Whole protein analyses of the samples from the titration experiment described in (A) and (C). (F) Relative amounts of LmHemQ without cross‐linked heme (black, 31 977.2 Da) and cross‐linked heme (red, 32 590.5) depending on the [H₂O₂]/coproheme‐HemQ ratio. Conditions: 50 mm phosphate buffer, pH 7.0.