Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp PCC 6803 is a true heme oxygenase Properties of the heme and enzyme complex Xuhong Zhang1, Catharina T Migita2, Michihiko Sato3, Masanao Sasahara1 and Tadashi Yoshida1 Department of Biochemistry, Yamagata University School of Medicine, Japan Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan Central Laboratory for Research and Education, Yamagata University School of Medicine, Japan Keywords biliverdin; cyanobacterium heme oxygenase; EPR; ferredoxin; heme oxygenase Correspondence T Yoshida, Department of Biochemistry, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan Fax: +81 23 6285225 Tel: +81 23 6285222 E-mail: tyoshida@med.id.yamagata-u.ac.jp (Received 25 August 2004, revised 10 December 2004, accepted 17 December 2004) doi:10.1111/j.1742-4658.2004.04535.x Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp PCC 6803 Isoform-1, Syn HO-1, has been characterized, while isoform-2, Syn HO-2, has not In this study, a full-length ho2 gene was cloned using synthetic DNA and Syn HO-2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein Like Syn HO-1, the purified Syn HO-2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXa, CO and iron in the presence of reducing systems such as NADPH ⁄ ferredoxin reductase ⁄ ferredoxin and sodium ascorbate The activity of Syn HO-2 was found to be comparable to that of Syn HO-1 by measuring the amount of bilirubin formed In the reaction with hydrogen peroxide, Syn HO-2 converted heme to verdoheme This shows that during the conversion of hemin to a-meso-hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions The absorption spectrum of the hemin–Syn HO-2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin-Syn HO-1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa-coordinate low spin species Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components These observations suggest that the structure of the heme pocket of the Syn HO-2 is different from that of Syn HO-1 Heme oxygenase (HO) was first reported in mammalian systems as a microsomal enzyme [1], in which the hydrophobic 26 amino acid residues at the C-terminus anchor the protein to the membrane [2,3] HO catalyzes the regiospecific oxidative degradation of heme to biliverdin IXa, iron, and CO via a-meso-hydroxyhemin, verdoheme, and the iron(III)–biliverdin IXa complex at the expense of three molecules of oxygen and seven electrons (Scheme 1) [4,5] The electrons are supplied from NADPH through microsomal NADPHcytochrome P450 reductase (CPR) Two isozymes of HO, denoted as HO-1 and HO-2, have been identified in mammalian systems [6] In general, HO-1 is responsible for the excretion of aged ⁄ disused heme as well as recycling of iron [7,8] and HO-2 is associated with signal transduction through the production of CO, where Abbreviations CPR, cytochrome P450 reductase; HO, heme oxygenase; Fd, ferredoxin; FNR, ferredoxin reductase; KPB, potassium phosphate buffer; rHO-1, heme oxygenase-1 of Rattus norvegicus; Syn HO-1, heme oxygenase-1 of Synechocistis sp PCC 6803; Syn HO-2, heme oxygenase-2 of Synechocystis sp PCC 6803; EPR, electron paramagnetic resonance 1012 FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS X Zhang et al Scheme Heme degradation pathway Conversion of heme to biliverdin IXa catalyzed by HO CO stimulates the formation of cGMP as a possible physiological messenger akin to NO [9] Additionally, HO plays an important role in the defense against oxidative stress, as lipophilic bilirubin IXa, the reduced form of biliverdin IXa, works as a potent endogenous antioxidant similar to vitamin E [10,11] HO is also found in some pathogenic bacteria, where it is essential for the heme-based iron acquisition needed to survive and produce proteinaceous poisons [12–14] In contrast to mammalian HO, bacterial HO is soluble owing to the lack of a C-terminal hydrophobic region The reaction mechanisms of bacterial HOs are essentially similar to those of mammalian HOs Prokaryotic plant heme oxygenase activity was first found in a red alga, Cyanidium caldarium, and then in cyanobacteria, Synechocystis sp PCC 6701 and PCC 6803, which have now been studied for 20 years [15– 