The N-terminal pre-A region of Mycobacterium tuberculosis 2/2HbN promotes NO-dioxygenase activity

A unique defense mechanisms by which M. tuberculosis protects itself from nitrosative stress is based on the O2 -dependent NO dioxygenase (NOD) activity of truncated hemoglobin 2/2HbN (Mt2/2HbN). The NOD activity largely depends on the efficiency of ligand migration to the heme cavity through a two-tunnel (long and short) system; recently, it has also been correlated to the presence at the Mt2/2HbN N-terminus of a short pre-A region, not conserved in most 2/2HbNs, whose deletion results in a drastic reduction of NO scavenging. Here we present the 1.53 Å resolution crystal structure of Mt2/2HbN-ΔpreA, lacking the pre-A region. We show that removal of the pre-A region results in long range effects on the protein C-terminus, promoting the assembly of a stable dimer, both in the crystals and in solution. In the Mt2/2HbN-ΔpreA dimer access of heme ligands to the short tunnel is hindered. MD simulations show that the long tunnel branch is the only accessible pathway for O2 -ligand migration to/from the heme, and that the gating residue Phe(62)E15 partly restricts the tunnel diameter. Accordingly, kinetic measurements indicate that the kon value for peroxynitrite isomerization by Mt2/2HbN-ΔpreA -Fe(III) is 4-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-ΔpreA-Fe(II)-O2 is reduced by 35-fold. Therefore, we speculate that Mt2/2HbN evolved to host the pre-A region as a mechanism to prevent dimerization, thus reinforcing survival of the microorganism against the reactive nitrosative stress in macrophages.