Mechanism of the Nitric Oxide Dioxygenase Reaction of Mycobacterium tuberculosis Hemoglobin N.

Many globins convert ^• NO to innocuous NO ₃ ^- through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O ₂ ^•- and ^• NO species and their OONO ^- and ^• NO ₂ derivatives) through the action of truncated hemoglobin N (trHbN), which catalyzes the NOD reaction with one of the highest rates among globins. The general NOD mechanism comprises the following steps: binding of O ₂ to the heme, diffusion of ^• NO into the heme pocket and formation of peroxynitrite (OONO ^- ), isomerization of OONO ^- , and release of NO ₃ ^- . Using quantum mechanics/molecular mechanics free-energy calculations, we show that the NOD reaction in trHbN follows a mechanism in which heme-bound OONO ^- undergoes homolytic cleavage to give Fe ^IV ═O ₂ ^- and the ^• NO ₂ radical but that these potentially harmful intermediates are short-lived and caged by the heme pocket residues. In particular, the simulations show that Tyr33(B10) side chain is shielded from Fe ^IV ═O ₂ ^- and ^• NO ₂ (and protected from irreversible oxidation and nitration) by forming stable hydrogen bonds with Gln58(E11) side chain and Leu54(E7) backbone. Aromatic residues Phe46(CD1), Phe32(B9), and Tyr33(B10) promote NO ₃ ^- dissociation via C-H···O bonding and provide stabilizing interactions for the anion along its egress route.