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Originally published In Press as doi:10.1074/jbc.M314199200 on February 23, 2004

J. Biol. Chem., Vol. 279, Issue 19, 20186-20193, May 7, 2004
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A Redox-controlled Molecular Switch Revealed by the Crystal Structure of a Bacterial Heme PAS Sensor*

Hirofumi Kurokawa{ddagger}§, Dong-Sun Lee§||, Miki Watanabe{ddagger}, Ikuko Sagami{ddagger}, Bunzo Mikami**, C. S. Raman||{ddagger}{ddagger}, and Toru Shimizu{ddagger}

From the {ddagger}Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan, ||Department of Biochemistry and Molecular Biology, Structural Biology Research Center, University of Texas Medical School, Houston, Texas 77030, and **Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received for publication, December 26, 2003 , and in revised form, February 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
PAS domains, which have been identified in over 1100 proteins from all three kingdoms of life, convert various input stimuli into signals that propagate to downstream components by modifying protein-protein interactions. One such protein is the Escherichia coli redox sensor, Ec DOS, a phosphodiesterase that degrades cyclic adenosine monophosphate in a redox-dependent manner. Here we report the crystal structures of the heme PAS domain of Ec DOS in both inactive Fe3+ and active Fe2+ forms at 1.32 and 1.9 Å resolution, respectively. The protein folds into a characteristic PAS domain structure and forms a homodimer. In the Fe3+ form, the heme iron is ligated to a His-77 side chain and a water molecule. Heme iron reduction is accompanied by heme-ligand switching from the water molecule to a side chain of Met-95 from the FG loop. Concomitantly, the flexible FG loop is significantly rigidified, along with a change in the hydrogen bonding pattern and rotation of subunits relative to each other. The present data led us to propose a novel redox-regulated molecular switch in which local heme-ligand switching may trigger a global "scissor-type" subunit movement that facilitates catalytic control.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The advantage of monitoring changes in small ligands, light, oxygen, and redox potential for cell survival is widely recognized. Numerous microorganisms adapt to living in response to such environmental changes. One widespread solution to sensing these changes is provided by Per-Arnt-Sim (PAS)1 domains, which have been identified in over 1100 proteins in all three kingdoms of life (1). PAS domains convert various input stimuli into signals that propagate to downstream components by altering intra- or intermolecular protein-protein interactions. The mechanisms whereby PAS domains transmit the input signal into the effector domain are of great current interest but are poorly understood.

One such protein is the Escherichia coli redox sensor, Ec DOS (2), a phosphodiesterase that degrades cAMP in a redox-dependent manner (3). The enzyme is thought to control the switch between aerobic and anaerobic metabolism. The protein comprises heme-bound PAS, nonheme PAS, GGDEF, and EAL domains (see Fig. 1a). The N-terminal heme-bound PAS domain (Ec DOSH) plays a key role in the regulation of phosphodiesterase (PDE) activity (4). Ec DOS is part of a growing family of proteins in which the heme is adapted for regulatory functions, including the NO receptor-soluble guanylyl cyclase, O2 sensors of FixL and HemAT, and CO sensor transcription activators, CooA and NPAS2 (5–9). The GGDEF and EAL domains are responsible for PDE activity (4). The GGDEF domain is homologous to adenylyl/guanylyl cyclase catalytic domain (10). These two motifs are widely present in bacteria (e.g. 19 copies of the GGDEF domain and 17 copies of the EAL domain are encoded in the E. coli genome) (11). Although these domains form one of the largest clusters of potential orthologous groups, little is known about their physiological function. Ec DOS is one of a few proteins that have been studied experimentally (2–4, 12, 13).



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  		FIG. 1.
a, Ec DOS domain structure depicting the N-terminal heme PAS domain (Ec DOSH), the nonheme PAS domain, and the C-terminal PDE domain comprising the GGDEF and EAL domains. b, stereo view of the Ec DOSH subunit I (thick line) and II (thin line) backbone with every 10th C{alpha} labeled. c, structure-based sequence alignment of PAS proteins: Ec DOSH (Fe3+ form), RMFIXL (Protein Data Bank code 1D06 [PDB] ) (22), PYP (2PHY [PDB] ) (23), LOV2 (1G28 [PDB] ) (24), and HERG (1BYW [PDB] ) (25). Boxed regions are used for superimposition of PAS proteins. The black bars and arrows represent {beta}-strands and helices, respectively. The figures were prepared with the programs Molscript (35), Bobscript (36), Raster3D (37), PyMOL (38), and MOLMOL (39).

