Biology Reference
In-Depth Information
G12, mostly fill the protein matrix tunnel space (
Bonamore et al., 2005;
Giangiacomo et al., 2005; Ilari et al., 2007; Milani et al., 2003; Pesce
et al., 2011
). In particular, in 2/2HbOs, the bulky side-chain of the con-
served TrpG8 obstructs the short tunnel branch and the deeper part of
the distal site pocket typical of 2/2HbNs. The 2/2HbN long tunnel branch
retains only two cavities in 2/2HbOs, both fully shielded from solvent con-
tact (
Milani et al., 2003
). Interestingly, in
M. tuberculosis
2/2HbO, residue
TrpG8 is also responsible for blocking the region corresponding to the
2/2HbN long tunnel and therefore key for ligand entry (
Boechi et al.,
2008; Ouellet et al., 2007
). The restriction of the cavities within the protein
matrix becomes extreme in
T. fusca
2/2HbO, where no internal cavities
are detected, due to substitutions with larger residues relative to other
2/2HbOs, or by conformational differences of conserved or similar size
residues (
Bonamore et al., 2005
).
The substantial absence of a protein matrix tunnel system is mirrored by
the conserved presence of a small distal site E7 residue in group II 2/2HbOs
(
Vuletich & Lecomte, 2006
), which does not hinder entrance to the haem
distal cavity. Therefore, in 2/2HbOs, diatomic ligands (such as O
2
, CO,
and NO) may preferably access the haem distal site through an E7 route.
Molecular dynamics simulations, however, showed that once the protein
is oxygenated, both the E7 route and the path corresponding to the
M. tuberculosis
2/2HbN tunnel long branch can contribute to ligand entry,
because they present similar barriers. This mechanism differs from the case
of 2/2HbN, in which each ligand has been proposed to migrate through a
separate pathway (
Bidon-Chanal et al., 2006
). The change in the free energy
barrier for the long tunnel is due to the TrpG8 interaction with the haem-
bound O
2
. The short-tunnel E7 barrier does not change significantly upon
oxygenation; consequently, the overall barrier presented by the short-tunnel
E7 is similar in the oxygenated and deoxygenated states of the protein.
This fact is consistent with the experimental kinetic constants for ligand
migration. The results highlight the importance of TrpG8 in regulating
ligand migration in 2/2HbO, since not only is it responsible for the high
barrier observed in the long tunnel, but it also blocks the short tunnel branch
displayed by group I 2/2HbNs. Furthermore, TrpG8 is important in
anchoring TyrCD1 and LeuE11 side chains, thereby allowing the stabiliza-
tion of the haem-bound ligand
via
hydrogen bonds donated from TrpG8
and TyrCD1. Following its dissociation, the ligand can migrate between
three temporary docking sites, which are modulated by the conformational
rearrangements of the side chains of several critical distal amino acids,
