Báo cáo khoa học: Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms doc

Spectroscopic characterization of the isolated
heme-bound PAS-B domain of neuronal PAS domain
protein 2 associated with circadian rhythms
Ryoji Koudo
1
, Hirofumi Kurokawa
1
, Emiko Sato
1
, Jotaro Igarashi
1
, Takeshi Uchida
2
,
Ikuko Sagami
1
*, Teizo Kitagawa
2
and Toru Shimizu
1
1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
2 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan
Circadian rhythms are composed of complicated feed-
back loops [1–10]. In the suprachiasmatic nucleus, two
proteins, CLOCK and BMAL1, form a heterodimer
that binds to a specific DNA sequence named the
E-box, which is located in the promoter region of
genes associated with clock oscillation, for example
Per and Cry. The binding of the CLOCK-BMAL1 het-
erodimer to the E-box initiates transcription, and the
translated proteins, in turn, negatively regulate the ini-

tial transcription, thus constituting a feedback loop. A
very similar type of feedback regulation is thought to
function in the forebrain [11], wherein a transcription
protein named neuronal PAS protein 2 (NPAS2), in
place of CLOCK, forms a heterodimer with BMAL1.
Keywords
circadian rhythms; heme-sensor protein;
PAS domain; resonance Raman
spectroscopy; transcription
Correspondence
T. Shimizu, Institute of Multidisciplinary
Research for Advanced Materials, Tohoku
University, 2-1-1 Katahira, Aoba-ku, Sendai
980-8577, Japan
Fax: +81 22 217 5604 ⁄ 5390
Tel: +81 22 217 5604 ⁄ 5605
E-mail:
*Present address
Graduate School of Agriculture, Kyoto
Prefectural University, Nakaragi-cho 1-5,
Shimogamo, Sakyo-ku, Kyoto 606-8522,
Japan
(Received 24 March 2005, revised 15 June
2005, accepted 20 June 2005)
doi:10.1111/j.1742-4658.2005.04828.x
Neuronal PAS domain protein 2 (NPAS2) is an important transcription
factor associated with circadian rhythms. This protein forms a heterodimer
with BMAL1, which binds to the E-box sequence to mediate circadian
rhythm-regulated transcription. NPAS2 has two PAS domains with heme-
binding sites in the N-terminal portion. In this study, we overexpressed

wild-type and His mutants of the PAS-B domain (residues 241–416) of
mouse NPAS2 and then purified and characterized the isolated heme-
bound proteins. Optical absorption spectra of the wild-type protein showed
that the Fe(III), Fe(II) and Fe(II)–CO complexes are 6-co-ordinated low-
spin complexes. On the other hand, resonance Raman spectra indicated
that both the Fe(III) and Fe(II) complexes contain mixtures of 5-co-ordi-
nated high-spin and 6-co-ordinated low-spin complexes. Based on inverse
correlation between m
Fe-CO
and m
C-O
of the resonance Raman spectra, it
appeared that the axial ligand trans to CO of the heme-bound PAS-B is
His. Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala,
His329Ala, and His335Ala) were generated, and their optical absorption
spectra were compared. The spectrum of the His335Ala mutant indicated
that its Fe(III) complex is the 5-co-ordinated high-spin complex, whereas,
like the wild-type, the complexes for the five other His mutants were
6-co-ordinated low-spin complexes. Thus, our results suggest that one of
the axial ligands of Fe(III) in PAS-B is His335. Also, binding kinetics sug-
gest that heme binding to the PAS-B domain of NPAS2 is relatively weak
compared with that of sperm whale myoglobin.
Abbreviations
BJ FixL, a heme-binding oxygen sensor kinase, FixL, from Bradyrhizobium japonicum; HIF2a, hypoxia-inducible factor 2a; NPAS2, neuronal
PAS domain protein 2; PAS, acronym formed from the names of Drosophila-period clock protein (PER), vertebrate aryl hydrocarbon receptor
nuclear translocator (ARNT) and Drosophila single-minded protein (SIM).
FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS 4153
The NPAS2–BMAL1 heterodimer binds to the E-box
and initiates transcription associated with circadian
rhythms. A mouse knockout of NPAS2 adapts poorly

