Báo cáo khoa học: Crystal structure of heme oxygenase-1 from cyanobacterium Synechocystis sp. PCC 6803 in complex with heme pot

Crystal structure of heme oxygenase-1 from cyanobacterium
Synechocystis
sp. PCC 6803 in complex with heme
Masakazu Sugishima
1
, Catharina T. Migita
2
, Xuhong Zhang
3
, Tadashi Yoshida
3
and Keiichi Fukuyama
1
1
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan;
2
Department of Biological
Chemistry, Faculty of Agriculture, Yamaguchi University, Yoshida, Yamaguchi, Japan;
3
Department of Biochemistry,
Yamagata University School of Medicine, Yamagata, Japan
Heme oxygenase (HO) catalyzes the oxidative degradation
of heme utilizing molecular oxygen and reducing equiva-
lents. In photosynthetic organisms, HO functions in the
biosynthesis of such open-chain tetrapyrroles a s phyto-
chromobilin and phycobilins, wh ich are involved in the sig-
nal t ransduction for light responses and light harvesting for
photosynthesis, respectively. We have determined the first
crystal structure of a HO-1 from a photosynthetic organism,
Synechocystis sp. PCC 6803 (Syn HO-1), in complex w ith
heme at 2.5 A

˚
resolution. Heme–Syn HO-1 shares a com-
mon folding with other heme–HOs. Although the heme
pocket of h eme–Syn HO-1 i s, for t he most part, similar to
that of mammalian HO-1, they differ in s uch features as the
flexibility of the distal helix and hydrophobicity. In addition,
2-propanol derived from the crystallization solution occu-
pied the hydrophobic cavity, which is proposed to be a C O
trapping site in rat HO-1 that suppresses product inhibition.
Although Syn HO-1 and mammalian HO-1 are similar in
overall s tructure and amino acid sequence (57% similarity
vs. human HO-1), their molecular surfaces differ in charge
distribution. The surfaces of the heme binding sides are both
positively charged, but this patch of Syn HO-1 is narrow
compared to that of mammalian HO-1. This feature is suited
to the selective binding of ferredoxin, the physiological redox
partner of Syn HO-1; the molecular size of f erredoxin is
 10 kDa whereas the size of NADPH-cytochrome P450
reductase, a reducing partner of mammalian HO-1, is
 77 kDa. A docking model of h eme–Syn H O-1 a nd ferre-
doxin suggests indirect electron transfer from an iron–sulfur
cluster i n ferredoxin to the heme iron of heme–Syn HO-1.
Keywords: crystal structure; cyanobacterium; heme oxy-
genase; light-harvesting pigment.
Heme oxygenase (HO) catalyzes the oxygen-dependent
cleavage of the porphyrin ring of heme, producing biliverdin
IXa, iron, and carbon monoxide utilizing reducing equiv-
alents [1]. In mammals, HO is mainly involved in heme
metabolism f or the purpose of recovering iron from waste
heme. Biliverdin IXa is further reduced by biliverdin

reductase to bilirubin IXa, a potent antioxidant that
protects cells from oxidative damage [2]. Another product,
carbon monoxide, has been proposed to function as a
neuronal or other signal transmitter [3]. Some pathogenic
bacteria utilize HO to obtain iron from the host [4]. The
reaction pathway involving mammalian HO consists of
three sequential oxidation steps that utilize O
2
and reducing
equivalents from NADPH-cytochrome P450 reductase
(CPR) [5–7]. In the first step, O
2
bound to the heme iron
is activated to form ferric-hydroperoxide, a nd electrophilic
addition of the terminal oxygen to the a-meso carbon
produces a-hydroxyheme. In the second step, a-hydroxy-
heme is converted to verdoheme with concomitant release
of the a-meso carbon as CO. Lastly, the oxygen bridge o f
verdoheme is cleaved to produce a biliverdin I Xa–iron
complex and ferrous iron is released p rior to the dissociation
of biliverdin IXa. The crystal structures of the human, rat,
Gram-negative pathogen Neisseria meningitidis,andGram-
positive pathogen Corynebacterium diphtheriae HOs in
complex with heme show that all HOs have similar overall
structures consisting mainly of a-helices [8–11].
Although major progress has been made in understand-
ing the nature of HO reactions based on HO structures from
several species, the structure o f HO from a p hotosynthetic
organism has not been determined. In contrast to the
physiological functions of HO in mammals, HO in photo-

synthetic organisms functions in the biosynthesis of such
open-chain tetrapyrroles as phytochromobilin [12] and
phycobilins [13]. Phytochromobilin is a pigment in phyto-
chrome involved in signal transduction of light responses in
higher plants and red algae [14]. Recently, an ortholog of
higher plant phytochrome was found in the cyanobac-
terium, Synechocystis sp. PCC 6803 [15], and was postulated
to be involved in phototaxis towards blue light [16].
Phycobilins are pigments in phycobiliproteins involved in
Correspondence to K. Fukuyama, Department of Biology, Graduate
School of Science, Osaka University, Toyonaka, Osaka 560-0043,
Japan. Fax: +81 6 6850 5425, Tel.: +81 6 6850 5422,
E-mail:
Abbreviations: CPR, NADPH-cytochrome P450 reductase; Fd, fer-
redoxin; HO, heme oxygenase; heme–HO, H O in complex with heme;
Syn HO-1, HO-1 from Synechocystis sp. PCC 6803; HmuO, HO from
Corynebacterium diphtheriae; HemO, HO from Neisseria meningitidis.
Enzyme: heme oxygenase (EC 1.14.99.3).
Note: Coordinates and structure factors have been deposited in t he
Protein Data B ank, accession code 1WE1.
(Received 1 1 August 2 004, revised 30 September 2004,
accepted 4 October 2004)
Eur. J. Biochem. 271, 4517–4525 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04411.x
light-harvesting f or photosynthesis in red algae, cyanobac-
teria, and cryptophytes. The characteristic blue-green and
red colors of cyanobacteria and red algae reflect the
presence of a light-harvesting complex called phycobilisome,
which is composed of phycobiliproteins [17].
Proteins possessing heme oxygenase activity have been
isolated from the red alga, Cyanidium caldarium,andthe