20] The HO of cyanobacteria and prokaryotic red algae is responsible for the biosynthesis of photoreceptive bilins such as phycocyanobilin and phycoerythrobilin, as these bilins are synthesized from biliverdin IXa, a product of the HO reaction [15] Phytochromobilin, one of the photo-sensing bilins required for the photomorphogenesis of higher plants, is also considered to come from biliverdin IXa [15,21–23] Like bacterial HOs, the plant HOs are soluble and supposed to need ferredoxin (Fd) as an electron donor In 1996, the entire genome sequence of Synechocystis sp PCC 6803 was published and two different HO genes, ho1 and ho2, were identified [24] Next, the molecular cloning of the HY1 gene of Arabidopsis was performed and the product of this gene expressed in Escherichia coli showed heme oxygenase activity [25] More recent success in decoding the genomes of plants such as tomato, soybean and pea also suggests the presence of HOs in these higher plants [26] Cornejo et al first FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS Cyanobacterium heme oxygenase-2 reported a bacterial expression and purification system for a protein (HO-1) encoded by the ho1 gene [27] and we also established an efficient E coli expression system to obtain highly purified protein (Syn HO-1) [28] This success has allowed for molecular-based studies on Syn HO-1 [28,29] Bacterial expression of the ho2 gene of Synechocystis sp PCC 6803 was also carried out by Cornejo et al and yielded a small amount of soluble fraction which did not show heme oxygenase activity [27] Nevertheless, the expected product of the ho2 gene, Syn HO-2, is highly homologous in amino acid sequence to Syn HO-1 (51%) and most of the residues critical for heme oxygenase activity in mammalian HO-1 are conserved [30–34] (Fig 1), so that Syn HO-2 is strongly suggested to be an active enzyme To obtain the active form of Syn HO-2 and clarify the enzymatic properties of this protein, we have constructed a bacterial expression system for the ho2 gene and successfully obtained purified Syn HO-2 protein Accordingly, it has been established that Syn HO-2 binds hemin stoichiometrically and converts it into biliverdin IXa, CO and iron in the presence of oxygen and electrons, demonstrating that Syn HO-2 is a true heme oxygenase This is the first report of the characterization of the cyanobacterial HO-2 protein and its heme complex Results and Discussion Expression and purification of Syn HO-2 Culturing the cells at two temperatures, first at 37 °C and then at 20 °C, we could avoid the accumulation of inclusion bodies of Syn HO-2 The harvested cells were pale green, indicative of the expression of a catalytically active Syn HO-2 We purified the Syn HO-2 from the soluble fraction by ammonium sulfate fractionation and subsequent column chromatography on Sephadex G-75, DE-52, and hydroxyapatite The final preparation after chromatography on a hydroxyapatite column was clear and colorless and gave a single band of 29 kDa with about 97% purity on SDS ⁄ PAGE (Fig 2), the size expected from the deduced Syn HO-2 amino acid sequence (28.5 kDa) About 10 mg of protein was obtained from L of culture Catalytic activity of Syn HO-2 First we measured the catalytic activities of Syn HO-2 and compared them with those of Syn HO-1 We used ferredoxin reductase (FNR) ⁄ Fd equivalent to seventenths of Syn HO-2 or sodium ascorbate as reducing reagents We added desferrioxamine and biliverdin 1013 Cyanobacterium heme oxygenase-2 X Zhang et al Fig Amino acid sequence of Syn HO-2 as compared with the sequences of Syn HO-1, rHO-1 and rHO-2 The shaded letters indicate residues with sequence identity reductase to the reaction mixture to facilitate the release of iron and biliverdin from the enzyme and to reduce the biliverdin to bilirubin, respectively Table indicates that Syn HO-2 is enzymatically active The specific activity for heme breakdown was comparable to that of Syn HO-1 in the presence of each of the reducing systems, although the ascorbate-supported activity of Syn HO-2 was stronger than that of Syn HO-1 The activity levels of both enzymes supported by NADPH ⁄ FNR ⁄ Fd were higher than those with ascorbate NADPH ⁄ CPR