to restricted feeding and becomes severely ill [12].
NPAS2 is an 816-amino-acid protein composed of
a basic–helix–loop–helix (bHLH) domain (residues
1–77), two heme-bound PAS domains, PAS-A (resi-
dues 78–240) and PAS-B (residues 241–354), and a
C-terminal nuclear receptor association region [resi-
dues 665–680 for human NPAS2 (or MOP4)] [13].
Both the PAS-A and PAS-B domains contain heme-
binding sites [13]. Transcription caused by binding of
the NPAS2–BMAL1 heterodimer is hampered by CO,
an axial heme ligand [13]. Therefore, it is likely that
CO binding to the heme in NPAS2 may impair het-
erodimer formation or its DNA binding. In addition,
heme biosynthesis has been reported to correlate with
the circadian clock system [15]. However, as NPAS2
has two heme-binding sites, it is unclear which heme
contributes more to the binding of CO and the inhibi-
tion of transcription. Also, the heme environment,
including the axial ligands and heme-binding proper-
ties of the apo-NPAS2 protein, is not known.
In this study, we attempted to characterize the
heme-binding environment of the isolated PAS-B
domain of NPAS2. Four PAS-B domains with differ-
ent sequences were expressed in Escherichia coli. The
most stable PAS-B domain binds one molecule of
heme per molecule of protein, but the domain
appeared to form a large multimer (larger than a hexa-
mer) in both the presence and absence of heme. Opti-
cal absorption spectra of both Fe(III) and Fe(II)
complexes indicate that both are 6-co-ordinated low-

spin complexes. Based on Raman spectra, one of the
axial ligands appeared to be His. Site-directed muta-
genesis indicated that His335 is one of the axial ligands
for heme in the isolated PAS-B protein.
Results
Protein expression and purification
We attempted to express four types of the PAS-B
domain with different lengths corresponding to amino-
acid residues 160–346, 218–416, 218–346, and 241–416.
Only the PAS-B domain with residues 241–416 was
properly overexpressed and folded in E. coli and dis-
played sufficient heme-binding affinity. Therefore, we
used the protein consisting of residues 241–416 for
further studies on the structure and function of the
PAS-B domain.
His-tagged PAS-B was expressed in E. coli cells and
purified as a heme-bound protein in the presence of
hemin. It was then treated with thrombin to eliminate
the His tag. Undigested His-tagged protein was
removed by Ni ⁄ nitrilotriacetate ⁄ agarose column chro-
matography. The yield of PAS-B protein was 1.6
mgÆL
)1
culture. Heme-free PAS-B was also expressed
and purified in the absence of hemin. Titration experi-
ments with hemin demonstrated that purified PAS-B
has one binding site per protein with a heme to protein
ratio of 1 : 0.91. We estimated the spectral dissociation
constant using concentrations of free heme in the solu-
tion to be  2 lm (Fig. S1 in Supplementary material).

Quantification of heme by the pyridine hemochromo-
gen method [16] confirmed that heme-saturated PAS-B
contains 1.23 hemes per monomer.
Optical absorption spectra
Figure 1A shows the optical absorption spectra of
Fe(III), Fe(II), and Fe(II)–CO complexes of heme-
bound wild-type PAS-B. Optical absorption spectra of
a His335Ala mutant are also shown in Fig. 1B, which
A
B
Fig. 1. Optical absorption spectra of Fe(III) (bold solid lines), Fe(II)
(thin solid lines), and Fe(II)–CO (broken lines) complexes of the
wild-type (A) and His335Ala mutant (B) proteins. Spectra were
obtained in 100 m
M Tris ⁄ HCl (pH 7.5). Spectra of the His266Ala,
His289Ala, His300Ala, His329Ala mutants were essentially the
same as that of the wild-type protein.
Spectra of PAS-B of NPAS2 R. Koudo et al.
4154 FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS
will be discussed in the last part of Results. Table 1
summarizes the spectral maxima of the wild-type and
His mutant PAS-B complexes. The Soret peaks of the
Fe(III) and Fe(II) complexes of wild-type PAS-B are
located at 419 and 424 nm, respectively. Comparison
with other 6-co-ordinated low-spin heme proteins sug-
gested that Fe(III), Fe(II), and Fe(II)–CO wild-type
PAS-B complexes are also 6-co-ordinated low-spin
complexes. In addition, the results implied that His is
probably one of the axial ligands of wild-type PAS-B.
Resonance Raman spectra

To further understand the heme environment of PAS-
B, resonance Raman spectra were obtained and com-
pared with those of other hemoproteins. Resonance
Raman spectra of wild-type PAS-B heme complexes in
the high-frequency region are shown in Fig. 2 and sum-
marized in Table 2. Bands at 1373 and 1359 cm
)1
for
the Fe(III) and Fe(II) complexes of PAS-B, respectively,
were assigned to redox-sensitive m
4
. In the Fe(III) PAS-
B complex, the spin-state and co-ordination-state mar-
ker band, m
3
, was located at 1468 and 1502 cm
)1
,
suggesting the presence of 6-co-ordinated high-spin and
low-spin complexes, respectively [21]. Two m
3
bands
observed at 1470 and 1492 cm
)1
for the Fe(II) PAS-B
complex represent 5-co-ordinated high-spin and 6-co-
ordinated low-spin complexes, respectively. Upon bind-
ing of CO to the Fe(II) complex, the m
3
band shifted to

