CO-dependent Activity-controlling Mechanism of Heme-containing CO-sensor Protein, Neuronal PAS Domain Protein 2*

Neuronal PAS domain protein 2, which was recently established to be a heme protein, acts as a CO-dependent transcription factor. The protein consists of the basic helix-loop-helix domain and two heme-containing PAS domains (PAS-A and PAS-B). In this study, we prepared wild type and mutants of the isolated PAS-A domain and measured resonance Raman spectra of these proteins. Upon excitation of the Raman spectrum at 363.8 nm, a band assignable to Fe3+-S stretching was observed at 334 cm–1 for the ferric wild type protein; in contrast, this band was drastically weaker in the spectrum of C170A, suggesting that Cys170 is an axial ligand of the ferric heme. The Raman spectrum of the reduced form of wild type was mainly of six-coordinate low spin, and the ν11 band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c3, suggesting coordination of a strong ligand and thus a deprotonated His. In the reduced forms of H119A and H171A, the five-coordinate species became more prevalent, whereas no such changes were observed for C170A, indicating that His119 and His171, but not Cys170, are axial ligands in the ferrous heme. This means that ligand replacement from Cys to His occurs upon heme reduction. The νFe-CO versus νC-O correlation indicates that a neutral His is a trans ligand of CO. Our results support a mechanism in which CO binding disrupts the hydrogen bonding of His171 with surrounding amino acids, which induces conformational changes in the His171-Cys170 moiety, leading to physiological signaling.

In recent years, a variety of heme-containing gas sensor proteins have been discovered in different species, from bacteria to mammals (1)(2)(3). In these proteins, a change in the concentration of gas molecules such as NO, O 2 , or CO is detected by a heme group and transduced to the functional domain as a signal, leading to modulation of protein activity. The hemebased gas sensor proteins discovered so far are listed in Table  I. NO is a signaling molecule involved in vasodilation and neuronal transmission (4,5). Soluble guanylate cyclase (sGC) 1 is a well known heme-based NO sensor protein. Upon binding of NO to sGC, the iron-histidine bond in the N-terminal region, the sole covalent linkage between the heme and protein, is cleaved. The bond cleavage induces conformational changes, resulting in a 400-fold increase in GC activity in the C-terminal region (6). Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) also forms a five-coordinated NO-heme complex via Fe-His bond cleavage, resulting in activation by NO (7). The O 2 -sensing proteins identified so far include FixL (8), DOS (9), PDEA1 (2), and HemAT (10,11). The sensory domains of FixL and Ec DOS belong to the PAS 2 superfamily. FixL is a hemebased oxygen sensor involved in the regulation of expression of nitrogen fixation genes in response to O 2 concentration (8,12,13). Under low O 2 concentrations, FixL is autophosphorylated at a histidine residue and transfers it to FixJ, whereas a high concentration of O 2 suppresses kinase activity (8). Ec DOS is also an O 2 (and/or redox) sensor protein identified in Escherichia coli that exhibits phosphodiesterase activity in an O 2dependent (and/or redox-dependent) manner (9,14). CooA was the first CO sensor protein identified from a purple nonsulfur photosynthetic bacterium, Rhodospirillum rubrum (15,16). When CO binds to heme, the accompanying conformational changes induce binding of CooA to its target DNA (17). Neuronal PAS domain protein 2 (NPAS2) was the second heme-based CO sensor protein discovered but the first one identified in mammals (18). It acts as a transcription factor for clock genes in a CO-dependent manner.
NPAS2 is a member of the basic helix-loop-helix (bHLH)-PAS family, including BMAL1 and Clock (19). As illustrated schematically in Fig. 1, the protein consists of the N-terminal bHLH domain and two PAS domains (PAS-A and PAS-B), structural modules that are present in widespread components of signal transduction proteins from organisms in all kingdoms of life (20). Both PAS-A and PAS-B bind one heme, although the protein can work as a transcriptional activator without heme (18). The PAS domains of NPSA2 display significant sequence similarity to Clock. The bHLH, PAS-A, and PAS-B domains are 84, 69, and 90% identical, respectively, to the corresponding domains of Clock sequence (21). Both NPAS2 and Clock form heterodimers with BMAL1 and activate the expression of per and cry genes, which are negative regulatory components of the circadian clock. NPAS2 performs the same function as Clock but is expressed in a different region of the body. Specifically, NPAS2 is expressed primarily in the forebrain (22,23), whereas Clock is present in the suprachiasmatic nucleus (21,24).