cyanobacteria, Synechocystis sp. PCC 6701 and P CC 6803
[18–22]. Two g enes (ho1, ORF sll1184, a nd ho2,ORF
sll1875) were annotated as HOs by genome sequence
analysis of Synechocystis sp. PCC 6803 [23]; however,
RNA blot analysis has suggested that ho2 is a silent gene
[24]. Reduced plant-type ferredoxin (Fd) is a possible
physiological electron donor for the ho1 gene product, Syn
HO-1, on the basis of in vitro assays, although CPR is
utilized by mammalian HO-1 [24]. In addition, it has been
reported that the Arabidopsis thaliana hy1 gene encodes a
protein related to HO [25], and could be genetically replaced
by ho1 from Synechocystis sp. P CC 6803 [26]. Recently, we
constructed a system to efficiently express Syn HO-1 in
Escherichia c oli and have c haracterized the spectroscopic
features of heme–Syn HO-1 [27]. P urified Syn HO-1 c an
catalyze heme degradation using ascorbate o r CPR as a
redox partner; however, catalysis using these redox partners
is slower than the catalysis of a rat HO-1 system.
We have determined the crystal structure of heme–Syn
HO-1 at 2.5 A
˚
resolution. This analysis demonstrates that
the surface features of Syn HO-1 differ markedly from the
features of mammalian HO-1, and may explain why Syn
HO-1 utilizes Fd as a redox partner, in contrast to the use of
CPR by mammalian HO-1. In addition, the heme pocket of
heme–Syn HO-1 differs from t hat of mammalian HO-1 in
the flexibility of the distal helix and hydrophobic ity.
Experimental procedures
Crystallization of the heme–Syn HO-1 complex

The expression and purification of Syn HO-1 and the
preparation o f its comple x with heme have b een described
previously [27]. Crystallization conditions for heme–Syn
HO-1 were screened using a Crystal Screen kit (Hampton
Research, Aliso Viejo, CA) and the hanging-drop vapor-
diffusion method. The protein solution was m ixed with an
equal volume of each reservoir solution and equilibrated.
Crystals of heme–Syn HO-1 were obtained at 293 K using a
reservoir solution containing 15% (v/v) 2-propanol, 15%
(w/v) P EG 4000, 2% (w/v) 1,5-diaminopentane dihydro-
chloride, and 0.1
M
sodium citrate (pH 5.9). The protein
concentration f or cryst allizat ion w as 20 mgÆmL
)1
in 0.1
M
potassium phosphate buffer (pH 7.4) containing 5 m
M
sodium cyanide. Plate-shaped crystals appeared after one
day (Fig. 1). Although the protein solution used for
crystallization changed color to bright red upon the addition
of potassium cyanide, the crystals were dark brown,
indicating that the cyanide in the s olution was not bound
tothehemeironinthesecrystals.
Data collection and processing
Heme–Syn HO-1 crystals were soaked in crystallization
solution co ntaining 10% (v/v) glycerol as a c ryo-protectant
and were flash-cooled with a nitrogen gas st ream at 100 K.
Diffraction d ata w ere collected at 100 K u sing synchrotron

radiation (k ¼ 1.500 A
˚
)fromtheBL41XUbeamlineat
SPring-8 and a marCCD detector (MarUSA, Evanston,
IL). The distance between the crystal and t he CCD was
130 mm. The crystal was rotated by 1.5° per frame with a
total measurement angle of 180°. Diffraction data were
processed, merged, and scaled with
MOSFLM
[28] and
SCALA
in the
CCP
4 package [29,30]. Crystallographic data and
diffraction statistics are given in Table 1.
Model building and refinement
The structure of heme–Syn HO-1 was determined by the
molecular replacement method with the program
MOLREP
[30,31], in which the protein moiety of the heme–rat
HO-1 (PDB code 1DVE) was used as the search model. A
cross-rotation and t ranslation search located four inde-
pendent Syn HO-1 molecules in an asymmetric unit.
Following rigid body refinement for the four polypeptide
chains, these chains were substituted with t he Syn HO-1
chain on the basis of the known sequence using the
GENEMINE
homology modeling software [32]. Simulated
annealing and temperature-factor refinements were
applied to Syn HO-1 models based on 20.0–2.5 A

