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D
investigated by comparing NO consumption of wild type,
HbN and the
complemented strains. Under aerobic conditions, after addition of
NO-saturated solution to wild-type cells, the NO signal generated showed
that NO is rapidly removed from solution; the case in the complemented
cells is more extreme, showing that trHbN can efficiently remove NO from
solution (
Ouellet et al., 2002
). However, in
HbN cells, NO removal
appears to be the same as buffer alone when exposed to 1
D
M NO, indicat-
ing that there is no NO detoxification activity in these cells; however, when
these cells are exposed to a lower concentration of NO (0.5
m
M), there is
some NO detoxification activity, indicating that there is another system
in
M. bovis
that can detoxify low concentrations of NO, which could be
trHbO. Interestingly, and agreeing with data discussed in
Section 3.1.1
,a
D
m
HbN mutant complemented with trHbN with TyrB10 replaced with
Phe showed no improvement in NO removal over the
HbN cells
(
Ouellet et al., 2002
), which is further proof that the TyrB10 residue is
essential for the function of trHbN (
Ouellet et al., 2006
). The same group
showed the stoichiometric production of nitrate by purified Mb trHbN,
showing that the reaction between NO and O
2
leads to this product
(
Ouellet et al., 2002
).
When Mtb (
Pathania, Navani, Gardner, et al., 2002
) or Ms trHbN was
expressed in an
E. coli hmp
strain, NO uptake was increased; Mtb trHbN
appears more efficient at NO detoxification than Ms trHbN, as determined
by NO uptake assays using an NO electrode (
Lama et al., 2006
). Under nor-
mal laboratory conditions, that is, without any NO stress, growth was nei-
ther improved nor impaired by the expression of the two globins under
aerobic conditions. The survival of these
E. coli
strains carrying the two
trHbN was determined after exposure to SNP, an NO donor. As expected,
expression of Mtb trHbN confers protection against SNP compared to wild-
type cells, and the protection afforded by Ms trHbN is almost negligible
(
Lama et al., 2006
). When the same two globins were over-expressed in
M. smegmatis
, there was an increase in the NO detoxification ability of
the cells, and again this ability was more improved in cells expressing
Mtb trHbN (18.6 nmol NO haem
1
s
1
) than Ms trHbN (5.1 nmol NO
haem
1
s
1
)(
Lama et al., 2006
). Wild-type
M. smegmatis
cells showed
low NO uptake (0.09 nmol NO haem
1
s
1
), perhaps again a reflection
of the lack of pre-A region and the fact that non-pathogenic Mycobacteria
might not require efficient NO removal activities. When the pre-A region
was removed from Mtb trHbN, NO consumption activity of the purified
protein was lower than wild type; expression of wild-type trHbN in
D
