Although the heme pocket of heme–Syn HO-1 is, for the most part, similar to that of mammalian HO-1, they differ in such features as the flexibility of the distal helix and hydrophobicity..
Trang 1Crystal structure of heme oxygenase-1 from cyanobacterium
Masakazu Sugishima1, Catharina T Migita2, Xuhong Zhang3, Tadashi Yoshida3and Keiichi Fukuyama1
1
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan;2Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yoshida, Yamaguchi, Japan;3Department 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 as
phyto-chromobilin and phycobilins, which are involved in the
sig-nal transduction for light responses and light harvesting for
photosynthesis, respectively We have determined the first
crystal structure of a HO-1 from a photosynthetic organism,
Synechocystissp PCC 6803 (Syn HO-1), in complex with
heme at 2.5 A˚ resolution Heme–Syn HO-1 shares a
com-mon folding with other heme–HOs Although the heme
pocket of heme–Syn HO-1 is, for the most part, similar to
that of mammalian HO-1, they differ in such 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 CO
trapping site in rat HO-1 that suppresses product inhibition
Although Syn HO-1 and mammalian HO-1 are similar in overall structure 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 ferredoxin 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 heme–Syn HO-1 and ferre-doxin suggests indirect electron transfer from an iron–sulfur cluster in 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 for 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 O2and reducing
equivalents from NADPH-cytochrome P450 reductase (CPR) [5–7] In the first step, O2bound to the heme iron
is activated to form ferric-hydroperoxide, and electrophilic addition of the terminal oxygen to the a-meso carbon produces a-hydroxyheme In the second step, a-hydroxy-heme is converted to verdoa-hydroxy-heme with concomitant release
of the a-meso carbon as CO Lastly, the oxygen bridge of verdoheme is cleaved to produce a biliverdin IXa–iron complex and ferrous iron is released prior to the dissociation
of biliverdin IXa The crystal structures of the human, rat, negative pathogen Neisseria meningitidis, and Gram-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 of HO from a photosynthetic 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: fukuyama@bio.sci.osaka-u.ac.jp
Abbreviations: CPR, NADPH-cytochrome P450 reductase; Fd,
fer-redoxin; HO, heme oxygenase; heme–HO, HO 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 the
Protein Data Bank, accession code 1WE1.
(Received 11 August 2004, revised 30 September 2004,
accepted 4 October 2004)
Trang 2light-harvesting for 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, and the
cyanobacteria, Synechocystis sp PCC 6701 and PCC 6803
[18–22] Two genes (ho1, ORF sll1184, and 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 PCC 6803 [26] Recently, we
constructed a system to efficiently express Syn HO-1 in
Escherichia coli and have characterized the spectroscopic
features of heme–Syn HO-1 [27] Purified Syn HO-1 can
catalyze heme degradation using ascorbate or 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 that of mammalian HO-1 in
the flexibility of the distal helix and hydrophobicity
Experimental procedures
Crystallization of the heme–Syn HO-1 complex
The expression and purification of Syn HO-1 and the
preparation of its complex with heme have been 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 mixed 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) PEG 4000, 2% (w/v) 1,5-diaminopentane
dihydro-chloride, and 0.1M sodium citrate (pH 5.9) The protein
concentration for crystallization was 20 mgÆmL)1in 0.1M
potassium phosphate buffer (pH 7.4) containing 5 mM
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 solution was not bound
to the heme iron in these crystals
Data collection and processing
Heme–Syn HO-1 crystals were soaked in crystallization
solution containing 10% (v/v) glycerol as a cryo-protectant
and were flash-cooled with a nitrogen gas stream at 100 K Diffraction data were collected at 100 K using synchrotron radiation (k¼ 1.500 A˚) from the BL41XU beamline at SPring-8 and a marCCD detector (MarUSA, Evanston, IL) The distance between the crystal and the 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 withMOSFLM[28] andSCALA
in the CCP4 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 programMOLREP [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 translation 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 the 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 the model with the programXFIT [33] The heme and the additionally ordered C-terminal
Fig 1 Photograph of heme–Syn HO-1 crystals.
Trang 3helix 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, and 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 programCNS[34] The stereochemical check
of the model was made with the programPROCHECK[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 The 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 HO-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 outermost shell (2.53–2.40 A˚) R free is the R-value calculated
for 10% of the dataset not included in the refinement.
Crystallographic data
Unit cell dimensions (A˚, ) a ¼ 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
Completeness (%) 94.7 (83.2)
R sym
a
0.064 (0.302) Refinement statistics
Resolution range (A˚) 20.0–2.5
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
Ramachandran plot
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. bR ¼
S|F obs (hkl) ) F calc (hkl)|/S|F obs (hkl)|.
