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octahedral geometry. Site 2 connects the DNA-binding domain with the
dimerization domain including the side chain of residues His
32
, Glu
80
,
His
89
and Glu
100
with a tetrahedral geometry (
Fig. 4.1
). The role of sites
1 (regulatory) and 2 (structural) for
Pseudomonas
Fur is subject to contro-
versy. Some authors propose that Fur from
P. aeruginosa
detects Fe
2+
through
the site initially described to accommodate the structural Zn
2+
(site 2) but
according to biochemical analyses, it lacks structural Zn
2+
. Site 1 could be
a metal-binding site of low affinity without biological significance (
Lee &
Helmann, 2007
).
The presence of a structural Zn
2+
-binding site has been found in most
Fur proteins crystallized to date (
Dian et al., 2011
;
Jacquamet et al., 2009
;
Lucarelli et al., 2007;
Sheikh & Tailor, 2009
). This structural site usually rep-
resents a regular tetrahedral co-ordination of four cysteine residues belong-
ing to two CX
2
C motifs. However, the presence of such motifs does not
seem to ensure the binding of structural zinc. In fact, Cys4-Zn appears not
to be essential for maintaining the DNA competent conformation and,
hence, for the DNA-binding activity of Nur (
An et al., 2009
). This also
seems to be the case of recombinant cyanobacterial FurA. Despite having
two pairs of CX
2
C (Cys
101
, Cys
104
, Cys
141
and Cys
144
), metal analysis and
electrospray ionization MS evidenced that neither zinc nor other metals
are present in this Fur homologue (
Hernández et al., 2002
). However, these
motifs are probably important in the cyanobacterial FurA regulatory mech-
anism since Cys
101
and Cys
141
correspond to residues Cys
93
and Cys
133
present in the dimerization domain in
Vibrio cholerae
Fur. They are con-
nected by a disulphide bond, which plays an analogous role to that of the
salt bridge between Asp
94
and Arg
131
that stabilizes the β3-β5 antiparallel
β-sheet in each subunit of the
Pseudomonas
Fur dimer (
Sheikh & Taylor,
2009
), and Cys
141
has been found to be involved in haeme co-ordination
by cyanobacterial FurA (
Pellicer et al., 2012
).
The metal-binding site that binds the regulatory metal seems to be con-
served in all Fur and Fur-like proteins although it shows some variabil-
ity in its co-ordination depending on the Fur homologue and the metal.
However, it always involves a histidine residue from the loop between α2
and α3 in the DNA-binding domain (
Fig. 4.1
). This histidine is also con-
served in most cyanobacterial FurA, FurC (
Fig. 4.2
) and Slr1738 (PerR)
(
Garcin et al., 2012
). In FurA from
Anabaena
PCC 7120, it corresponds
to His
39
that according to a monomer, three-dimensional model belongs
to a buried core formed by polar residues His
39
, His
85
, His
96
, His
98
, Glu
87
and Glu
109
(
Hernández et al., 2005
). This histidine is noticeably absent