1498 cm
)1
, and the presence of a band at 1467 cm
)1
indicated partial photodissociation of CO from heme.
The CO-isotope dependences of the resonance
Raman spectra of the Fe(II)–CO complex of PAS-B
are shown in Fig. 3. The lower and middle spectra in
the 300–700 cm
)1
region in the left panel and the
1700–2000 cm
)1
region in the right panel represent
12
CO
16
O-PAS-B and
13
C
18
O-PAS-B, respectively,
whereas the upper spectra in both panels show the
Table 1. Optical absorption spectra (nm) of the wild-type and His mutant proteins of the isolated heme-bound PAS-B domain of NPAS2. Cyt
b
5
, Cytochrome b
5
;Cytb
562

, cytochrome b
562
; Sw Mb, sperm whale metmyoglobin.
Proteins
Fe(III) Fe(II) Fe(II)CO
ReferenceSoret Visible Soret Visible Soret Visible
Wild-type 419 536 424 529, 557 420 536, 571 This work
Wild-type 426 530, 561 426 530, 561 [14]
His266Ala 418 539 425 527, 556 420 536, 561 This work
His289Ala 419 538 424 530, 558 420 535, 564 This work
His300Ala 416 534 426 531, 559 421 537, 564 This work
His302Ala 417 536 425 530, 558 421 538, 562 This work
His329Ala 414 534 426 530, 559 421 539, 568 This work
His335Ala 395 536 421 559 420 542, 567 This work
Cyt b
5
412
(His ⁄ His 6cLS)
533, 562 423
(His ⁄ His 6cLS)
525, 556 [19]
Cyt b
562
418
(His ⁄ Met 6cLS)
230, 564 427
(His ⁄ Met6 cLS)
531, 562 [19]
Sw Mb 410
(His ⁄ H

2
O 6cLS)
505, 635 434
(His 5cHS)
556
(His ⁄ CO 6cLS)
423 542, 579 [20]
Fig. 2. Resonance Raman spectra (excited at 413 cm
)1
) of Fe(III)
(bottom), Fe(II) (middle), and Fe(II)-CO (top) complexes of PAS-B.
R. Koudo et al. Spectra of PAS-B of NPAS2
FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS 4155
isotope difference. It is clear from the difference spec-
tra that the 497-cm
)1
and 1961-cm
)1
bands of
12
C
16
O-
PAS-B are shifted to 489 and 1864 cm
)1
, respectively,
upon binding of
13
C
18

O. Accordingly, we assigned the
497-cm
)1
and 1961-cm
)1
bands to the m
Fe-CO
and m
C-O
modes, respectively. In Fig. 3 (left panel), the Fe-C-O
bending mode, m
Fe-C-O
, was also identified at 577 cm
)1
.
Figure 4 shows inverse correlations between m
Fe-C
and m
C-O
observed in the resonance Raman spectra.
The plot for PAS-B falls on the line of the histidine-
co-ordinated heme proteins, indicating a His-Fe-CO
6-co-ordinated adduct with PAS-B.
Heme association and dissociation
As shown in Fig. 5A, we obtained the association rate
constant for binding of the Fe(II)–CO heme complex
to apo-PAS-B. The spectral change monitored at
420 nm was composed of fast (73%) and slow (27%)
phases (Fig. 5B). The rate constant of the fast phase
for the heme association was dependent on the apo-

protein concentration (Fig. 5C), and the rate constant
of even the fast phase, 7.7 · 10
5
m
)1
Æs
)1
, was lower
than those of other heme-binding proteins (Table 3).
Note that SOUL and p22HBP are tentatively consid-
ered to be heme-transporting proteins, but their phy-
siological roles are not yet certain.
We also determined the dissociation rate of heme
from Fe(III) PAS-B by adding an excess of the His64-
Tyr ⁄ Val68Phe apomyoglobin mutant [18]. The forma-
tion of Fe(III)-bound myoglobin was monitored by
following the increase in A
410
(Fig. 6). The spectral
change was also composed of fast (32%) and slow
(68%) phases. The rate of the major slow change,
3.0 · 10
)4
s
)1
, is one order of magnitude lower than
those of other hemoproteins (Table 3).
Site-directed mutagenesis
His is probably the ligand trans to CO in the PAS-B
Fe(II)–CO complex. To identify which His is the axial

