Biology Reference
In-Depth Information
the haem induces the reconstruction of hydrogen-bond network around the
haem with the conformational change of the FG loop, which results in trig-
gering the conformational changes of FixL for the functional regulation
of FixL.
4.1.4 Ligand-binding properties of the haem in FixL
Resonance Raman and UV-vis spectroscopy reveal that the ferric and fer-
rous haems are 5-coordinate and high-spin state (
Balland et al., 2006; Lukat-
Rodgers, Rexine, & Rodgers, 1998; Tomita et al., 2002
). The ferric and
ferrous haem in FixL can bind an external ligand at the vacant site of the
haem as an axial ligand. Rate constants of ligand binding/dissociation for
FixL are summarised in
Table 7.1
.O
2
-binding affinity of FixL is lower com-
pared with a typical O
2
-binding protein, sperm whale myoglobin (
Sw
Mb),
which is due to lower
k
on
by about two orders of magnitude.
O
2
-binding affinity of
Bj
FixL increases by a truncation of the C-terminal
kinase domain by ca. fourfold, where the
K
d
values for O
2
are 142 and 33
m
M
for
Bj
FixL with and without the kinase domain, respectively. Truncation of
the kinase domain results in the increase and decrease of
k
on
and
k
off
,respec-
tively, in
Bj
FixL. These differences may be caused by conformational changes
of the PAS domain induced by the truncation of the kinase domain.
4.1.5 Ligand-binding and protein dynamics revealed by
time-resolved spectroscopy
Time-resolved spectroscopy is a useful tool to elucidate the dynamic prop-
erties of ligand binding and protein conformational changes. Time-resolved
resonance Raman spectroscopy with subpicosecond time resolution reveals
that no sizeable doming of the haem is observed in photolysis of O
2
-bound
Bj
FixLH, suggesting that efficient ultrafast O
2
rebinding to the haem occurs
on the femtosecond timescale (
Kruglik et al., 2007
). This property is differ-
ent from that for ligand-bound globins, in which haem doming occurs faster
than 1 ps after photolysis of the haem-bound ligand. The dissociated O
2
can-
not move away substantially from the haem because of steric constraints and
remain in a favourable position for rebinding in
Bj
FixLH (
Kruglik et al.,
2007
). On the other hand, the recombination of CO to the haem does
not occur on the time scale up to 4 ns (
Jasaitis et al., 2006; Liebl,
Bouzhir-Sima, Negrerie, Martin, & Vos, 2002
). Time-resolved infrared
spectroscopy reveals that photolyzed CO is trapped in the docking site,
in which CO is oriented at a large angle with respect to the haem normal
(
Nuernberger et al., 2011
). Escape of CO from the docking site to the
