Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes

Abstract

Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2μM (circular dichroism spectroscopy) and 16.8μM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis.

Keywords: Chlorite dismutase; HemQ; Heme binding; Heme biosynthesis; Protein stability; X-ray crystallography.

Graphical abstract

Fig. 1

Subunit size and oligomeric structure in solution. SDS–PAGE (A), Western blot (B) of purified LmCld. (C) HPLC-chromatogram of holo-LmCld (red), apo-LmCld (black) and Bio-Rad Gel Filtration Standard (#151-1901) (gray), chromatograms are shifted for clarity. UV–vis spectra of peaks eluted at 11.0 (pink), 13.3 (green), and 15.9 min (black) of holo-LmCld (D) and apo-LmCld (E). Multi-angle-light-scattering (MALS) data of holo-LmCld (F) and apo-LmCld (G), molecular mass distributions over the elution peak of the samples are represented as dashed lines, average molar masses are written next to respective size distributions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Crystal structure of apo-LmCld. (A) Overlay of pentameric apo-LmCld (green) and pentameric holo-NdCld (pdb-code: 3NN1) (yellow). (B) Overlay of the heme-binding sites of apo-LmCld (green) and holo-NdCld (yellow); residue-labels of holo-NdCld are underlined. Ribbon representation of apo-LmCld (C) and holo-NdCld (E); for better orientation the heme group of holo-NdCld was placed to the heme binding site of apo-LmCld (gray). Representation of the surfaces of apo-LmCld (D) and holo-NdCld (F). Substrate-channel forming secondary structural elements of holo-NdCld and respective residues of apo-LmCld are depicted in red (C–F). Figures were generated using PyMOL (http://www.pymol.org/). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Hemin binding to apo-LmCld. Hemin binding followed spectroscopically by ECD and UV–vis (A and B) and calorimetrically by ITC (C). 35 μM apo-LmCld were titrated with hemin up to a molar ratio of 1.0 (A and B) and 2.5 (C) in 100 mM phosphate buffer, pH 7.0. (A) represents ECD spectra, the starting spectrum is shown in black, the resulting spectrum in red, intermediate spectra in gray, free hemin is depicted in cyan, the inset shows intensities at the ECD absorption minimum of (black dots); the sigmoidal fit is depicted as a red line. (B) shows UV–vis absorption spectra of the same experiment as in (A); spectra on the left are uncorrected measured spectra, spectra on the right side are corrected for excess hemin; the inset shows intensities at the absorption maximum (black dots) and a sigmoidal fit (red line). (C) depicts exotherms derived from each injection of the titrant, hemin, to the protein; the inset shows the plot of the integrated peak areas from the exotherms (black dots) with a sigmoidal fit (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Effect of pH on heme binding to apo-LmCld. (A) shows 3 μM holo-LmCld at different pH values; conditions: 50 mM citrate/phosphate buffer, pH 4.0–7.0; 50 mM phosphate buffer, pH 6.5–8.0; 50 mM glycine buffer, pH 7.5–10.0. The inset depicts the pH dependence of the Reinheitszahl (R_Z) of holo-LmCld; black dots are in absence of and gray triangles in presence of 150 mM NaCl. In (B)–(D) 4 μM holo LmCld were present in 5 mM phosphate buffer pH 7.0 and diluted rapidly using stopped-flow technique with 100 mM citrate/phosphate buffer, pH 4.0 (B), 100 mM phosphate buffer, pH 7.0 (C), and 100 mM glycine buffer, pH 10.0 (D). The starting spectra are depicted in black, spectra after 1 ms in cyan and resulting spectra in red, intermediate spectra are represented in gray. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Thermal stability of LmCld. ECD spectra of 12 μM holo-LmCld at (A) pH 7.0 (50 mM phosphate buffer), (B) pH 10.0 (50 mM glycine buffer), and (C) pH 4.0 (50 mM citrate/phosphate buffer), at 20 °C (black lines), after heating to 85 °C (red lines), and after cooling again to 20 °C (green lines). (D) Temperature mediated unfolding of 12 μM holo-LmCld followed at 222 nm at pH 4.0 (black), pH 7.0 (red), and pH 10.0. (E) DSC thermograms of 35 μM apo-LmCld (black) and 35 μM holo-LmCld (red) in 50 mM phosphate buffer, pH 7.0. The inset depicts UV–vis spectra of samples used in the DSC experiment; apo-LmCld (black), holo-LmCld (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Reversibility of heme binding and dissociation. UV–vis spectral transitions when heating (A) from 25 °C and 70 °C and cooling from 70 °C to 25 °C (C) of 4 μM holo-LmCld. Plot of the Reinheitzahl (B) and A_Soret/A₃₈₀ ratio (D) during heating (black dots and labels) and cooling (gray dots and labels). (E) ECD spectra of 6 μM holo-LmCld at 20 °C (black), 90 °C (red), and 20 °C after cooling (green); (F) Temperature mediated release of the prosthetic group followed at the Soret minimum at 414 nm (black line) with its sigmoid fit (red line). Conditions: 100 mM phosphate buffer, pH 7.0. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Heme transfer of LmCld to apomyoglobin. The transfer of the prosthetic group of 1 μM holo-LmCld to 5, 7.5, 10, and 15 μM apo-Mb was observed by the increase in absorbance at 409 nm. (A) Time traces when 1 μM holo-LmCld was mixed with 5–15 μM apo-LmCld in 100 mM phosphate buffer, pH 7.0. Double exponential fits are depicted in gray. The inset shows the linear dependence of k_obs1 values from the apo-Mb concentration. The apparent association rate constant, k_on, was calculated from the slope and the apparent dissociation rate constant, k_off, from the intercept. (B) UV–vis spectra of undiluted holo-LmCld (black) prior to the measurement and one representative spectrum of the mixture of LmCld and Mb after 1200 s (red). Holo-Mb is depicted as gray dashed line for comparison. The inset depicts holo-Mb and apo-Mb after heme extraction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Cyanide binding to LmCld. Transient-state kinetics of binding of cyanide to holo-LmCld. (A) Spectral changes upon reaction of 3 μM ferric holo-LmCld with 1 mM cyanide measured in the conventional stopped-flow mode. (B) Typical time trace at 412 nm with a single-exponential fit. (C) Linear dependence of k_obs values from the cyanide concentration. (D) UV–vis spectra of 8 μM ferric (black solid line), ferrous (red solid line), cyanide-bound ferric (black dotted line) and cyanide-bound ferrous (red dotted line) of holo-LmCld. Conditions: 100 mM phosphate buffer, pH 7.0; 10 mM sodium dithionite (freshly prepared). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)