˚
resolution data with posing restraints on the four mole-
cules with noncrystallographic symmetry. The structure
was revised by adjusting t he model w ith the program
XFIT
[33]. The heme and the additionally ordered C-terminal
Fig. 1. Photograph of heme–Syn HO-1 crystals.
4518 M. Sugishima et al.(Eur. J. Biochem. 271) Ó FEBS 2004
helix were clearly seen in the electron density map and
included in the subsequent refinements. After a few cycles
of water picking and energy minimization refinements
without restraints by noncrystallographic symmetry,
2-propanol molecules, and phosphate and chloride ions
were identified on the basis of the distribution of electron
density and the chemical environments of the sites. Eight
2-propanol molecules, two phosphate ions, a nd five
chloride ions were included in the final refinement. Bond
distances among the heme iron and its ligands were
weakly restrained as 2.0 A
˚
. All refinements were carried
out with the program
CNS
[34]. The stereochemical check
of the model was made with the program
PROCHECK
[35].
Refinement statistics are given in Table 1.
Docking simulation of heme–Syn HO-1 and Fd
Fd I from Synechocystis sp. PCC 6803 [36] was used for a

docking simulation with heme–Syn HO-1. T he simulation
was performed using the program
HEX
[37] based on the
surface complementarities and electrostatic interactions. In
the four best candidates derived from the simulation, Fd
was bound to the heme binding side of the heme–Syn H O-1
with similar orientation. The most probable docking model
was selected from these candidates on the basis of the
Table 1. Summary of crystallographic statistics. Values in parentheses
are for the outerm ost shell (2.53–2.40 A
˚
). R
free
is the R-value calculated
for 10% of th e dataset not included in t h e refinement.
Crystallographic data
Space group C2
Unit cell dimensions (A
˚
, °)a¼ 110.79, b ¼ 113.73,
c ¼ 109.70, b ¼ 112.26
No. of molecules in an
asymmetric unit
4
Diffraction statistics
Resolution range (A
˚
) 50–2.4
No. of observations 153703

No. of unique reflections 46584
Redundancy 3.3
Completeness (%) 94.7 (83.2)
Mean I
o
/r 7.2 (2.4)
R
sym
a
0.064 (0.302)
Refinement statistics
Resolution range (A
˚
) 20.0–2.5
R/R
free
b
0.222/0.268
No. of protein/heme atoms 7040/172
No. of water molecules 204
No. of atoms of 2-propanol/
chloride/phosphate
32/5/10
Root mean square deviations from ideality
Bond lengths (A
˚
) 0.008
Angles (degrees) 1.21
Ramachandran plot
Most favored (%) 91.2

Additionally allowed (%) 8.5
Generously allowed (%) 0.2
a
R
sym
¼ S
hkl
S
i
|I
i
(hkl) ) <I(hkl)>|/S
hkl
S
i
I
i
(hkl), where <I(hkl)>
is the mean intensity for multiple recorded reflections.
b
R ¼
S|F
obs
(hkl) ) F
calc
(hkl)|/S|F
obs
(hkl)|.
Fig. 2. Crystal structure of heme–Syn HO-1. (A) R ibbon diagram of
heme–Syn HO-1. H e me, its ligands, and h etero c ompou nds i ncluded i n

the m odel are superimp osed on the ribbon diagram (A, Ala6–Gly24;
B, Phe25–Gly32; C, Asn37–Phe60; D, Lys77–Phe86; E, Ala100–
Thr115; F, Leu120–Met146; G, Asp163–Leu178; H, Thr184–Arg222).
Yellow sp heres indicate chloride ions. A phosphate ion and one of t he
chloride ions are bound by two molecu les in the crystal. (B) Backbone
stabilizationinSynHO-1.Thedistalhelixisshowninorangefor
clarity. Residues involving distal helix conformation stabilization are
shown a s ball-and-stick models. This figure was prepared using the
programs
MOLSCRIPT
[48],
RASTER
3
D
[49], and
VMD
[50].
Ó FEBS 2004 Structure of cyanobacterium heme–HO complex (Eur. J. Biochem. 271) 4519
distances between the Fd iron–sulfur cluster and the heme
iron in the heme–Syn HO-1.
Results and Discussion
Overall structure
The structure of heme–Syn HO-1 has been r efined using
2.5 A
˚
resolution d ata to an R-factor of 0 .222 and a free
R-factor of 0.268. The segments from Ser2 to Thr223 in
the four heme–Syn HO-1s are ordered in the crystal.
Heme–Syn HO-1 consists of eight a-helices (Fig. 2A).
The structures of the four complexes in an asymmetric

unit are very similar, with an overall root mean square
deviation (rmsd) for main chain atoms of 0.35 A
˚
.The
folding of heme–Syn HO-1 is similar to the folding
observedinotherheme–HOs[rmsdofCas are 0.94–
1.06 A
˚
for human HO-1, 1.06–1.16 A
˚
for rat HO-1,
1.17–1.40 A
˚
for HO from the Gram-positive bacterium,
Corynebacterium diphtheriae (HmuO), 1.69–1.86 A
˚
for
HO from the Gram-negative bacterium, Neisseria men-
ingitidis (HemO)]. One apparent characteristic of Syn
HO-1 is that its H-helix is extended by five turns; the
segment from Gly208 protrudes from the globular part
of the molecule. In the crystal, this segment hydropho-
bically interacts with the corresponding helix in another
molecule; t hese interactions are identical to each other.
Thus, it is likely that intermolecular interactions force
this segment into a fixed orientation in the crystal;
however, the segment may fluctuate o r take on another
orientation in solution.
Conformation of the distal helix
As in the previously reported s tructures of o ther heme–