ligand, we examined the conserved His residues in
sequence alignments of PAS-B and PAS-A of NPAS2,
Table 2. Resonance Raman spectra of Fe(III) and Fe(II) complexes
of PAS-B.
Proteins m
4
m
3
State
Fe(III) 1373 1468 ⁄ 1502 6cHS ⁄ 6cLS
Fe(II) 1359 1470 ⁄ 1492 5cHS ⁄ 6cLS
Fe(II)–CO 1372 1467 ⁄ 1498 5cHS ⁄ 6cLS
Fig. 3. Resonance Raman spectra (excited at 413 cm
)1
) of the Fe(II)–CO complex of isolated PAS-B in the low (left) and high (right) fre-
quency regions. The lower spectra are of the Fe(II)–
12
C
16
O complexes, the middle spectra are of the Fe(II)–
13
C
18
O complexes, and the upper
spectra are difference spectra between those of the
12
C
16
O and
13

C
18
O complexes.
Spectra of PAS-B of NPAS2 R. Koudo et al.
4156 FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS
PAS-B of hypoxia-inducible factor 2a (HIF2a), and
the heme-binding PAS domains of BJ FixL and Ec
DOS (Fig. S2 in Supplementary material). However,
the sequence alignments indicated that, except for
His302 in PAS-B of NPAS2, the His residues are not
well conserved in the heme-binding PAS domains.
Optical absorption and resonance Raman spectral ana-
lyses suggested that PAS-B Fe(III) is a 6-co-ordinated
complex similar to PAS-A. In PAS-A NPAS2, His119
and Cys170 should be the axial ligands of the Fe(III)
complex, whereas His119 and His171 should be the
axial ligands for the Fe(II) complex [31]. We examined
the effects of mutating six His residues in PAS-A cor-
responding to those between His119 and His171 and
those spanning the Hb-sheet. Thus, we generated the
following mutants: His335Ala, His266Ala, His289Ala,
His300Ala, His302Ala, and His329Ala. As summarized
in Table 1, only the His335Ala mutant had an optical
absorption spectrum different from the wild-type. For
example, the spectrum of the Fe(III) His302Ala
mutant (data not shown) was essentially the same as
that of the Fe(III) wild-type (Fig. 1A), whereas that of
the Fe(III) His335Ala mutant (Fig. 1B) was distinct
and indicated a 5-co-ordinated high-spin Fe(III) com-
plex. Therefore, the results suggest that His335 is one

of the axial ligands of heme for both the Fe(III) and
Fe(II) PAS-B proteins.
Discussion
In this study, we characterized the isolated heme-
bound PAS-B domain of NPAS2 and identified His335
as one of the axial ligands. We found that the isolated
PAS-B domain from NPAS2 forms a large multimer
(larger than a hexamer; see Experimental procedures),
even though SDS ⁄ PAGE indicates that the protein is
more than 95% homogeneous (data not shown).
Although the PAS-B domain of the intact NPAS2 pro-
tein may play a critical role in heterodimer formation
with BMALl, the multimer found in this study appears
to be an artifact due to the truncation. Nevertheless,
optical absorption spectra of the isolated PAS-B
domain indicate one species for the Fe(III), Fe(II), and
Fe(II)–CO complexes. Resonance Raman spectra indi-
cate an apparent mixture of 5-co-ordinated high-spin
A
B
C
Fig. 5. Changes in the optical absorption spectra accompanying the
association of CO-heme (0.5 l
M) with heme-free PAS-B (3 lM)
upon mixing in a stopped-flow spectrometer (A). The spectral chan-
ges at 420 nm are shown in (B), and the correlation between k
obs
and the concentration of isolated heme-free PAS-B is shown in (C).
The spectral change was composed of fast (73%) and slow (27%)
phases, and the k

obs
values were taken from the fast phase.
Fig. 4. Inverse correlations between m
Fe-C
vs. m
C-O
in resonance
Raman spectra.
R. Koudo et al. Spectra of PAS-B of NPAS2
FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS 4157
and 6-co-ordinated low-spin complexes in the Fe(III)
and Fe(II) states. Two phases were found for heme
association and dissociation processes, which may
reflect heterogeneity of the oligomeric states.
Dioum et al. [14] first reported heme-binding and
spectroscopic characteristics of the isolated PAS-B
domain as well as those of the bHLH-PAS-A and
bHLH-PAS-A-PAS-B domains. They found that
absorption peaks of Fe(III) PAS-B are located at 426,
530 and 561 nm, and those of Fe(II)–CO PAS-B are
located at 426, 530 and 561 nm. These spectral max-
ima are different from those that we found (Table 1).
This discrepancy may be due, in part, to differences in
the method used to prepare the isolated domains.
Dioum et al. prepared the isolated domains by dena-
turation of an insoluble suspension of E. coli cells
overexpressing the protein and then subsequently
refolding the protein in the presence of heme. In con-
trast, our method does not use denaturation–refolding
procedures. Furthermore, our PAS-B protein contains

amino acids 241–416, whereas that of Dioum et al.is
composed of residues 160–346, which contain part of
the PAS-A domain and lack part of the C-terminus of
PAS-B (Fig. 7). These differences in protein prepar-
ation and domain architecture may result in differences
in spectroscopic parameters. We also attempted to
overexpress and isolate PAS-B with different amino-
acid residues (i.e. residues 160–346, 218–416, or 218–
346; Fig. 7), but we found that they had very low
heme-binding affinities, were much less stable, and had
lower expression than the PAS-B protein containing
amino acids 241–416 (our unpublished observations).
Recent crystal and solution structures of PAS proteins
and analysis sequence homologies have shown that
residues between amino acids 347 and 354 of mouse
NPAS2, which were absent in the studies by Dioum
et al. make up part of the core of the PAS-B domain
[27,28]. Therefore, the lack of these residues in the
study by Dioum et al. may have led to instability of
their protein [14].
Dioum et al. [14] also examined heme dissociation
from the bHLH-PAS-A-PAS-B domain. However,
because the protein they examined has two heme-bind-
ing sites, it is difficult to obtain independent values for
Table 3. Association and dissociation rate constants for heme bind-
ing to the isolated PAS-B domain and other heme proteins. Note
that k
on
values were obtained for the Fe(II)–CO complex, whereas
k

off
values were obtained for the Fe(III) complex. SOUL, a hexa-
meric heme-binding protein expressed in the retina and pineal
gland; p22HBP, a heme-binding protein isolated from mammalian
liver; Sw Mb, sperm whale metmyoglobin.
Proteins k
on
(M
)1
Æs
)1
) k
off
(s
)1
) Reference
PAS-B 7.7 · 10
5
(fast: 73%)
3.2 · 10
)3
(fast: 32%)
This work
<10
5
(slow: 27%)
3.0 · 10
)4
(slow: 68%))
SOUL 1.9 · 10

6
6.1 · 10
)3
[21]
p22HBP 1.0 · 10
8
4.4 · 10
)3
[21]
Sw Mb 7.6 · 10
7
8.4 · 10
)7
[17, 18]
A
B
Fig. 6. Changes in the optical absorption spectra accompanying the
dissociation of heme from isolated PAS-B and association with the
H64Y ⁄ V68F apomyoglobin mutant (A). The formation of Fe(II)–myo-
globin was monitored by measuring the increase in A
410
in the mix-
ture of Fe(III)–PAS-B and H64Y ⁄ V68F apomyoglobin (B).
Fig. 7. Constructs of PAS-B domains generated in this study. The
PAS-B construct containing residues 241–416 had appropriate
heme-binding affinity, whereas the PAS-B constructs consisting of
amino acids 160–346, 218–416, and 218–346 had low heme-binding
affinity and were much less stable than PAS-B containing residues
241–416.
Spectra of PAS-B of NPAS2 R. Koudo et al.

4158 FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS
heme binding to each PAS domain. The present study
is the first report of heme binding and dissociation
kinetic values of the isolated PAS-B domain.
Recent structural studies of heme-bound PAS pro-
teins suggest that ligand binding or a change in the
heme redox state causes profound structural changes
in the heme environment [22,23]. These structural
changes in the heme-bound PAS domain are trans-
mitted to the functional domain to regulate catalysis
or DNA binding for transcription. Structural changes
in the PAS domain of phototropins induced by light
have also been reported [24,25]. PAS domains are
probably very flexible in solution and change their
conformation in response to external signals, allowing
them to transmit the signal to downstream transducer
proteins [26].
Interestingly, it has been suggested that PAS-A, a
heme-binding PAS domain in NPAS2, has His119 and
Cys170 as axial ligands in the Fe(III) complex and that
ligand switching occurs from Cys170 to His171 upon
the reduction of heme [31]. Therefore, it is possible
that axial ligand switching of the heme in PAS-B also
occurs upon a change in redox state [23] because the
His335 mutation affected the Fe(III) form but not the
Fe(II) form. Because Fe(II)–CO heme was used to
evaluate heme binding and Fe(III) heme was used to
study heme dissociation, the large discrepancy between
the heme-binding rate constant ( 3s
)1

