Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study

Abstract

Nitrobindins (Nbs) are all-β-barrel heme proteins present along theevolutionary ladder. They display a highly solvent-exposed ferric heme group with theiron atom being coordinated by the proximal His residue and a water molecule at thedistal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxicperoxynitrite (ONOO − ) to harmless nitrate, with the value of the second-order rateconstant being similar to those of most heme proteins. The value of the second-orderrate constant of Nbs increases as the pH decreases; this suggests that Nb(III)preferentially reacts with peroxynitrous acid (ONOOH), although ONOO − is morenucleophilic. In this work, we shed light on the molecular basis of the ONOO − andONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissectingthe ligand migration toward the active site, the water molecule release, and the ligandbinding process by computer simulations. Classical molecular dynamics simulationswere performed by employing a steered molecular dynamics approach and theJarzynski equality to obtain ligand migration free energy profiles for both ONOO − and ONOOH. Our results indicate that ONOO −and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligandbinding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using ahybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO − exhibits a muchlarger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leavinggroup by the incoming ONOOH.