The Hemophore HasA from <i>Yersinia pestis</i> (HasA_yp) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change

Hemophores from <i>Serratia marcescens</i> (HasA_sm) and <i>Pseudomonas aeruginosa</i> (HasA_p) bind hemin between two loops, which harbor the axial ligands H32 and Y75. Hemin binding to the Y75 loop triggers closing of the H32 loop and enables binding of H32. Because <i>Yersinia pestis</i> HasA (HasA_yp) presents a Gln at position 32, we determined the structures of apo- and holo-HasA_yp. Surprisingly, the Q32 loop in apo-HasA_yp is already in the closed conformation, but no residue from the Q32 loop binds hemin in holo-HasA_yp. In agreement with the minimal reorganization between the apo- and holo-structures, the hemin on-rate is too fast to detect by conventional stopped-flow measurements

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182535#pone.0182535.g005" target="_blank">Fig 5</a>.</p

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

<p>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.</p

Crystallographic structure of the ferric heme LepHO complex.

<p>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: <u><b>1WE1</b></u>, green) from <i>Synechocystis</i> sp. PCC6803. D) Final m<i>F</i>_o−D<i>F</i>_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.</p

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

<p>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 (○).</p

LepHO heme binding pocket and hydrogen bond network.

<p>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.</p

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

<p>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 (---).</p

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

<p>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 (―).</p

Replacing the Axial Ligand Tyrosine 75 or Its Hydrogen Bond Partner Histidine 83 Minimally Affects Hemin Acquisition by the Hemophore HasAp from <i>Pseudomonas aeruginosa</i>

Hemophores from <i>Pseudomonas aeruginosa</i> (HasAp), <i>Serratia marcescens</i> (HasA_sm), and <i>Yersinia pestis</i> (HasA_yp) bind hemin between two loops. One of the loops harbors conserved axial ligand Tyr75 (Y75 loop) in all three structures, whereas the second loop (H32 loop) contains axial ligand His32 in HasAp and HasA_sm, but a noncoordinating Gln32 in HasA_yp. Binding of hemin to the Y75 loop of HasAp or HasA_sm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasA_yp is already in the closed conformation, such that binding of hemin to the conserved Y75 loop occurs with minimal structural rearrangement and without coordinative interaction with the Q32 loop. In this study, structural and spectroscopic investigations of the hemophore HasAp were conducted to probe (i) the role of the conserved Tyr75 loop in hemin binding and (ii) the proposed requirement of the His83–Tyr75 hydrogen bond to allow the coordination of hemin by Tyr75. High-resolution crystal structures of H83A holo-HasAp obtained at pH 6.5 (0.89 Å) and pH 5.4 (1.25 Å) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for N_δ of His83 to interact with the O_η atom of Tyr75, likely stabilizing the Tyr75–Fe interaction. Nuclear magnetic resonance spectroscopy revealed that in apo-Y75A and apo-H83A HasAp, the Y75 loop is disordered, and that disorder propagates to nearby elements of secondary structure, suggesting that His83 N_δ–Tyr75 O_η interaction is important to the organization of the Y75 loop in apo-HasA. Kinetic analysis of hemin loading conducted via stopped-flow UV–vis and rapid-freeze-quench resonance Raman shows that both mutants load hemin with biphasic kinetic parameters that are not significantly dissimilar from those previously observed for wild-type HasAp. When the structural and kinetic data are taken together, a tentative model emerges, which suggests that HasA hemophores utilize hydrophobic, π–π stacking, and van der Waals interactions to load hemin efficiently, while axial ligation likely functions to slow hemin release, thus allowing the hemophore to meet the challenge of capturing hemin under inhospitable conditions and delivering it selectively to its cognate receptor