Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water

Yannick H. Ouellet, Richard Daigle, Patrick Lagüe, David Dantsker, Mario Milani, Martino Bolognesi, Joel M. Friedman, Michel Guertin

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k′NOD ≈ 745 × 10 6 M-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109 M-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photo-product of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).

Original languageEnglish (US)
Pages (from-to)27270-27278
Number of pages9
JournalJournal of Biological Chemistry
Volume283
Issue number40
DOIs
StatePublished - Oct 3 2008

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Truncated Hemoglobins
Heme
Mycobacterium tuberculosis
Ligands
Iron
Water
Molecules
Hemeproteins
Detoxification
Kinetics
Myoglobin
Molecular Dynamics Simulation
Hydrogen Bonding
Carbon Monoxide
Nitrates
Horses
Molecular dynamics
Rate constants
Hydrogen bonds
Nitric Oxide

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water. / Ouellet, Yannick H.; Daigle, Richard; Lagüe, Patrick; Dantsker, David; Milani, Mario; Bolognesi, Martino; Friedman, Joel M.; Guertin, Michel.

In: Journal of Biological Chemistry, Vol. 283, No. 40, 03.10.2008, p. 27270-27278.

Research output: Contribution to journalArticle

Ouellet, Yannick H. ; Daigle, Richard ; Lagüe, Patrick ; Dantsker, David ; Milani, Mario ; Bolognesi, Martino ; Friedman, Joel M. ; Guertin, Michel. / Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 40. pp. 27270-27278.
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T1 - Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water

AU - Ouellet, Yannick H.

AU - Daigle, Richard

AU - Lagüe, Patrick

AU - Dantsker, David

AU - Milani, Mario

AU - Bolognesi, Martino

AU - Friedman, Joel M.

AU - Guertin, Michel

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N2 - The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k′NOD ≈ 745 × 10 6 M-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109 M-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photo-product of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).

AB - The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k′NOD ≈ 745 × 10 6 M-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109 M-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photo-product of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).

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