Biology Reference
In-Depth Information
discovered that in deoxy trHbN, PheE15 can only be in the closed position,
preventing NO access to the haem cavity. Another molecular dynamics
study, previously discussed, proposed that TyrB10 has slow motions, which
may play a role in how this residue modulates trHbN function (
Savard et al.,
2011
). With respect to NO binding to trHbN, quantum mechanics/molec-
ular mechanics calculations showed that NO interacts with TyrB10 and
GlnE11, and that NO oxidation is a favourable process in the trHbN haem
environment (
Crespo et al., 2005
). The same calculations with the
TyrB10Phe mutant had similar results to wild type, which suggest that this
residue is not important in the chemical reaction.
Another important residue for the function of trHbN is PheE15, thought
to be involved in controlling ligand migration. PheE15 is located in the main
tunnel, and in the crystal structure was present in two different conforma-
tions (
Milani et al., 2001
), leading to a suggested function as a 'gate' to the
haem (
Bidon-Chanal et al., 2007
) controlling ligand access to the haem
pocket; this function is supported by a 2006 paper which implicates both
TyrB10 and GlnE11 in the ability of PheE15 to influence the protein tunnel
dynamics (
Ouellet et al., 2006
). PheE15 exists in two conformations: molec-
ular dynamics simulations showed that when in the closed conformation, the
phenyl ring blocks the tunnel, whereas in the open conformation, there is no
blockage and the ligand can move freely into the tunnel (
Mishra &Meuwly,
2009
). The authors investigated when such a change in conformation was
likely to occur and found a greater than 70% probability that PheE15 is
in the open conformation when the protein is in the oxy form. They also
concluded that there is a large energy barrier preventing the switch from
open to closed state (greater than 3 kcal mol
1
), and this change is more rare
than the switch from closed to open (around 1.2 kcal mol
1
)(
Mishra &
Meuwly, 2009
).
Daigle et al. (2009)
state that the orientation of PheE15
defines the open or closed state of the protein, as determined by both the
redox state of the haem and whether O
2
is bound, whereas
Bidon-
Chanal et al. (2007)
concluded that this state is controlled solely by
O
2
binding.
To further study this gate residue, site-directed mutants were made
where PheE15 was replaced by Ala, Ile, Tyr or Trp. None of these mutants
showed any differences in the binding of O
2
or CO; however, all mutants
showed significant decreases in NO detoxification activity even in the con-
servative PheE15Tyr mutation, suggesting that specifically Phe at this posi-
tion is important for the function of the protein (
Oliveira et al., 2012
). This
reduction in activity was mirrored in the resistance of the mutant proteins to
