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: fukuyama@bio.sci.osaka-u.ac.jp 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. 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PCC 6803 in complex with heme Masakazu Sugishima 1 , Catharina T. Migita 2 ,. 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