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FG loop in the PAS domain. Thus, the dissociation of Met104 upon CO
binding will cause conformational changes at the distal haem pocket as is
the case of FixL and
Ec
Dos, which is responsible for the activation of RcoM2
by CO.
3.3. Neuronal PAS domain 2 protein
Neuronal PAS domain 2 protein (NPAS2) is a mammalian transcriptional
factor that binds DNA as a heterodimer with brain and muscle Arnt-like
protein (BMAL1), which is thought to be involved in the regulation of cir-
cadian rhythm. NPAS2 knockout mice fail to exhibit rhythmic
per2
gene
expression (
Reick, Garcia, Dudley, & McKnight, 2001
). NPAS2/BMAL1
heterodimer activates the expression of
per
and
cry
genes that are negative
regulatory components of the circadian clock (
King et al., 1997
).
NPAS2 is a member of the basic helix-loop-helix (bHLH)-PAS family,
which consists of the N-terminal bHLH domain and two PAS domains
(PAS-A and PAS-B). Though both of the recombinant PAS-A and PAS-
B bind a b-type haem, DNA-binding activity of NPAS2 is independent
of presence or absence of haem (
Dioum et al., 2002
). NPAS2/BMAL1
heterodimer can bind to DNA regardless of whether NPAS2 is apo- or
holo-forms (
Dioum et al., 2002
). On the other hand, CO regulates the
DNA-binding activity only for holo-NPAS2, but not for apo-NAPS2
(
Dioum et al., 2002
). CO-dependent regulation of DNA binding for
NPAS2 will be caused by the inhibition of the NPAS2/BMAL1
heterodimer formation, which will be induced by CO-binding to the haem
in NPAS2. Haem oxygenase 2 (HO-2), which produces CO as a reaction
product of haem degradation, is expressed in the same region where NPAS2
is expressed. These results suggest that CO generated by HO-2 acts as a
physiological effector of NPAS2 (
Dioum et al., 2002
).
The resonance Raman spectra of the ferric and ferrous haems in PAS-A
domain consist of a mixture of 6-coordinate and 5-coordinate states (
Uchida
et al., 2005
). The coordination structures of the haem in the predominant
species are proposed as shown in
Fig. 7.2
, which are elucidated by site-
directed mutagenesis and resonance Raman spectroscopy. Mutation of
His119 or Cys170 to Ala significantly increases the intensity of the
n
3 band
at 1490 cm
1
for a 5-coordinate, high-spin haem compared with that of the
n
3 band at 1504 cm
1
for a 6-coordinate, low-spin haem, whereas the
mutation of His148 or His171 induces a moderate increase (
Uchida et al.,
2005
). These results suggest that His119 and Cys170 are axial ligands of
