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water molecules. In particular, the haem-coordinated water molecule is
directly hydrogen bonded to a distal site intervening water molecule and
to TrpG8 (
Pesce et al., 2011
). Thus, overall, the group II-conserved TrpG8
seems to be the residue playing a crucial/pivotal role in stabilizing the ligand
and modulating its escape rate out of the distal pocket (
Bonamore et al.,
2005; Giangiacomo et al., 2005; Ilari et al., 2007; Milani et al., 2003;
Pesce et al., 2011
).
2/2HbOs also tend to contain a small residue at the E7 site, typically Ala,
Ser, or Thr. A small residue at the E7 position may suggest the presence of
an E7 route entry system to facilitate the accessibility of diatomic ligands to
the 2/2HbO haem distal site (see below).
Group III sequences (all bacterial) display the largest extent of conserva-
tion (
Wittenberg et al., 2002
), all containing PheB9, TyrB10, PheCD1,
HisE7, PheE14, TrpE15, and TrpG8. Such a large number of strictly con-
served residues near the haem group suggest a narrow range of chemical
properties and group III 2/2HbPs form a single homogeneous class. It
should be noted, however, that recent sequence analysis based on an
expanded and corrected bacterial genome database containing 181 group
III 2/2HbPs in eight
phyla
showed group III to be less homogeneous than
originally thought and raised the possibility that diverse chemical behaviours
may be exhibited by its members (
Nothnagel, Winer, et al., 2011
).
In group III
C. jejuni
2/2HbP, the haem distal pocket residues TyrB10,
PheCD1, HisE7, IleE11, PheE14, and TrpG8 surround the haem-bound
ligand (
Nardini et al., 2006
). Similar residues are also conserved in the
2/2HbP from
Helicobacter hepaticus
(
Nothnagel, Winer, et al., 2011
). Among
these, only HisE7 is specific (and fully conserved) in group III (
Vuletich &
Lecomte, 2006
), although the hydrophobic character of the haem pocket
distal side is a highly conserved feature of group III globins. Contrary
to group II, group III 2/2HbPs display Phe (or hydrophobic) residue
at position CD1 and a hydrophobic residue at site E11. In
C. jejuni
2/2HbP, the only hydrogen-bonding residues involved in ligand stabiliza-
tion are TyrB10 and TrpG8, while no ligand-stabilizing interactions may be
provided by residues at CD1 and E11 positions, nor by HisE7. Mutagenesis
and molecular dynamics studies revealed that in the wild-type protein, the
main residue responsible for oxygen stabilization is TyrB10. Bound oxygen
is further stabilized by a hydrogen bond from either TrpG8 or HisE7,
depending on the orientation of the Fe-O-O moiety, the hydrogen bond
to TrpG8 being stronger than to HisE7. Most importantly, the coexistence
of multiple conformations
for the residues
in the distal cavity, each