HOs, the heme of heme–Syn HO-1 is sandwiched
between the A- and F-helices and the F-helix kinks at
the distal side o f the heme [8–11 ]. Three types of
conformation of the F-helix were reported: ÔopenÕ,
ÔclosedÕ,andÔmore closedÕ, depending on the kinking
angles of the F-helices and the hydrogen bonds stabil-
izing these conformations (Table 2, Fig. 3). The ÔopenÕ
and ÔclosedÕ conformations were first reported in the
human heme–HO-1 [8]. Each conformation is stabilized
by a hydrogen bond between the amide group of Gly143
and the carbonyl group of Gly139, although the distance
between Gly143 and the heme iron differs in the two
conformations. ÔClosedÕ conformations were also reported
in HemO and HmuO [8,11]. A Ômore closed Õ conforma-
tion was reported in rat heme–HO-1 [9]. In contrast to
the two conformations described above, in the Ômore
closedÕ confor mation, the hydrogen bond partner of the
carbonyl group of Gly139 is the amide group of Gly144,
and the amide group of Gly143 is hydrogen-bonded to
the distal ligand of the heme iron (the residue numbers
of the three glycines are the same in rat HO-1 and in
human HO-1). The Gly139–Gly143 segment (Gly130–
Gly134 in Syn HO-1) takes on an a-helical conformation
in the ÔopenÕ and ÔclosedÕ conformations, whereas i n the
Ômore closedÕ conformation, the segment takes on a
p-h elical conformation. This structural feature suggests
that the ÔopenÕ and ÔclosedÕ conformations are more
stable than the Ômore closedÕ conformation and that
stabilization by the hydrogen bonding of glycine (Gly143
in mammalian HO-1) to the distal ligand of the heme

iron is required f or the formation of the Ômore closedÕ
conformation. Indeed, the distal helix conformation is
bidirectionally converted during the reaction process of
rat HO-1 [38] and O
2
binding to the heme iron of
HmuO [39]. Following this d efinition of helix conforma-
Table 2. Selected distances (A
˚
) between atoms at the di stal heme poc ket. Residue n umbers shown in the first line are those in Syn HO-1. Gly130,
Gly134, Gly135, and Asp131 correspond to Gly139, Gly143, Gly144, and Asp140 in human and rat HO-1s and to Gly135, Gly139, Gly140, and
Asp136 in HmuO.
F-helix confomation Gly130 O–Fe Gly134 N–Fe Gly130 O–Gly134 N Gly130 O–Gly135 N Asp131 O–Gly135 N
Syn HO-1 closed 4.6–4.9 5.1–5.4 2.8–3.0 4.0–4.1 2.9–3.0
Human HO-1 closed 4.8 5.3 3.2 4.3 3.1
Human HO-1 open 4.9 6.0 3.1 4.8 2.9
Rat HO-1 more closed 5.1 4.3 3.1 2.9 3.8
HmuO closed 4.7–4.8 4.9–5.4 2.9–3.0 3.1–3.6 3.0–3.4
Fig. 3. Schematic diagram of the distal helix conformations. The heme
iron, distal ligand, and the two conserved glycine residues are shown.
Dashed lines indicate hydrogen bon ds. Residue nu mbers of Syn HO-1
and mammalian HO- 1 (in parentheses) are shown. Hydrogen bonding
patterns in the ÔopenÕ and ÔclosedÕ conformations and in the Ômore
closedÕ conformation are characterized by a-helix and p-helix,
respectively.
4520 M. Sugishima et al.(Eur. J. Biochem. 271) Ó FEBS 2004
tion, all F-helices in heme–Syn HO- 1s i n t he cry stal are
in the ÔclosedÕ conformation. The fact that all heme–Syn
HO-1s i n t he asym metric unit form identical distal helix
conformations indicates that the distal helix of Syn HO-1

is more rigid than that of human HO-1, in which two
conformations were present in the crystal. The distal
helix of HmuO also seems more rigid than that of
mammalian HO-1; it has been proposed that two
aromatic residues in HmuO, His150 and Tyr151, sup-
press the flexibility of the distal helix by tight hydro-
phobic interactions [11]. However, these residues are
substituted by Ala and Met, respectively, in Syn HO-1,
and the tight hydrophobic i nteractions seen in HmuO are
absent in Syn HO-1. These residues in Syn HO-1 are
identical to rat HO-1 (Met is substituted by Leu in
human HO-1). Therefore, the mechanism by which the
distal helix is stabilized differs i n HmuO and Syn HO-1.
In the structures of mammalian HO-1 and HmuO, a
hydrophobic aromatic cluster is located near th e heme
pocket and supports the distal helix conformation. In
Syn HO-1, this hydrophobic cluster is made more rigid
due to substitution of Phe at residue 39 (Phe47 in human
HO-1) for Tyr, which forms a hydrogen bond to Tyr156
(Fig. 2 B). The rigidity of the hydrophobic cluster may
contribute to stabilization of the distal helix conforma-
tion. In addition, Lys139 in the distal helix forms a salt-
bridge to Glu157 in Syn HO-1 (Fig. 2B), which also
stabilizes the distal helix o f Syn HO-1.
Fig. 4. Heme pocket structure of heme–Syn
HO-1. (A) r
A
-weighted 2F
o
–F

c
map (blue;
contoured at 1.0 r)andF
o
–F
c
map omitted
the distal ligand of heme (red; contoured at
5.0 r). The e lectron density map is superim-
posed on the ball-and-stick model o f heme–
Syn HO-1 around the heme p oc ket. (B) The
detailed structure around th e heme is s hown
by ball-and-stick models. (C) 2 -propanol
binding in the hydrop ho bic cavity. r
A
-weigh-
ted 2F
o
–F
c
map (blue; c ontoured at 1.0 r)is
superimposed on the ball-and-stick model of
heme–Syn HO-1 around the distal hydro-
phobic cavity. This figure w as prepared using
the programs
MOLSCRIPT
[48],
RASTER
3
D