 
To obtain structure-based insight into the mechanism by which the redox state regulates Ec DOS catalytic activity, we have determined the crystal structures of Ec DOSH in both the inactive Fe3+ and active Fe2+ forms at 1.32 and 1.9 Å resolution, respectively. In this report, we present the structure of the redox-sensing PAS domain for the first time. These results lead us to propose a possible link between local conformational changes caused by a single electron and a global "scissor-type" motion of dimer subunits that may be important for downstream signaling control.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Purification and Crystallography—Ec DOSH was purified from a recombinant E. coli system, as described previously (3). Most of the sample was obtained in the oxy form after lysis of E. coli. and finally purified as the Fe3+ form. Crystals of Fe3+ Ec DOS were grown using the hanging drop vapor-diffusion method at 22 °C. The reservoir solution consisted of 100 mM MES, pH 6.0, and 1.6 M ammonium sulfate as a precipitant. Initial droplets contained 1.5 µl of 24 mg/ml protein solution and 1.5 µl of a reservoir solution and were equilibrated against 500-µl reservoir solutions. Crystals were transferred into reservoir buffer containing 20% (v/v) glycerol. A crystal was picked up with a nylon loop and immediately frozen in liquid nitrogen. Crystals of the Fe2+ form were prepared by soaking the Fe3+ crystal in the above 20% glycerol reservoir buffer saturated with sodium dithionite for 5 min and then frozen in liquid nitrogen.

Data Collection and Structure Determination—Single wavelength anomalous diffraction data were collected to 1.9 Å resolution on an R-AXIS VII imaging plate using a Rigaku rotating anode x-ray generator equipped with confocal mirror optics. Intensities were integrated and scaled using CrystalClear (Rigaku/MSC). The space group and unit cell parameter are similar to those reported previously (14). The heme iron position was determined by means of a Bijvoet difference Patterson map using Crystallography and NMR System program (15). Further calculation and density modification by crystallography NMR software revealed an electron density map of interpretable quality. Model building was performed using the O program (16) and XtalView (17). High resolution native data were collected at beamlines BL41XU, equipped with a MarCCD detector for the Fe3+ form, and BL44XU, equipped with a DIP2040 detector for the Fe2+ form of SPring-8, respectively. Data were processed, merged, and scaled using the HKL2000 package (18) for the Fe3+ form and with MOSFLM and SCALA in the CCP4 package (19) for the Fe2+ form. With high resolution data on the Fe3+ form, the anisotropic temperature factor refinement was applied using SHELXL (20). The structure of the Fe2+ sample was determined using the Fe3+ structure as the first model. Further refinement was performed by crystallography NMR software. Statistics of data collection and refinement are summarized in Table I.


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  		TABLE I
 
Comparison with Other PAS Domains—The secondary structure of Ec DOSH was assigned using Procheck (21). A least square fit analysis of the Ec DOSH structure against four known structures of the PAS superfamily was performed with the residues shown boxed in Fig. 1c. The secondary structure-based alignment led to r.m.s.d. values of 1.19 Å for RmFixL (an oxygen sensor, FixL, from Rhizobium meliloti) (22), 1.72 Å for PYP (23), 2.88 Å for LOV2 (24), and 3.32 Å for the HERG (25).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure—Ec DOSH exhibits a PAS fold characterized by several {alpha}-helices flanking a five-stranded antiparallel {beta}-sheet scaffold (see supplementary Fig. 1). Two characteristic differences are evident in the secondary structure compared with other PAS domains. Specifically, an additional short helix (F'{alpha}) is present, and the F{alpha} helix is shorter than that of Rm-FixL (see supplementary Fig. 1) (22). This may be due to the existence of two proline residues (Pro-74 and Pro-78), which act as a helix breaker (Fig. 1c). As expected from the size exclusion chromatography (3), the structure shows a homodimer (Fig. 1b). Two subunits in an asymmetric unit show similar structure (r.m.s.d. of C{alpha} atoms is 0.42 Å) and are related by a pseudo 2-fold axis (Fig. 2a). The heme planes are approximately parallel to each other (Fig. 2a), with an iron–iron distance of 30.4 Å. Heme distal sites face each other and are located near the dimer interface, which includes a short N-terminal helix (II{alpha}) and two {beta}-strands (H and I). The II{alpha} helices in the subunits interact with each other, leading to stabilization of the dimer via hydrophobic interactions between Phe-22, Phe-23, Leu-26, and Phe-44 and a large total buried surface area of 2440 Å2.



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  		FIG. 2.
Dimer of Ec DOSH and redox-induced changes at the heme distal site. a, left, Fe3+ form of the Ec DOSH dimer. The 2-fold axis of subunits is specified as a blue stick. Right, the view after 90° rotation of the left figure. Heme and proximal histidine 77 are shown as a ball-and-stick model. Disordered FG loop regions are indicated by blue broken lines. Water molecules are represented by spheres (cyan). N, N terminus; C, C terminus. b, close-up view of the heme site of the Fe3+ form of Ec DOSH (left); the same region of the Fe2+ form (right). FG loop regions are rigidified in the Fe2+ form (cyan). The distal axial ligand is replaced by Met-95, as indicated in the sphere model.

 
Heme Binding Site—An initial single wavelength anomalous diffraction map clearly shows that Fe3+ Ec DOSH contains a hexa-coordinate heme iron. Fig. 2b shows a close-up view of the heme distal site. The heme ligand in the distal site is a water molecule, W1. This is critical for Ec DOS function, because the distal axial heme ligand must be displaced in the allosteric switch from the "off" to the "on" state (12). The water molecule W1 is in a hydrophobic environment defined by Leu-99, Phe-113, and Leu-115 and is stabilized by another water molecule, W2, which is hydrogen-bonded to the heme propionate oxygen (Fig. 3a). Previous resonance Raman studies showed that Fe3+ Ec DOSH contains a low spin heme. The spectra do not change in the range of pH 4.0–10.0 (12). Earlier data and the current structure collectively suggest that the distal water molecule W1 is in the OH- form with an unusual pKa value below pH 4.0.



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  		FIG. 3.
The heme site of Ec DOSH. a, stereo figures of the heme site of the Fe3+ form of Ec DOSH subunit I. The 1.32 Å resolution 2Fo - Fc electron density map was contoured at the 1.6 {sigma} level. His-77 and the water molecule W1 coordinate to the heme iron. W1 is surrounded by three hydrophobic residues, Leu-99, Phe-113, and Leu-115, and forms a hydrogen bond with W2, which is also hydrogen-bonded to a heme propionate oxygen atom. The heme proximal ligand His-77 forms hydrogen bonds with a water molecule, W3. The hydrogen bond network in the proximal site is stabilized by Asp-40 and Asn-64. b, heme and the FG loop regions of the Fe2+ form of Ec DOSH subunit II. The heme distal ligand is replaced by Met-95. The FG loop region (residues 86–96), which was disordered in the Fe3+ form, displays clear electron density. Main chain nitrogen atoms of Gly-94 and Met-95 form hydrogen bonds with heme propionate oxygen atoms.

 
In the heme proximal site, His-77 axially coordinates with the heme iron. The Fe–N bond distance is 1.97 Å (Fig. 3a). The imidazole ND1 atom of His-77 is hydrogen-bonded to the water molecule, W3, with a distance of 2.78 Å. W3 is additionally hydrogen-bonded to W4 and W5. W3 and W4 form hydrogen bonds with Asp-40 and Asn-64, respectively, consequently fixing the orientation of the imidazole ring of His-77 to the plane of the heme group. One of the structural factors responsible for the Fe–N bond character is the basicity of the imidazole ring of proximal His, which is modulated by the strength of the hydrogen bond between imidazole NH and its surroundings. His-77 does not form a strong hydrogen bond to the surrounding residues. This appears to be consistent with the relatively low Fe–NHis stretching frequency for Ec DOSH (214 cm-1) in resonance Raman spectra (12).

