Role of Pre-A motif in nitric oxide scavenging by truncated hemoglobin, HbN, of Mycobacterium tuberculosis.

TitleRole of Pre-A motif in nitric oxide scavenging by truncated hemoglobin, HbN, of Mycobacterium tuberculosis.
Publication TypeJournal Article
Year of Publication2009
AuthorsLama, Amrita, Pawaria Sudesh, Bidon-Chanal Axel, Anand Arvind, Gelpí Josep-Lluis, Arya Swati, Martí Marcelo, Estrin Darío A., F Luque Javier, and Dikshit Kanak L.
JournalJ Biol Chem
Date Published2009 May 22
KeywordsAmino Acid Motifs, Amino Acid Sequence, Circular Dichroism, Computer Simulation, Crystallography, Escherichia coli, Free Radical Scavengers, Models, Molecular, Molecular Sequence Data, Mutant Proteins, Mycobacterium smegmatis, Mycobacterium tuberculosis, Nitric Oxide, Oxidation-Reduction, Pliability, Protein Structure, Secondary, Sequence Deletion, Structure-Activity Relationship, Thermodynamics, Truncated Hemoglobins, X-Ray

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability.