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(
Fig. 4.3
C). However, deletion of
ctb
does not induce sensitivity to a variety
of peroxides (
Wainwright et al., 2005
), indicating an alternative function for
Ctb other than peroxide decomposition.
6.3. Biophysical and mechanistic characterisation
As for Cgb, resonance Raman spectroscopy with CO as a structural probe
(see
Section 5.4
) was used to confirm that Ctb also has a positively polar distal
pocket (
Lu, Egawa, et al., 2007
). In addition, this approach was used to
probe the role of B10Tyr and E7His residues in ligand stabilisation: these
residues were proposed to contribute to the high affinity for oxygen
exhibited by Ctb (
K
d
¼
5 nM). Substitution of these residues for non-
hydrogen-bonding amino acids combined with oxygen association/dissoci-
ation measurements and Raman spectroscopy yielded data consistent with
the G8Trp residue providing the major contribution towards ligand
stabilisation. Indeed, this was later confirmed by combining similar reso-
nance Raman techniques with molecular dynamics to conclude that, when
oxygen is bound, Ctb may exist in one of two conformations whereby both
conformers rely upon the G8Trp for stabilising interactions (
Arroyo Manez
et al., 2011
). In line with this important role for the G8Trp residue, mutation
to Phe resulted in a large increase in the oxygen dissociation rate.
7. CONTROL OF GLOBIN EXPRESSION
7.1. Control of globin expression by NssR
The inducibility of the
cgb
gene was first described in cells of
C. coli
carrying a
vector containing a reporter gene (
astA
) under control of the
cgb
promoter
(
Hendrixson & DiRita, 2003
). Induction of Cgb expression by GSNO and
SPN, but not by methyl viologen, was reported. This result and immuno-
blotting tests using Cgb polyclonal antibodies were consistent with Cgb
expression under nitrosative stress conditions (
Elvers et al., 2004
).
Screening of the
C. jejuni
genome (
Parkhill et al., 2000
) revealed the
presence of three potential proteins that could sense NO and induce the
expression of Cgb (
Elvers et al., 2004
); Fur, an iron sensor containing a
Fe
2
þ
cofactor that, in
C. jejuni
, is associated with iron acquisition, flagellar
biogenesis and non-iron ion transport (
Butcher, Sarvan, Brunzelle,
Couture, & Stintzi, 2012
), PerR, a Fe
2
þ
-containing metalloregulator
involved in peroxide stress responses (
Butcher et al., 2012; Mongkolsuk &
Helmann, 2002
) and Cj0466, belonging to the Crp-Fnr superfamily of
