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involving Lysine 46 in the D-region (not shown). As a result of this
stabilising hydrogen-bonding network, Cgb exhibits higher ligand affinities
compared to mammalian globins such as Mb. This is evidenced by the dis-
sociation constants for oxygen binding being 6 nM and 0.86
M for Cgb
(
Lu, Mukai, et al., 2007
) and swMb (
Gibson, Olson, McKinnie, &
Rohlfs, 1986
), respectively. The presence of such a hydrogen-bonding net-
work is not unique to Cgb, as both Hmp and Vgb also possess tyrosine
and glutamine residues at the B10 and E7 positions, respectively, although
neither Hmp nor Vgb structures exhibit hydrogen-bonding interactions
between these residues (
Shepherd et al., 2010
). This is possibly because
the region surrounding the E7 residue is disordered in the Vgb structures
and is interacting with the reductase domain in Hmp. In addition, the
organisation of hydrogen-bonding residues in Cgb is reminiscent of cyto-
chrome
c
peroxidase (CcP;
Fig. 4.3
C), which is unsurprising since the
NOD mechanism of Cgb has been proposed to proceed via a peroxidase-
like mechanism that requires a positively polar distal environment
(
Lu, Mukai, et al., 2007
).
In addition to the hydrogen-bonding network found in the distal pocket
of Cgb, another network of hydrogen bonds is found in the proximal
pocket, where the haem cofactor is coordinated to the F8 histidine
(
Fig. 4.3
A). The 'catalytic triad' of H23Glu, G5Tyr and F8His form a stable
network of hydrogen bonds, resulting in a 90
rotation in the imidazole ring
of the axial F8His ligand, similar to that of CcP (
Fig. 4.3
C). This contrasts
to the F8His residue of Mb (
Fig. 4.3
B), which does not interact with
other amino acid side chains (
Fig. 4.3
B). The presence of a hydrogen-
bonding network in the proximal pocket is proposed to impose imidazolate
(negatively charged) character upon the F8His residue, which is also impli-
cated in the peroxidase-like mechanism of Cgb catalysis (
Lu, Mukai,
et al., 2007
).
m
5.4. Biophysical and mechanistic characterisation
The structure of Cgb is consistent with an imidazolate character of the axial
F8His and a positively polar distal environment, both necessary for the
well-characterised'push-pull' model for peroxidase-like enzymes (
Poulos,
1996
). Such a mechanism was proposed for O
d
O bond cleavage of the
proposed peroxynitrite intermediate (Eq.
4.2
) of Hmp during an NOD
reaction (
Mukai, Mills, Poole, & Yeh, 2001
) and is depicted in
Fig. 4.4
.
Fe
2
þ
Fe
3
þ
NO
Fe
3
þ
þ
NO
3
þ
¼
!
!
ð
:
Þ
O
O
N
O
O
O
4
2
