title: Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water.
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) approximately 745 x 10(6) m(-1) x s(-1)), which is approximately 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 O(2) binding is very rapid with rates approaching 1-2 x 10(9) m(-1) x 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 photoproduct of the *NO derivative of met-trHbN, where both the *NO and water can be directly followed, revealed that water rebinding is quite fast (approximately 1.49 x 10(8) 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).
authors.affiliation: Department of Biochemistry and Microbiology, Laval University, Quebec, Canada, G1K 7P4.
authors.forename: Yannick H
authors.initials: YH
authors.lastname: Ouellet
authors.forename: Richard
authors.initials: R
authors.lastname: Daigle
authors.forename: Patrick
authors.initials: P
authors.lastname: Lagüe
authors.forename: David
authors.initials: D
authors.lastname: Dantsker
authors.forename: Mario
authors.initials: M
authors.lastname: Milani
authors.forename: Martino
authors.initials: M
authors.lastname: Bolognesi
authors.forename: Joel M
authors.initials: JM
authors.lastname: Friedman
authors.forename: Michel
authors.initials: M
authors.lastname: Guertin
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006736:Horses / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000737:chemistry* / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D054793:Truncated Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C102539:GlbN protein, bacteria; D008024:Ligands; D009211:Myoglobin; D009566:Nitrates; D054793:Truncated Hemoglobins; D014867:Water; D009569:Nitric Oxide; D006418:Heme; D007501:Iron