Both proteins bind to the same DNA sequence as heterodimers with BMAL1 (25,26). DNA binding of NPAS2-BMAL1 is modulated by the concentration ratio of oxidized and reduced NAD. NAD(P)H enhances the DNA binding ability of NPAS2-BMAL1 by interacting with the bHLH domain, whereas NAD(P) ϩ inhibits DNA binding (26). Furthermore, CO molecules produced by heme oxygenase-2 also modulate DNA binding of NPAS2-BMAL1 in the presence of a heme bound to the PAS domains, as reported by Gilles-Gonzalez and Mc-Knight (18). At low micromolar levels of CO, heme forms a complex with CO, resulting in inhibition of DNA binding of NPAS2. In this study, we perform a resonance Raman (RR) investigation of the PAS-A domain of mouse NPAS2 to elucidate the signal transduction mechanism induced by CO binding. Although CO binds to both the PAS-A and PAS-B domains, the CO association rate constant for the PAS-A domain is about 10-fold higher than that for the PAS-B domain (18). This indicates that CO initially binds to the PAS-A domain. Our data show that the heme in the PAS-A domain exists as a mixture of penta-and hexacoordinate forms in both ferric and ferrous states, consistent with absorption spectra (18), and that axial ligand switching of heme between Cys 170 and His 171 occurs in a redox-dependent manner. Furthermore, our data indicated that the hydrogen bonding of the proximal histidine to surrounding amino acids is disrupted by CO binding. We discuss the CO-dependent signal transduction mechanism of NPAS2 in comparison with those of other heme-PAS sensor proteins.

EXPERIMENTAL PROCEDURES
Materials-Mouse livers were obtained from C57BL/6 mice. The mRNA purification and reverse transcription-PCR kits were purchased from Amersham Biosciences and Roche Applied Science, respectively. Oligonucleotides were synthesized at Nihon Gene Research Laboratory (Sendai, Japan). The cloning vector, pBluescript SK II(ϩ), and an expression vector, pET28a(ϩ), were purchased from Toyobo (Osaka, Japan) and Novagen (Darmstadt, Germany), respectively. E. coli competent cells, XL1-blue (for cloning) and BL21 (for protein expression), were purchased from Novagen and Stratagene (La Jolla, CA), respectively. Restriction and modifying enzymes for DNA recombination were obtained from Takara Bio Inc., Toyobo, New England BioLabs (Beverly, MA), and Nippon Roche (Tokyo, Japan). 13 C 18 O and 13 CO were acquired from Cambridge Isotope Laboratories, Inc. (Andover, MA). 54 Fe-labeled heme was purchased from Frontier Scientific, Inc. (Logan, UT). Other chemicals were from Wako Pure Chemicals (Osaka, Japan).
Construction of the Isolated PAS-A Domain of NPAS2-His-tagged expression plasmids of the isolated PAS-A domain of NPAS2 (containing amino acid residues 78 -240) were generated by subcloning into the pET28a(ϩ) expression vector. cDNA encoding the PAS-A domain was generated by reverse transcription-PCR using RNA isolated from mouse livers. The primers employed for reverse transcription-PCR are 5Ј-CGGGATCCCATATGTCATTCCTCAGTAACG-3Ј and 5Ј-GCAAGC-TTGTCGACTTATTCCTTTAAGAACTGC-3Ј. Primers for the 5Ј-ends contained BamHI and NdeI restriction sites, and those for 3Ј ends contained SalI restriction sites for subcloning. PCR products were digested with BamHI and SalI and inserted in the corresponding sites of the cloning vector, pBluescript SK II(ϩ). The clones obtained were confirmed by determination of the nucleotide sequence by Sanger's method using an automatic sequencer, DSQ-2000L (Shimadzu Co., Kyoto, Japan). The pBluescript-PASA plasmid was digested with NdeI and SacI and subcloned into the E. coli expression vector, pET28a(ϩ), which introduces a His 6 tag at the N terminus of expressed proteins.
To create mutants of the PAS-A domain, PCR-based mutagenesis was performed using the QuikChange mutagenesis kit from Stratagene with pET28 containing wild type PAS-A as a template. The desired mutation was confirmed by sequencing.
Protein Expression and Purification-The His-tagged PAS-A domain was expressed in E. coli BL21(DE3)-CodonPlus harboring each expression vector. Protein expression was induced at A 600 ϭ 0.6 by the addition of isopropyl ␤-D-thiogalactopyranoside (final concentration of 50 M). Cells were further incubated for 20 -24 h after the addition of isopropyl ␤-D-thiogalactopyranoside. E. coli cells expressing the PAS-A domain were suspended in buffer A (50 mM sodium phosphate buffer, pH 7.8, 50 mM NaCl, 2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml pepstatin, 2 mM 2-mercaptoethanol). Cells were crushed by pulsed sonication for 2 min (3 times with 2-min intervals) on ice using the ULTRASONIC DISRUPTOR UD-201 (Tomy Seiko, Tokyo, Japan) and centrifuged at 35,000 rpm for 35 min at 4°C. Purification of the PAS-A domain using buffer without hemin resulted in a mixture of apo-and heme-bound proteins. To reconstitute PAS-A with hemin, a final concentration of 100 M hemin was added to the crude cell extract after sonication and incubated for 30 min at 4°C. Ammonium sulfate was added to the resulting mixture up to 70% saturation. The precipitate was collected and dissolved in buffer A. Next, the solution was passed through a Sephadex G-25 column (4 ϫ 20 cm) pre-equilibrated with the same buffer. The eluted solution was applied to a Ni 2ϩ -nitrilotriacetic acidagarose column (Qiagen, Hilden, Germany) pre-equilibrated with buffer B (50 mM sodium phosphate (pH 7.8), 50 mM NaCl, and 2 mM 2-mercaptoethanol). The column was washed sequentially with buffer B containing 20 mM and 50 mM imidazole. Protein was eluted with buffer B containing 100 mM imidazole. Protein fractions were pooled and concentrated. After concentration, purified proteins were immediately frozen in liquid nitrogen and stored at Ϫ80°C until use.
