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arrangements: (i) a closed distal site conformation, whereby residue Trp(60)
B9 points towards the Fe-bound ligand and shuts tunnel 1 and (ii) an open
distal site conformation, where the Trp(60)B9 side chain points away from
the distal pocket, thus keeping tunnel 1 in its open state. In addition, con-
sidering that for a ligand such as nicotinamide, the ligation state of the pro-
tein does not result in the relocation of the Trp(60)B9 side chain into
the haem distal cavity, nor in the transition of tunnel 1 from the open to
the closed state, the reactivity of
Ma
Pgb appears to be regulated also by
the nature of the haem ligand (
Pesce, Tilleman, et al., 2013
).
Another interesting point is that the insertion of Trp(60)B9 into the
haem distal cavity upon ligand binding is coupled to a change in the back-
bone conformation of the neighbouring 149-154 region, located in the sec-
ond half of the G-helix, at the subunit interface of the
Ma
Pgb
*
homodimer.
Within the homodimeric
Ma
Pgb
*
G-H four-helix bundle (
Fig. 3.1C
), tight
packing involves specifically the N-terminal half of the G-helices and the
C-terminal half of the H-helices, while the remaining interface regions
are mostly solvent exposed, thus allowing the 149-154 region to afford some
flexibility to compensate the structural changes transmitted from reshaping
of the haem distal site upon ligand binding. Alternatively, the 149-154
region might also be able to influence/modulate the architecture and
ligand-binding properties of the haem distal cavity through association of
an (unknown) effector molecule or even a partner protein. Therefore,
the 149-154 region has been proposed to be a potential allosteric regulation
site (
Pesce, Tilleman, et al., 2013
), although (negative) co-operativity has
only been reported for O
2
but not for CO binding (
Abbruzzetti et al.,
2012; Nardini et al., 2008
).
5. BIOCHEMICAL AND FUNCTIONAL
CHARACTERISATION
Originally, Pgbs were identified in the genomes of the
Actinobacterium
Thermobifida fusca
, the green non-sulphur bacterium
Chloroflexus aurantiacus
and the two Archaea
A. pernix
and
M. acetivorans
.
Pgbs from the latter two Archaea were characterised and found to purify
in the oxidised state; generation of reduced Pgb proved to require an envi-
ronment free of O
2
, as they autoxidise rapidly, at the rates of 0.0032 and
0.0027 s
1
, corresponding to half-lives of 3.6 and 4.3 min for oxy-
Ma
Pgb
and oxy-
Ap
Pgb, respectively (
Freitas et al., 2004, 2005
). Globins with such
high oxidation rates and long reduction times are usually not suitable for