Biology Reference
In-Depth Information
the S
d
S bond, if it is present, to restore kinase activity.
Tuckerman et al.
(2002)
and
Akimoto et al. (2003)
use dithionite and DTT, respectively,
to reduce met
Sm
FixL, which may be a reason of the above discrepancies.
Though CO, NO, CN
,F
, and imidazole (Im) can be bound to the
haem in FixL as is O
2
, their regulatory effects on the FixL activity are not
identical in all ligands. CO, NO, and F
do not significantly inhibit the
kinase activity of FixL, while CN
and Im inhibit the ferric FixL activity
as effective as O
2
(
Akimoto et al., 2003; Tuckerman et al., 2002
). These
results clearly indicate that FixL discriminates O
2
from CO and NO. Spec-
troscopic and structural analyses of the ligand-bound FixLs with these
ligands give useful information to elucidate the molecular mechanisms of
selective O
2
sensing by FixL and of O
2
-dependent signal transduction,
which are described in the following sections.
4.1.2 X-ray crystal structures of the PAS domain in FixL
The X-ray crystal structures of the PAS domain have been determined in the
met and deoxy forms for
Sm
FixL (
Miyatake et al., 2000
). The overall
structures of the met and deoxy forms are almost same, and there is no
functionally important structural difference between these forms.
A protohaem (b-type haem) is accommodated between the F helix and five
antiparalleled
b
strands (A
b
,B
b
,H
b
,I
b
, and G
b
strands). His194 on the
F helix in
Sm
FixL (His200 in
Bj
FixL) is the proximal ligand of the haem.
The ferric haem is 5-coordinate with a vacant site at the sixth position,
which is out-of-plane configuration with a doming of haem plane. The
iron is displaced by 0.49
˚
from the pyrrole nitrogen (
Miyatake et al.,
2000
). The Fe-N distance between the haem iron and the proximal Im is
2.14
˚
, which is consistent with the distance determined by EXAFS
(2.11
˚
)(
Miyatake et al., 1999a,b
). The orientation of His194 is fixed
by a hydrogen-bond network among His194, carboxylate of Asp195, the
amide group of Asn181, and a water molecule in the proximal haem pocket
(
Miyatake et al., 2000
).
There are several hydrophobic residues (Ile209, Leu230, and Val232 in
Sm
FixL) in the vicinity of the distal side on the haem. The distal haem
pocket is packed so densely with these hydrophobic residues that O
2
could
not bind to the haem iron without collisions with these residues, indicating
that a conformational change in the distal haem pocket should be required to
bind O
2
(
Miyatake et al., 2000; Mukai, Nakamura, Nakamura, Iizuka, &
Shiro, 2000
). Based on these structural properties, the “hydrophobic triad
model” is proposed for the signal transduction mechanism of FixL. In this
