Biology Reference
In-Depth Information
Figure 4.4 The
model for the activation of the peroxynitrite-bound interme-
diate of Cgb. (A) The active site of yeast cytochrome
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c
peroxidase (PDB id
¼
2PCC;
Pelletier & Kraut, 1992
). (B) The
model for the cleavage of O
d
O bonds in
peroxidases. (C) The haem-binding cleft of Cgb (PDB id¼2WY4;
Shepherd et al., 2010
).
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Evidence for the imidazolate character of the axial F8His ligand was pro-
vided by resonance Raman spectroscopy, where the iron-histidine-
stretching mode was found to be 251 cm
1
(
Lu, Egawa, Wainwright,
Poole, & Yeh, 2007
), an unusually high frequency for a globin protein. This
spectral feature was dependent upon pH, suggesting that protonation of the
H23Glu residue perturbs the imidazolate character of the F8His residue.
The requirement for this proximal hydrogen-bonding network for the
imidazolate character of the F8His residue was later confirmed by mutagen-
esis, whereby substitution of either H23Glu or G5Tyr decreased the iron-
histidine-stretching frequency to 225 cm
1
(
Shepherd et al., 2010
), close to
that for the neutral F8His of Mb. Intriguingly, mutation of the G5Tyr res-
idue to Phe also resulted in the partial formation of a six coordinate haem
species, which was attributed to the axial coordination of the haem iron
by the B10Tyr in the distal pocket, highlighting the potential influence
of the proximal hydrogen-bonding network upon ligand binding. To fur-
ther demonstrate the effect of the proximal residues upon ligand binding, the
E134A mutant (H23) was shown to have a higher
on
rate for negatively
charged CN
ions: mutation of the H23Glu will diminish the imidazolate
character (negative charge) of the F8His residue, raising the formal charge
(positive) of the haem iron.
The positively polar distal environment was characterised through the
analysis of a CO-bound derivative via a resonance Raman approach. CO
has been widely used to probe the electronic environments of haem