supported activities of both HOs were considerably low Our recent study on Syn HO-1 crystals indicated that the positively charged surface interacting with an electron donor was narrower than that of mammalian HO-1 [29] Then, the electrons from CPR might not be transferred efficiently to Syn HO-1 or Syn HO-2 Properties of the heme-Syn HO-2 complex Fig SDS ⁄ PAGE of the purified Syn HO-2 protein Lane 1, molecular mass marker; lane 2, 10 lg of purified protein 1014 All HOs studied to date bind heme stoichiometrically to form a substrate–enzyme complex Like other HOs, Syn HO-2 also binds hemin, with a dissociation constant of about 8.87 ± 2.1 lm to form a : stoichiometric complex (Fig 3, inset) The complex was stable and purified as described in Experimental procedures FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS X Zhang et al Cyanobacterium heme oxygenase-2 Table Activities of the purified Syn HO-2 The HO activity was determined from the initial rate of bilirubin formation with the NADPH ⁄ FNR ⁄ Fd, NADPH ⁄ CPR or sodium ascorbate system at 30 °C, pH 7.4 All the measurements were done in triplicate Bilirubin formation (nmolỈmg protein)1Ỉh)1) Reaction system Syn HO-2 Syn HO-1 NADPH ⁄ FNR ⁄ Fd NADPH ⁄ CPR Sodium ascorbate 622 ± 21 81 ± 424 ± 20 603 ± 27 69 ± 273 ± 23 Fig Absorption spectra of various forms of the Syn HO-2–heme complex The concentration of the complex was lm The spectra are the iron(III) (solid line), iron(II) (dotted line), iron(II)-CO (dashed line), and oxy (dotted-dashed line) complexes Inset, titration of Syn HO-2 (9 lm) with hemin as monitored by the increase in absorbance at 412 nm ()) The increase in absorbance because of the addition of hemin in the absence of Syn HO-2 is indicated (*) Absorption spectra of the complexes of heme and HOs resemble the spectrum of myoglobin with one exception, the HO of Drosophila melanogaster In the HO of fruit fly, the iron of heme was not involved in binding to the enzyme, resulting in a different spectrum from the other HOs [35] Figure exhibits optical absorption spectra of the iron(III), iron(II), oxy, and CO-bound forms of the heme-Syn HO-2 complex at pH 7.4 Interestingly, the spectrum of the iron(III) complex has two peaks at 576 and 540 nm besides the Soret band at 412 nm in the visible region, which resembles that of the complex of iron(III) heme and Syn HO-1 at alkaline pH [28], suggesting that the major species of the iron(III) heme iron of Syn HO-2 at neutral pH is a hexa-coordinate low spin species This is quite different from other known HOs The Soret peak of the iron(III) complex was slightly redshifted at pH 8.0 with a slight decrease in its absorbance and slightly blue-shifted at pH 6.0 with a slight increase in its absorbance However, the dependence FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS Fig EPR spectra of the heme–Syn HO-2 complexes (A) The iron(III) resting state complex in pH 7.4 solution (solid line) and in pH 8.0 solution (dotted line) EPR conditions were microwave frequency, 9.35 GHz, microwave power, mW, field modulation frequencies, 100 kHz, field modulation amplitude, 10 G, and sample temperature, 10 K (B) The 15NO bound iron(II) complex measured with microwave power, 0.2 mW, field modulation amplitude, G, at 25K, where other conditions are the same as those described in (A) on pH was not fully reversible because of denaturation of the protein By the pyridine hemochrome method, the extinction coefficient at 412 nm for the iron(III) heme–Syn HO-2 complex is calculated to be 110 mm)1Ỉcm)1 The electron paramagnetic resonance (EPR) spectrum of the iron(III) resting state of heme-Syn HO-2 in pH 7.4 solution reveals that the heme in Syn HO-2 is in an admixture of high-spin and low-spin states (Fig 4A) Though the high spin species exhibits an apparently axially symmetric type of spectrum with g^ ¼ and gII ¼ 2, small rhombicity is observed at the perpendicular component of the spectrum, which is not seen in the spectrum of the high-spin heme Syn 1015 Cyanobacterium heme oxygenase-2 X Zhang et al HO-1 complex This suggests that the in-plane anisotropy of the ligand field to the heme iron in Syn HO-2 is relatively large The seating of