[49],
VMD
[50], and
CONSCRIPT
[51].
Ó FEBS 2004 Structure of cyanobacterium heme–HO complex (Eur. J. Biochem. 271) 4521
Fig. 5. Molecular interactions between Syn HO-1 and Fd. (A) Electrostatic potentials of heme–Syn HO-1, heme–rat HO-1, Fd I from Synechocystis
sp. PCC 6803, and C PR. Positive a nd negative su rfaces are shown in blue (+12.0 kTÆe
)1
for Fd an d + 5.0 kTÆe
)1
for other prote ins) a nd re d
()12.0 kTÆe
)1
for Fd and )5.0 k TÆe
)1
for other proteins), respect ively. Electrostatic potential calculations included only fully charged residues (Asp,
Glu, Arg, and Lys) using diele ctric constants of 80 for the exterior of the protein an d 2 f o r the interior o f the protein. The redox c enter of each
molecule is shown as a w ire-fr ame model (2Fe)2S cluster of Fd I is located inside). (B) Putative docking model of heme–Syn HO-1 and Fd I from
Synechocystis sp.PCC6803.A
CPK
model o f th e red ox c enters is superimposed on the ribbo n model of the putative docking mod el (Syn HO-1, gray;
Fd I, yellow). The distance be tween the heme iron and iron–sulfur cluster is also shown. T his figure was prepared using the program
VMD
[50].
4522 M. Sugishima et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Heme pocket structure
An electron density map and a model around the heme
pocket of heme–Syn H O-1 are shown in Fig. 4A,B. His17
(corresponding to His25 in mammalian HO-1) and a

water molecule are coordinated to the heme iron in heme–
Syn HO-1. Glu21 would hydrogen-bond to the proximal
histidine ( 3.2 A
˚
). The carbonyl group of Gly130
(corresponding to Gly139 in mammalian HO-1) would
hydrogen-bond to the distal ligand ( 3.1 A
˚
). The heme
orientation along the a–c axis is stabilize d by the
electrostatic interaction s of basic residues (Arg10,
Lys168, and Arg172) and a hydrogen bond of Tyr125
to the heme propionate groups. These electrostatic
interactions and hydrogen bond are c onserved in human
and r at HO- 1s, and HmuO, but not in HemO. Asp140 in
mammalian H O-1 (Asp131 in Syn H O-1) is thought to be
a key residue for HO catalysis on the basis of mutation
analysis [40,41]. The carboxyl group of Asp140 interacts
with O
2
coordinated t o the heme iron via water molecules
in HmuO [39]. In human and rat HO-1s, nitric oxide and
azide bound forms show similar structural features [42–
44]. In Syn HO-1, the conformation of Asp131 is similar
to the conformations of the corresponding residues in
other heme–HOs. Several water molecules are located on
the distal side of the heme and form a hydrogen bond
network that is also observed in other heme–HOs. The
amino acid residues constituting the heme pocket are
highly conserved in Syn HO-1 and mammalian HO-1;

however, Gln38 in rat HO-1 is substituted by Leu30 in
Syn HO-1, indicating that the heme pocket of Syn HO-1
may be more hydrophobic than that of mammalian
HO-1. Leu30 is adjacent to the porphyrin macrocycle.
Hydrophobic cavity near the heme pocket
A small hydrophobic cavity surrounded by Phe25, Val26,
Phe29, Tyr39, Leu42, Leu46, Leu138, Phe155, Tyr158, and
Phe203 is located near the distal heme pocket of S yn HO-1,
similar to mammalian HO-1. The cavity volume in S yn
HO-1 is 52.8–61.5 A
˚
3
, which is similar to the size of the
cavities in human and rat HO-1s (human HO-1; 43.6–
59.7 A
˚
3
, rat HO-1; 44.9 A
˚
3
). Recently, a cryo-trapped
intermediate structure of the photolysis of rat HO-1 in
complex with heme and CO has shown that photolyzed CO
can be trapped in this cavity; this suggests that the cavity
may trap CO produced in the reaction step generating
verdoheme from a-hydroxyheme, which facilitates the
subsequent reaction step from verdoheme to biliverdin
[45]. Interestingly, in heme–Syn HO-1, a broad electron
density appeared in each cavity of the f our molecules in the
asymmetric unit (Fig. 4C). Such density was not found in

rat h eme–HO-1. The hydrophobicity of this cavity suggests
that water would not bind to this site. In fact, assuming that
a water molecule occupied this cavity, residual density was
observed in the difference Fourier map. However, a model
of 2-propanol, which was present in the crystallization
solution, fitted well to t he electron density. In addition, the
hydroxyl group of 2-propanol is within hydrogen bonding
distance of Tyr156 (Phe167 in rat HO-1) and/or a water
molecule. Thus, we conclude th at 2-propanol present in t he
crystallization solution i s bound to he me–Syn HO-1 in this
cavity. T his raises t he possibility that 2-propanol affects the
Syn HO-1 reaction by occupying the CO escape route.
Molecular recognition of the redox partner
In the physiological HO reaction, heme–Syn HO-1 is
probably reduced by the reduced form of Fd [22,24], a
small, acidic iron–sulfur protein; in contrast, CPR is the
redox partner i n mammalian systems [24,27]. Fd would be
recognized by Syn HO-1 through electrostatic interactions,
as is proposed between CPR and mammalian HO-1. The
surface electrostatic potentials of heme–Syn HO-1, heme–
rat HO-1, Fd I from Synechocystis sp. PCC 6803 (PDB
code 1OFF) [36], and CPR (PDB code 1AMO) [46] are
shown in Fig. 5A. The surfaces of the heme binding sides
of both HOs are positively charged whereas the opposite
sides are negatively charged. This feature indicates that
the docking surfaces of both HOs are the heme bin ding
sides. However, it should be noted that the positively
charged surface of Syn HO-1 is narrower t han that of rat
HO-1. This may reflect the size of the physiological
counterparts. Indeed, the in vitro single turnover reaction

