Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis Properties of the heme complex of recombinant active enzyme Catharina T. Migita 1 , Xuhong Zhang 2 and Tadashi Yoshida 2 1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan; 2 Department of Biochemistry, Yamagata University School of Medicine, Japan An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-bili- verdin IX a in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH- cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high- spin component in the ferric form spectrum. The heme- enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, Hmu O. Keywords: cyanobacterium heme oxygenase isoform-1; EPR; heme complex; protein expression; spectroscopy. Photoreceptor chromophores in the plant kingdom are categorized into two groups of chlorophyll and phycobilin. Chlorophyll, which is contained in all plants including cyanobacteria and protoflorideophyceae, is synthesized from protoporphyrin IX, a precursor of heme. Phycobilins of open-chain tetrapyrroles are produced from biliverdin that is a product of heme degradation. Accordingly, the chlorophyll and phycobilin syntheses share the pathway of protoporohyrin synthesis from d-aminolevulinic acid [1,2]. Phycobilins work as the main photoreceptor of photosyn- thesis in procaryophyta, cyanobacteria and other primitive eucaryotic algae. Phycobilin synthesis branches from chlo- rophyll synthesis at the iron insertion to protoporphyrin IX to form heme that is catalyzed by ferrochelatase. Then, heme is converted to biliverdin IX a by an enzyme named heme oxygenase (HO). Biliverdin IX a is further reduced and isomerized to produce phycobilins such as phycoerythro- bilin and phycocyanobilin [3–5]. The enzymes catalyzing these reactions, phycobilin synthase(s), have not been identified yet. In higher plants, phytochromobilins are also supposed to be synthesized from biliverdin IX a [6]. The phytochromobilins are precursors of the chromophore of phytochromes, which are photo-reversible light signal- transducing biliproteins and have closely related structure with phycobilins [7]. HO was first established in mammalian systems as a membrane-bound microsomal enzyme that catalyzed the regiospecific oxidative degradation of heme [8]. The enzymatic reaction requires three molecules of oxygen and six electrons to convert ferric heme to the ferric- biliverdin complex and CO [9–13]. NADPH coupled with cytochrome P450 reductase supplies the electrons in mammalian systems. The mammalian HOs (inducible isoform-1 and conserved isoform-2 are known) and their heme complexes have been characterized relatively well Correspondences to C. T. Migita, the Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515, Japan. Fax/Tel: + 81 83 933 5863, E-mail: ctmigita@po.cc.yamaguchi-u.ac.jp Abbreviations: hemin, ferric protoporphyrin IX; heme, iron proto- porphyrin IX either ferrous or ferric form; hydroxyheme, iron meso-hydroxyl protoporphyrin IX; HO, heme oxygenase; Syn HO-1, Synechocystis heme oxygenase isoform 1. Enzymes: heme oxygenase (EC 1.14.99.3); NADPH cytochrome P450 reductase (EC 1.6.2.4). (Received 17 September 2002, revised 9 December 2002, accepted 10 December 2002) Eur. J. Biochem. 270, 687–698 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03421.x using recombinant proteins [14–16]. The crystal structures of the human HO-1 [17] and rat HO-1 [18] have been published. The reaction mechanism of heme degradation, especially that of the first oxygenation step from heme to a-hydroxyheme, has been clarified by recent works [19,20]. Recently, heme oxygenase has also been found in some pathogenic bacteria [21–28]. They are water-soluble pro- teins and mainly function to release iron from heme of the host cells, which is necessary for the survival of bacteria and for causing diseases. The crystal structure of the bacterial HO (Hem O) has been reported [27]. The reaction mechanism is supposed to be analogous to those of mammalian HOs [22,23,25]. Algal and cyanobacterial HOs have been studied as a cell extract for the last 20 years [29–33]. The proteins possessing heme oxygenase activity have been obtained from red alga, Cyanidium caldarium, and cyanobacterium, Synechocystis sp. PCC 6701 and PCC 6803 [33], to date. The algal HO is a soluble protein, localized in the plastids. The in vitro heme oxygenase activity is supposed to need reduced ferredoxin and the second reductant such as ascorbate. In 1993, the HO gene of rhodophyta Porphyra purpurea (pbsA)was isolated [34] and in 1996, the entire genome sequence of Synechocystis sp. PCC 6803 was determined, identifying two different HO genes (ho1 and ho2) [35]. Trials of cloning and expression of these genes in Escherichia coli yielded single active protein, HO-1, from the ho1 gene [36]. Another cyanobacterial HO gene was identified in the complete genomic sequence of Nostoc (Anabaena) sp. PCC 7120, very recently [37]. On the other hand, recent research has reported that, in higher plants, the Arabidopsis thaliana hy1 gene encords a protein related to HO [38–40]. Thus, HO seems to present ubiquitously in the plant kingdom, as a key enzyme for the synthesis of photon-accepting chromo- phores. However, knowledge