) and the heme
dissociation rate constant (3 · 10
)4
s
)1
; Table 3) sug-
gest that axial ligand switching may occur upon a
change in redox state. Also, His residues that we did
not examine by site-directed mutagenesis could be
additional axial ligands for heme in PAS-B.
His300 of PAS-B in NPAS2 is conserved in the PAS
proteins examined (Fig. S2, Supplementary material).
However, the corresponding His of Ec DOS, His83, is
not the axial ligand; rather, the axial ligand is His77.
Therefore, it is possible that co-ordination of heme by
PAS-B and PAS-A of NPAS2 is different from that in
Ec DOS.
Based on amino-acid alignments (Fig. S2, Supple-
mentary material), it appears that His335 of PAS-B in
NPAS2 is located at a turn between the Hb- and
Ib-sheets. This His is not conserved in the heme-bind-
ing PAS proteins. If the PAS-B domain functions only
in the heme binding, then it can be speculated that the
regulation of transcription by NPAS2 may be modula-
ted not only by CO binding but also by the redox state
of the heme iron. In this regard, structural changes
dependent on oxygen binding were observed at the
same turn between the Hb-sheets and Ib-sheets of Ec
DOS [32]. For BJ FixL, it has been suggested that CO
binding regulates intermolecular interactions through

the Hb and Ib sheets [33]. NMR analysis of PAS-B of
HIF2a has also suggested that the Hb-sheets and
Ib-sheets interact with ARNT in heterodimer forma-
tion [28]. Therefore, the turn between Hb-sheets and
Ib-sheets appears to play a critical role in various func-
tions of PAS proteins, including the interaction of
NPAS2 with BMAL1.
The PAS domain is critical for the ability of PAS
proteins to bind other proteins [27,28]. The small
chemical compound-binding sites and the flexibility of
the PAS domain have been examined by NMR spectr-
oscopy [29,30]. However, the specific role of the PAS-B
domain of NPAS2 remains elusive. In separate studies,
we have found that the heme-binding affinity of the
isolated PAS-A domain is much higher than that of
the isolated PAS-B domain (our unpublished results).
We speculate therefore that the heme in the PAS-A
domain plays a more important role than the PAS-B
domain in the regulation of transcription by CO. In
addition, the possible redox-dependent ligand switch-
ing of the PAS-B domain could modulate heterodimer
formation with BMAL1, leading to altered transcrip-
tion.
Although CO binding to the NPAS2–BMAL1 hetero-
dimer abolishes its ability to promote transcription,
the binding of CN, NO, or O
2
to the heterodimer
could promote heme-dependent transcription, but whe-
ther PAS-B is involved in the regulation of transcrip-

tion by these ligands remains to be determined.
Further studies are needed to address the specific
role of the hemes in PAS-B and PAS-B in heterodimer
formation with BMAL1, DNA binding, and transcrip-
tional inhibition by CO [14].
Experimental procedures
Materials
Mouse brain was obtained from C57BL ⁄ 6 mice. An mRNA
purification kit was purchased from Amersham Biosciences
(Uppsala, Sweden), and an RT-PCR kit was purchased
from Roche Diagostics Japan (Tokyo, Japan). Oligonucleo-
tides were synthesized at the Nihon Gene Research Labor-
atory (Sendai, Japan). The cloning vector, pBluescript SK
II(+), was purchased from Toyobo (Osaka, Japan), and
the expression vector, pET28a(+), was from Novagen
(Darmstadt, Germany). E. coli competent cells XL1-blue
for cloning were purchased from Novagen, and BL21-
CodonPlus(DE3)-RIL cells for protein expression were
from Stratagene (La Jolla, CA, USA). Restriction and
modifying enzymes for DNA recombination were pur-
chased from Takara Shuzo Co. (Otsu, Japan), Toyobo,
New England Biolabs (Beverly, MA, USA), and Roche
R. Koudo et al. Spectra of PAS-B of NPAS2
FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS 4159
Diagnostics Japan. All other chemicals were purchased
from Wako Pure Chemicals (Osaka, Japan).
Construction of NPAS2 PAS-B expression
plasmid
The His-tagged NPAS2 PAS-B expression plasmid was cre-
ated by subcloning into the pET28a(+) expression vector.

