Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

Ana Oliveira,Marcelo A Martí,Leonardo Boechi,F Javier Luque,Flavio Forti,Axel Bidon-Chanal,Sandeep Singh,Dario A Estrin,Kanak L Dikshit,Paolo Carloni

doi:10.1371/journal.pone.0049291

Abstract

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O2/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Highlights

Mycobacterium tuberculosis (Mtb) poses a serious threat to the public health worldwide, infecting nearly one third of the global population
Compared to horse heart myoglobin, the nitric oxide dioxygenase (NOD) reaction catalyzed by Mtb hemoglobin N (HbN) is,15-fold faster, suggesting that it may be crucial in relieving nitrosative stress [4]
It was expected that the distinct chemical nature of the side chains would translate into differences in the ligand migration through the long tunnel, which in turn should lead to differences in the NOD activity measured for the mutants

Summary

Introduction

Mycobacterium tuberculosis (Mtb) poses a serious threat to the public health worldwide, infecting nearly one third of the global population. The remarkable adaptability of tubercle bacillus to cope with hazardous level of reactive nitrogen/oxygen species within the intracellular environment contributes to its pathogenicity. An enhanced level of nitric oxide (NO) and reactive nitrogen species produced within activated macrophages during infection act as a vital part of host defense, limit the intracellular survival of Mtb, and contributes in restricting the bacteria to latency. One of the unique defense mechanisms by which Mtb protects itself from the toxicity of NO relies on the oxygenated form of truncated hemoglobin N (HbN), which catalyzes the rapid oxidation of NO to harmless nitrate [1,2,3]. Compared to horse heart myoglobin, the nitric oxide dioxygenase (NOD) reaction catalyzed by Mtb HbN is ,15-fold faster, suggesting that it may be crucial in relieving nitrosative stress [4]

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Results

Conclusion