of the characteristics of plant HO is limited because large amounts of purified protein have not been available so far. In this study, we have constructed an efficient bacterial expression system of the HO-1 protein, based on the ho1 gene sequence of cyanobacteria, Synechocystis sp. PCC 6803 [35] and have succeeded in obtaining highly purified soluble protein, Syn HO-1, in a large scale. This is the first report of the characterization of the isolated cyanobacterial HO-1 protein and its heme complexes, applying the optical absorption and electron paramagnetic resonance (EPR) spectroscopies. Experimental procedures Construction of Synechocystis heme oxygenase-1 expression plasmid, pMWSynHO1 Plasmid purification, subcloning, and bacterial transfor- mations were carried out as previously described [23]. A T7 promoter-based expression vector, pMW172 (a gift from K. Nagai, MRC Laboratory of Molecular Biology, Cambridge, UK) was used to make the expression plasmid pMWSynHO1 for the recombinant Synechocystis heme oxygenase-1 by incorporating a double-stranded synthetic oligonucleotide with unique restriction enzyme sites for SpeI, SacI, AvrII, ClaI, and MluI between the NdeIandHindIII sites. A 720-base pair synthetic gene coding for the entire Syn HO-1 was synthesized from nine oligonucleotides and their complements constructed by 55–99mer nucleotides. Double strand DNAs, Oligo I to Oligo IX, were ligated step by step into the restriction enzyme sites of the plasmid, by use of T 4 ligase. Oligo I and Oligo II were inserted between the sites of NdeIand SpeI; Oligo III, between the sites of SpeI and SacI; Oligo IV and Oligo V, between SacIandAvrII; Oligo VI, between AvrII and ClaI; Oligo VII and VIII, between ClaIandMluI; Oligo IX, between MluIandHindIII. Escherichia coli strain JM109 was used for DNA manipulation. The nucleotide sequence of the expression plasmid, pMWSynHO1, was determined by an Applied Biosystems 373A DNA sequencer. Protein expression and purification A 10-mL inoculumin Luria–Bertani medium (+ 50 lgÆmL )1 ampicillin : 0.1% glucose) was prepared from plates of transformed E. coli BL21 (DE3) cells carrying pMWSyn- HO1. Cultures (500 mL) were inoculated with 1 mL of the inocula and grown in Luria–Bertani medium (+ 200 lgÆmL )1 ampicillin) at 37 °CuntilD 600 reached 0.8–1.0. The cells were grown for an additional 16 h at 25 °C, harvested by centrifugation, and stored at )80 °C until use. Typical yield of cells from a 500-mL culture was 2 g. The E. coli cells (10 g), resuspended into 90 mL Tris buffer (pH 7.4, + 2 m M EDTA), were lyzed (2 mg lyso- zyme per g cells) with stirring at 4 °C for 30 min. After sonication (Branson 450 Sonifire) and centrifugation at 39 000 g for 1 h, the resulting supernatant was converted into 35–60% (NH 4 ) 2 SO 4 fraction and centrifuged. The subsequent precipitates, containing the Syn HO-1 protein, were dissolved in 20 m M potassium phosphate buffer (pH 7.4) and applied to a Sephadex G75 column (3.6 · 50 cm), pre-equilibrated with the same buffer. The protein fractions eluted in the potassium phosphate buffer, with the intense 27 kDa band on SDS/PAGE, were gathered and directly loaded onto a DEAE-cellulose (DE-52) column (2.6 · 28 cm). After washing the column with 50 mL of 20 m M potassium phosphate (pH 7.4)- 50 m M KCl, the protein was eluted with 400 mL of 20 m M potassium phosphate (pH 7.4) using a linear gradient, 50–250 m M KCl. Collected fractions with high protein content were further run through a hydroxylapatite column (2.6 · 20 cm). The protein was eluted with 400 mL of potassium phosphate (pH 7.4) using a linear gradient, 20–200 m M . Only fractions with the single band at 27 kDa on SDS/PAGE were finally collected. The protein concen- trations were estimated by Lowry’s method using crystalline bovine serum albumin as standard. Reconstitution of Syn HO-1 with hemin An alkaline-hemin solution of 0.86 m M in 4.6 lL increments was added to the 10 l M solution of Syn HO-1 in 2 mL of 0.1 M potassium phosphate buffer (pH 7.0). Optical absor- bance at 402 nm was monitored for each addition of the hemin solution and plotted against the volume of added hemin solutions to construct titration curves. The heme–Syn HO-1 complex was purified by Sephadex G-25 and DEAE-cellulose (DE-52) column chromatography. 688 C. T. Migita et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Optical absorption spectroscopy Optical absorption spectra were recorded on a Shimadzu UV-2200 spectrophotometer at 25 °C. The ferrous heme- Syn HO-1 complex was prepared in a sealed cuvette by the addition of dithionite to the 0.1 M potassium phosphate (pH 7.0) solution of hemin-Syn HO-1 that was previously deoxygenated by use of oxygen absorber (Iuchi, A500–50S) and saturated with argon. The CO complex of heme-Syn HO-1 was prepared by displacing argon filled in the space of a sealed cuvette containing the ferrous-Syn HO-1 solution with CO. The oxy complex was prepared by introducing air into the anaerobic sample of ferrous heme-Syn HO-1 generated by the reduction of the ferric complex with NADPH/reductase. pH titration of the hemin-Syn HO-1 complex was conducted with the 1 M Tris solution from pH 6.0–8.5 