Biology Reference
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were analysed; results showed that when TrpG8 was replaced by a smaller
amino acid, the long tunnel was unblocked and was able to connect the
haem pocket to the solvent (
Boechi et al., 2008
). As trHbN undergoes
changes in conformation following O
2
binding to the haem, allowing
NO passage into the active site, simulations were performed to determine
whether trHbO also undergoes such changes. In Mtb trHbO, after O
2
has bound to the haem, TrpG8 is displaced—moving away from LeuE11
and forming a hydrogen bond with O
2
—which leads to the opening of
the long tunnel; this leads to a decrease in the free energy barrier for ligand
diffusion towards the haem (
Boechi et al., 2008
). The authors suggest that
the first ligand (i.e. O
2
) gains access to the deoxy haem via the short tunnel,
which opens the long tunnel and allows the second ligand (NO) into the
haem pocket via either the long or the short tunnels.
3.2.2 Biochemical characteristics
The trHbO from
M. tuberculosis
has also been studied in great detail by a
number of different groups. Its expression in
E. coli
lends the cells a reddish
brown colour (
Pathania, Navani, Rajamohan, et al., 2002
), as does trHbN
(
Lama et al., 2006
), giving researchers an easy visual test for the over-
expression of these proteins in heterologous hosts. TrHbO usually purifies
as a monomer (
Ouellet et al., 2003
) and was identified as an iron-
protoporphyrin IX (
Mukai et al., 2002
); analysis of cells over-expressing
Mtb trHbO showed a 14.5-kDa protein which corresponds to the predicted
size of trHbO (
Pathania, Navani, Rajamohan, et al., 2002
). Optical absorp-
tion spectra showed that Mtb trHbO has a Soret peak at 413 nm with peaks
in the
region at 544 and 581 nm, suggesting that, again similarly to
trHbN, trHbO is expressed and purified in the oxy-ferrous form. Upon
reduction using dithionite, the peak in the Soret region shifts to 422 nm
and the two distinct peaks in the
a
/
b
region merge to give a single, broad
peak at 554 nm; this behaviour is consistent with other globins (
Pathania,
Navani, Rajamohan, et al., 2002
). The function of trHbO is thought to
be an involvement in O
2
transport or delivery, proposed due to its associ-
ation with the cell membrane during purification from over-expression in
E. coli
(
Pathania, Navani, Rajamohan, et al., 2002
). However, trHbO has
a very high affinity for O
2
compared with other globins (
Mukai et al.,
2002
). The protein also displays low O
2
dissociation rate constants
(
Ouellet et al., 2003
) and extremely fast ligand re-binding after photoexci-
tation (
Jasaitis et al., 2012
), suggesting that O
2
is tightly bound to the haem
and also that it will re-bind with extreme efficiency; this is perhaps evidence
a
/
b