the heme in the heme pocket might be less homogeneous than in Syn HO-1 As the pH of the solution rises from 7.4 to 8.0, the signal intensity of the high-spin species decreases and alternatively that of the low-spin species increases without a change in the g-values The low-spin component of the spectrum shows that there are two kinds of low-spin species: the major species has g-values of 2.69, 2.20, and 1.79 and the minor species has more anisotropic g-values (Fig 4A, asterisks) The g-values of the major species (2.69, 2.20, 1.79) are very close to those (2.68, 2.20, 1.80) of the minor alkaline component of heme–Syn HO-1 as well as to the values (2.67, 2.21, 1.79) of the alkaline form of heme-rHO-1 [28] The g-values of the minor component of heme–Syn HO-2 seem to be similar to those of the major lowspin component (2.78, 2.14, 1.74) of heme–Syn HO-1 in alkaline solution [28] The low-spin species of heme– Syn HO-2 therefore probably is the same species as the alkaline forms of known heme–HO The existence of alkaline forms is evidence that a water molecule is possessed at the distal site of the heme iron and strongly interacts with the distal helix main chain or its dissociable residues The close similarity of the g-values suggests that the heme pocket milieu of heme-Syn HO-2 in alkaline solution more resembles that of rHO-1 than that of Syn HO-1 The 15NO-bound form of heme-Syn HO-2 shows a typical six-coordinated nitrosyl heme spectrum (Fig 4B) The hyperfine splitting pattern at the g2 component indicates that nitrogen nuclei both of 15NO and of 14N-proximal ligand cause the splitting The heme proximal ligand is considered to be His16, which corresponds to His17 of the proximal ligand of hemeSyn HO-1 EPR parameters of 15NO-heme–Syn HO-2 were compared with those of the Syn HO-1 and rHO1 complexes (Table 2) Hyperfine coupling constants, A(15N-O) and A(14N-His), of the Syn HO-2 complex are closer to those of the rHO-1 complex than Syn HO-1 complex, indicating that the axial ligand coordination structure of heme-Syn HO-2 rather resembles Table EPR parameters of the iron(II) 15NO bound heme complexes of Syn HO-2, Syn HO-1, and rat HO-1 Data from Syn HO-1 and rat HO-1 are taken from reference [28] Protein g3 g2 g1 A([15N]NO) ⁄ G A([14N]His) ⁄ G Syn HO-2 Syn HO-1 rat HO-1 2.082 2.079 2.086 2.006 2.003 2.008 1.965 1.962 1.986 27.2 31.1 26.0 7.4 7.1 7.4 1016 that of heme-rHO-1 In conclusion, EPR reveals that the heme-Syn HO-2 complex is in dynamic equilibrium between high- and low-spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components Further, part of the heme pocket structure of Syn HO-2 more resembles that of rHO-1 than that of Syn HO-1 Degradation of hemin bound to Syn HO-2 to biliverdin by the NADPH/FNR/Fd or sodium ascorbate systems To reduce FNR-mediated heme degradation leading to nonbiliverdin products, we used FNR ⁄ Fd equivalent to one-twenty fourth of Syn HO-2 in this study With the addition of NADPH to the reaction mixture, the absorption showing a peak at 412 nm decreased gradually until finally, broad absorption bands centered near 380 and 690 nm appeared, indicative of the conversion of hemin to biliverdin (Fig 5A) The formation of biliverdin is supported by the decrease in absorbance around 690 nm and concomitant increase in absorbance around 450 nm after the addition of biliverdin reductase, reflecting the conversion of biliverdin to bilirubin Similar spectrophotometric changes were observed in Fig 5B, where sodium ascorbate was used as a reductant The decrease in absorbance at 412 nm supported by FNR ⁄ Fd was slower than that with ascorbate This is because a relatively small amount of FNR ⁄ Fd compared to Syn HO-2 was added to the reaction system Again, the absorption due to biliverdin was converted to that of bilirubin by the addition of biliverdin reductase and NADPH A previous study on rHO-1 indicated that when ascorbate was used as a reductant, the final heme degradation product was not biliverdin but iron(III) biliverdin bound to the enzyme [36] Then, we conducted similar experiments without desferrioxamine The rates of heme degradation did not differ in the presence or absence of desferrioxamine However, the