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

Abstract

Many globins convert ^•NO to innocuous NO₃^– through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O₂^•– and ^•NO species and their OONO^– and ^•NO₂ 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 O₂ to the heme, diffusion of ^•NO into the heme pocket and formation of peroxynitrite (OONO^–), isomerization of OONO^–, and release of NO₃^–. 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O₂^– and the ^•NO₂ 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O₂^– and ^•NO₂ (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 NO₃^– dissociation via C–H···O bonding and provide stabilizing interactions for the anion along its egress route