The cDNAs encoding the full-length mouse NPAS2 PAS-B
domain (residues 241–416) was generated by RT-PCR using
mouse brain RNA. The primers for RT-PCR were 5¢-
CGGGATCCCATATGTGTGTTAGCTGACG-3¢ and 5¢-
ACGCGTCGACTTAGTGGGAACTCCTTGAG-3¢. The
sequences of the products were confirmed using a DSQ-
2000 L automatic sequencer (Shimadzu Co., Kyoto, Japan)
and subcloned into the NdeI and SalI sites of the E. coli
expression vector, pET28a(+), which introduces a His
6
tag
at the N-terminus of expressed proteins.
To create the His266Ala, His289Ala, His300Ala,
His302Ala, His329Ala, and His335Ala mutants of NPAS2
PAS-B, PCR-based mutagenesis was performed using the
QuikChange mutagenesis kit from Stratagene using
pET28a(+) containing wild-type NPAS2 PAS-B cDNA as
a template. The primers used for mutations were as fol-
lows:
5¢-ATTTCTGGATGCCAGAGCTCCTCCAATC-3¢ and
5¢-GATTGGAGGAGCTCTGGCATCCAGAAAT-3¢ for
the His266Ala mutant, 5¢-GGCTACGACTACTACGC
CATTGATGACC-3¢ and 5¢-GGTCATCAATGGCGTAG
TAGTCGTAGCC-3¢ for the His289Ala mutant, 5¢-CTGG
CCAGGTGCGCCCAGCATCTGATG-3¢ and 5¢-CATCA
GATGCTGGGCGCACCTGGCCAG-3¢ for the His300Ala
mutant, 5¢-GTGCCACCAGGCTCTGATGCAGTTTGG-3¢
and 5¢-CCAAACTGCATCAGAGCCTGGTGGCAC-3¢ for
the His302Ala mutant, 5¢-GGTTGCAAACCGCCTACTA
CATCACCTAC-3¢ and 5¢-GTAGGTGATGTAGTAGGC

GGTTTGCAACC-3¢ for the His329Ala mutant, and 5¢-
CTACATCACCTACGCCCAATGGAACTCC-3¢ and 5¢-
GGAGTTCCATTGGGCGTAGGTGATGTAG-3¢ for the
His335Ala mutant.
The insertion of these mutations was confirmed by
sequencing.
Protein expression and purification
His-tagged NPAS2 PAS-B was expressed in E. coli BL21-
CodonPlus(DE3)-RIL cells. When the A
600
reached 0.6,
protein expression was induced by adjusting the culture to
0.05 mm isopropyl b-d-thiogalactopyranoside and incuba-
ting for 20–24 h. The E. coil cells expressing NPAS2 PAS-B
were suspended in buffer A (50 mm sodium phosphate,
pH 7.8, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol,
1mm phenylmethanesulfonyl fluoride, 2 lgÆmL
)1
aprotinin,
2 lgÆmL
)1
leupeptin, 2 lgÆmL
)1
pepstatin, and 2 mm
2-mercaptoethanol) and were crushed by pulsed sonication
for 2 min (three times with a 2-min interval between each
pulse) on ice using a UD-201 ultrasonic disruptor (Tomy
Seiko, Tokyo, Japan). Apo-PAS-B in crushed cells was
reconstituted with heme by incubating it with buffer A con-
taining 100 lm heme. After centrifugation at 142 000 g for

35 min at 4 °C, ammonium sulfate was added to the result-
ing supernatant up to 50% saturation. Precipitates were
collected and then dissolved in buffer A. The solution was
passed through a Sephadex G-25 column (4 · 20 cm) pre-
equilibrated with buffer A. The eluted solution was applied
to a Ni ⁄ nitrilotriacetate ⁄ agarose column pre-equilibrated
with buffer B (50 mm sodium phosphate, pH 7.8, 50 mm
NaCl, and 2 mm 2-mercaptoethanol). The column was
washed with sequential steps of buffer B containing 20 and
50 mm imidazole. The NPAS2 PAS-B protein was then
eluted with buffer A containing 100 mm imidazole. The
protein fractions were pooled and concentrated. The buffer
in the collected protein was exchanged with 100 mm
Tris ⁄ HCl (pH 8.0) using a HiTrap desalting column (Amer-
sham Biosciences). The purified protein was quickly frozen
in liquid nitrogen and stored at )80 °C. Protein concen-
trations were determined using the CBB dye binding
method (Nacalai Tesque, Kyoto, Japan), and heme content
was determined by the pyridine hemochromogen method
[16].
Removal of His tag by digestion with thrombin
His-tagged NPAS2 PAS-B was incubated with 2 eq heme
for 3 h at 0 °C in 100 mm Tris ⁄ HCl buffer (pH 8.0). Excess
heme was then removed using a Bio-Gel P-6 column
(Bio-Rad, Hercules, CA, USA) under the same buffer con-
ditions. Thrombin protease [10 UÆ (mg NPAS2 PAS-B
protein)
)1
] was added to the heme-saturated His-tagged
NPAS2 PAS-B and incubated for 4 h at 16 °C. The solu-