Heme-Ligand Switching and Refolding of the FG Loop—We analyzed the physiologically active Fe2+ form of Ec DOSH. Upon heme iron reduction, the distal water molecule W1 was displaced by Met-95 of the FG loop (encompassing residues 86–97 between the F{alpha}-helix and G{beta}-strand) (see supplementary Fig. 2). Heme-ligand switching is one of the most important findings in this study. The redox-dependent allosteric switch observed here is consistent with our previous conclusion that the PDE activity of Ec DOSH is controlled by the heme redox state (3). The heme-ligand switching found here is also consistent with our previous finding that the distal heme ligand in Fe3+ form, which is now identified as a water molecule, is displaced by Met-95 in Fe2+ form (12). In the Fe2+ state the side chain of His-77, the heme ligand at the proximal site, did not display significant movement (Figs. 2b, right, and 3b). Additionally, no significant effect was observed on the hydrogen bonding network around the His-77 side chain. Thus, heme reduction appears to affect only the distal site near the dimer interface (Fig. 2b).

Heme-ligand switching does not induce porphyrin movement (Fig. 3b) but is accompanied by refolding of the FG loop region (Fig. 2b). FG loops are located near the heme distal site. This region is highly flexible in the Fe3+ form, in view of its noninterpretable electron density. However, the reduced (Fe2+) form of Ec DOSH exhibits unambiguous electron density (Fig. 3b). We conclude that upon heme reduction, the FG loop is significantly rigidified. Gly-94 and Met-95 main chains form hydrogen bonds with heme propionate oxygen, which appear to fix the FG loop regions. Gly-94 may be important for accommodating large conformational changes, because the small side chain is preferred to avoid steric hindrance. These observations indicate a direct link between coordination of Met-95 with the heme iron and refolding of the FG loop region.

The CO-sensing transcriptional activator, CooA, displayed similar heme-ligand switching from the Cys-75/Pro-2 to His-77/Pro-2 ligand pair upon heme reduction (8, 26). Additionally, heme-ligand switching was observed from His-17/His-69 to Met-106/His-69 in the cytochrome c domain of cytochrome cd1 nitrite reductase (27). Both cases involved anomalous redox chemistry, i.e. hysteresis (26, 28). However, Ec DOS has similar oxidation and redox potentials (3). This finding suggests that the activation energy upon heme-ligand switching of Ec DOS is much smaller than that in CooA or cytochrome cd1 nitrite reductase.

Redox-induced protein folding is observed in cytochrome c, which also contains a His/Met heme-ligand pair (29). This is possibly because of stabilized Met-iron coordination in the reduced form. Selective formation of the His/Met ligand pair in the reduced form is additionally observed in the cytochrome c domain of cytochrome cd1 nitrite reductase, as described above (27). The data indicate a general mechanism by which redox energy is converted into conformational energy using a His/Met heme-ligand pair.

Rearrangement of the Hydrogen Bond Network—Heme-ligand switching from His-77/water to a His-77/Met-95 ligand pair induces conformational adaptations, including rearrangement of the hydrogen bond pattern and fine-tuning of side chains in the heme vicinity. Fig. 4a depicts the hydrogen bond network around the heme in the Fe3+ and Fe2+ forms of Ec DOSH. Two water molecules, W1 and W2, are lost upon coordination of Met-95 with the heme iron (Fig. 4a). The hydrogen bond between W2 and heme propionate is replaced by a bond between main chain nitrogen of Met-95 and heme propionate oxygen. Main chain oxygen of Gly-94 of the rigidified FG loop forms hydrogen bonds with the main chain oxygen of Phe-113 and nitrogen of Leu-115 via a water molecule. In addition to hydrogen bond rearrangement, fine-tuning of the side chains of Phe-113 and Leu-115 occurs in response to heme coordination with Met-95 (Fig. 4a). Ile-238 of the oxygen sensor, BjFixL (FixL from Bradyrhizobium japonicum), corresponding to Leu-115 of Ec DOSH, displays similar side chain adaptations upon binding of the exogenous heme ligand of O2 (30). These changes in the heme distal site appear to stabilize the formation of an intersubunit hydrogen bond between the main chain oxygen of Phe-113 and Arg-131 via a water molecule (Fig. 4a). Fig. 4, b and c, depicts the buried surface area of subunit I in the Fe3+ and Fe2+ forms. As shown in Fig. 4a, Ser-116 forms intersubunit hydrogen bonds with the main chain of Met-30. This region is in close contact in both the Fe2+ and Fe3+ forms and is therefore not significantly affected (Fig. 4b). The contact area of Phe-113 and Arg-131 is located in the peripheral region of the dimer interface. This region shifts closer in the Fe2+ form (Fig. 4, b and c). Upon heme reduction, Met-95 coordination at the heme distal site locks the dimer into a tighter complex by bringing Phe-113 of one subunit and Arg-131 of the other subunit closer together (Fig. 4a). Therefore, redox-induced adaptation at the heme distal site triggers changes at the dimer interface.



