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

doi:10.1074/jbc.m804215200

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).

Highlights

In Mycobacterium tuberculosis the glbN gene encodes the truncated hemoglobin N4 (Fig. 1)
Our results indicate that both the main barrier to ligand binding to deoxy-truncated hemoglobin N (trHbN) and the origin of the low geminate yield are due to the presence of Tyr(B10)-stabilized water within the distal heme pocket (DHP) at a site that blocks access to the heme iron
The present kinetic data and MD simulations indicate that the main barrier to ligand binding from solvent and geminate phase to deoxy-trHbN is the displacement of a non-coordinated distal site water molecule, which is mainly stabilized by the Tyr(B10) residue

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis, Expression, and Purification—Recombinant trHbN and mutants were expressed and purified as previously described [31]. The isosbestic point at 408.5 nm was used to follow the water coordination subsequent to trHbN(Fe3ϩ-NO) photolysis At this wavelength we expect an increase in absorbance (Abs) when H2O binds to the heme iron and a decrease when 1⁄7NO replaces H2O. The water molecule was arbitrarily positioned in the DHP in a cavity located between the heme iron and the B10 residue, and the initial position was optimized with a short energy minimization, keeping all other coordinates constrained. Because of their high structural similarities [31], mutant systems were built from equilibrated wild-type coordinates. One coordinate set every 10 ps was used for analysis

RESULTS AND DISCUSSION

Geminate and Solvent Phase Recombination

Water Controls Ligand Binding to Ferric trHbN

Molecular Dynamics Simulations Suggest That Water May

Cavity formation frequency and volume over the iron atom