Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism

Abstract

Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins.

Fig 1. Crystallographic structure of the ferric heme LepHO complex.

A) Cartoon representation of the LepHO-C26S-stop structure. B) Arrangement of the most relevant amino acid residues involved in heme binding to LepHO. C) Overall structural overlay of LepHO (cyan) with the HO-1 (PDB ID: 1WE1, green) from Synechocystis sp. PCC6803. D) Final mF_o−DF_c electron density map (green mesh) around heme ligand contoured at the 3σ level. The two orientations of the heme are displayed in yellow and light brown stick, respectively with oxygen atoms in red, and nitrogen atoms in blue. The Fe atoms are represented as orange spheres. Water molecules are depicted as blue spheres, and the heme ligand is represented in sticks. The amino acid residues in the recombinant LepHO are numbered according to the sequence of the wild type enzyme. The methionine in position 8 (numbers in parenthesis) of the recombinant protein has been labeled as 1, and so on for the subsequent amino acids. The recombinant protein contains an extra amino-terminal sequence (GHMASGS) which has remained from the construct for expression and purification of the protein.

Fig 2. LepHO heme binding pocket and hydrogen bond network.

A) Distinctive hydrophobic residues facing the α-meso carbon atom of heme in LepHO. B) LepHO residues, water molecules (blue spheres) and coordination bonds (broken lines) involved in the hydrogen bond distal site network. C) Detailed view of a LepHO structure in surface representation, where it can be seen that the hydrogen bonded network of structural waters reaches the surface of the protein and suggests a possible proton entry site.

Fig 3. Optical absorption spectra of the purified wild type LepHO, F157I and F157A variants.

Spectra of the different LepHO variants purified by metal affinity chromatography before (A) and after (B) ferric heme complex formation: wild type LepHO (―); F157A (…) and F157I (---).

Fig 4. Absorption spectral changes of the LepHO-heme complex during the NADPH/LepFNR-supported heme degradation.

Time dependent absorption spectra of wild type LepHO (A), F157I (B) and F157A (C), before (---) and after (―) the addition of LepFNR and NADPH: Experimental conditions are as indicated in Materials and methods. The inset shows an enlargement of the spectral region between 500 and 800 nm. The time-dependent decay of the intensity at 403 nm (D) and the increase at 680 nm (E) were obtained from the spectra shown in panels (A) to (C). Wild type LepHO (●); F157I (▼) and F157A (○).

Fig 5. Reduction of the ferric LepHO-heme complex in anaerobic conditions and subsequent autooxidation in air.

Absorption spectral changes of 6 μM wild-type LepHO (A) or F157I mutant (B) before (---) and after the addition of 1.2 μM LepFNR (―) in the presence of NADPH. Autoxidation of the ferrous LepHO-heme complex in air. Wild type LepHO (C) and F157I mutant (D) before (---) and after bubbling with O₂ (―).

Fig 6. Reduction of the ferric LepHO-heme complex in anaerobic conditions and spontaneous reoxidation.

Time dependent formation (A) and autoxidation (B) of the ferrous heme complex of wild type LepHO (●) and F157I mutant (○) as monitored by variations in absorbance at 426 and 403 nm, respectively. Data extracted from the spectra shown in Fig 5.

Fig 7. Conversion of ferric LepHO-heme complex to verdoheme by addition of H₂O₂.

Time dependent heme hydroxylation of wild type LepHO (●) and F157I mutant (○) by H₂O₂ in aerobic conditions as monitored by increase in absorbance at 671 nm (A) or under anaerobiosis as monitored by decrease in absorbance at 403 nm.

Fig 8. Models of the complex between LepHO and LepFNR and distal modulation of the LepHO activity.

Electrostatic surface potential of LepFNR (A) and LepHO (B) as obtained by using the PDB2PQR service [51], the APBS plugin [52] and Pymol 1.8. The color scale was set from -4 kT/e (red) to +4 kT/e (blue). Proteins were slightly rotated to show the surfaces involved in complex formation. Putative model of the complex LepHO-LepFNR (C) obtained with ClusPro 2.0 [53]. The CD-loop homologous to that found in human HO-1 [54] is depicted in magenta (D). The region containing F157 (red) is presented in light blue and heme in light brown.