To eliminate His tags, PAS-A was treated with thrombin. Undigested His tag protein was removed by passing through an Ni 2ϩ -nitrilotriacetic acid-agarose column. Before analysis, the protein buffer was altered to 100 mM Tris-HCl (pH 8.0) using a Sephadex G-25 column or a Bio-Gel P-6 column (Bio-Rad). Concentrations were determined using the Coomassie Brilliant Blue dye binding method for protein (Nacalai Tesque, Kyoto, Japan) and the pyridine hemochromogen method for heme.
Resonance Raman Measurements-Static RR spectra were obtained with a single polychromator (SPEX750M; Jobin Yvon) equipped with a liquid nitrogen-cooled CCD detector (Spec10:400BLN; Roper Scientific). The excitation wavelengths employed were 413.1 and 568.2 nm from a krypton ion laser (BeamLok 2060; Spectra Physics) and 363.8 nm from an argon ion laser (BeamLok 2080; Spectra Physics). The laser power at the sample point was adjusted to ϳ5 milliwatts for air-oxidized and dithionite-reduced forms and to 0.1 milliwatts for the CO-bound form to prevent photodissociation. Raman shifts were calibrated with indene, CCl 4 Fig. 2 depicts the RR spectra of ferric (A and D), ferrous (B and E), and CO-bound (C and F) PAS-A domains of NPAS2 at pH 8.0 in the low (left) and high (right) frequency regions. It is well established that the Raman spectra of heme proteins in the high frequency region comprise porphyrin in-plane modes, which are sensitive to oxidation, spin, and coordination states of the heme iron (27)(28)(29). The oxidation state marker band, 4 , of the oxidized protein appeared at 1373 cm Ϫ1 , typical of the ferric state of heme proteins (Fig. 2, spectrum D). The spin and coordination marker band, 3 , is composed of an intense 1504cm Ϫ1 peak with a small shoulder at 1490 cm Ϫ1 and a weak 1471-cm Ϫ1 peak, suggesting the co-existence of six-coordinate low spin (6cLS), five-coordinate high spin (5cHS), and sixcoordinate high spin (6cHS) hemes in the ferric form. The 6cLS species (1504 cm Ϫ1 ) is dominant at neutral pH. A mixture of five-and six-coordinate species is also observed in the ferrous form ( Fig. 2, spectrum E), for which 3 bands observed at 1471 and 1493 cm Ϫ1 correspond to the 5cHS and 6cLS species, respectively. The addition of CO to the ferrous protein shifts the 4 band from 1360 to 1372 cm Ϫ1 , indicating the formation of CO-bound heme. The frequencies of the marker bands of NPAS2 are compared with those of other heme proteins in Table II.

Soret-excited Resonance Raman Spectra of the PAS-A Domain-
Low frequency RR spectra of the PAS-A domain are additionally illustrated in Fig. 2 (left panel). Spectra in this region are very useful for identifying a ligand, since metal-ligand vibrations directly demonstrate the presence of a particular ligand and the nature of its interactions in the heme pocket. Fe 3ϩ -OH stretching ( Fe-OH ) bands have been observed for alkaline ferric myoglobin (Mb), hemoglobin, and horseradish peroxide in the 450 -555-cm Ϫ1 region, which shift upon isotope substitution of solvent (D 2 O and H 2 18 O) (30 -32). However, for the ferric PAS-A domain, no isotope-sensitive bands were observed in the Fe-OH region (data not shown). This finding suggests that the sixth ligand of the heme iron in the ferric state is not a hydroxide at pH 8.0, although the results do not necessarily exclude the possibility of water coordination in the 6cHS species. Generally, the Fe-His stretching mode ( Fe-His ) is evident in the 200 -250-cm Ϫ1 region for the ferrous five-coordinate species (33). However, RR spectra of the ferrous PAS-A domain excited at 413.1 and 441.6 nm exhibited a weak feature at around 220 cm Ϫ1 , probably due to the predominance of 6cLS species. The CO-bound form (Fig. 2, spectrum F) gives 4 at 1372 cm Ϫ1 , but the spectral pattern in the lower frequency region (spectrum C) is similar to that of the ferrous form (spectrum B) except for a prominent band at 496 cm Ϫ1 (spectrum C), which is assigned to the Fe-CO stretching mode ( Fe-CO ), as examined later.