Fig 2 Crystal structure of heme–Syn HO-1 (A) Ribbon diagram of heme–Syn HO-1 Heme, its ligands, and hetero compounds included in the model are superimposed 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 spheres indicate chloride ions A phosphate ion and one of the chloride ions are bound by two molecules in the crystal (B) Backbone stabilization in Syn HO-1 The distal helix is shown in orange for clarity Residues involving distal helix conformation stabilization are shown as ball-and-stick models This figure was prepared using the programs [48], 3 [49], and [50].
Trang 4distances 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 refined using
2.5 A˚ resolution data 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
observed in other heme–HOs [rmsd of Cas 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; these 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 or take on another
orientation in solution
Conformation of the distal helix
As in the previously reported structures of other 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 of the heme [8–11] Three types of
conformation of the F-helix were reported: open,
closed, and more closed, depending on the kinking
angles of the F-helices and the hydrogen bonds
stabil-izing these conformations (Table 2, Fig 3) The open
and closed 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 Closed conformations were also reported
in HemO and HmuO [8,11] A more closed
conforma-tion was reported in rat heme–HO-1 [9] In contrast to the two conformations described above, in the more closed conformation, 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 open and closed conformations, whereas in the
more closed conformation, the segment takes on a p-helical conformation This structural feature suggests that the open and closed conformations are more stable than the more closed 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 for the formation of the more closed conformation Indeed, the distal helix conformation is bidirectionally converted during the reaction process of rat HO-1 [38] and O2 binding to the heme iron of HmuO [39] Following this definition of helix
conforma-Table 2 Selected distances (A˚) between atoms at the distal heme pocket Residue numbers 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
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 bonds Residue numbers of Syn HO-1 and mammalian HO-1 (in parentheses) are shown Hydrogen bonding patterns in the open and closed conformations and in the more closed conformation are characterized by a-helix and p-helix, respectively.
Trang 5tion, all F-helices in heme–Syn HO-1s in the crystal are
in the closed conformation The fact that all heme–Syn
HO-1s in the asymmetric 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 interactions 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 in HmuO and Syn HO-1
In the structures of mammalian HO-1 and HmuO, a hydrophobic aromatic cluster is located near the 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 2B) 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 of 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) and F o –F c map omitted
the distal ligand of heme (red; contoured at
5.0 r) The electron density map is
superim-posed on the ball-and-stick model of heme–
Syn HO-1 around the heme pocket (B) The
detailed structure around the heme is shown
by ball-and-stick models (C) 2-propanol
binding in the hydrophobic cavity r A
-weigh-ted 2F o –F c map (blue; contoured 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 was prepared using
the programs MOLSCRIPT [48], RASTER 3 D [49],
VMD [50], and CONSCRIPT [51].
Trang 6Fig 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 CPR Positive and negative surfaces are shown in blue (+12.0 kTÆe)1for Fd and +5.0 kTÆe)1for other proteins) and red ( )12.0 kTÆe )1 for Fd and )5.0 kTÆe )1 for other proteins), respectively Electrostatic potential calculations included only fully charged residues (Asp, Glu, Arg, and Lys) using dielectric constants of 80 for the exterior of the protein and 2 for the interior of the protein The redox center of each molecule is shown as a wire-frame 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 PCC 6803 A CPK model of the redox centers is superimposed on the ribbon model of the putative docking model (Syn HO-1, gray;
Fd I, yellow) The distance between the heme iron and iron–sulfur cluster is also shown This figure was prepared using the program VMD [50].
Trang 7Heme pocket structure
An electron density map and a model around the heme
pocket of heme–Syn HO-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 stabilized by the
electrostatic interactions 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 conserved in human
and rat HO-1s, and HmuO, but not in HemO Asp140 in
mammalian HO-1 (Asp131 in Syn HO-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 O2coordinated to 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 Syn HO-1,
similar to mammalian HO-1 The cavity volume in Syn
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 four molecules in the
asymmetric unit (Fig 4C) Such density was not found in
rat heme–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 the 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 that 2-propanol present in the
crystallization solution is bound to heme–Syn HO-1 in this
cavity This raises the 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 in 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 binding sides However, it should be noted that the positively charged surface of Syn HO-1 is narrower than 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 and 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 Kawamoto and Hisanobu Sakai of JASRI for their aid with data collection using the synchrotron radiation at
SPring-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, and Technology of Japan.
References
1 Tenhunen, R., Marver, H.S & Schmid, R (1968) The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase Proc Natl Acad Sci USA 61, 748–755.
Trang 82 Stocker, R., Yamamoto, Y., McDonagh, A.F., Glazer, A.N &
Ames, B.N (1987) Bilirubin is an antioxidant of possible
physio-logical importance Science 235, 1043–1046.
3 Ryter, S.W., Otterbein, L.E., Morse, D & Choi, A.M (2002)
Heme oxygenase/carbon monoxide signaling pathways:
regula-tion and funcregula-tional significance 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 Chem Rev 96, 2841–2888.