tion was then applied to a Ni ⁄ nitrilotriacetate column
pre-equilibrated with 100 mm Tris ⁄ HCl (pH 8.0). The flow-
through fractions were collected as purified NPAS2 PAS-B
lacking the His tag.
Size exclusion column chromatography
We used size-exclusion chromatography, Superdex 75, for
evaluation of the oligomeric state of the purified protein.
Apparent molecular masses determined for both heme-bound
and heme-free PAS-B proteins were higher than 120 kDa.
Optical absorption spectra
Optical absorption spectra were collected under aerobic
conditions using a Shimadzu UV-2500 and a Shimadzu
Multi Spec 1500 spectrophotometer maintained at 25 °C.
To ensure that the temperature of the solution was appro-
priate, the reaction mixture was incubated for 10 min
Spectra of PAS-B of NPAS2 R. Koudo et al.
4160 FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS
before spectroscopic measurements. Experiments were per-
formed at least three times for each complex.
Heme quantification
To quantify heme, 10 lm protein was treated with 30%
(v ⁄ v) pyridine in 0.1 m NaOH, after which a few grains of
sodium dithionite were added. The heme concentration was
calculated from the difference in A
556
and A
540
and using
an absorption coefficient of 22.1 mm
)1

Æcm
)1
[16].
Resonance Raman measurements
The Fe(III)–PAS-B complex (25 lm in 100 mm Tris ⁄ HCl,
pH 8.0) was placed in an airtight spinning cell with a
rubber septum and reduced by the addition of sodium
dithionite (10 mm final concentration).
12
C
16
Oor
13
C
18
O
(Cambridge Isotope Laboratories, Andover, MA, USA) gas
was introduced into the Raman cell with an airtight syr-
inge. Raman scattering was excited at 413.1 nm with a Kr
ion laser (BeamLok 2060; Spectra-Physics, Mountain View,
CA, USA). The excitation light was focused into the cell at
a laser power of 5 mW for the Fe(III) and Fe(II) com-
plexes. For the Fe(II)–CO complexes, to avoid photolysis,
the laser power was 0.1–0.2 mW. Raman spectra were
detected with a N
2
-cooled CCD camera (Spec10: 400BLN;
Roper Scientific, Inc., Trenton, NJ, USA) attached to a sin-
gle polychromator (SPEX750M; Jobin Yvon, Longjumeau,
France). Raman shifts were calibrated with indene, acetone,

CCl
4
, and an aqueous solution of ferrocyanide.
Stopped-flow measurements
Stopped-flow absorbance measurements for obtaining heme
association rate constants were conducted using an RSP-
1000 stopped-flow apparatus (Unisoku, Osaka, Japan)
maintained at 25 °C. Association of CO-heme with heme-
free PAS-B was monitored at 420 nm. The reaction was ini-
tiated by mixing with excess apoprotein. Data were fitted
using the data analysis program Igor-Pro (Wavemetrics,
Inc., Oswego, OR, USA) [17].
Heme dissociation experiments
All heme dissociation experiments were carried out in
1-cm path length, 1-mL volume cuvettes containing
800 lL of reaction mixture at 25 °C. For most experi-
ments, this mixture consisted of 0.15 m potassium phos-
phate (pH 7.0), 0.45 m sucrose, 30 lm apomyoglobin
His64Tyr ⁄ Val68Phe, and 3.0 lm stock PAS-B holoprotein
solution. The pH values of these reaction mixtures never
deviated more than 0.02 pH unit from that of the original
solution. The reactions were initiated by adding holopro-
tein to the buffer ⁄ apomyoglobin mixture. When multiple
reactions were carried out, absorbance time course data
were collected at a single wavelength (410 nm) as des-
cribed previously [18].
Acknowledgements
This work was supported in part by Grants-in-Aid
from the Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan (to T. S. and H. K.).

We thank Dr John S. Olson (Rice University) for
kindly providing the expression plasmid for His64-
Tyr ⁄ Val64Phe apomyoglobin.
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Supplementary material
The following material is available online.
Fig. S1. Spectral changes caused by adding heme to
apo-PAS-B. The spectral change was composed of two
phases. It is difficult to estimate the exact dissociation

constants from the two phases. We tentatively estima-
ted the apparent spectral dissociation constant to be
 2 lm.
Fig. S2. Sequence alignments of PAS-B and PAS-A of
NPAS2, PAS-B of HIF2a, a heme-binding PAS
domain of BJ FixL and PAS-A of Ec DOS. Amino
acids at 266, 289, 300, 302, 329, and 335 with bold H
of PAS-B of NPAS2 were mutated to Ala in this
study. The secondary structure is based on the solution
structure of HIF2a PAS-B domain [28].
Spectra of PAS-B of NPAS2 R. Koudo et al.
4162 FEBS Journal 272 (2005) 4153–4162 ª 2005 FEBS