Propionate bending modes, ␦(C ␤ C c C d ), for the PAS-A domain in the ferrous and CO-bound forms were observed at 382 and 379 cm Ϫ1 , respectively (Fig. 2, spectra B and C). The frequency of the propionate bending mode is correlated with the strength of the hydrogen bond between heme propionate and surrounding amino acid residues (34 -36). For instance, the heme-7propionate group of Mb is hydrogen-bonded to His 97 and Ser 92 , and its ␦(C ␤ C c C d ) mode appears at 376 cm Ϫ1 . Disruption of this hydrogen bond in H97F and H97A/S92A mutants results in a downshift to 366 and 365 cm Ϫ1 , respectively (36). Therefore, the higher frequencies of ␦(C ␤ C c C d ) observed for the PAS-A domains in both ferrous and CO-bound forms suggest strong hydrogen bonding between the heme propionate group and nearby amino acid residues.
Near UV-excited Resonance Raman Spectra of the PAS-A Domain-With near UV excitation for the 5cHS heme possessing Cys as a proximal ligand, the Fe-S stretching mode is intensity-enhanced upon excitation within the Fe-S charge transfer band and in fact is observed around 350 cm Ϫ1 for P450 and nitric-oxide synthase (37,38). A low frequency RR spec-trum of the ferric PAS-A domain at pH 8.0 excited at 363.8 nm is shown in Fig. 3. A broad band was observed around 340 cm Ϫ1 for the wild type (WT) protein (spectrum A). When the band is fit with two Gaussian bands, the calculated Raman shifts were located at 334 and 347 cm Ϫ1 . With decreasing pH from 8.0 to 7.0, the intensity of the 334 cm Ϫ1 band decreased drastically, whereas the 347-cm Ϫ1 band was little affected (spectrum B). At pH 7.0, Soret-excited RR spectrum showed the disappearance of the 5cHS species as shown by the loss of the 3 band at 1491 cm Ϫ1 (spectra E and F, left inset). Since the Fe-S stretching mode is observable for the 5cHS species, the intensity decrease in the 334-cm Ϫ1 band at pH 7.0 suggests that the band is derived from the 5cHS species.
Among four cysteine residues contained in the PAS-A domain, only Cys 170 is inferred to be close to the heme-binding region, and accordingly, we replaced Cys 170 with Ala to confirm Cys coordination. The corresponding Raman spectrum of the C170A mutant is depicted by spectrum C. The Raman spectrum of the C170A mutant is similar to that of WT protein at pH 7.0 (spectrum B). Furthermore, the band at 334 cm Ϫ1 was upshifted by ϳ2.0 cm Ϫ1 in the 54 Fe-labeled protein (Fig. 3, right inset, spectrum H). Although the 334-cm Ϫ1 band did not disappear completely in the C170A mutant and WT at pH 7.0, it is presumably due to a porphyrin mode of 6cLS heme present around 330 cm Ϫ1 and accidentally overlapped with the Fe-S stretching mode. These results indicate that a major part of the Raman band at 334 cm Ϫ1 is derived from the Fe-S stretching mode, and Cys 170 is an axial ligand of the ferric heme in the PAS-A domain. On the other hand, the 347-cm Ϫ1 band was assigned to the porphyrin 8 mode (35), since the 347-cm Ϫ1 band was also observed with Soret excitation (spectrum D). Resonance Raman Spectrum of the Photodissociated CObound PAS-A Domain-Due to the predominance of the 6cLS species, the Fe-His stretching mode was weak in RR spectra of the ferrous form (Fig. 2, spectrum B). Although the hemoproteins with six-coordinate structure in the ferrous form do not exhibit the Fe-His mode, the photoproduct formed transiently by CO dissociation from the CO-heme complex under high laser power conditions, which adopts the 5cHS state, displays the Fe-His mode in some cases. To analyze the Fe-His stretching mode, the RR spectrum of the photoproduct of the CO-heme complex was recorded. Unfortunately, the Fe-His stretching mode of the PAS-A domain was not identified with continuous wave excitation at 413.1, 422.6, and 441.6 nm, even with high laser power (Ͼ20 milliwatts), probably due to rapid rebinding of photodissociated CO. An example of excitation at 441.6 nm is depicted in spectrum A in Fig. 4. On the other hand, a single color pulse excitation with 10-ns width was applied to get the spectrum of transient species present in a period of 10 ns following the CO dissociation, and the resultant spectrum is depicted in Fig. 4, spectrum B. An intense band is clearly observed at 220 cm Ϫ1 , which is assigned to the Fe-His stretching mode of the transient 5cHS species. Thus, it is evident that His is a trans ligand of CO in the CO-bound PAS-A domain.
