Crystal structure of human indoleamine
2,3-dioxygenase: Catalytic mechanism of O2
incorporation by a heme-containing dioxygenase
Hiroshi Sugimoto*, Shun-ichiro Oda*†, Takashi Otsuki*†, Tomoya Hino*, Tadashi Yoshida‡, and Yoshitsugu Shiro*†§
*Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148, Japan;†Department of Life Science, Graduate School
of Science, Himeji Institute of Technology, Hyogo 678-1297, Japan; and‡Department of Biochemistry, Yamagata University School of Medicine,
Yamagata 990-9585, Japan
Edited by Osamu Hayaishi, Osaka Bioscience Institute, Osaka, Japan, and approved December 26, 2005 (received for review October 14, 2005)
Human indoleamine 2,3-dioxygenase (IDO) catalyzes the cleavage
of the pyrrol ring of L-Trp and incorporates both atoms of a
molecule of oxygen (O2). Here we report on the x-ray crystal
structure of human IDO, complexed with the ligand inhibitor
4-phenylimidazole and cyanide. The overall structure of IDO shows
two ?-helical domains with the heme between them. A264 of the
flexible loop in the heme distal side is in close proximity to the iron.
in the distal heme pocket are essential for activity, suggesting that,
unlike the heme-containing monooxygenases (i.e., peroxidase and
cytochrome P450), no protein group of IDO is essential in dioxygen
activation or proton abstraction. These characteristics of the IDO
structure provide support for a reaction mechanism involving the
abstraction of a proton from the substrate by iron-bound dioxy-
gen. Inactive mutants (F226A, F227A, and R231A) retain substrate-
binding affinity, and an electron density map reveals that 2-(N-
cyclohexylamino)ethane sulfonic acid is bound to these residues,
mimicking the substrate. These findings suggest that strict shape
complementarities between the indole ring of the substrate and
the protein side chains are required, not for binding, but, rather, to
permit the interaction between the substrate and iron-bound
dioxygen in the first step of the reaction. This study provides the
structural basis for a heme-containing dioxygenase mechanism, a
missing piece in our understanding of heme chemistry.
iron ? kynurenine ? tryptophan ? x-ray crystallography
substrate, and thus play a crucial role in the metabolism and
synthesis of a variety of biological substances. Two types of
oxygenase are currently known: monooxygenases (scheme I) and
dioxygenases (scheme II):
xygenases (1) are metal-containing enzymes that catalyze
the incorporation of a molecule of oxygen (O2) into the
RH ? O2? 2e?? 2H?3 ROH ? H2O
R ? O23 R(O)2.
In the 1950s and 1960s, Hayaishi and coworkers reported that
two heme-containing dioxygenases, indoleamine 2,3-dioxygen-
ase (IDO) (2) and tryptophan 2,3-dioxygenase (TDO) (3),
catalyze the initial and rate-limiting step of L-Trp catabolism in
the kynurenine (Kyn) pathway (4). This step involves the oxi-
dative cleavage of the 2,3 double bond in the indole moiety of
L-Trp, resulting in the production of N-formyl Kyn. Increased
levels of the Kyn pathway metabolites quinolinic acid and
3-hydroxykynurenine (3OHKyn) have been observed in a num-
ber of neurological or psychiatric disorders. L-Trp-derived UV
filters (Kyn and 3OHKyn glucoside) can bind to the lens protein
and appear to be mainly responsible for the nuclear cataract (5).
L-Trp also serves as a precursor for the synthesis of the neuro-
transmitter serotonin and the hormone melatonin. IDO exhibits
a broader substrate specificity than TDO, because the former
can degrade indoleamines, including L-Trp, D-Trp, serotonin,
melatonin, and tryptamine (6). In addition to its role as a
L-Trp-catabolizing enzyme, IDO is involved in the immuno-
regulating system [review by Mellor and Munn (7)]. IDO-
dependent T cell suppression and tolerance induction by den-
dritic cells suggests that L-Trp catabolism has profound effects
on T cell proliferation and differentiation, which implicates the
immunotherapeutic manipulations designed for patients with
cancer and chronic infectious diseases.
reaction is poorly understood, primarily because of a lack of
structural information. On the other hand, the nature of the
intermediates (8–10) and structure?function correlations for
heme-containing monooxygenases (11) [i.e., cytochrome P450
(P450) and peroxidase] or nonheme type dioxygenase (12, 13)
have been extensively studied on the basis of the model system
and crystal structures. The issue of how the active-site structure
of heme-containing dioxygenases is related to their functions
and how the substrate or dioxygen is activated has stimulated a
great deal of interest.
Here we report on the crystal structures of recombinant
human IDO (45 kDa), complexed with the ligand inhibitor
4-phenylimidazole (PI) (14) and cyanide (CN?) at resolutions of
2.3 and 3.4 Å, respectively. The tertiary structure of IDO
provides structural insights into the catalytic reaction of heme-
containing dioxygenase enzyme.
Results and Discussion
Overall Structure of IDO.Theoverallstructure(Fig.1A)showsthat
IDO is folded into two distinct domains (small and large). The
large domain is an all-helical domain and is comprised of 13
?-helices and two 310helices. Four long helices (G, I, Q, and S)
in the large domain run parallel to the heme plane and interact
with the neighboring helix by hydrophobic interactions. Helix Q
provides an endogenous ligand (H346 imidazole) for the heme
iron at the fifth coordination position (proximal side) (Fig. 1B).
The heme-binding pocket is created mainly by these four helices
N also contribute to heme–protein interactions and connect the
two domains. The small domain and a long loop (residues
250–267) connecting the two domains above the sixth-
coordination site of the heme (distal side) cover the top of the
short ?-sheets, and three 310helices. Contact between these two
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
CHES, 2-(N-cyclohexylamino)ethane sulfonic acid; P450, cytochrome P450.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 2D0T and 2D0U).
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
February 21, 2006 ?
vol. 103 ?
no. 8 ?
domains is very extensive with a buried surface area of 3,100 Å2.
The interface is formed by a combination of hydrophobic
interactions, salt bridges, or H bonds mediated by the side chains
of amino acid residues. The folding of each domain of IDO was
not detected among other known protein structures in the
similarity-searching database DALI (15).
The entrance for the ligand or substrate entrance toward the
pocket is indicated from the molecular surface representation
shown in Fig. 2. It was not possible to construct an atomic model
for the region between R and S helices (residues 360–380)
because of the disordered electron density. This region is
presumably a flexible loop outside the heme pocket.
Heme Environment. The proximal side of the heme is occupied by
only side chains from the large domain. Fig. 3 shows the heme
with the surrounding residues in the PI-bound form. The heme
6-propionate contacts to the water molecule (wa2) and R343 in
the proximal side (Fig. 3A). wa2 also interacts with wat1 and
L388 carbonyl. Previous site-directed mutagenesis studies (16)
have suggested that D274 is a key residue in the IDO catalytic
proximal side, creating a salt bridge with R343, which is in
contact with the 6-propionate. Therefore, the large decrease in
enzymatic activity upon the mutation of D274 is likely derived
from the structural instability and failure to maintain the heme
position. The proximal ligand H346 is H-bonded to a water
molecule (wa1) (Fig. 3A). The environment of the heme prox-
imal side suggests that the H346 imidazole of IDO is partially
anionic by the 6-propionate and L388 carbonyl with the medi-
ation of two waters (wa1 and wa2). It is consistent with the
previously reported resonance Raman spectroscopic data (17) in
which the Fe-His stretching frequency (?Fe-His) was observed at
231 cm?1. This value is in contrast to the highly anionic character
of the proximal His imidazole in peroxidases (248 cm?1) and the
neutral one in myoglobin (220 cm?1). The 7-propionate of the
heme is pointed upward from the heme plane and interacts with
the hydroxyl group of S263.
The distal heme pocket, which is a coordination site of the
heme iron for the sixth external ligand (PI and CN?), is
comprised of a combination of the small domain, the large
domain, and the loop connecting the two domains (Fig. 1).
Because molecular oxygen (O2) binds at this site, the structural
characteristics of the distal heme pocket should be responsible
for the dioxygenase reaction catalyzed by IDO. His residue has
been predicted to be a distal and catalytic residue on the basis
of EPR spectroscopic data (18). However, the structures in
complex with PI and CN?reveal that His is not present in the
heme distal side, and that neither polar residues nor water
molecules are available to interact with the iron-bound ligand
(Fig. 3B). Whereas PI binds to the iron as a noncompetitive
inhibitor in the ferric and ferrous forms, the small ligand CN?
and L-Trp markedly enhance the affinity of each other for the
ferric form. Although we cannot describe the ligand geometry of
and green ribbons, respectively. The helices A–S are named in the order of
appearance in the primary sequence. The connecting helices (K-L and N) are
colored in cyan. The long loop connecting the two domains is colored in red.
