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2008; Yeh, Couture, Ouellet, Guertin, & Rousseau, 2000
); these indicate
that the ligand binding is largely controlled by a pair of interacting amino
acids (GlnE11 and Tyr10) in the haem distal site that participate in hydrogen
bonding with the haem-bound diatomic O
2
ligand. Indeed, in 2/2HbN
from
M. tuberculosis
, a direct hydrogen bond occurs between TyrB10 side
chain and the ligand (O
2
or cyanide, in the ferrous or ferric haem states,
respectively), stabilized by GlnE11 that interacts with TyrB10 (
Couture,
Yeh, et al., 1999; Milani et al., 2001; Milani, Ouellet, et al., 2004; Yeh
et al., 2000
). It has been shown that the main barrier to ligand binding to
deoxy
M. tuberculosis
2/2HbN is the displacement of a distal cavity water
molecule, which is mainly stabilized by residue TyrB10, but not coordinated
to the haem iron. As observed in the TyrB10/GlnE11 apolar mutants
(TyrB10Phe/Val and GlnE11Val/Ala, respectively), once this kinetic bar-
rier is lowered, CO and O
2
binding is very fast with rates approaching
1
2
10
9
M
1
s
1
. These large values almost certainly represent the
upper limit for ligand binding to a haem protein and also indicate that the
iron atom in 2/2HbN is highly reactive (
Ouellet et al., 2008
). In
P. caudatum
2/2HbN and in
C. eugametos
2/2HbN, residue TyrB10, buried
in the inner part of the haem pocket, is properly oriented through hydrogen
bonds towards residues GlnE7 and Thr/GlnE11, to provide stabilization
of the haem-bound distal ligand (
Pesce et al., 2000
). In
T. pyriformis
2/2HbN, TyrB10 and GlnE7 are hydrogen bonded to the haem-bound
O
2
molecule. Furthermore, TyrB10 is hydrogen bonded to GlnE7
and GlnE11 residues. Mutation of these residues results in fast O
2
dissociation
and autoxidation (
Igarashi et al., 2011
). In all cases, the strongly conserved
TyrB10 plays a pivotal role in ligand stabilization through a direct hydrogen
bond to the haem ligand. In general, when in group I a hydrogen bonding
residue is present at B10, a Gln is located at E7 or E11, or at both these sites,
likely completing the distal site hydrogen-bonded network. On the contrary,
when a side chain devoid of hydrogen-bonding capabilities is (rarely) hosted at
B10, then large hydrophobic residues are coupled at the E7 and E11 sites
(
Nardini et al., 2007; Vuletich & Lecomte, 2006
).
In group I 2/2Hbs, an example of
bis
-histidine hexacoordination has
been reported for group I 2/2HbN from the cyanobacterium
Synechococcus
sp. strain PCC 7002 and PCC 6803 (involving the proximal/distal residues
HisF8 and HisE10, respectively), where binding of an exogenous ligand to
the haem requires the dissociation of the Fe-coordinated HisE10 from the
haem and a large conformational change of the B- and E-helices (
Couture