Resonance Raman Spectra of the CO-bound PAS-A Domain-Low frequency RR spectra of the CO-bound PAS-A domain are shown in Fig. 5 (left). Two isotope-sensitive bands were observed at 496 and 572 cm Ϫ1 for 12 C 16 O (spectrum A), which were shifted to 491 and 557 cm Ϫ1 for 13 C 18 O, and are thus assigned to the Fe-CO stretching ( Fe-CO ) and Fe-C-O bending (␦ Fe-C-O ) modes, respectively. The C-O stretching mode ( C-O ) was observed in the high frequency region at 1962 cm Ϫ1 (Fig. 5, spectrum D), which shifted to 1917 cm Ϫ1 upon 13 CO substitution (spectrum E). Their difference spectrum (spectrum F) confirms the assignment. These assignments are summarized in Table III, along with those of other heme-based sensor proteins.
There is an inverse linear correlation between the frequencies of the Fe-CO and C-O stretching modes of heme proteins and model compounds (39,40). The corresponding frequencies of the PAS-A domain fall on the same line as that for proteins with proximal histidine, as illustrated in Fig. 6. This finding suggests that the trans ligand of iron-bound CO is a neutral histidine, in accordance with the frequencies of Fe-His detected for transient CO photoproducts (Fig. 4, spectrum B).
Resonance Raman Spectra of the Mutant PAS-A Domain-To determine the coordination structure of the heme, histidine and cysteine residues were replaced with Ala. In total, six mutants (H119A, H138A, H148A, H171A, H217A, and C170A) were generated. Although absorption spectra of the mutant PAS-A domain changed slightly, CD spectra showed that the protein folds remain unchanged after the mutation. 3 Since 3 is sensitive to the spin and coordination states of the heme iron (29), the 3 regions of Raman spectra of the mutant PAS-A domains in the ferric (left panel) and ferrous (right panel) states are depicted in Fig. 7. As described earlier, the 3 band of the ferric WT PAS-A domain consists of three peaks at 1471, 1490, and 1504 cm Ϫ1 , and their relative intensities are altered by mutation without changes in frequency (Supplemental Fig. 1S and Table IS

FIG. 4. Resonance Raman spectra of nonphotodissociated (A) and photodissociated products (B) of CO adducts of the PAS-A domain.
A 10-ns width pulse at 435.7 nm was used to obtain spectrum B, whereas the continuous wave 441.6-nm line was employed to obtain spectrum A.
H148A and H171A induced a moderate increase. This finding indicates an increase in the 5cHS species at the expense of the 6cLS species for these mutants. This spectral change is understandable if His 119 and Cys 170 are axial ligands of heme in the 6cLS species of the ferric PAS-A domain. Cys 170 ligation is consistent with the observed Fe-Cys stretching mode by 363.8-nm excitation (Fig. 3). On the other hand, the intensity of the 1471-cm Ϫ1 band increased significantly for ferric H138A and moderately for H119A and H148A mutants. The data signify that mutation of three His residues results in an increase in the 6cHS species, thus allowing coordination of a weak ligand to the heme iron. These observations are compatible with coordination of His 119 or His 138 and Cys 170 to the ferric iron in the PAS-A domain.
In contrast to the ferric form, ferrous C170A and H119A mutants displayed no significant differences from WT regarding the 3 mode. In all of the spectra, the 5cHS species at 1471 cm Ϫ1 is the main component. A new band appeared at 1501 cm Ϫ1 for the ferrous H119A and H171A mutants, for which the 1493-cm Ϫ1 band appeared weaker than that of WT (Fig. 7A). This frequency is similar to that observed previously for fourcoordinate ferrous hemes (41,42), suggesting that His 119 and His 171 are axial ligands in 6cLS species of the ferrous form. Fig. 8 shows the Fe-CO stretching mode of WT and the above mutants in the CO-bound forms. Except for the H217A mutant whose Fe-CO mode increased by 2 cm Ϫ1 , none of the His and Cys mutants exhibited a change in Fe-CO frequency. Thus, the polarity around iron-bound CO as well as the trans ligand of CO is not affected by the mutation. The specific axial histidine that is replaced by CO in the 6cLS species and the His serving as the trans ligand of CO remain to be identified.