The heme (yellow), proximal ligand H346 (white), and heme inhibitor 4-phy-
nylimidazole (white) are shown in a ball-and-stick model. The helices of the
large domain create the cavity for the heme. The connecting loop (red) and
small domain above the sixth-coordination site (heme distal side) cover the
top of cavity on the heme. (B) The four proximal helices I, G, Q, and S run in
parallel. The helices N (blue) and K-L (cyan) connect the two domains. The
connecting loop (red) and small domain above the sixth-coordination site of
the heme cover the top of the cavity on the heme.
Positive potentials are drawn in blue, negative are in red. The heme is shown
as a ball-and-stick model. Unlike as in monooxygenase P450, an asymmetric
distribution of positively and negatively charged areas is not observed in IDO.
The 7-propionate is partially exposed to the solvent.
The solvent-accessible surface with an electrostatic potential of IDO.
www.pnas.org?cgi?doi?10.1073?pnas.0508996103Sugimoto et al.
the CN?-bound form in detail at 3.4-Å resolution, the N and C?
atoms of A264 in the connecting loop are estimated to be located
within 3 Å from the CN?. In a structural comparison between
the PI and the CN?-bound forms (Fig. 4), the connecting loop
exhibits a large conformational change in the main chain with a
1.3-Å displacement, whereas rms deviation values for all com-
mon C?atoms is 0.2 Å. The highly conserved sequences
(AGGSAG for residues 260–265) in this loop appear to provide
flexibility. We suggest that the ligand and?or substrate binding
induce a conformational change in the connecting loop.
In the PI-bound form, Phe-163 interacts with the phenyl group
of PI in the ?-? stacking. Ser-167, which is located 3.7 Å above
to the iron. At the back of the pocket, the side chains of Phe-164,
Val-130, and Cys-129 also contribute to the wall but are far from
the iron (?10 Å).
In the heme distal pocket of both crystal forms, we observed
clear density (Fig. 5) for two molecules of 2-(N-cyclohexylami-
no)ethane sulfonic acid (CHES), a component of the crystalli-
zation buffer. The cyclohexan-ring of CHES-1 is very close to the
ligand (4.0 Å) and iron (5.6 Å). The side chain of F226 and the
alkyl chain of R231 are in contact with the cyclohexan ring of
CHES-1 at distances of 3.8 and 4.2 Å, respectively (see Figs. 3
and 5). G261 and G262 in the connecting loop also interact with
the ethanesulfonic group of CHES-1. CHES-2 is adjacent (4–5
Å apart) and antiparallel to CHES-1. The amino group of each
CHES interacts with 7-propionate of the heme. Despite these
of CHES (?50 mM). It seems reasonable to consider that the
high concentration of CHES binds to the putative substrate-
Site-Directed Mutagenesis. To identify the important amino acid
residue for the catalytic reaction, we replaced the residues that
make up the wall of the distal pocket to Ala by site-directed
mutagenesis. As shown in Table 1, the mutations for polar amino
acids (S167A, C129A, and S263A) retain the dioxygenase activ-
ity. The reason the activity of the mutant of S263A is reduced to
15% is that the side chain of S263 appears to interact with the
heme 7-propionate to stabilize the heme position. In contrast,
the mutants of F226A, F227A, and R231A have drastically
reduced the dioxygenase activity. This result supports the hy-
in the crystal. It is possible that F226 and R231 are directly
involved in substrate recognition by hydrophobic interactions.
The side chain of F227 interacts with the guanidium group of
R231 by cation–? interactions in the present structures, which
suggests an indirect contribution of F227 to substrate recogni-
tion by stabilizing the conformation of R231. It is noteworthy
that the mutation of F163A does not affect the activity and other
parameters, whereas F163 forms a ?-? stacking interaction with
PI in the PI-bound form.
Reaction Mechanism. A range of substrates and inhibitors are
known for IDO. It suggests that the inhibitory effect of meth-
ylation (1-methyl D-Trp) or substitutions of the indole N with O
is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 N?. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226,
F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of
CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational
analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted.
Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346
Sugimoto et al.
February 21, 2006 ?
vol. 103 ?
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or S (furan or thiophene analogs) are caused by the lack of H at
the 1-position of the indole derivatives. In addition, the electron-
donating group at C-5 or C-6 of L-Trp has been known to be a
good substrate. Based on these analyses, several reaction mech-
anisms have been proposed [reviewed by Sono et al. (11)].
Regarding the step after the formation of the ternary complex
(IDO Fe2?-O2:substrate), two types of schemes have been
proposed (Fig. 6). The earlier proposed mechanisms involve the
protein base-assisted abstraction of proton from the indole NH
group (Fig. 6, scheme B). Terentis et al. (17) recently proposed
a mechanism involving proton abstraction by the iron-bound O2,
followed by the electrophilic addition of the dioxygen-attacking
double bond between C-2 and C-3 of the pyrrole ring (Fig. 6,
scheme A). Because the present crystal structure and mutational
analysis demonstrate that no polar?charged protein side chains
act as a catalytic base, we conclude that proton abstraction by
iron-bound dioxygen (Fig. 6, scheme A) is the most plausible
event for the reaction mechanism of IDO. The interaction of an
indole NH group with the proximal oxygen atom (? oxygen
atom bonded to iron) (Fig. 6, 2) would lead to the rearrangement
of electrons of the substrate and weaken the FeOO2bond. It
blue, respectively. The ligand exchange (from PI to CN?) induces a conforma-
is observed in the A264–G265 region with a 1.3-Å shift toward the center of
difference Fourier map calculated from the phase of PI form. The negative
density at ?3.0 ? and the positive density at 3.0 ? are shown in light blue and
Comparison of the PI- and CN?-bound IDO viewed from the distal
of PI form at 2.3-Å resolution is contoured at 1.2 ?. The final refined model is
The electron density of a 2 Fobs? Fcalcsimulated annealing composite
bond between 2-C and 3-C of its substrate L-Trp. The trigger for the reaction
must be the abstraction of a proton from 1-N. There have been two possible
a pathway that involves proton abstraction by iron-bound dioxygen. Their
model is modified based on the tertiary structure in our proposed model
(scheme A). The binding of O2and the substrate L-Trp, whose orientation is
restricted by F226 and R231, enables an interaction between the NH group of
indole and the proximal atom of dioxygen (2). The proton is then abstracted
from 1-N by dioxygen. The rearrangement of the electronic structure of the
indole ring induces an electrophilic reaction, which involves the formation of
subsequent cleavage of the FeOO bond results in the formation of the
3-hydroperoxyindolenine intermediate (3). In scheme B, a protein base ab-
stracts the proton of 1-NH. The dioxetane (4) has been proposed as the
(5) is converted to Kyn (6) nonenzymatically or by Kyn formamidase (33).
The proposed catalytic mechanism. IDO catalyzes the cleavage of the
Table 1. Comparison of activity and dissociation constants (Kd)
for L-Trp of wild type and mutants
126 ? 12
134 ? 6
148 ? 9
117 ? 5
1.3 ? 0.3
1.2 ? 0.5
2.3 ? 1.0
19 ? 7
0.32 ? 0.03
0.40 ? 0.04
1.08 ? 0.10
1.37 ? 0.15
0.80 ? 0.12
0.65 ? 0.07
0.77 ? 0.11
42 ? 5
0.53 ? 0.05
0.46 ? 0.06
0.87 ? 0.05
1.01 ? 0.12
0.93 ? 0.10
0.53 ? 0.09
0.43 ? 0.01
0.33 ? 0.03
0.63 ? 0.05
0.53 ? 0.04
0.41 ? 0.02
*Mol of product?mol of holoenzyme per min.
†ND, spectrum change was not detectable.
www.pnas.org?cgi?doi?10.1073?pnas.0508996103Sugimoto et al.
of the bound dioxygen to the relatively electron-rich C-3 of
indole to form the intermediate Fe2?:3-hydroperoxyindolenin
complex (Fig. 6, 3). It has been speculated that the product
N-formyl Kyn is converted by the intermediate dioxetane (Fig.
6, 4) (17).
