Replacing the axial ligand tyrosine 75 or its hydrogen bond partner histidine 83 minimally affects hemin acquisition by the hemophore HasAp from pseudomonas aeruginosa

Ritesh Kumar, Hirotoshi Matsumura, Scott Lovell, Huili Yao, Juan C. Rodríguez, Kevin P. Battaile, Pierre Moenne-Loccoz, Mario Rivera

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Hemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) 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 HasAsm, but a noncoordinating Gln32 in HasA yp. Binding of hemin to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasAyp 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 Nδ 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 Nδ-Tyr75 Oη 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.

Original languageEnglish (US)
Pages (from-to)2112-2125
Number of pages14
JournalBiochemistry
Volume53
Issue number13
DOIs
StatePublished - Apr 8 2014

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Hemin
Histidine
Pseudomonas aeruginosa
Tyrosine
Hydrogen
Hydrogen bonds
Ligands
Serratia marcescens
Yersinia pestis
Kinetics
Ports and harbors
Heme
Kinetic parameters
Nuclear magnetic resonance spectroscopy
Ligation
Conformations
Magnetic Resonance Spectroscopy
Iron
Crystal structure
Atoms

ASJC Scopus subject areas

  • Biochemistry

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Replacing the axial ligand tyrosine 75 or its hydrogen bond partner histidine 83 minimally affects hemin acquisition by the hemophore HasAp from pseudomonas aeruginosa. / Kumar, Ritesh; Matsumura, Hirotoshi; Lovell, Scott; Yao, Huili; Rodríguez, Juan C.; Battaile, Kevin P.; Moenne-Loccoz, Pierre; Rivera, Mario.

In: Biochemistry, Vol. 53, No. 13, 08.04.2014, p. 2112-2125.

Research output: Contribution to journalArticle

Kumar, Ritesh ; Matsumura, Hirotoshi ; Lovell, Scott ; Yao, Huili ; Rodríguez, Juan C. ; Battaile, Kevin P. ; Moenne-Loccoz, Pierre ; Rivera, Mario. / Replacing the axial ligand tyrosine 75 or its hydrogen bond partner histidine 83 minimally affects hemin acquisition by the hemophore HasAp from pseudomonas aeruginosa. In: Biochemistry. 2014 ; Vol. 53, No. 13. pp. 2112-2125.
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abstract = "Hemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) 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 HasAsm, but a noncoordinating Gln32 in HasA yp. Binding of hemin to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasAyp 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 {\AA}) and pH 5.4 (1.25 {\AA}) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for Nδ 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 Nδ-Tyr75 Oη 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.",
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AU - Matsumura, Hirotoshi

AU - Lovell, Scott

AU - Yao, Huili

AU - Rodríguez, Juan C.

AU - Battaile, Kevin P.

AU - Moenne-Loccoz, Pierre

AU - Rivera, Mario

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N2 - Hemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) 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 HasAsm, but a noncoordinating Gln32 in HasA yp. Binding of hemin to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasAyp 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 Nδ 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 Nδ-Tyr75 Oη 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.

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