intensities of the absorbance around 690 nm in the NADPH ⁄ FNR ⁄ Fd system (Fig 5C) and ascorbate system (Fig 5D) were about two-thirds and one-half of those observed in the presence of desferrioxamine, respectively These results suggest that the final product of the Syn HO-2 reaction supported by both reducing systems is biliverdin and that the release of iron(III)biliverdin from Syn HO-2 is slow In independent experiments, we analyzed the stereoselectivity of the products of the Syn HO-2 reaction supported by the NADPH ⁄ FNR ⁄ Fd system and ascorbate system using HPLC In both cases, only FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS X Zhang et al Cyanobacterium heme oxygenase-2 Fig Reaction of hemin bound to Syn HO-2 with the NADPH ⁄ FNR ⁄ Fd system or sodium ascorbate in the presence or absence of desferrioxamine (DFO) Spectrum of the hemin–Syn HO-2 complex (solid line); 10 after the start of the reaction depicted between 350 and 450 nm (dotted line); 50 after the start of the reaction (dashed line); after the addition of biliverdin reductase (dotted-dashed line) In the ascorbate system (B and D), NADPH was added together with biliverdin reductase Inset, five-fold enlarged spectra between 500 and 750 nm the a-isomer of biliverdin was detected (data not shown), indicating a-specificity of the Syn HO-2 reaction Reaction of hemin bound to Syn HO-2 with hydrogen peroxide In the case of mammalian HO, an iron(III) hydroperoxide species was speculated to be an intermediate in the first oxygenation step, as hydrogen peroxide converted hemin to iron(III) verdoheme via a-mesohydroxyhemin (Scheme 1) [37] This was confirmed experimentally [38–40] Then, we reacted the hemin– Syn HO-2 complex with hydrogen peroxide and found that a iron(III) verdoheme–Syn HO-2 complex was formed (data not shown) This result shows that a iron(III) hydroperoxide species must be an active intermediate in the first oxygenation step from hemin to FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS a-meso-hydroxyhemin in the Syn HO-2 reaction, like for other HOs studied Verdoheme formation during the course of heme degradation With rHO-1, hemin bound to the enzyme was converted into iron(II)-CO form of verdoheme under O2 and CO, and the reaction was arrested at this stage because CO inhibits the further conversion of verdoheme to iron(III)-biliverdin (Scheme 1) [41] To detect the iron(II)–CO form of the verdoheme–Syn HO-2 complex, we carried out similar experiments in an atmosphere of approximately 5% CO, 5% O2 and 90% N2 (v ⁄ v ⁄ v) The spectrum (Fig 6, dotted line) recorded after the start of the reaction has four peaks at 415, 540, 570, and 637 nm in the visible region The first peak is attributable to the iron(III)–Syn HO-2 1017 Cyanobacterium heme oxygenase-2 Fig Reaction of hemin bound to Syn HO-2 with the NADPH ⁄ FNR ⁄ Fd system under O2 and CO Spectrum of the hemin–Syn HO-2 complex with FNR ⁄ Fd (solid line); after the addition of NADPH to start the reaction (dotted line); after the start of the reaction (dashed line) Inset, fivefold enlarged spectra between 500 and 750 nm complex and the peaks at 540 and 570 nm are due to an admixture of the iron(III) and the iron(II)-CO complexes The peak at 637 nm is attributable to the CO-bound verdoheme–Syn HO-2 complex The absorption at 637 nm increased with time and reached a maximum after the start of the reaction The broken line, a spectrum recorded a further later, has a decreased absorbance at 637 nm and increased absorbance around 690 nm, indicative of the conversion of verdoheme to biliverdin The spectral changes depicted in Fig together with the result described in the preceding section indicate that verdoheme is an intermediate of the Syn HO-2 reaction, like other HO reactions Detection of CO during the Syn HO-2 reaction Difference absorption spectroscopy in the presence of mutated myoglobin, H64L, which has a high affinity for CO, was used to detect CO formed during the NADPH ⁄ FNR ⁄ Fd-supported reaction The Soret band of myoglobin was monitored at 1-min intervals after the addition of NADPH to both the sample and the reference cuvette As depicted in Fig 7, the myoglobin Soret band shifted