Biology Reference
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from ferrous to ferric (
Ouellet et al., 2002
). When NO was added to puri-
fied, fully oxygenated Mtb trHbN, spectral analysis clearly showed oxida-
tion of the protein, with the disappearance of peaks in the
a
/
b
region
(
Lama et al., 2009; Ouellet et al., 2002
). In addition, when NO was added
to fully oxygenated trHbN, the wild-type protein was oxidised as deduced
by a flattening of the
region, whereas a mutant with the pre-A region
removed showed no change in this region, suggesting that the protein can-
not either bind NO or proceed with the reaction without the pre-A region
intact (
Lama et al., 2009
). Resonance Raman spectroscopy predicted that
the ligand on-rate for trHbN would be fast due to the unstrained His res-
idue acting as the proximal haem ligand; consequently, the iron atom can
be brought into a fully planar conformation upon ligand binding, thus
avoiding the constraint that is present in other globins (
Couture et al.,
1999
). Indeed, the same authors present experimental data which show that
trHbN has a high affinity for O
2
, with the binding process being cooper-
ative (
Couture et al., 1999
). The authors speculate that this property could
be useful for the protein to function in the low O
2
environments that
Mycobacteria operate in, for example, in the hypoxic granuloma environ-
ment in the lung, as it will be able to capture O
2
for the reaction with NO
even when O
2
concentrations are relatively low. When the kinetics of CO
re-binding to trHbN after photodissociation were investigated, it was
shown that CO re-binds in a single exponential phase, assigned to solvent
phase recombination (
Ouellet et al., 2008
).
Analysis of the formation of nitrate by Mb trHbN in the presence of NO
and O
2
showed a stoichiometric formation of nitrate when protein was pre-
sent in excess of NO, and stopped-flow spectroscopy detected the produc-
tion of the aquomet form of Mb trHbN without detection of any
intermediates (
Ouellet et al., 2002
). Similar to Mtb trHbN, Ms trHbN
was purified in the oxy-ferrous form when expressed in
E. coli
, albeit smaller
at 12.8 kDa as predicted by the amino acid sequence, particularly due to the
lack of pre-A region (
Lama et al., 2006
). In addition, antibodies raised against
Mtb trHbN can also react with Ms trHbN, reiterating their similarities. As is
the case with
M. leprae
, where reductive evolution has led to the loss of all
trHbs except trHbO (
Wittenberg et al., 2002
), perhaps the loss of the pre-A
region is a result of the non-pathogenic lifestyle of
M. smegmatis
compared
with
M. tuberculosis
(
Lama et al., 2006
). Its optical spectra are also very similar
to that of Mtb trHbN, with peaks in the Soret region at 416 nm and in the
a
a
/
b
region at 543 and 580 nm. Other values obtained with the deoxy fer-
rous and CO-bound forms also conformed to known values for the trHbs.
/
b