In the Fe2?-CO:L-Trp and Fe3?-OH?:L-Trp complex forms
of IDO, close contact of the substrate to the iron-bound ligand
has been implicated by resonance Raman spectroscopy (17). It
should also be noted that, in the case of the O2-bound form of
the bacterial hemoglobin (19), the interaction between the
proximal oxygen and the side chain of the distal Tyr residue
weakens the FeOO2bond, suggesting that, in addition to the
moderately strong FeONHisbond of IDO, the interaction of the
proximal oxygen atom with the NH of the indole ring might also
be important for the dioxygen-releasing mechanism from heme
iron without or before the cleavage of the OOO bond.
Interestingly, the mutations of F226A, F227A, and R231A do
not affect the Kdvalues (Table 1). Nevertheless, they dramati-
cally reduce activity. These mutants suggest that a necessary
condition for the reaction is the proper geometry between the
substrate and dioxygen for proton abstraction, which is brought
about by the strict complementarities between the indole ring of
the substrate and a hydrophobic moiety of the protein. It is also
suggested that strict complementarities are not required for
L-Trp binding, which may reflect the broader substrate speci-
ficity of IDO. The structural differences between indoleamine
derivatives may be absorbed by the flexibility of the connecting
Comparison of IDO with Heme-Containing Protein. The mechanism
for the reaction of IDO proposed above can be comparable to
that for heme monooxygenases (20). The P450 family consists of
typical monoxygenase enzymes that contain heme-iron in their
active site for dioxygen activation (21). In the case of P450, as
well as nitric oxide synthases and heme oxygenase, the elec-
tropositive patches on the proximal surface are important for the
binding of its redox partner (22). In the distal side of P450,
hydroxyl or carboxyl groups in the conserved Thr or acidic
residues play a crucial role in the formation of a specific
H-bonding network to deliver the protons from solvent water to
the active site during the catalytic reaction of P450. In peroxi-
dase, the presence of a strong H bond between the proximal His
and an Asp leads to anionic character in the proximal His. The
distal Arg polarizes the OOO bond and distal His acts as an
acid-base catalyst for the heterolytic cleavage of the OOO bond.
In the case of myoglobin, the absence of the distal Arg and H
bond in proximal His is indicative of the reversible binding of O2.
In contrast, IDO has a symmetric charge distribution on the
the possible reductase can be identified in the proximal surface
(Fig. 2). Furthermore, the significant polar amino acid or water
molecules are absent from the active site of IDO, suggesting that
the H-bonding network connecting the active site to the solvent
region is not present in the distal pocket of the IDO structure.
These structural characteristics around the heme pocket of IDO
are in good agreement with the proposed catalytic reaction, in
that it does not require either protons or electrons supply.
The nonheme Fe2?-containing extradiol dioxygenases cleave
the COC bond adjacent to the -OH of catecholic substrates by
activating dioxygen and incorporating both oxygen atoms. The
crystal structures of the substrate-bound form indicate that the
chains and water, bind directly to Fe2?(13, 23). In the case of
naphthalene dioxygenase, the dioxygen binds side-on with both
oxygen atoms coordinated to the nonheme iron (12). Thus, the
reaction mechanism for nonheme dioxygenases is quite different
from that of IDO, which is allowed by the flexible coordination
geometry and the accessible space around the nonheme iron.
The structure of human IDO provides evidence to show that
the proposed reaction mechanism involves the proton abstrac-
tion by iron-bound dixoygen. This scheme is quite different
from the oxygen activation process in monooxygenases. The
analysis of the site-directed mutants strongly suggests that not
only substrate binding but also the proper geometry between
the substrate and iron-bound dioxygen is required for the
reaction. The recognition of the substrate likely involves strict
complementarities between the indole ring of the substrate
and protein groups. A comparison with the structure of other
well known heme protein suggests that the OOO bond is
precisely controlled by the heme proximal and distal environ-
ment and is not cleaved before the incorporation of both
oxygen atoms into the substrate.
Materials and Methods
Protein Expression, Purification, and Kinetics. The coding region for
human IDO was cloned into the pET-15b vector (Novagen). The
wild-type and mutant IDO were expressed in transformed
Escherichia coli BL21(DE3) cells and purified by using a Ni
affinity column and anion exchange chromatography. The assays
Table 2. X-ray data and refinement statistics
Multiwavelength anomalous dispersion data (PI form)
PI form CN?formPeakEdgeHigh LowPre-edge
rms deviation bond, Å
rms deviation angle, °
Numbers in parentheses are for the highest-resolution shell.
