Biology Reference
In-Depth Information
contracts in the crystal structure, but do not deny its potential importance in
the function of Mtb (and other) trHbN (
Savard et al., 2011
).
In addition to Mtb trHbN, the trHb from
M. smegmatis
(Ms trHbN) has
also been studied. As already mentioned, Ms trHbN is smaller than Mtb
trHbNdue to the deletion of the pre-A region; adding the pre-A region from
Mtb trHbN onto Ms trHbN improves NO detoxification activity when
over-expressed in
E. coli
(8.9 nmol NO haem
1
s
1
compared to 3.8 nmol
NO haem
1
s
1
) and
M. smegmatis
(11.8 nmol NO haem
1
s
1
compared to
4.9 nmol NO haem
1
s
1
)
Lama et al., 2009
). Bidon-Chanal and
co-authors speculate that the difference between the abilities of wild-type
trHbN from
M. tuberculosis
and
M. smegmatis
to detoxify NO may be due
to the pre-A region, with the proviso that until the function of the pre-A
region is determined, this is speculation (
Bidon-Chanal et al., 2007
). How-
ever, Ms trHbN retains all of the already mentioned important structural fea-
tures for the function of trHbN and for the preservation of the globin fold
(
Lama et al., 2006
), lending further proof that the pre-A region is important
for
the function of
trHbN proteins
that are found in pathogenic
Mycobacteria species.
3.1.2 Biochemical characteristics
An early paper expressed Mtb trHbN in
E. coli
and then purified the protein
(
Couture et al., 1999
). Analysis by SDS-PAGE determined that the protein
is
14.4 kDa in size. It purified as a dimer, shown by gel filtration analysis, and
was an iron-protoporphyrin IX protein, as judged by maxima at 525 and
556 nm and a minimum at 539 nm in the oxidised minus reduced
pyridine-haemochromogen spectra, with the haem non-covalently bound
to the protein (
Couture et al., 1999
). Optical spectra showed that Mtb
trHbN can bind a number of ligands, including O
2
, CO and NO. In the
ferric state, the haem is six-coordinate high-spin with a water molecule act-
ing as the sixth ligand, and in the ligand-free ferrous form, the haem is in the
five-coordinate high-spin state (
Couture et al., 1999
). An optical spectrum
of the oxy-ferrous form shows a maximum in the Soret region of 416 nm,
with peaks in the
region at 545 and 581 nm. These conclusions were
confirmed by a Resonance Raman study, which also showed that the ferric
form changes from high- to low-spin state upon increase in pH
(
Yeh et al., 2000
).
TrHbN has been shown to act in an NO detoxification reaction, turn-
ing harmful NO into nitrate via reaction with O
2
(
Pathania, Navani,
Gardner, et al., 2002
). During this reaction, the protein itself is oxidised
a
/
b