from 393 to 425 nm with the appearance of a ⁄ b bands at 568 and 538 nm and absorbance at 425 nm increased These results indicate that the iron(III) form of myoglobin was reduced to the iron(II) form by the NADPH ⁄ FNR ⁄ Fd system and this was followed by CO binding to yield the iron(II)-CO form, the authentic absorption spectrum of which is depicted in the inset of Fig This experiment clearly demonstrates the formation of CO during 1018 X Zhang et al Fig Detection of CO produced during the Syn HO-2 reaction The sample solution contained the hemin–Syn HO-2 complex, FNR ⁄ Fd and the H64L mutant of myoglobin Myoglobin was omitted from the reference solution The reaction was started by the addition of NADPH to both solutions and the difference spectrum was recorded Difference spectrum before the start of the reaction (dashed line); 0.5 (dotted-dashed line); 1.5 (dotted line); 4.5 (solid line) after the start of the reaction Inset: Absorption spectra of various forms of the H64L mutant of myoglobin CO-iron(II) form (solid line); iron(II) form (dotted line); iron(III) form (dashed line) the degradation of heme by Syn HO-2 in line with the mechanism shown in Scheme Conclusive remarks Although a previous effort to express the ho2 gene in E coli cells by Cornejo et al resulted in mostly insoluble protein [27], we fortunately obtained soluble Syn HO-2 protein on a large scale As the conditions of cell culture seemed to be similar in both cases, the difference in the plasmid used might be the cause of the discrepancy In spite that an RNA blot analysis of cyanobacteria grown in light suggested that ho2 is silent [27], the Syn HO-2 obtained in this study has shown heme oxygenase activity Similar to mammalian HO-1 and HO-2 as well as bacterial HOs (Hmu O, Hem O, Pig A) and Syn HO-1, Syn HO-2 binds a stoichiometric amount of heme to form a stable heme–HO complex Optical absorption and EPR spectroscopy reveal that heme–Syn HO-2 is mostly in the iron(III) low-spin state, which is a unique feature of this complex In spite of the lowspin resting state, the bound heme is converted to biliverdin IXa, CO and free iron in the presence of reducing equivalents such as ascorbate or NADPH ⁄ FNR ⁄ Fd and oxygen The activity of Syn HO-2 for the catabolism of heme is comparable to that of Syn HO-1 as determined by the initial rate FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS X Zhang et al of bilirubin formation The a-selectivity of the product is strictly retained The second intermediate of the HO reaction, verdoheme, has also been detected in the Syn HO-2 reaction and hydrogen peroxide has been ascertained to substitute for O2 and electrons in the conversion of hemin to verdoheme, thereby implying that the chemistry of heme degradation by Syn HO-2 is similar to that by other HOs Accordingly, we conclude that Syn HO-2 is a true heme oxygenase even though the physiological importance of this isoform is unknown at this stage It is noticeable that the EPR spectrum of nitrosyl heme–Syn HO-2, in which the minute difference in the ligand field around the heme is saliently represented, is explicitly different from that of heme–Syn HO-1 As 85% of the amino acids composing of the distal-site helix in Syn HO-1 are conserved in Syn HO-2, some factor(s) other than the primary structure, may be responsible for the difference in the heme-pocket structure We are now in the process of analyzing the crystal structure of Syn HO-2 Experimental procedures Construction of Synechocystis heme oxygenase-2 expression vector, pMWSynHO2 A 50 base pair double-stranded synthetic oligonucleotide with unique sites for the restriction enzymes NdeI, Bsu36I, NheI, EcoRI, XhoI, and HindIII was ligated between NdeI and HindIII sites of a T7-promotor-based bacterial expression vector, pMW172, to make a plasmid tentatively referred to as pMW-A Ten oligonucleotides and their complements, 59–88 nucleotides in length, were synthesized to construct a 752 base pair synthetic gene coding for the entire Syn HO-2 from the ATG initiation codon to the TAA stop codon Each nucleotide was phosphorylated with T4 polynucleotide kinase, then annealed with its complement to make a double-stranded