and with the 1 M NaOH solution from pH 8.5–11.0. Determination of the pK a value was per- formed by a curve fitting with the calculated curves of the fraction of alkaline form vs. pH for given pK a values in the Henderson–Hasselbalch equation. EPR spectroscopy EPR spectra were measured by a Bruker E500 spectrometer, operating at 9.35–9.55 GHz, with an Oxford liquid helium cryostat. The 15 NO-bound heme–Syn HO-1 complex was prepared by adding dithionite to the argon-saturated solution of Syn HO-1 and 15 NaNO 2 in EPR tubes. Reaction of the heme–Syn HO-1 complex with ascorbic acid and NADPH-cytochrome P450 reductase Ascorbic acid (final concentration 10 m M ) was added to an optical cell containing heme–Syn HO-1 (8.4 l M )in2mL of 0.1 M potassium phosphate buffer (pH 7) at 25 °C. Spectral changes between 240 and 900 nm were recorded until the reaction was completed by monitoring the maximum loss of the Soret band (A 402 ) and the formation of biliverdin (A 730 ). In the experiments using NADPH- reductase, the 14 equivalent of NADPH was added to the solution of heme–Syn HO (8.5 l M ) containing 55 n M of the reductase. Soluble cytochrome P450 reductase is a recombinant human enzyme, which lacks hydrophobic region consisting of N-terminal 50 amino acid residues. For construction of expression plasmid, we used the gene gifted from F. J. Gonzalez of the National Institute of Health (Bethesda, Maryland, USA), to be published elsewhere. HPLC analysis of the heme–Syn HO-1 reaction products For the ascorbate-assisted reaction, ascorbate (final con- centration 100 m M ) was added to a mixture of heme–Syn HO-1 (20 l M ) and desferrioxamine (2 m M )in2mLof 0.1 M Tris/HCl buffer (pH 7.4). For the NADPH/reduc- tase supported reaction, NADPH (final concentration, 0.5 m M ) was added to the solution of heme–Syn HO-1 (20 l M ) and reductase (55 n M )in2mLof0.1 M Tris/HCl buffer (pH 7.4). After 2 h, the reactants were hydrolyzed with HCl to ensure the full conversion into free biliverdin. Each solution was subjected to a Supelclean LC-18 solid phase extraction column prewashed with acetonitrile/water (1 : 9, v/v) and eluted with acetonitrile/water (1 : 1, v/v). Lyophilization of the collected fractions gave green pigment, which was then dissolved in 5% HCl/methanol and kept at 4 °C overnight. The product was extracted with chloroform and analyzed by a column of Capcell Pak C18 (SG 120, 4.6 · 150 mm) pre-equilibrated with degassed acetonitrile/water (3 : 2, v/v) at a flow rate of 1mLÆmin )1 . The biliverdin standards were eluted in the order a (21.5 min), d (23.0 min), b (24.7 min), and c (37.0 min). Results Expression and purification of Syn HO-1 We have successfully expressed a recombinant cyanobac- terial Syn HO-1 protein using a synthetic gene constructed from nine oligonucleotides. Amino acid sequence of the Synechocystis sp. PCC 6803 heme oxygenase (Syn HO-1) has been compared with those of related mammalian and bacterial HOs in Fig. 1. The harvested BL21 cells carrying the Syn HO-1 expression vector, pMWSynHO1, were green, same as reported for the cloned protein from Synechocystis sp. PCC 6803 ORF sll1184 [36] and for other mammalian and bacterial heme oxygenase proteins [23,25,28,42]. Accumulation of a green pigment strongly suggests the production of biliverdin, so that the Syn HO-1 is supposed to be expressed as a catalytically active protein, which has been confirmed as described later. Recombinant Syn HO-1 was obtained as a soluble protein. The purified protein through a hydroxylapatite column showed a single band at 27 kDa on the SDS/ PAGE (Fig. 2, lane 2 and 3). Three litters of cell-cultured solution ( 12 g of cell) yielded 30 mg of the purified protein. Formation of the heme–Syn HO-1 complex When an aliquot of the alkaline-hemin solution is added into the solution containing Syn HO-1, the resultant solution gives the optical absorption spectrum which has a Soret band at 402 nm, that is apparently distinguishable from the Soret band of free hemin. Utilizing this difference, the stoichiometry of the heme binding reaction ratio to Syn HO-1 was examined. The inset of Fig. 3 illustrates obtained titration plots. It clearly indicates that the Syn HO-1 protein (10 l M ) is saturated at a ratio of 1 : 1 hemin to protein, thereby establishing that Syn HO-1 binds equimolar hemin to form the hemin-enzyme complex, same as mammalian and bacterial HOs [14,23,25]. Spectroscopic characterization Figure 3 exhibits optical absorption spectra of the ferric-, ferrous-, oxy-, and CO-bound forms of heme–Syn HO-1. The Soret bands of the ferric and ferrous forms, having maxima at 402 and 427 nm, respectively, are slightly blue-shifted compared with those of mammalian (404 and 431 nm) and bacterial (404, 406 and 434 nm) heme– HO complexes. By contrast, absorption maxima of the Ó FEBS 2003 Recombinant of cyanobacterium heme oxygenase (Eur. J. Biochem. 