of Syn HO-1 using Fd is more rapid than that using
mammalian CPR (C. T. Migita & T. Yoshida, unpub-
lished data). One of the reasons why Syn HO-1 cannot
efficiently accept electrons from CPR is due to its
narrower patch of positively charged surface. The large
neutral surface of the heme binding side of Syn HO-1
would retard the proper binding of CPR.
Based on the charge distributions of the molecular
surfaces, the complementarities of the molecular surfaces,
and the distance between the iron–sulfur cluster of Fd and
the heme iron of heme–Syn HO-1, we constructed a
docking model of Fd a nd heme–Syn HO-1 (Fig. 5B). The
distance between the Fd iron–sulfur cluster and the heme
iron of heme–Syn HO-1 ( 15 A
˚
in this docking model)
seems too far to transfer electrons directly from the iron–
sulfur cluster to the heme iron. Several Fd residues
(Tyr24, Arg41, and Tyr81 in this docking model) interact
with heme and Glu21 of Syn HO-1, which hydrogen
bonds to the proximal histidine. This glutamic acid is
conserved in other HOs. In addition, the corresponding
Glu29 in human HO-1 is in the docking site with
cytochrome P450 reductase [47]. Thus, those residues on
the docking surfaces may be involved in indirect electron
transfer from Fd to heme–Syn HO-1.
Acknowledgements
We thank Drs Masahide Kawam oto and Hisan obu Sakai of JASRI for
their aid with data c ollectio n using th e synchrotr on radiation at SPr ing-
8. This work was supported in part by Grants-in-Aid for Scientific

Research (C) to K.F. (No. 16570095) and to T.Y. (Nos 14580641 and
16570108) and by a grant of the National Project on Protein Structural
and Functional Analyses from the Ministry of Education, Culture,
Sports, Science, a nd Technology of J apan.
References
1. Tenhunen, R., Marver, H.S. & Sch mid, R. (1968) The enzymatic
conversion of heme to bilirubin by microsomal heme oxygenase.
Proc.NatlAcad.Sci.USA61, 748–755.
Ó FEBS 2004 Structure of cyanobacterium heme–HO complex (Eur. J. Biochem. 271) 4523
2. Stocker,R.,Yamamoto,Y.,McDonagh,A.F.,Glazer,A.N.&
Ames, B.N. (1987) Bilirubin is an antioxidant of p ossible physio-
logical importance. Science 23 5 , 1043–1046.
3. Ryter, S.W., Otterbein, L.E., Morse, D. & Choi, A.M. (2002)
Heme oxygenase/carbon monoxide signaling pathways: regula-
tion and functional significa nce. Mol. Cell. Biochem. 234–235,
249–263.
4. Schmitt, M.P. (1997) Utilization of host iron sources by Coryne-
bacterium diphtheriae: identification of a gene whose product is
homologous to eukaryotic heme oxygenases and is required for
acquisition of iron from heme and hemoglobin. J. Bacteriol. 179,
838–845.
5. Sono, M., Roach, M.P., Coulter, E.D. & Dawson, J.H . (1996)
Heme-containing oxygenases. Ch em. Rev. 96, 2841 –2888.
6. Ortiz de Montellano, P.R. (2000) The mechanism of he me oxy-
genase. Curr. Opin. Chem. Biol. 4, 221–227.
7. Yoshida, T. & Migita, C.T. (2000) Mechanism of heme degrada-
tion by heme oxygenase. J. Inorg. Biochem. 82, 33–41.
8. Schuller, D .J., Wilks, A., O rtiz de Montellano, P .R. & Poulos,
T.L. (1999) Cry stal structure of human heme oxygenase-1. Nat.
Struct. Biol. 6, 860–867.