Q-band Excited Resonance Raman Spectra of WT and Mutant PAS-A Domains-Q-band
excitation selectively enhances nonsymmetric modes, 10 , 11 , and 19 , in the 1400 -1700-cm Ϫ1 region (29). 11 is a -electron marker band sensitive to the donor strength of the axial ligand, particularly in the ferrous state (43)(44)(45). The high frequency RR spectra of the ferrous WT and mutant PAS-A domains obtained with 568.2-nm excitation are illustrated in Fig. 9. The WT species (bottom) produced 11 , 19 , and 10 bands at 1533, 1585, and 1621 cm Ϫ1 , respectively. The 11 frequency of the PAS-A domain is lower than that of cytochrome c 3 (1540 cm Ϫ1 ) (46), which has two neutral histidines as axial ligands. Since the 11 frequency decreases with increasing electron donation from an axial ligand (43), the low frequency shift of the 11 mode of the PAS-A domain indicates coordination of an anionic ligand to heme in the ferrous state.  The frequency of 11 was not affected by mutation at His 148 and His 217 but upshifted by 2-3 cm Ϫ1 for H119A, H138A, and H171A mutants. Unexpectedly, the C170A mutant displayed the largest high frequency shift of 11 . Presumably, Cys 170 is not an axial ligand of the ferrous heme (discussed below) but interacts directly or indirectly with the axial histidine so strongly as to deprotonate it. Therefore, its removal affects the donation of the His residue. Time-resolved Resonance Raman Spectra after CO Photodissociation-To investigate the conformational dynamics induced by CO binding to the PAS-A domain, we measured nano-second time-resolved RR spectra. Fig. 10 (left) illustrates the raw time-resolved RR spectra in the low frequency region of the CO-bound PAS-A domain after photolysis. The spectrum at the bottom was observed in the absence of the pump beam (hereafter referred as "probe-only" spectrum). The probe-only spectrum was almost identical to the continuous wave-excited RR spectrum for the CO-bound form and contained no bands in the 200 -250-cm Ϫ1 region as shown in Fig. 2 (spectrum C). This implies that the probe pulse is weak enough to protect the sample from photolysis. In the spectrum for ⌬t ϭ 0, a new band appeared, which was identical to that observed in Fig. 4 (spectrum B). This band is located at 220 cm Ϫ1 and is more apparent in the difference spectra obtained by subtracting the probe-only spectrum from the pump-probe spectra depicted on the right. The band gradually decayed and completely disappeared at ϳ500 s. Since the intensity decrease of this band is in parallel with the increase of the 496-cm Ϫ1 negative band, which is the Fe-CO stretching mode, the 220-cm Ϫ1 band is assigned to the Fe-His stretching mode of the transiently formed five-coordinate species. This means that the photodissociated CO rebinds to the heme in 500 s. Vinyl and propionate bending modes were also observed at 413 and 379 cm Ϫ1 , respectively, in the raw time-resolved RR spectra (left panel), but these bands exhibited no shifts during CO rebinding (right panel). This implies that the structural changes of heme propionate and vinyl side chains are not involved in CO binding, in contrast to that observed for sGC (47).

DISCUSSION
Coordination Structure of the PAS-A Domain-RR spectra in the high frequency region provide information on the coordination and spin state of heme (29). The present Raman spectra demonstrate that both the ferric and ferrous PAS-A domains consist of a mixture of five-and six-coordinate heme (Fig. 2). Since a potent Fe-S vibration appeared at 334 cm Ϫ1 upon excitation at 363.8 nm, which was dramatically weakened by replacement of Cys 170 with alanine (Fig. 3), Cys 170 is considered to be one of the axial ligands of ferric heme. The observed increase in the five-coordinate species for the C170A mutant (Fig. 7) is consistent with this conclusion.
On the other hand, Cys 170 is not one of the axial ligands in the ferrous form, indicating replacement of the axial ligand. In high frequency RR spectra of the ferrous H119A and H171A mutants, a new 3 band appeared at 1501 cm Ϫ1 , whereas the C170A mutant displayed an identical spectrum to that of WT (Fig. 7B). The frequency of the new 3 band is higher than that of six-coordinate species and identical to that observed for the ferrous four-coordinate heme (41,42). In RR spectra of the heme complexes of heme oxygenase mutants in which the proximal His 25 was replaced with Ala or Tyr, the Raman bands derived from the four-coordinate species were observed at 1500 cm Ϫ1 in the ferrous form (48,49). Therefore, the Raman band at 1501 cm Ϫ1 in ferrous H119A and H171A mutants of the PAS-A domain is indicative of the presence of the ferrous fourcoordinate heme. Furthermore, the intensity of the 1471-cm Ϫ1 band, which is typical of the ferrous five-coordinate species, increased in both mutants. Therefore, replacement of His 119 or His 171 with Ala converts the five-or six-coordinate heme to four-or five-coordinate heme. These results suggest that His 119 and His 171 , not Cys 170 , are axial ligands of heme in the WT ferrous form. Fig. 11 represents a sequence alignment of the PAS-A domain with other heme PAS proteins (FixL and Ec DOS). Cys 170 and His 171 of the PAS-A domain are located in the early G ␤ strand following the FG loop. In Ec DOS, Met 95 , an axial ligand of the ferrous heme, exists in the FG loop, and is replaced by O 2 (50,51). Arg 220 in Bradyrhizobium japonicum FixL, which corresponds to Met 95 in Ec DOS, is located at the same position as the distal histidine of Mb and forms a hydrogen bond with iron-bound O 2 (52,53). Accordingly, Cys 170 and His 171 of the PAS-A domain are present in the so-called distal heme pocket, and either of them may be the sixth ligand of heme.