270) 689 Soret and Q (a and b) bands of the oxy form and the CO-bound form do not show specific differences from those of mammalian or bacterial HOs. The small band at 634 nm seen in the spectrum of the CO-bound form is thought to come from the verdoheme-CO complex generated by the reaction of ferrous Syn HO-1 with oxygen contaminated. Compared with the visible spec- trum of the ferric form of heme-rat HO-1, the spectrum of ferric heme–Syn HO-1 shows distinctively stronger CT bands (498 and 630 nm) and weaker Q-bands (575 and 535 nm) in pH 7.0 potassium phosphate solutions (data not shown). Optical absorption data of the heme-HO complexes with different taxonomical origins are sum- marized in Table 1. The absorption coefficient at 402 nm for the ferric heme–Syn HO-1 complex is determined to be 128 m M )1 Æcm )1 by the pyridine hemochrome method [23], which is the smallest among the values reported for the mammalian and bacterial HOs. EPR of the heme–Syn HO-1 complex at pH 7 exhibits an axially symmetric high-spin spectrum originated from the ferric ion in approximately tetragonal ligand field (g ¼ 6andg ¼ 2, upper spectrum in Fig. 4). The axially symmetric spectra and g-values are similar to the ferric high-spin state of mammalian and bacterial heme-HO complexes [14,24]. pH dependence of the heme–Syn HO-1 complex The optical absorption spectrum of the ferric heme–Syn HO-1 complexvaries depending on pH. AspHincreases from 6 to 10, the Soret peak shifts from 402 to 418 nm gradually and the peaks at 498 and 630 nm in the visible region are alternated with the peaks at 537 and 575 nm, as shown in Fig. 5, panel A. The expanded visible region spectrum shows that the CT bands derived from the high-spin species remain at pH 10. This pH-dependent alteration is reversible between pH 6 and 10. The pK a value of this acid–base transition is estimated based on the increase of absorbance at 418 nm as pH increases. Curve fitting of the fraction of thealkaline form to the calculated values using the Henderson–Hasselbalch equation yielded the best-fitted result with pK a ¼ 8.9 (Fig. 5B). EPR spectra of the heme–Syn HO-1 complex also show the pH dependency. As the pH increases from 7 to 10, intensity of the axially symmetric spectrum is reduced and instead, the low-spin signals newly appear. This change is also reversible. Apparently two types of low-spin signals are observed (Fig. 4, the lower spectrum). The major species (denoted as A) with g 1 ¼ 2.78, g 2 ¼ 2.14, and g 3 ¼ 1.74 shows larger anisotropy than the minor species (denoted as B) with g 1 ¼ 2.68, g 2 ¼ 2.20, and g 3 ¼ 1.80. Two kinds of Fig. 1. Amino acid sequence alignment of Synechocystis, mammalian, and bacterial heme oxygenases. The plus sign indicates similar, while the asterisk indicates identical amino acid residues. Nostocho, cyanobacterial Nostoc sp.PCC7120 [37], Hmu O, C. diphtheriae [23], and Hem O, N. meningitidis A 2855 [25]. 690 C. T. Migita et al.(Eur. J. Biochem. 270) Ó FEBS 2003 alkaline forms have been also observed for the heme complexes of bacterial HO (Hmu O), while the single alkaline species detected for the heme–rat HO-1 complex [14,24]. The g-values of the alkaline forms of heme-HO complexes are presented in Table 2. EPR of the nitrosyl heme–Syn HO-1 complex The EPR spectrum of the 15 NO complex of ferrous heme– Syn HO-1 is represented in Fig. 6. The rhombic spectrum typical of a six-coordinated nitrosyl heme complex exhibits a triplet of doublet splitting at the g 2 component, that comes from the interaction between nuclear spins of 14 N(I ¼ 1) and 15 N(I¼ ½) and an electron spin, respectively. By compa- rison of the spectra of known nitrosyl heme–HO complexes [14,24,41], the doublet component with a hyperfine coupling constant of 31.1 gauss is reasonably assigned to the 15 N nucleus of 15 NO on the distal site of heme. Similarly, the triplet component with the hyperfine splitting of 7.1 gauss is attributable to the 14 N nuclei of the axial ligand trans to the nitrosyl ligand. This firmly establishes that the proximal ligand of the heme–Syn HO-1 complex is a nitrogenous base. The close value of hyperfine coupling constant of the proximal 14 N nuclei to those of established histidyl axial ligand in heme–HOs strongly suggests that the nitrogenous proximal ligand of the heme–Syn HO-1 complex also has histidyl origin in the proximal site (Table 3). Catalytic turnover of the heme–Syn HO-1 complex The time course spectra of the heme-conversion reaction in the presence of ascorbate are depicted in Fig. 7, panel A. Addition of ascorbate to the heme–Syn HO-1 complex commences the reaction, which is monitored by the steady decrease of the Soret and 498 nm bands and the shift of the band at 630 nm to the longer-wavelength direction. At the same time, a broad band with the maximum at approxi- mately 690 nm appears and increases with time. The newly Fig. 2. SDS/PAGE of purified Syn HO-1 protein. Lane 1, molecular mass markers; lane 2, 2.4 lg of protein and lane 3, 24 lgofprotein. Fig. 3. Optical absorption spectra of the heme–Syn HO-1 complexes. The spectra are the ferric (–-–), ferrous (– –), ferrous-CO (––), and oxy (- - - -) complexes, respectively. Inset, titration of Syn HO-1 (10 l M ) with hemin detected by the absorbance increase at 402 nm. The background absorbance shown in Ôwithout Syn HO-1Õ comes from added free hemin. [Heme-Syn HO-1] ¼ 9.7 l M ,in0.1 M potassium phosphate (pH ¼ 7.0). The ferrous form was made by the addition of dithionite (150 l M ) under anaerobic condition and the spectrum was recorded after 15 min of incubation. The oxy form was produced by the addition of air in the ferrous complex produced with NADPH (120 l M ) and the spectrum was taken after 5 min of incubation. Ó FEBS 2003 Recombinant of cyanobacterium heme oxygenase (Eur. J. Biochem. 