*Rmerge? ?hkl?i?Ii(hkl) ? ?I(hkl)????hkl?iIi(hkl), where ?I (hkl)? is the average intensity of the i observations.
†Rcryst? ?hkl?Fobs(hkl)? ? ?Fcalc(hkl)???hkl?Fobs(hkl)?. Rfreeis calculated for 5% of reflections randomly selected and excluded from refinement. Rcrystis calculated for
the remaining 95% of reflections used for structure refinement.
Sugimoto et al.
February 21, 2006 ?
vol. 103 ?
no. 8 ?
for the wild-type and mutant IDO were performed as described Download full-text
(24), except that the buffer with 0.1 M potassium phosphate
buffer and 0.1 M NaCl (pH 8.0) was used at 37C°. The rate of
product formation was determined from the slope of the initial
increase in absorbance at 480 nm derived from Kyn as a function
of time. Kdvalues of L-Trp for ferric, ferric CN?, and ferrous
forms of IDO were determined by monitoring the peak height of
the Soret region (404, 418, and 423 nm, respectively) of optical
absorption spectra in various concentrations (0–5 mM) of L-Trp.
The Kd values of L-Trp for the ferrous CO complex were
deduced from the CO binding rates that were measured in laser
flash photolysis experiments, which were performed in 0.1 M
potassium phosphate buffer (pH 8.0), 0.1 M NaCl, and 2 mM
L-Trp at 20°C by using an instrument constructed by UNISOKU
(Osaka). The emission from a dye solution (rhodamine 6G in
methanol at a concentration of 0.24 g?liter) was used. Changes
in absorption after CO photolysis were monitored at 435.4 nm
by a light from a xenon lamp.
Crystallization, Structure Determination, and Refinement. IDO was
concentrated to 40 mg?ml in a buffer containing 10 mM Mes
(pH 6.5), 25 mM NaCl, and 1 mM PI. Crystals of IDO–PI
complex were obtained by vapor diffusion from hanging drops
(6 ?l) containing a 1:1 (vol?vol) ratio of protein solution to
reservoir solution (10% polyethylene glycol 8000, 200 mM
ammonium acetate, 100 mM CHES, pH 9.0). Crystals formed
in 2 weeks at 20°C in the space group P212121(cell dimensions
of a ? 86.1, b ? 98.0, and c ? 131.0 Å) with two IDO
monomers in the asymmetric unit and a solvent content of
55%. The crystals were transferred to a cryo buffer consisting
of the reservoir with an additional 30% xilitol. X-ray diffrac-
tion data of PI form were collected by using a Jupiter210
charge-coupled device detector (Rigaku, Tokyo) in BL26B1 at
SPring-8. Data collection statistics are shown in Table 2.
Multiwavelength anomalous dispersion data of the PI form
were measured at the absorption edge of the Fe atom. The
determination of the iron position and initial phasing was
performed by using the AUTOSHARP program (25). The initial
2.8-Å phases were improved, and the poly(A) models were
partially built with the ARP?WARP program (26). The protein
and heme model was then built manually with the program O
(27). This model was refined with the CNS program (28) by
using higher-resolution data (20–2.3 Å). The positions of the
S atom in the CHES molecules in the active site were con-
firmed by an anomalous difference Fourier map. The N-
terminal three residues (G-S-H) from a cloning artifact and
residues 1–10 and 360–380 of IDO could not be defined in
electron density. Noncrystal symmetry (NCS) restrains were
not applied in the final stage of the refinement. The diffraction
data for the CN?-bound form with the unit cell dimensions of
a ? 86.4, b ? 97.1, and c ? 129.4 Å were collected in BL44B2
at SPring-8. The form was prepared by soaking the PI-bound
crystal in the cryo buffer containing 5 mM KCN for 5 min.
Because the soaking experiments damaged the diffraction
quality, we searched various conditions of soaking time and
concentration of KCN. Five minutes are a minimum to exclude
the electron density of PI. NCS restrains were applied in the
whole refinement process for the CN?-bound form. The
refinement statistics are shown in Table 2. Figures were
created by O (27), MOLSCRIPT (29), RASTER3D (30), PYMOL
(31), and GRASP (32).
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