9. Sugishima, M., Omata, Y., Kakuta, Y., Sakamoto, H., Noguchi,
M. & Fukuyama, K. (2000) Crystal structure of rat heme oxy-
genase-1 in complex with heme. FEBS Lett. 471, 61–66.
10. Schuller, D .J., Zhu, W., Stojiljkovic, I., Wilks, A. & Poulos, T.L.
(2001) Crystal structure of heme oxygenase from the gram-nega-
tive p athogen Neisseria meningitidis and a comparison with
mammalian h eme oxygenase-1. Bioc hemistry 40, 11552–11558.
11. Hirotsu, S., Chu, G.C., Unno, M., Lee, D.S., Yoshida, T., Park,
S.Y., Shiro, Y. & Ikeda-Saito, M. (2004) The crystal structures of
the ferric and ferrous forms o fthehemecomplexofHmuO,a
heme oxygenase of Corynebacterium diphtheriae. J. Biol. Chem.
279, 11937–11947.
12. Terry, M.J., McDowell, M.T. & Lagarias, J.C. (1995) (3 Z)- and
(3E)-phytochromobilin are intermediates in the biosynthesis of the
phytochrome chromophore. J. Biol. Chem. 270, 11111–11118.
13. Tooley, A.J., Cai, Y .A. & Glazer, A.N. (2001) Biosynthesis of a
fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a
heterologous host. Proc. N atl Acad. Sci. USA 98, 105 60–10565.
14. Schafer, E. & Bowle, C. (2002) Phytochrome-mediated photo-
perception and signal trans duction in higher plants. EMBO
Report 3, 1042–1048.
15. Lamparter, T., Mittmann, F., Gartner, W., Borner, T., Hart-
mann, E . & Hughes, J. (1997) Characterization of recombinant
phytochrome from the cyanobacterium Synechocystis. Proc. Natl
Acad. Sc i. USA 94 , 11792–11797.
16. Wilde, A., Fiedler, B. & Borner, T. (2002) The cyanobacterial
phytochrome Cph2 inhibits p hototaxis towards blue light. Mol.
Microbiol. 44, 981–988.
17. Grossman, A.R., Schaefer, M.R., Chiang, G.G. & Collier, J.L.
(1993) The phycobilisome, a light-harvesting complex responsive

to environmental conditions. Microbiol. Rev. 57, 725–749.
18. Beale, S.I. & Cornejo, J. (1984) Enzymatic heme oxygenase
activity in soluble extracts of the unicellular r ed alga, Cyanidium
caldarium. Arch. Biochem. Biophys. 235, 371–384.
19. Cornejo, J. & Beale, S.I. ( 1988) Algal he me oxyge nase from
Cyanidium caldarium: partial purification and fractionation i nto
three required protein co mpon ents. J. Biol. Chem. 263, 11915–
11921.
20. Rhie, G. & Beale, S.I. (1992) Biosynthesis of phycobilins. Ferre-
doxin-supported NADPH-independent heme oxygenase and
phycobilin-forming activities from Cyanidium c aldarium. J. Biol.
Chem. 267, 16088–16093.
21. Rhie, G. & Beale, S.I. (1995) Phy cobilin biosynthesis: reductant
requirements and product identification for heme oxyge nase from
Cyanidium caldarium. Arch. Biochem. Biophys. 320, 182–194.
22. Cornejo, J. & Beale, S.I. (1997) Phycobilin biosynthetic reaction in
extracts of cyanobacteria. Photosynth. Res. 51, 223–230.
23. Kaneko, T., Sato, S., Kotani, H., T anaka, A., Asamizu, E .,
Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasa-
moto, S., Kimura, T., Hosouchi, T., Matsuno, A., M uraki, A.,
Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C.,
Wada, T., Watanabe, A., Yamada,M.,Yasuda,M.&Tabata,S.
(1996) Seque nce analysis of the genome of the unicellular cyano-
bacterium Synechocystis sp. strain PCC6803. II: sequence de-
termination of the entire genome and assignment of potential
protein-coding regions. DNA Res. 3, 109–136.
24. Cornejo, J., Willows, R.D. & Beale, S.I. (1 998) Phytobilin bio-
synthesis: c lon ing and expressio n of a ge ne encoding solub le fer-
redoxin-dependent heme o xygenase from Synechocystis sp. P C C
6803. Pla nt J. 15 , 99–107.

25. Davis, S.J., Kurepa, J. & Vierstra, R.D. (1999) The Arabidopsis
thaliana HY1 locus, required for phytochro me-chro mophore
biosynthesis, encodes a protein related to heme oxygenases. Proc.
NatlAcad.Sci.USA96, 6 541–6546.
26. Willows, R.D., Mayer, S.M., Foulk, M.S., D eLong, A., Hanson,
K., Chory, J. & Beale, S.I. (2000) Phytobilin biosynthesis: the
Synechocystis sp. P CC 6803 heme oxygenase-encoding ho1 gene
complements a phytochrome-deficie nt Arabidopsis thalianna hy1
mutant. Plant Mol. Biol. 43, 113–120.
27. Migita, C.T., Zhang, X. & Yoshida, T. (2003) Expression and
characterization of cyanobacterium heme oxygenase, a key
enzyme in the phycobilin synthe sis. Properties of the heme
complex o f r ecomb inant ac tive enzyme. Eur. J. Biochem. 270, 687–
698.
28. Leslie, A.G.W. (1992) Rec ent changes to the MOSFLM package
for processing film and image plate data. Joint C CP4 + ESF-
EAMCB Newsletter o n P rotein Crystallography,no.26.Daresbury
Laboratories, W arrington, UK.
29. Kabsch, W. (1988) Evaluation of single-crystal X-ray diffraction
data from a position-sensitive detector. J. App l. Crystallogr. 21,
916–924.
30. Collaborative Computational Project, Number, 4 (1994) The
CCP4 suite: p rogram s f or pr otein crystallography. Acta Crystal-
logr. D Biol. Crystallogr. 50 , 760–763.
31. Vagin, A. & Teplyakov, A . (1997) MOLREP: a n automated
program for molecular r eplacement. J. Appl. Crystallogr. 30,
1022–1025.
32. Lee, C. & Iriz arry, K. (2001) The G eneMine System for ge nome/
proteome an notatio n an d collaborative data mining. IBM S ys-
tems Journal 40, 5 92–603.