270) 691 Table 1. Optical absorption data for the heme-heme oxygenase complexes with different taxonomical sources. Syn HO-1, cyanobacterial Synechocystis sp. PCC 6803; rat HO-1, taken from refs [9] and [14]; Pig A, Pseudomonas aeruginosa, taken from ref [28]; Hmu O, Corynebacterium diphtheriae, taken from ref [23]; Hem O, Neisseriae meningitidis, taken from ref [25]. Protein Syn HO-1 rat HO-1 Pig A HO Hmu O Hem O Ferric form k max (Soret) (e (m M )1 )) 402 (128) 404 (140) 406 (129) 404 (150) 406 (179) k max (visible) 630, 498 631, 500 632 630, 500 Ferrous deoxy form k max (Soret) 427 431 434 434 k max (visible) 555 554 550 Oxy form k max (Soret) 410 410 410 410 k max (a, b) 574, 537 575, 539 570, 540 570, 540 CO form k max (Soret) 418 419 419 421 421 k max (a, b) 566, 536 568, 535 567, 537 568, 538 568, 538 Alkaline form k max (Soret) 418 413 k max (a, b) 575, 537 575, 540 pK a 8.9 7.6 8.0 9.0 Fig. 4. EPR spectra of ferric heme–Syn HO-1 complexes in neutral and basic solutions. Measuring conditions: T ¼ 8 K, microwave frequency 9.55 GHz, field modulation 100 kHz, modulation amplitude 10 G, microwave power 0.5 mW. In the pH 10.6 spectrum, the low-spin region is expanded fivefold. The sample at pH ¼ 7.0, 100 lL of heme– Syn HO-1 (400 l M )in0.1 M potassium phosphate; the sample at pH ¼ 10.6, 120 lL of heme–Syn HO-1 (300 l M )in1m M potassium phosphate whose pH was adjusted with NaOH (1 M ). Fig. 5. Determination of pK a for the heme–Syn HO-1 complex. (A) Absorption difference spectra of the alkaline solutions, [heme-Syn HO-1] ¼ 7.9 l M , referring to the spectrum at pH 6.0. The visible- region is shown in the enlarged absorption spectra. (B) The fraction of the alkaline form at given pH calculated for each value of pK a ,8.7 (–-–), 8.9 (––), 9.1(- - -), and 9.3 (– –), based on the Henderson–Has- selbalch equation. The heavy dots are the fractions estimated from the experimentally obtained absorbance at 418 nm that is normalized against the value at pH 10.2. 692 C. T. Migita et al.(Eur. J. Biochem. 270) Ó FEBS 2003 appeared visible band is suggestive of verdoheme or biliverdin formation, or of their admixture. Then, after 2 h, the reaction mixture was separately analyzed by HPLC, confirming that the final product was biliverdin IX a (data not shown). To examine the formation of the verdoheme intermediate, this reaction was performed under the limited oxygen condition. As exhibited in Panel B in Fig. 7, the spectrum recorded after 2 h (the dashed-and-dotted line) has peaks at 534, 637, and 686 nm other than the Soret peak. The solid-line spectrum recorded after 4 h shows new peaks at the Soret region (416 nm) and at 566 nm and indicates the 686 nm band further increased. The combined double peaks at 600–750 nm are commonly observed in the heme degradation by mammalian HO, which are markers of verdoheme formation. Peaks at 534 and 686 nm are attributable to the ferrous–verdoheme complex and the peak at 637 nm to the CO-bound verdoheme complex due to the trapping of CO concomitantly produced. The peaks at 416 and 566 nm are attributable to the CO-bound heme– Syn HO-1 complex (Table 1). Addition of CO transforms the solid-line spectrum into the broken-line spectrum, in which the peak at 637 nm is much enhanced and new peaks Table 2. g-Values and g-anisotropy of alkaline forms of heme–heme oxygenase complexes. Data for Hmu O and rat HO-1 are taken from refs [24] and [14], respectively. g-Anisotropy is defined as Dg ¼ g 1 ) g 3 . Protein Species Syn HO-1 Hmu O ABA¢ B¢ rat HO-1 g 1 2.776 2.675 2.72 2.67 2.67 g 2 2.144 2.203 2.16 2.21 2.21 g 3 1.737 1.795 1.76 1.80 1.79 Dg 1.039 0.880 0.96 0.87 0.88 Fig. 6. EPR spectrum of the 15 N-nitrosyl heme–Syn HO-1 complex. Measuring conditions: T ¼ 30 K, microwave frequency 9.35 GHz, field modulation 100 kHz, microwave power 0.2 mW, field modula- tion amplitude 2G. [heme–Syn HO-1] ¼ 430 l M ,in0.1 M potassium phosphate (pH ¼7.0). Table 3. EPR parameters of the ferrous 15 N-nitrosyl heme–heme oxyg- enase complexes. Data for Hmu O and rat HO-1 are taken from refer- ence [24] and [14], respectively. g-Anisotropy is defined as Dg ¼ g 3 –g 1 . Protein Syn HO-1 Hmu O rat HO-1 g 3 2.079 2.082 2.086 g 2 2.003 2.004 2.008 g 1 1.962 1.966 1.986 Dg 0.117 0.116 0.100 A( 15 N-NO) 31.1 G 30 26 A( 14 N-His) 7.1 G a 6.8 7.4 a Value of A( 14 N-L). Fig. 7. Heme conversion by Syn HO-1 initiated by the addition of ascorbate. (A) Spectra were recorded at the indicated time after the addition of ascorbate solution (10 m M ) to the heme–Syn HO-1 solu- tion (8.4 l M in 0.1 M potassium phosphate at pH 7.0). The Soret and 498 nm bands decrease with time, while the band at 680 nm appears and increases. The spectrum recorded after 4 h indicates the formation of mixture: free biliverdin, verdoheme, and verdoheme-CO. (B) The reaction was conducted under argon atmosphere. Spectra were recorded 2 h after the addition of ascorbate (–-–), 4 h after (––), and after the replacement of Ar in the space of the sealed cell with CO (– –). Ó FEBS 2003 Recombinant of cyanobacterium heme oxygenase (Eur. J. Biochem. 