Biology Reference
In-Depth Information
(
Couture et al., 1999
). Complementing a
Mycobacterium bovis glbN
mutant
with
M. bovis
trHbN (Mb trHbN) containing the amino acid substitution
TyrB10Phe did not restore NO detoxifying activity, which points towards
the important role of TyrB10 in the function of trHbN (
Ouellet et al.,
2002
). Studies using Resonance Raman spectroscopy, optical spectroscopy
and X-ray crystallography have shown that TyrB10, along with GlnE11,
controls the binding of water molecules to ferric Mtb trHbN, plus its
ionisation state (
Ouellet, Milani, Couture, Bolognesi, & Guertin, 2006
).
These properties are important as they ensure that the sixth coordination
position is kept empty in deoxy trHbN, in order to allow the binding of
O
2
to the haem. Laser flash-photolysis analysis of Mtb trHbN TyrB10Phe
and TyrB10Leu mutants showed that substitution of the Tyr at this position
increases the
k
on
value for O
2
by approximately 10-fold, indicating that
TyrB10 is a barrier to O
2
binding to the haem (
Ouellet et al., 2008
). The
k
on
for COwas also increased in these mutants. Using kinetics and molecular
dynamics, the authors showed that ligand binding and geminate re-binding
are discouraged by the displacement of a non-coordinated distal site water
molecule, stabilised mostly by TyrB10. When TyrB10 is changed to Phe,
geminate re-binding of both O
2
and CO is increased; along with additional
data which showed increased NO binding rates to the ferric haem of a
TyrB10Leu/GlnE11Val double mutant, these suggest that TyrB10 plays a
major role in access of ligands to the haem iron (
Ouellet et al., 2008
).
Molecular dynamics studies of the TyrB10Phe mutant showed a decrease
in binding energy compared with the wild type (
Crespo et al., 2005
). Two
studies in 2009 found extensive hydrogen bonding between haem-bound
O
2
, TyrB10 and GlnE11 (
Daigle et al., 2009; Mishra &Meuwly, 2009
); they
are dynamic in nature with the bonds being broken and reformed many
times during the simulation. These bonds may contribute to the ligand
chemistry by supporting the different protein conformations required in
the NOD reaction (
Mishra & Meuwly, 2009
). Indeed, there is hydrogen
bond formation between TyrB10 and GlnE11; GlnE11 may therefore inter-
act with NO and allow it to be positioned correctly with respect to O
2
, lead-
ing to the chemistry required for the detoxification to nitrate (
Crespo et al.,
2005
). A molecular dynamics study of Mtb trHbN in explicit water inves-
tigating TyrB10 and GlnE11 showed that these two amino acids and the
hydrogen bonds they form exist in two different configurations, controlled
by O
2
coordination to the haem (
Daigle et al., 2009
). These changes in con-
formation, especially of GlnE11, control the positioning of PheE15, and
therefore whether the large tunnel is open or closed; in addition, it was