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  		FIG. 4.
Reorganization of the hydrogen bond network at the heme distal site near the dimer interface. a, comparison of heme distal sites between Fe3+ (cyan) and Fe2+ (yellow) forms of Ec DOSH. Residues 31–85 and 97–132 of subunit I in each form were superimposed. Residues in subunit II are colored in blue for the Fe3+ form and orange for the Fe2+ form. Hydrogen bonds are presented as dotted lines. Met-95 coordination to the heme iron upon reduction induces reorganization of the hydrogen bond network at the heme distal site. Side chains of Phe-113 and Leu-115 move slightly to adjust to heme-ligand switching. These changes induce hydrogen bond formation between Phe-113 and Arg-131. No effects were observed on the hydrogen bond between Ser-116 and Met-30. The buried dimer surface areas of the Fe3+ form (b) and Fe2+ form (c) of subunit I are shown. Distances between the surfaces of each subunit were calculated with MOLMOL and ranged from <2 Å (red) to >7 Å (blue). Met-30 and Arg-131 of subunit II are presented as a stick model.

 
Scissor-type Subunit Movement—To characterize changes at the dimer interface, the relative orientation of the two subunits was compared between the Fe3+ and Fe2+ forms. Fig. 5a depicts the superimposition of Fe3+ and Fe2+ forms of Ec DOSH. Superimposition of the two forms (PAS core regions, residues 31–88 and 97–132 of the two subunits) revealed r.m.s.d. values of 0.49 Å for 188 C{alpha} atoms. When each subunit was superimposed independently, the r.m.s.d. values of C{alpha} atoms were smaller (0.40 Å for subunit I and 0.37 Å for subunit II). The data suggest that each subunit moves as a rigid body following heme reduction. This unexpected redox-induced subunit movement appears critical for catalytic control. To define subunit movement more precisely, the rotation axis was calculated (Fig. 5a, circle with dot in it, b, red stick) (31). The rotation axis runs close to and is approximately perpendicular to the pseudo 2-fold axis (Fig. 5, a and b, blue stick). For superimposition of subunit I (depicted in blue for the Fe3+ form and orange for the Fe2+ form) following that of subunit II (depicted in cyan for the Fe3+ form and yellow for the Fe2+ form), a 3° rotation around the axis was required. Although no significant changes were observed in most residues near the rotation axis (hinge region) upon reduction, this scissor-type motion led to maximum displacement of Ser-107 of subunit I (1.3 Å), which is 30 Å away from the hinge.



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  		FIG. 5.
Redox-induced scissor-type subunit motion of Ec DOSH. a, to compare the relative position of each monomer of Ec DOSH in the Fe3+ (cyan) and Fe2+ (yellow) forms, core regions of the subunits (residues 31–85 and 97–132) were superimposed. For superimposition of subunit I after that of subunit II, a 3° rotation around the screw axis (a circle with a dot in it) was required. The 2-fold axis of the Fe3+ form of Ec DOSH is depicted as a blue stick. The rotation axis runs close and is almost perpendicular to the 2-fold axis. b, the view after a 90° rotation of (a). The rotation axis is indicated as a red stick. c, proposed model for the regulation of PDE activity by Ec DOSH. The redox-controlled scissor-type motion alters the relative position of the PDE domain to switch catalytic activity on and off.

 
Redox-controlled Signal Transduction—The crystal structures of the Fe3+ and Fe2+ forms of Ec DOSH reported here propose a novel heme-redox switch, which may provide important insight into redox-dependent signal transduction. Although the structure discussed in this report only represents part of Ec DOS (Fig. 1a), the data reveal a possible connection between heme-ligand switching and the scissor-type subunit movement, which may be important for the catalytic control of Ec DOSH. The overall transition upon heme reduction is likely to be initiated by heme-ligand switching from a water molecule to a Met-95 side chain at the heme distal site. This step appears to be strictly regulated by the heme redox state and is one of the most important findings in this report. Significant changes were observed in the FG loop of Ec DOSH only, indicating that conformational alterations upon reduction are limited to this region. Similar conformational changes in this loop region are observed in PAS kinase, a mammalian serine/threonine kinase with the PAS sensor domain. Refolding of the FG loop in PAS kinase is induced by binding to a small organic molecule, which results in activation of the kinase domain (32). This is of interest because two PAS sensors from bacteria (Ec DOSH) and mammals (PAS kinase) may share a similar sensory mechanism in which refolding of the FG loop plays a key role in converting environmental stimuli into biological signals by modifying protein-protein interactions.