DNA, e.g Oligo I to Oligo X Oligo I was designed so that the 5¢ end could be ligated to the NdeI site, whereas its 3¢ cohesive end was complementary to the 5¢ end of Oligo II The 3¢ end of Oligo II could be ligated to the Bsu36I site Similarly, the 5¢ ends of Oligos III, V, VII and IX were designed to ligate to the Bsu36I, NheI, EcoRI, and XhoI sites, respectively, and their 3¢ ends had sequences for ligation to the 5¢ ends of Oligo IV, VI, VIII and X The 3¢ end of Oligo X had a sequence designed to ligate to the HindIII site To complete the Syn HO-2 expression vector pMWSynHO2, double-stranded Oligo I to Oligo X were ligated step by step into the restriction enzyme sites of pMW-A The nucleotide sequence of the thus constructed pMWSynHO2 was determined with an Applied Biosystems 373A DNA sequencer FEBS Journal 272 (2005) 1012–1022 ª 2005 FEBS Cyanobacterium heme oxygenase-2 Syn HO-2 expression and purification A mL inoculum in Luria–Bertani medium (+ 50 lgỈmL)1 ampicillin ⁄ 0.1% glucose) was prepared from a plate of transformed E coli BL21 (DE3) cells carrying pMWSynHO2 Five-hundred milliliter cultures were inoculated with 300 lL of the inocula and grown in Luria–Bertani medium (+ 200 lgỈmL)1 ampicillin) at 37 °C until the D600 reached 0.8–1.0 The cells were grown for an additional 24 h at 20 °C, harvested by centrifugation, and stored at )80 °C prior to use The typical yield of cells from a 500 mL culture was g The E coli cells (10 g), resuspended in 90 mL of Tris ⁄ HCl buffer (pH 7.4, +2 mm EDTA), were lysed (2 mg lysozyme per g cells) with stirring at °C for 30 After sonication (Branson 450 Sonifire) and centrifugation at 100 000 g for h, the resulting supernatant was covered with a 20–50% ammonium sulfate fraction and centrifuged The subsequent precipitates, containing the Syn HO-2 protein, were dissolved in 20 mm potassium phosphate buffer (KPB) (pH 7.4) and applied to a Sephadex G-75 column (3.6 · 50 cm), pre-equilibrated with the same buffer The protein fractions eluted in the KPB, with an intense 29 kDa band on SDS ⁄ PAGE, were collected and directly loaded onto a column of DE-52 (2.6 · 28 cm) The column was washed with 50 mL of 20 mm KPB (pH 7.4)-50 mm KCl, and the protein was eluted with 400 mL of 20 mm KPB (pH 7.4) using a linear gradient, 50–400 mm KCl Collected fractions with a high protein content were further run through a column of hydroxyapatite (2.6 · 20 cm) Again the column was washed with 50 mL of 20 mm KPB (pH 7.4), and the protein was eluted with 200 mL of 50 mm KPB (pH 7.4) Only fractions with a single band at 29 kDa on SDS ⁄ PAGE were gathered Heme binding study Heme binding of Syn HO-2 was tested by adding hemin to lm Syn HO-2 in mL of 50 mm KPB (pH 7.4) The reference cuvette contained mL of 50 mm KPB (pH 7.4) alone A solution of mm hemin was added in lL aliquots to both test and reference cuvettes with equilibration between additions at 25 °C The absorbance between 350 and 750 nm was measured on a Beckman DU7400 single-beam spectrophotometer Preparation of a complex of Syn HO-2 and hemin Syn HO-2 in 50 mm KPB (pH 7.4) was added to a 1.2 equivalent excess of hemin This solution was passed through a column of Sephadex G-25 equilibrated with 50 mm KPB (pH 7.4) Fractions colored brown were loaded onto a column of hydroxyapatite equilibrated with 50 mm KPB (pH 7.4) The passed fraction colored brown was collected and used as a complex of hemin and Syn HO-2 for spectrophotometric experiments 1019 Cyanobacterium heme oxygenase-2 Optical absorption spectroscopy Optical absorption spectra were recorded on a Beckmann, DU7400 spectrophotometer at 25 °C The iron(II) heme– Syn HO-2 complex was prepared in a sealed cuvette by the addition of dithionite to a 0.1 m KPB (pH 7.4) solution of hemin–Syn HO-2 saturated with argon The CO complex of heme–Syn HO-2 was prepared by displacing the argon in the space of a sealed cuvette containing the iron(II)–Syn HO-2 solution with CO The oxy complex was prepared by introducing air into the anaerobic sample of iron(II) heme–Syn HO-2 generated by the reduction of the iron(III) complex with a stoichiometric amount of sodium dithionite Assay of heme oxygenase activity by measuring