270) 693 appears at 350 and 541 nm while the peaks at 534 and 686 nm almost disappear. The peaks at 350, 404, 541, 637 nm are very close to those reported for the CO bound verdoheme–rat HO-1 complex [16]. Accordingly, it can be concluded that verdoheme is produced during the course of heme degradation by Syn HO-1, accompanied by releasing CO simultaneously. The overall rate of the heme degrada- tion by Syn HO-1 with ascorbate is roughly estimated to be one-fifth of that by rat HO-1 when the same amount of enzyme and ascorbate are used. Time course of the heme catabolic reaction by Syn HO-1 in the presence of NADPH cytochrome P450 reductase was also examined. As illustrated in Fig. 8, the obtained spectra are clearly discriminated from those of the ascorbate- supported reaction. Although addition of 14 equivalent of NADPH to heme–Syn HO-1 in the presence of reductase initiates the reaction, the reaction is almost at a standstill from 6 to 15 min after addition of NADPH. Shift of the Soret maximum to 410 nm and appearance of the 534 and 573 nm bands in the visible region indicate that the oxy complex of heme–Syn HO-1 is produced within 3 min and accumulated. Decomposition of the oxy complex appears much slower than its formation and does not end even after 210 min, exhibiting the bands of the remaining oxy complex. The 340 nm-band of NADPH decreases in proportion to the decrease of Soret band at 410 nm and to the increases of broad band spreading 600–700 nm. The latter band was confirmed to belong to biliverdin IX a by the HPLC analysis (data not shown). Discussion Overall structure and heme binding The primary structure of Syn HO-1 has very high identity (38%) and similarity (67%) to that of human HO-1 [26]. Such resemblance is higher than the 57.4% homology of cyanobacterial Nostoc sp. PCC7120 [37]. Other prokaryotic HOs bear less resemblance to Syn HO-1: Hmu O, 31% identity and 59% similarity; Hem O, 19% identity and 42% similarity. Then, the tertiary structure of Syn HO-1 protein is expected to resemble that of mammalian HO-1. Overall holdings of bacterial HO (Hem O) [27] and mammalian HO-1 [17,18] are known to be similar though their primary structure are less similar than that between Syn HO-1 and mammalian HO-1. Syn HO-1 binds equimolar hemin to form the stable heme–Syn HO-1 complex. EPR of the nitrosyl heme–Syn HO-1 complex has established that the proximal ligand of the heme–enzyme complex is a nitro- genous base. The aligned sequence depicted in Fig. 1 designates His17 as a potential candidate for the proximal ligand of heme–Syn HO-1, that corresponds to the estab- lished proximal ligand of His25 in mammalian HO-1 and His20 in bacterial Hmu O [15,43]. Axial coordination structure of heme The cryogenic EPR has revealed that the resting state of heme–Syn HO-1 is in the axially symmetric ferric high- spin state at pH 7.0. At alkaline pH values, the high-spin state is partially converted into the low spin state. This pH-dependent spin-state conversion is also observed at room temperatures (Fig. 5). The alkaline forms of heme– Syn HO-1 have g-values that are close to those of the alkaline forms of heme–rat HO-1 and Hmu O (Table 1), which are established to be the hydroxide-bound form generated by deprotonation of the axially ligated water. Therefore, the alkaline forms of heme–Syn HO-1 are also thought to be the hydroxide-bound forms, which are produced by the coordination of hydroxide originated from dissociation of the heme-bound or nearby water, correlating to the change of protonic equilibria of protic residues in the distal heme pocket. As illustrated in Fig. 5, the transition to the alkaline form is not completed even at pH 10. The determined pK a value of 8.9 for the heme–Syn HO-1 complex is higher than that of rat heme–HO-1 and close to that of bacterial heme–Hmu O (Table 1). Hence, it follows that the proton dissociation of distal water in Syn HO-1 is less favorable than that in rat HO-1 but is similar to that in Hmu O. The amino acid residues constructing the distal helix in rat HO-1 (Leu129 to Met155) are almost all Fig. 8. Heme conversion by Syn HO-1 initi- ated by the addition of NADPH. Spectra were recorded at the indicated time after the addi- tion of NADPH (final concentration 120 l M ) to the solution of heme–Syn HO-1 (8.5 l M , in 0.1 M potassium phosphate at pH 7.0) and reductase (55 n M ). 694 C. T. Migita et al.(Eur. J. Biochem. 