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Abstract
Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation
of nitric oxide (NO) to nitrate with a second-order rate constant k approximately
745 microM(-1) s(-1) at 23 degrees C (nitric oxide dioxygenase reaction). It was proposed
that this high efficiency is associated with the presence of hydrophobic tunnels inside
trHbN structure that allow substrate diffusion to the distal heme pocket. In this
work, we investigated the mechanisms of NO diffusion within trHbN tunnels in the context
of the nitric oxide dioxygenase reaction using two independent approaches. Molecular
dynamics simulations of trHbN were performed in the presence of explicit NO molecules.
Successful NO diffusion from the bulk solvent to the distal heme pocket was observed
in all simulations performed. The simulations revealed that NO interacts with trHbN
at specific surface sites, composed of hydrophobic residues located at tunnel entrances.
The entry and the internal diffusion of NO inside trHbN were performed using the Long,
Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by
NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic
funnel-shape entrance, covering a large area extending far from the tunnel entrance.
This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities
capable of hosting up to three NO molecules, thereby accelerating NO capture and entry.
The importance of hydrophobicity of entrances for NO capture is highlighted by a comparison
with a polar mutant for which residues at entrances were mutated with polar residues.
A complete map of NO diffusion pathways inside trHbN matrix was calculated, and NO
molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect
agreement with the three-dimensional free-energy distribution calculated using implicit
ligand sampling. The trajectories showed that NO significantly alters the dynamics
of the key amino acids of Phe(62)(E15), a residue proposed to act as a gate controlling
ligand traffic inside the Long tunnel, and also of Ile(119)(H11), at the entrance
of the Short tunnel. It is noteworthy that NO diffusion inside trHbN tunnels is much
faster than that reported previously for myoglobin. The results presented in this
work shed light on the diffusion mechanism of apolar gaseous substrates inside protein
matrix.