Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ

Abstract

A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out.

Figure 1. Characteristic spectra of SaHemQ in complex with hemes

(A) UV/vis spectra of SaHemQ in complex with ferric coproheme III (red), harderohemes III and IV (dark and light green, respectively), and heme b (blue) (5 µM coproheme, 10 µM harderoheme isomers and heme, 50 mM KPi, pH 7.4). The inset shows the visible bands on an expanded scale. (B) Low frequency rR spectra of SaHemQ in complex with the various hemes are shown in the same colors as in A. Spectra were obtained with 406.7 nm excitation of 20 µM samples in 50 mM NaPi, pH 6.8; the propionate and vinyl bending modes are labeled.

Figure 2. Spectroscopic changes upon titration of the SaHemQ-coproheme III complex with H₂O₂

Reactions were monitored by (A) UV/vis spectra following the addition of 0-10 eq H₂O₂ (2 eq increments) to 5 μM SaHemQ-coproheme III, 50 mM KPi, pH 7.4. The lack of clean isosbestic points through most of the spectrum is consistent with the involvement of more than two heme complexes in the reaction. (B) Low-frequency rR spectral titration of 15 mM SaHemQ-coproheme III under the same buffer conditions as A with 406.7-nm excitation. The labeling scheme used to describe the band labels in panels B and C and throughout the narrative is found in Scheme 1. (C) High-frequency range of the rR spectra shown in B. Molar equivalents of H₂O₂ are marked on the respective difference spectra. For A, B and C, spectra of the SaHemQ-coproheme III complexes are in red. Partially decarboxylated species are in purple with the final spectrum [λ_max (Soret) = 406 nm)] in blue. The difference spectra in B and C were generated by subtracting the spectrum of SaHemQ-coproheme III from the spectrum of the equilibrium reaction mixture recorded with the indicated molar eq of H₂O₂.

Figure 3. HPLC analysis of the products of the reaction of the SaHemQ-coproheme III complex with H₂O₂

(A) Representative HPLC traces showing protein-free products of the reaction with 0, 2, 8, 10, and 100 eq H₂O₂ (a–e) are plotted. Comparison of the retention times to standards (Figure S1B) identified harderoheme isomer III as the reactive intermediate. Note that the absorbance wavelength (400 nm) used for detection is closer to the λ_max for free coproheme III, which also has a higher extinction coefficient. (B) Quantitation of heme species involved in the HemQ reaction are plotted as percent of total heme versus eq H₂O₂ added: harderoheme (green triangles), coproheme (red squares) and heme b (circles). Points are averages of 3 values (standard deviations all within ± 15%). Solid lines are spline curves intended to qualitatively illustrate trends in the data.

Figure 4. Time resolved reactions between the SaHemQ-coproheme III and 12 eq H₂O₂

(A) Reaction monitored by UV/vis (8 μM enzyme, 50 mM KPi, pH 7.4). SaHemQ-coproheme III complex (red), spectra measured every 0.25 min (purple), and final spectrum (Soret band λ_max = 406 nm, blue) are shown. The absorbance at 394 nm versus time and fitted to a single exponential (k_Soret) is shown as an inset. (B) Reaction monitored discontinuously by HPLC following a chemical quench: red squares, coproheme III; green triangles, harderoheme III; blue circles, heme b. The data describing coproheme decay were fit to a single exponential equation (red line) to obtain a first order rate constant (k_c). The harderoheme III progress of reaction was fit (green line) to a two-term exponential decay function, which was used to determine t at maximum [harderoheme III]. A first order rate constant for harderoheme conversion to heme b (k_h) was computed as described in the text and SI. (C) Reaction (15 μM enzyme) monitored continuously by 406.7-nm excited rR scattering. Difference spectra recorded at the indicated times are plotted. The inset shows ΔΙ_t/ΔI_total versus time at 416 cm⁻¹ where increasing rR intensity reports vinyl formation; ΔI_t = change in rR intensity at time t, ΔI_total = total change in rR intensity over the course of the reaction. Red line: single-term exponential fit (first order rate constant k_v); open circles: residuals.

Figure 5. Titration of SaHemQ-coproheme III complex with peracetic acid

(A) HPLC analyses of the products of the reaction of the SaHemQ-coproheme III complex with 2, 8, or 10 eq of peracetic acid are shown. Full conversion of coproheme III to products followed addition of 10 eq of PAA. Comparison of the retention times to pure standards (Figure S1B) identified harderoheme isomer III as the reactive intermediate and heme b as the final product. Note that the absorbance wavelength (400 nm) used for detection is closer to the λ_max for free coproheme III, which also has a higher extinction coefficient. (B) Peracetic acid titration monitored by changes in the Soret (406.7 nm) excited rR spectrum. The spectrum for SaHemQ-coproheme III is red; partially decarboxylated complexes are shown in purple and the final spectrum is blue. Difference spectra were generated by subtracting the spectrum of SaHemQ-coproheme III from the spectrum obtained from the reaction mixture at equilibrium with the given molar equivalents of PAA.

Figure 6. Overlay of the structures of a representative heme b-bound chlorite dismutase (carbon green, PDB ID 3Q08) and solvent-bound HemQ (carbon cyan, PDB ID 1T0T).

(A) The monomeric subunit structures are very similar with the exception of a loop-helix region, highlighted in darker shades of blue/green. This region is located on the exterior of the HemQ homopentamer. (B) Key residues surrounding the heme b/coproheme III binding sites are indicated in this view overlooking the distal pocket. The placement of tetrapyrrole rings A-D in the expected orientation of the coproheme III is shown. K151, W155, N117, and Y113 (Cld numbering) are conserved in both HemQs and Clds and form contacts to the ring C/D propionates. Y113 (HemQ) is at the same sequence position as Y118 (Cld). It is part of the loop-helix region in HemQ. (C) Conserved active site Cld/HemQ residues (side view).

Scheme 1. Biosynthetic pathways leading to heme and other tetrapyrroles^†

^†The genes encoding catalysts for the canonical steps common to eukaryotes and many gram-negative bacteria are designated in blue. The pathway used by Archaea and sulfur-reducing bacteria are in orange. The recently proposed terminus of the pathway, found in gram-positive members of Actinobacteria and Firmicutes, is shown in purple. Note that HemF or HemN can catalyze the indicated step in different organisms under aerobic and anaerobic conditions, respectively. Similarly, HemJ, G, or Y catalyze the removal of 2 H-atoms ([H]).

Scheme 2. Substrate, possible intermediates, and product of the HemQ-catalyzed reaction.^‡

^‡Heme rings are labeled A-D and substituted pyrrole carbons 1-8. Vinyl and propionate carbons are labeled a-d.

Scheme 3

Possible reaction pathways following from Compound 0