270) Ó FEBS 2003 conserved both in Syn HO-1 and Hmu O though the entire primary structure of Syn HO-1 is much closer to that of rat HO-1 than to that of Hmu O. The crystal structure of the hydroxide-bound heme–rat HO-1 showed that Gly143N is located within hydrogen bonding distance (2.60 A ˚ )withthe heme-coordinating hydroxide [18]. Recently, we have found that the alanine mutation of residues on the distal helix of rat HO-1 alters the pK a value in order of 8.8 (S142A) > 8.6 (D140A) > 8.5 (R136A) > 8.0 (T135A) [44]. It appears that the closer is the mutated residue to G143, the higher is the pK a value, independent of the nature of the displaced amino acid. One possible explanation for the high pK a value of heme–Syn HO-1, and of Hmu O, is that the distal ionizable group(s) that is responsible for the deprotonation of the distal water is more distant from the heme axial site than in rat HO-1. Multiple alkaline forms The major component (species A) of the two alkaline forms of heme–Syn HO-1 is present at an approximately threefold larger quantity than the minor component (species B) (Fig. 4, Table 2). The bacterial heme-Hmu O complex also forms two low-spin species, of which one is far more predominant than the other (species B¢ and A¢ in Table 2, respectively) [24]. For the low-spin ferric heme complexes in the ground electronic states with dp spin orbitals, the small but definite differences in the coordina- tion circumstances are discriminated by g-values and g-anisotropy [45]. Species B from Syn HO-1, species B¢ from Hmu O, and the species from rat HO-1 which has only one alkaline form have very similar g-values and g-anisotropy. In these species, then, the distal hydroxide protons are possibly fixed to the same direction relative to the heme plain. In rat HO-1, Gly143N resides in the d-meso direction of heme, where the heme-coordinated hydroxide least destabilizes dp orbitals of the heme iron, resulting in smaller g-anisotropy. Consistently, g-aniso- tropy of these species is smaller than that of Species A (Syn HO-1) and Species A¢ (Hmu O). In the latter species with the larger g-anisotropy, the hydroxyl ligand might more lean to the direction of the counter pyrrole N a –N a axis, where the dp orbitals are the most destabilized. Coordination structure of the nitrosyl heme complex There are considerable numbers of studies aimed at characterizing the coordination structure of the nitrosyl heme complexes and heme proteins. The rhombic type of spectra obtained for the ferrous nitrosyl heme–HO com- plexes is classified to Ôtype IÕ and supposed to contain a bent Fe–N–O bond with an angle of 120–150° [46]. The nitrosyl heme complex of Syn HO-1 has larger nitrogen hyperfine coupling constant of the nitrosyl-nitrogen nuc- leus, A N ( 15 NO), and the smaller one of the proximal- ligand nitrogen nucleus, A N ( 14 N-L), compared with those of the nitrosyl heme–rat HO-1 complex (Table 3). In addition, each of the g-values of the nitrosyl heme–Syn HO-1 complex is smaller than that of the rat HO-1 or Hmu O complexes. The hyperfine interaction in the nitrosyl heme complex arises from an unpaired electron that originally occupies the 2 pp* orbital of nitrogen oxide and is delocalized into the metal d-orbitals through r-and p-interaction. The larger A N ( 15 NO) and the smaller A N ( 14 N-L) mean that the r-delocalization from the NO p* orbital to the iron d orbitals is reduced. This phenomena can be interpreted on assumption that the Fe–N(O) distance is elongated due to the shortening of Fe–L bond. Recent analysis of g-tensors of the six-coordinated nitrosyl iron(II) porphyrins with the imidazole ligand by density function theory describes that g-tensors of the type I complexes are sensitive to the Fe–N(Im) bond length as well as to the orientation of the NO ligand (but not to the orientation of the imidazole ligand) [47]. Changes in the Fe–N(Im) bond length less than 0.5 A ˚ is reflected in deviations of the g-component up to 0.02, where the shorter are the distance, the smaller are the g-tensor components (g 1 , g 2 ,andg 3 ). Such small variation in the bond lengths is detectable only by ultra-high resolution X- ray crystallography [48]. According to this theoretical estimation, our observation that all of the g 1 , g 2 ,andg 3 components of nitrosyl heme–Syn HO-1 are smaller than those of the rat HO-1 complex (Table 3) implies that the Fe–N(L) bond length in nitrosylheme–Syn HO-1 is shorter than that in nitrosyl heme–rat HO-1, in accordance with the aforementioned assumption deduced from the consid- erationonA N . The rhombic g-anisotropy thus reflects the difference of the heme pocket structures that perturb the coordination structure of the nitrosyl heme complexes. In this meaning, the structure of either proximal or distal sites of the heme pocket of heme–Syn HO-1 differs from that of heme–rat HO-1, and rather resembles that of bacterial heme–Hmu O. Protein modification of coordination geometry in the heme–Syn HO-1 complex Among the heme complexes of Syn HO-1 and other wild type HOs reported so far, the Syn HO-1 complex has distinctively small absorption maximum of the Soret band with a small absorption coefficient (Table 1). On the other hand, relative intensities of the 498 and 630 nm bands compared with those of the 575 and 535 nm bands in the visible region spectrum are larger than those observed in the spectrum of heme–rat HO-1. The former bands, referred as CT bands, are commonly distinctive in high-spin derivatives of ferric hemoproteins, while the latter bands (a-andb-bands) are usually weak in the high-spin derivatives but are distinctly observed in the low-spin derivatives [49]. As the position and the intensity of these bands are dependent not only on the spin state or thermal spin-state equilibria but also on the nature of the sixth ligand and the type of apoprotein, we could not attribute these features to one of the possible causes at present. However, it can be mentioned that the protein modification of coordination geometry in the heme–Syn HO-1 complex apparently differs from that in the known heme–HO complexes. Heme catabolism by Syn HO-1 The heme bound to Syn HO-1 is transformed into biliverdin IX a regioselectively in the presence of oxygen and electrons. In the course of reaction, verdoheme Ó FEBS 2003 Recombinant of cyanobacterium heme oxygenase (Eur. J. Biochem. 