The H138A mutant exhibited a slightly different Raman spectrum in the ferric state, compared with that of WT (Fig.  7A). The intensity of the 3 band at 1470 cm Ϫ1 , which is derived from the 6cHS species, increased at the expense of the 1503cm Ϫ1 band of the 6cLS species in this mutant. This increase in the 6cHS species for the H138A mutant may be explained by the coordination of a water molecule to heme instead of His 138 . Furthermore, a sequence alignment of the PAS-A domain with FixL and Ec DOS shows that His 138 is in the F ␤ helix in which proximal histidine (His 77 for Ec DOS and His 194 for FixL) is located (Fig. 11). Therefore, His 138 may be possibly an axial ligand of heme in the ferric state, similar to His 119 . However, it is unlikely that both axial ligands are replaced upon heme reduction as discussed below. Therefore, it seems more plausible at this point that His 119 is an axial ligand of the ferric heme, which is retained upon heme reduction (Fig. 12).
Heme Ligand Switching between Oxidized and Reduced Heme-In the ferric heme, Cys 170 serves as one of the axial ligands, as discussed above, whereas the ferrous PAS-A domain displays bis-histidine (His 119 /His 171 ) coordination as illustrated in Fig. 12. This requires ligand exchange upon reduction of the heme iron, probably between Cys 170 and His 171 . Such redox-dependent ligand switching is also observed in CooA, the first CO-sensing hemoprotein discovered (15,16), and Ec DOS (51). In the case of CooA, ligand switching occurs between Cys 75 and His 77 during changes in the oxidation state of the heme iron (11,54). Interestingly, the PAS-A domain of NPAS2, another CO-sensing hemoprotein, shares the same type of ligand-switching mechanism, although the physiological role of the redox-dependent ligand switching is unknown.
As discussed above, His 119 and Cys 170 are possibly axial ligands in the ferric heme. However, the dominant species for H119A and C170A mutants in the ferric state are still sixcoordinate ( 3 ϭ 1504 cm Ϫ1 for 6cLS). These results suggest that amino acid residues near the intrinsic axial ligands can bind to heme when His 119 or Cys 170 is replaced with Ala. In view of the increase in the 6cHS species in the ferric form of the H138A mutant (Fig. 7A), His 138 is a possible candidate. Our findings indicate a flexible heme environment in the PAS-A domain. This kind of flexibility in the structure of heme vicinity is also observed for CooA and HRI (7). N-terminal Pro2 is the trans ligand to Cys 75 or His 77 in CooA (17). A truncated CooA mutant, in which four residues from the N terminus were deleted, still contained a six-coordinate heme and fully maintained DNA binding affinity (55,56). Furthermore, His 77 3 Ala mutant CooA had little effect on the coordination structure of heme (11). These results characterize the flexible nature of the heme environment of CooA. In the case of the PAS-A domain, no mutants displayed complete conversion from a six-to fivecoordinate heme. This finding may be accounted for by a flexible heme environment, which allows the retention of the six-coordinate heme even after the intrinsic axial ligand is removed.
Conformational Change of NPAS2 upon CO Binding and Signal Transduction Mechanism-It is well established that correlation between the Fe-C and C-O stretching frequencies provides information on the proximal amino acid in the CObound form (40). The frequencies of Fe-CO at 497 cm Ϫ1 and C-O at 1962 cm Ϫ1 for the PAS-A domain fall in the region observed for heme proteins containing a neutral imidazole ligand (Fig.  6). Therefore, a CO adduct of the PAS-A domain has a neutral histidine as an axial ligand in the CO-bound form. The frequencies of the Fe-His stretching modes reflect the electrostatic properties of the axial histidine governed by the hydrogenbonding network (33). For example, cytochrome c peroxidase, in which proximal histidine forms a strong hydrogen bond with the adjacent Asp 235 , has a frequency of Fe-His at 246 cm Ϫ1 (57). Elimination of this hydrogen bond by the Asp 235 mutation significantly decreases the value to 205 cm Ϫ1 (58,59). The Fe-His value at 220 cm Ϫ1 for the PAS-A photoproduct of the CO-complex is similar to those of deoxy-Mb and R-type deoxyhemoglobin, which possess relatively weak hydrogen bonding between axial histidine and surrounding amino acid residues. Accordingly, the Fe-His frequency of the photodissociated PAS-A domain at 220 cm Ϫ1 signifies that the axial histidine of the CO-bound PAS-A domain is in the neutral form and has no or weak hydrogen bonding. This finding is consistent with results derived from the correlation plot.
