Biology Reference
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the high affinity for O
2
that the protein displays (
Ouellet et al., 2003
). Upon
escape from the haem pocket, the ligand has three docking sites to choose
from upon dissociation, where it can then either re-bind to the haem or
escape through the protein into solution (
Guallar et al., 2009
). The unique
hydrogen bond network displayed in trHbO in comparison with trHbN
shows that the distal haem pocket in trHbO is crowded and rigid and quite
distinct from the haem region in trHbN (
Ouellet et al., 2003
), adding further
support for the difference in functions.
Additional information is available about the importance of TrpG8 in the
stability and structure of trHbO. Protein stability was investigated and it was
shown that when TrpG8 was mutated to Phe, the protein was less stable than
wild-type protein (
Ouellet, Milani, et al., 2007
). Analysis of wild-type
trHbO and both TyrCD1Phe and TrpG8Phe mutants by Resonance
Raman spectroscopy showed that bound CO potentially interacts with
TyrCD1 and TrpG8 through hydrogen bonds; however, no polar interac-
tions between the distal residues and the bound CO were detected, further
characterising the haem pocket in trHbO (
Ouellet, Milani, et al., 2007
).
Studies were also made of the O
2
-bound ferrous complex of wild-type
and mutant proteins; data suggest that TrpG8 interacts with the proximal
O
2
atom bound to the haem and is involved in stabilisation of bound O
2
(
Ouellet, Milani, et al., 2007
).
The tunnel system in trHbO is more restricted than in trHbN due to the
orientation of the G and H helices and six amino acid changes; in trHbO, the
short tunnel is inhabited by TrpG8 and the long tunnel is split into two
smaller cavities which inhabit the same space as in trHbN (
Milani,
Savard, et al., 2003
). Entrance into the tunnel by ligands appears to be con-
trolled by the AlaE7 gate (
Boechi et al., 2008; Milani, Savard, et al., 2003
).
Molecular dynamics investigations of the entrance of O
2
into deoxy Mtb
trHbO found there to be two access tunnels for O
2
from the solvent into
the haem: a long tunnel (16
˚
long) was also identified in the crystal struc-
ture (
Milani, Savard, et al., 2003
) along with a short tunnel (around 10
˚
long), thought to be blocked in the crystal structure (
Boechi et al., 2008
).
The long tunnel connects the haem with the solvent between the B,
E and G helices, and ligand movement through the tunnel appears to be con-
trolled by the distance between PheB2 and LeuH3—when these are close
together, the ligand cannot pass between them and is forced to choose
another path (
Boechi et al., 2008
). Following from the suggestion that
TrpG8 is involved in blocking the short tunnel and the distal haem pocket
(
Milani, Savard, et al., 2003
),
in silico
mutants (TrpG8Phe and TrpG8Ala)