Met-95 ligation and refolding of the FG loop in Ec DOSH tightens the hydrogen bond network at the heme distal site. Reorganization of the hydrogen bond network, in turn, may induce alterations at the dimer interface. These changes appear to be mediated by residues in the I{beta}-strand, specifically Phe-113, Leu-115, and Ser-116. Recent NMR studies (33) on the photoreceptor PAS domain, AsLOV2, revealed that an additional C-terminal J{alpha} helix associates with the PAS core region of G{beta}, H{beta}, and I{beta} strands in the dark state. Photoinduced changes disrupt interactions of these strands with the J{alpha} helix, which in turn enhances the autophosphorylation activity of the C-terminal kinase domain. The I{beta}-strand appears important for regulating the signaling of both Ec DOS and AsLOV2, because this region mediates the conversion of local conformational changes sensed by small organic molecules (heme for Ec DOSH and FMN for AsLOV2) into global subunit-subunit interaction modifications (scissor-type motion for Ec DOSH and the release of the J{alpha} helix from the PAS core for AsLOV2) in both proteins.

The subunit movement of Ec DOSH is characteristic of a scissor-type hinge motion, as shown in Fig. 5. Similar subunit movements were observed in the aspartate receptor of chemotaxis, which also involves a small subunit rotation (4°) upon ligand binding (34). This inhibits the activity of histidine kinase CheA, a downstream signaling molecule. Milburn et al. (34) suggested that a minor rotation at the periplasmic sensor domain of the aspartate receptor is amplified at the cytoplasmic domain more than 100 Å away. Because dimer or tetramer formation of Ec DOS is a prerequisite for PDE activity (4), subunit-subunit interactions at the PDE domain appear to be critical for catalytic regulation. Possibly, the scissor-type motion of Ec DOS may affect PDE activity (Fig. 5c), because hinge rotation at the PAS domain is likely to be amplified at the distant PDE domain. Note that the catalytic control by the scissor-type subunit movement shown in Fig. 5c is only a proposal. To verify the current proposed model, further experiments are needed to examine the interactions between the Ec DOSH domain and the PDE domain in both the Fe3+ and Fe2+ forms. Mutational and crystallographic studies of the entire Ec DOS enzyme are currently underway.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1V9Y and 1V9Z) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to H. K. and T. S.), Grant AU-1524 from the Robert A. Welch Foundation (to C. S. R.), and a grant from Pew Charitable Trusts (to C. S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. Back

§ These authors contributed equally to this work. Back

{ddagger}{ddagger} A Pew scholar. Back

To whom correspondence should be addressed. Tel.: 81-22-217-5604; -5606; Fax: 81-22-217-5604; -5390; E-mail: kurokawa{at}tagen.tohoku.ac.jp.

1 The abbreviations used are: PAS, an acronym formed from the names of Drosophila period clock protein (Per), vertebrate aryl hydrocarbon receptor nuclear translocator (Arnt), and Drosophila single-minded protein (Sim); Ec DOS, a heme-regulated phosphodiesterase or a redox sensor from Escherichia coli; Ec DOSH, isolated heme domain of Ec DOS; PYP, photoactive yellow protein; HERG, human ether-a-go-go-related gene; LOV, a domain sensitive to light, oxygen, or voltage; PDE, phosphodiesterase; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviation. Back


   ACKNOWLEDGMENTS
 
We thank M. Kawamoto and E. Yamashita for help on data collection at beamlines BL41XU and BL44XU and C. Vonrhein and G. Bricogne for timely help with SHARP. We are also grateful to the Stanford Synchrotron Radiation Laboratories for prompt access to beam time (Proposal 6A98) and the staff of BL 9-1, 9-2, and 1-5 for help with MAD data collection.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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