270) 695 intermediate is produced accompanied by CO release. Therefore, the mechanism of heme conversion by Syn HO-1 is found to be fundamentally the same as that by mammalian HOs, i.e. heme is converted to biliverdin IX a , carbon monoxide, and iron through the three-step reaction with the intermediates of a-meso-hydroxyheme and verdoheme [13]. In the heme–Syn HO-1 reaction, the final product is free biliverdin even under the ascorbate-supported reaction, differing from the product, ferric-biliverdin IX a , in the ascorbate-supported heme catabolism by rat HO-1 and similar to that in the heme catabolism of bacterial Hmu O under ascorbate [23]. Though both NADPH cytochrome P450 reductase and ascorbate can support this reaction, the overall rate of heme degradation is considerably slow in both systems compared with that by mammalian HO, even slower than that by bacterial Hmu O [23]. Notably, the heme conversion with NADPH cytochrome P450 reductase is retarded at the oxy- complex, which has been observed for the heme catabolism neither by mammalian nor bybacterial HOs. The collation of time-course spectra of Panel A in Fig. 7 with those of Fig. 8 makes us realize that reduction of the ferric heme–Syn HO-1 complex followed by the oxy complex formation is unfa- vorable in the ascorbate-supported reaction as evidenced by no accumulation of the oxy form. By contrast, conversion of the oxy complex (to the hydroxyheme complex) is extremely slow in the NADPH-reductase supported reaction although reduction of the ferric heme is sufficiently fast. The slow reduction rate of the ferric heme–Syn HO-1 complex by ascorbate seems to imply the lower oxidation-reduction potential of the heme iron in the Syn HO-1 complex compared with that of the one in the rat HO-1 complex, that might limit the overall reaction rate. As for the retardation of heme conversion under NADPH/reductase, the electron transfer from NADPH cytochrome P450 reductase to the oxy complex appears to be quite inefficient in the heme–Syn HO-1 complex. We have observed that the presence of 30 equivalents of ascorbate together with reductase in the reaction mixture avoids the retardation (data not shown). This makes us speculate that the long-range electron tunneling pathway through the Syn HO-1 protein, from the binding site of reductase to the heme edge, is not the Ôright pathÕ. The cyanobacterial cell must provide an effective and successive electron-transfer system for Syn HO-1. Searches for the inherent reducing system that works in the physio- logical Syn HO-1 reaction are currently underway. Conclusive remarks An effective bacterial expression system of cyanobacterial Synechocystis heme oxygenase protein, was constructed for the first time and the highly purified protein, Syn HO-1, was obtained successfully. Syn HO-1 binds equimolar hemin to form the heme–Syn HO-1 complex. The resultant complex is converted to biliverdin IX a bythereactionwithoxygeninthe presence of ascorbate or NADPH cytochrome P450 reduc- tase, forming detectable intermediates, the oxy-heme and verdoheme complexes. However, the overall reaction rate of heme conversion is relatively slow. Characteristics of the heme–Syn HO-1 complex discriminate from those of the other heme–HO complexes. The resting state of the heme–enzyme complex, which has a nitrogenous proximal ligand, is in the ferric high-spin state. The complex exhibits an acid–base transition with the pK a value of 8.9, which is larger than that of the heme–rat HO-1 complex, suggesting that the proton dissociation of the distal water is less efficient. The heme–enzyme complex generates two kinds of the alkaline form. The nitrosyl heme–Syn HO-1 complex of type I is generated, which has a relatively large nitrosyl A N ,small proximal ligand A N ,andsmallg components with large anisotropy. These characters strongly suggests that the heme pocket structure is different from that of mammalian HO and somewhat resembles that of bacterial Hmu O, in spite of very high conservation of the amino acid residues constitu- ting the heme pocket among these HOs. Acknowledgements This work was supported in part by a grant-in-aid for Scientific Research 12680625 and 14580641 (to T.Y.) from the Ministry of Education, Science, Sports and Culture of Japan. References 1. Beale, S.I. & Cornejo, J. (1983) Biosynthesis of phycocyanobilin from exogenous labeled biliverdinin Cyanidium caldarium. Arch. Biochem. Biophys. 227, 279–286. 2. Beale, S.I. (1993) Biosynthessis of phycobilins. Chem. Rev. 93, 785–802. 3. Beale, S.I. & Cornejo, J. (1991) Biosynthessis of phycobilins. 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