Role of the pre-A motif in nitric oxide scavenging by truncated hemoglobin HbN of Mycobacterium Tuberculosis

A. LAMA; S. PAWARTA; A. BIDON CHANAL; A. ANAND; J.L. GELPI; S. ARYA; M.A. MARTI; D.A. ESTRIN; F.J. LUQUE; K.L. DIKSHIT

JOURNAL OF BIOLOGICAL CHEMISTRY

American Society for Biochemistry and Molecular Biology

Año: 2009 vol. 284 p. 14457 - 14468

0021-9258

<!-- @page { size: 21cm 29.7cm; margin: 2cm } P { margin-bottom: 0.21cm } --> Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric oxide dioxygenase (NOD) 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 M.smegmatis improved its NOD activity. Titration of <!-- @page { size: 21cm 29.7cm; margin: 2cm } P { margin-bottom: 0.21cm } --> oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is several fold slower when 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 towards 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.