33. McRee, D.E. (1999) XtalVi ew/Xfit – A versatile program for
manipulating atomic c oordinates a nd elec tron density. J. Struct.
Biol. 125, 156–165.
34. Bru
¨
nger, A.T., Adams , P.D., Clore, G.M., DeLano, W.L., Gros,
P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M.,
Pannu, N.S., Read, R.J., Rice, L.M., S imonson, T. & Warren,
G.L. (1998) Crystallography & NMR system: a new software su ite
for m acromolecular structure d etermination. Acta Crystallogr.
D Biol. Crystallogr. 54 , 905–921.
35. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton,
J.M. (1993) PROCHECK: a program to check the stere ochemical
quality of p rotein structures. J. Appl. Crystallogr. 26, 283–291.
36. van den Heuvel, R.H., Svergun, D.I., Petoukhov, M.V., Coda, A.,
Curti, B., R avasio, S., V anoni, M.A. & M attevi, A. (2003) The
active conformation of g lutamate synthase and its binding to
ferredoxin. J. Mol. Biol. 330, 1 13–128.
37. Ritchie, D.W. (2003) Evaluation of protein d ocking predictions
using Hex 3.1 in CAPRI rounds 1 a nd 2. Proteins 52, 9 8–106.
38. Sugishima, M., Sakamoto, H ., Higashimoto, Y ., Noguchi, M. &
Fukuyama, K. (2003) Crystal structure of rat heme oxygenase-1 in
complex with biliverdin-iron chelate: conformational change of
4524 M. Sugishima et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the distal helix during the heme cleavage reaction. J. Biol. Chem.
278, 32352–32358.
39. Unno, M., Matsui, T., Chu, G.C., Couture, M., Yoshida, T.,
Rousseau, D.L., Olson, J.S. & Ikeda-Saito, M. (2004) Crystal
structure of the dioxygen-bound heme oxygenase from
Corynebacterium diphtheriae: I mplica tions for h eme oxygenase

function. J. Biol. Chem. 279, 21055–21061.
40. Lightning, L.K., Huang, H., Moenne-Loccoz, P ., L oehr, T.M.,
Schuller, D.J., Poulos, T.L. & de Montellano, P.R. (2001) Dis-
ruption of an active site h ydrogen b ond c onverts human heme
oxygenase-1 into a peroxidase. J. Biol. Chem. 276, 10612–10619.
41. Fujii, H., Zhang, X., Tomita, T., Ikeda-Saito, M. & Yoshida, T.
(2001) A role for hig hly co nserved carboxylate, a spartate-140, in
oxygen activation and heme degradation by heme o xygenase-1.
J. Am. Chem. So c . 123, 6475–6484.
42. Sugishima, M., Sakamoto, H., Higashimoto, Y., Omata, Y.,
Hayashi, S., Noguchi, M . & Fukuyama, K. (2002) Crystal struc-
ture of rat heme oxygenase-1 in complex with heme bound to
azide: implication f or regio specific hydro xylation of hem e at the
a-meso ca rbo n. J. Biol. Chem. 277, 4 5086–45090.
43. Sugishima, M., Sakamoto, H., Noguchi, M. & Fukuyama, K.
(2003) Crystal structures of ferrous and CO-, CN
–
-
,
and
NO-bound forms of rat heme oxygenase-1 (HO-1) in complex
with heme: structural implications for discrim ination between CO
and O
2
in HO-1. Bioc hemistry 42, 9898–9905.
44. Lad,L.,Wang,J.,Li,H.,Friedman,J.,Bhaskar,B.,Ortizde
Montellano, P.R. & Poulos, T.L. ( 2003) Crystal structures of the
ferric, ferrous, and ferrous-NO forms of the Asp140Ala mutant of
human heme oxygenase-1: catalytic implications. J. Mol. Biol. 330,
527–538.

45. Sugishima, M., Sakamoto, H., Noguchi, M. & Fukuyama, K.
(2004) CO-trapping site in heme o xygenase revealed by photolysis
of its CO-bo und heme complex: mechanism of escaping from
product inhibition. J. Mol. Biol. 341, 7 –13.
46. Wang, M., Roberts, D.L., Paschke, R., Shea, T.M., Masters,
B.S. & Kim, J.J. ( 1997) Three-dimensional structure of
NADPH-cytochrome P450 reductase: prototype for FMN- and
FAD-containing enzymes. Proc.NatlAcad.Sci.USA94, 8411–
8416.
47. Wang, J . & Ortiz de Mon tellano, P.R. (2003) The b inding sites on
human heme oxygenase-1 for cytochrome P450 reductase and
biliverdin reductase. J . Biol. Chem. 278, 2 0069–20076.
48. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both
detailed a nd schematic plots of protein structures. J. Appl. Crys-
tallogr. 24 , 946–950.
49. Merritt, E.A. & Bacon, D.J. (1997) Raster3D: Photorealistic
Molecular Graphics. Methods Enzymol. 277, 505–524.
50. Humphrey, W., Dalke, A. & Schulten, K. (1996) VMD – Visual
Molecular Dynamics. J. Mol. Graph. 14, 33–38.
51. Lawrence, M.C. & Bourke, P. ( 2000) CONSCRIPT: a program
for g enerat ing electron density isosurfaces for presentation in
protein crystallography. J. Ap pl. Crystallogr. 33 , 990–991.
Ó FEBS 2004 Structure of cyanobacterium heme–HO complex (Eur. J. Biochem. 271) 4525