6 Ortiz de Montellano, P.R (2000) The mechanism of heme
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., Ortiz de Montellano, P.R & Poulos,
T.L (1999) Crystal 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 pathogen Neisseria meningitidis and a comparison with
mammalian heme oxygenase-1 Biochemistry 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 of the heme complex of Hmu O, a
heme oxygenase of Corynebacterium diphtheriae J Biol Chem.
279, 11937–11947.
12 Terry, M.J., McDowell, M.T & Lagarias, J.C (1995) (3Z)- 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 Natl Acad Sci USA 98, 10560–10565.
14 Schafer, E & Bowle, C (2002) Phytochrome-mediated
photo-perception and signal transduction 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 Sci USA 94, 11792–11797.
16 Wilde, A., Fiedler, B & Borner, T (2002) The cyanobacterial
phytochrome Cph2 inhibits phototaxis 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 red alga, Cyanidium
caldarium Arch Biochem Biophys 235, 371–384.
19 Cornejo, J & Beale, S.I (1988) Algal heme oxygenase from
Cyanidium caldarium: partial purification and fractionation into
three required protein components 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 caldarium J Biol.
Chem 267, 16088–16093.
21 Rhie, G & Beale, S.I (1995) Phycobilin biosynthesis: reductant
requirements and product identification for heme oxygenase 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., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasa-moto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M & Tabata, S (1996) Sequence 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 (1998) Phytobilin bio-synthesis: cloning and expression of a gene encoding soluble fer-redoxin-dependent heme oxygenase from Synechocystis sp PCC
6803 Plant J 15, 99–107.
25 Davis, S.J., Kurepa, J & Vierstra, R.D (1999) The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases Proc Natl Acad Sci USA 96, 6541–6546.
26 Willows, R.D., Mayer, S.M., Foulk, M.S., DeLong, A., Hanson, K., Chory, J & Beale, S.I (2000) Phytobilin biosynthesis: the Synechocystis sp PCC 6803 heme oxygenase-encoding ho1 gene complements a phytochrome-deficient 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 synthesis Properties of the heme complex of recombinant active enzyme Eur J Biochem 270, 687– 698.
28 Leslie, A.G.W (1992) Recent changes to the MOSFLM package for processing film and image plate data Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, no 26 Daresbury Laboratories, Warrington, UK.
29 Kabsch, W (1988) Evaluation of single-crystal X-ray diffraction data from a position-sensitive detector J Appl Crystallogr 21, 916–924.
30 Collaborative Computational Project, Number, 4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystal-logr D Biol CrystalCrystal-logr 50, 760–763.
31 Vagin, A & Teplyakov, A (1997) MOLREP: an automated program for molecular replacement J Appl Crystallogr 30, 1022–1025.
32 Lee, C & Irizarry, K (2001) The GeneMine System for genome/ proteome annotation and collaborative data mining IBM Sys-tems Journal 40, 592–603.
33 McRee, D.E (1999) XtalView/Xfit – A versatile program for manipulating atomic coordinates and electron 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., Simonson, T & Warren, G.L (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination 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 stereochemical quality of protein structures J Appl Crystallogr 26, 283–291.
36 van den Heuvel, R.H., Svergun, D.I., Petoukhov, M.V., Coda, A., Curti, B., Ravasio, S., Vanoni, M.A & Mattevi, A (2003) The active conformation of glutamate synthase and its binding to ferredoxin J Mol Biol 330, 113–128.
37 Ritchie, D.W (2003) Evaluation of protein docking predictions using Hex 3.1 in CAPRI rounds 1 and 2 Proteins 52, 98–106.
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
Trang 9the 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: Implications for heme oxygenase
function J Biol Chem 279, 21055–21061.
40 Lightning, L.K., Huang, H., Moenne-Loccoz, P., Loehr, T.M.,
Schuller, D.J., Poulos, T.L & de Montellano, P.R (2001)
Dis-ruption of an active site hydrogen bond converts 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 highly conserved carboxylate, aspartate-140, in
oxygen activation and heme degradation by heme oxygenase-1.
J Am Chem Soc 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 for regiospecific hydroxylation of heme at the
a-meso carbon J Biol Chem 277, 45086–45090.
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 discrimination between CO
and O 2 in HO-1 Biochemistry 42, 9898–9905.
44 Lad, L., Wang, J., Li, H., Friedman, J., Bhaskar, B., Ortiz de
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 oxygenase revealed by photolysis
of its CO-bound 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 Natl Acad Sci USA 94, 8411– 8416.
47 Wang, J & Ortiz de Montellano, P.R (2003) The binding sites on human heme oxygenase-1 for cytochrome P450 reductase and biliverdin reductase J Biol Chem 278, 20069–20076.
48 Kraulis, P.J (1991) MOLSCRIPT: a program to produce both detailed and 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 generating electron density isosurfaces for presentation in protein crystallography J Appl Crystallogr 33, 990–991.