bilirubin formation The catalytic activity of Syn HO-2 and Syn HO-1 was determined after the conversion of biliverdin IXa produced by the enzyme, to bilirubin by biliverdin reductase The reaction mixture of the NADPH ⁄ FNR ⁄ Fd system contained in a final volume of 1.5 mL; 50 mm KPB (pH 7.4), 0.5 mg of bovine serum albumin, 20 lm hemin, lm enzyme, 0.7 lm maize FNR ⁄ maize Fd III, 140 lm NADPH, mm desferrioxamine, and lm recombinant rat biliverdin reductase [42] NADPH was omitted in the control system The reaction was started by the addition of NADPH after 3-min preincubation at 30 °C, and monitored at 468 nm for 10 The value of 43.5 mm)1Ỉcm)1 was used as the extinction coefficient for bilirubin at 468 nm [43] Assay in the presence of NADPH ⁄ CPR was performed by the similar way except that 0.7 lm of rat liver CPR was used instead of 0.7 lm of FNR ⁄ Fd The ascorbate system contained in a final volume of 1.5 mL; 50 mm KPB (pH 7.4), 0.5 mg of bovine serum albumin, 20 lm hemin, lm enzyme, 13.3 mm sodium ascorbate, 70 lm NADPH, mm desferrioxamine, and lm biliverdin reductase Ascorbate was omitted from the control system The reaction was initiated by the addition of ascorbate Other conditions were the same as those for the NADPH ⁄ FNR ⁄ Fd system Reaction of hemin bound to Syn HO-2 with NADPH/FNR/Fd, sodium ascorbate, and hydrogen peroxide systems in the presence or absence of desferrioxamine Spectral changes were recorded at 30 °C between 350 and 750 nm We used three electron donor systems, NADPH ⁄ FNR ⁄ Fd, ascorbate, and H2O2 The standard reaction mixture for the NADPH ⁄ FNR ⁄ Fd system consisted of lm Syn HO-2–hemin complex and 0.33 lm FNR ⁄ Fd in a final volume of 1.5 mL of 50 mm KPB 1020 X Zhang et al (pH 7.4) After preincubation, the reaction was started by the addition of 15 lL of 10 mm NADPH (final concentration, 0.1 mm) The reaction mixture for the ascorbate system contained lm Syn HO-2–hemin complex in a final volume of 1.5 mL of 50 mm KPB (pH 7.4) After preincubation, the reaction was initiated by the addition of 15 lL of m sodium ascorbate (final concentration, 10 mm) When desferrioxamine was added, a final concentration of mm was used The H2O2 system consisted of lm Syn HO-2–hemin complex in a final volume of 1.5 mL of 50 mm KPB (pH 7.4) After preincubation, the reaction was started by the addition of 15 lL of mm H2O2 in water (final concentration, 10 lm) The concentration of H2O2 in the original aqueous reagent solution was determined spectroscopically using a value of 43.6 m)1Ỉcm)1 for the extinction coefficient at 240 nm [44] EPR spectroscopy EPR measurements were performed with a Bruker E500 spectrometer, operating at 9.35–9.55 GHz, with an Oxford ESR 900 liquid helium cryostat The 15NO-bound form of the heme–Syn HO-2 complex was prepared by adding dithionite to the argon-saturated hemin–protein solution, containing Na15NO2 in an EPR tube Detection of carbon monoxide To detect CO produced during the reaction supported by a system of NADPH ⁄ FNR ⁄ Fd, a myoglobin mutant, H64L, which has higher affinity for CO than the wild type [45], was included in the reaction mixture The reaction solutions contained lm hemin–Syn HO-2 complex, and 0.67 lm FNR ⁄ Fd in 1.5 mL of 50 mm KPB (pH 7.4) H64L, at a final concentration of 6.5 lm, was included in the test solution After the addition of NADPH (final concentration, 0.1 mm) to both cuvettes, the spectrum was recorded 30 s after the start of the reaction and then recorded at 1-min intervals between 350 and 750 nm Other procedures Sequence translation and sequence alignment were performed using the Wisconsin Package from the Genetic Computer Group (Madison, WI, USA) and clustalw multiple sequence alignment program at the EBI (EMBL-EBI) HPLC analysis of reaction products was done as previously described [35] Purified Syn HO-1 was obtained by a 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This is the first report of the characterization of the cyanobacterial HO-2 protein and its heme. .. chromatography on Sephadex G-75, DE-52, and hydroxyapatite The final preparation after chromatography on a hydroxyapatite column was clear and colorless and gave a single band of 29 kDa with about