In contrast to the observed neutral histidine coordination in the CO-bound form, the 11 frequency of the native ferrous form suggests a different heme environment. The 11 band is a good -electron density marker and is sensitive to the donor strength of the axial ligand, especially in the ferrous state (43). The 11 mode of the bis(imidazole) complex of iron(II)-protoporphyrin (Fe(II)PP(ImH) 2 ) is observed at 1533 cm Ϫ1 , which is significantly downshifted upon ionization of one or both coordinated imidazoles ( 11 ϭ 1526 cm Ϫ1 for Fe(II)PP(ImH)(Im Ϫ ) and 11 ϭ 1517 cm Ϫ1 for Fe(II)PP(Im Ϫ ) 2 ), since the deprotonated imidazole increases the back-donation from the d orbital of the heme iron to the e g * porphyrin orbital. The increase in electron density of the e g * orbital with anti-bonding character about the C ␤ -C ␤ bond results in reduction of the 11 frequency. The 11 value of the PAS-A domain (1533 cm Ϫ1 ) is lower than that of reduced cytochrome c 3 (1540 cm Ϫ1 ) (46), which implies that one of the coordinated histidines is strongly hydrogen-bonded in the ferrous form. Therefore, the electrostatic character of the iron-coordinated His is different between the ferrous and CO-bound forms.
Although mutational analysis revealed that His 119 and His 171 are probable axial ligands in the ferrous form, it is not clear which His is replaced by CO, since the Fe-C stretching modes remain unaffected by replacement of His 119 and His 171 with Ala (Fig. 8). In view of the finding that the 11 band shifts from 1533 to ϳ1539 cm Ϫ1 when Cys 170 is replaced with Ala, we propose that Cys 170 acts as a hydrogen bond acceptor of His 171 (Figs. 9 and 12). If His 171 is replaced by CO and His 119 is retained, release of His 171 from the heme iron would trigger conformational changes relevant to signaling (Fig. 12A). On the other hand, if His 119 is replaced by CO and His 171 is retained, the Fig. 6 data indicate that the hydrogen bonding between His 171 and Cys 170 would be disrupted upon CO binding (Fig.  12B). Sequential alignment with Ec DOS and FixL suggests that the former case is more plausible as shown in Fig. 11. However, in any case, His 171 appears to play a crucial role in signal transduction for dissociation of NPAS2 from BMAL1 accompanied by CO binding.
Comparison of the Signal Transduction Mechanism with Those of Other Sensory PAS Proteins-In FixL, conformational changes at the distal site are important for signal transduction to the kinase domain, whereas the proximal site is not included in the signaling pathway (1,60). The x-ray crystal structure of the B. japonicum FixL heme domain shows that hydrogen bonding between heme 7-propionate and Arg 220 is disrupted upon O 2 binding. Instead, Arg 220 forms a hydrogen bond with heme-bound O 2 (52,53). This type of hydrogen bonding rearrangement in the distal site induces conformational changes to deactivate the kinase domain. The frequency of ␦(C ␤ C c C d ) of the ferrous PAS-A domain of NPAS2 is slightly downshifted from 382 to 379 cm Ϫ1 upon CO binding (Fig. 2), suggesting that the strength of hydrogen bonding of the heme propionate is weakened by CO binding. However, the frequency of ␦(C ␤ C c C d ) at 379 cm Ϫ1 still indicates strong hydrogen bonding. Moreover, the frequency does not change during the CO rebinding process in time-resolved RR spectra (Fig. 10). Although we cannot rule out the possibility of rearrangement of hydrogen bonding upon CO binding, it seems that hydrogen bonding of the propionate group is not essential for the signal transduction mechanism of the PAS-A domain.
In Ec DOS, the sixth ligand of the reduced heme is Met 95 (50,51). In the presence of O 2 , Met 95 is replaced by O 2 , and Arg 97 , which corresponds to Arg 220 in B. japonicum FixL, orients to the heme distal pocket from the protein surface to form a hydrogen bond with iron-bound O 2 (50). Thus, the conformational changes accompanied by O 2 ligation are localized to the FG loop, which serves as a trigger for initial signal transduction in Ec DOS. This mechanism indicates that His 171 , located close to the FG loop of the PAS-A domain, may play the same role as Met 95 in Ec DOS.
In both FixL and Ec DOS, changes in the heme distal pocket initiate signal transduction, as discussed above. His 171 in the PAS-A domain, which is located in the early G ␤ strand, may be involved in the signal transduction pathway. Our results thus collectively implicate a conserved mechanism of signal transduction in the heme PAS superfamily.
In summary, RR and mutational studies demonstrate that Cys 170 and His 119 are axial ligands of the ferric heme in the PAS-A domain. Cys 170 may be replaced by His 171 upon reduction of heme. In the ferrous heme, one of the axial histidines (possibly His 171 ) is deprotonated, whereas a neutral histidine coordinates to heme in the CO-bound form. These results suggest that CO binding alters the structure around the protonated histidine, which triggers a signal to the dimer interface with BMAL1.