Tài liệu Báo cáo khoa học: Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied docx

Eur J Biochem 271, 1713–1724 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04077.x Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied Xuhong Zhang1, Michihiko Sato2, Masanao Sasahara1, Catharina T Migita3 and Tadashi Yoshida1 Department of Biochemistry and 2Central Laboratory for Research and Education, Yamagata University School of Medicine, Japan; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form of D melanogaster HO (DmDHO) The purified DmDHO degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P450 reductase and sodium ascorbate, although the reaction rate was slower than that of mammalian HOs Some properties of DmHO, however, are quite different from other known HOs Thus DmDHO bound hemin stoichiometrically to form a hemin–enzyme complex like other HOs, but this complex did not show an absorption spectrum of hexacoordinated heme protein The absorption spectrum of the ferric complex was not influenced by changing the pH of the solution Interestingly, an EPR study revealed that the iron of heme was not involved in binding heme to the enzyme Hydrogen peroxide failed to convert it into verdoheme A spectrum of the ferrous–CO form of verdoheme was not detected during the reaction from hemin under oxygen and CO Degradation of hemin catalyzed by DmDHO yielded three isomers of biliverdin, of which biliverdin IXa and two other isomers (IXb and IXd) accounted for 75% and 25%, respectively Taken together, we conclude that, although DmHO acts as a real HO in D melanogaster, its active-site structure is quite different from those of other known HOs Heme oxygenase (HO, EC 1.14.99.3) was first characterized in mammals as a microsomal enzyme that catalyzes the three-step oxidation of hemin to biliverdin IXa, CO, and free iron, via a-meso-hydroxyhemin, verdoheme, and ferric iron–biliverdin complex [1–3] (Scheme 1) To date two mammalian isozymes of HO have been identified [4]: HO-1, an inducible enzyme that is highly expressed in the spleen and liver; HO-2, a constitutive enzyme found abundantly in the brain and testes The two isozymes have about 43% similarity at amino acid level, and both have a C-terminal hydrophobic domain that is involved in binding to microsomal membrane Both HO-1 and HO-2 have been demonstrated to play important roles in physiological iron homeostasis [5,6], antioxidant defense [7,8], and possibly the cGMP signaling pathway [9,10] Although HO-3 was once reported as an isozyme of HO, its function is not yet well defined [11] HO has also been found and characterized in bacteria [12–14] and plants [15–18] and other species such as Rhodophyta [19] In contrast with mammalian HO, these HOs are water-soluble enzymes because they lack a membrane-anchoring domain at the C-termini of their sequences In pathogenic bacteria, HO is thought to help bacteria to acquire iron from heme-containing proteins found in their host cells for survival and toxin production In plants, biliverdin is used for the biosynthesis of photoresponsive bilins such as phycobilins and phytochromobilins [15–19] Although the HOs have been characterized structurally and functionally in most species, very little is known about HO in insects Heme is extremely important in insects It is a vital nutrient for most, if not all, insects for their embryonic development [20], although they not use it as a transport vehicle or storage vessel for oxygen Heme also serves as the prosthetic moiety of hemoproteins, such as hemoglobin [21,22], catalase [23] and nitric oxide synthase [24], which are essential for biological function However, heme is potentially toxic to insects, particularly blood-sucking insects such as mosquitoes, because it catalyzes oxidative reactions that can damage membranes and destroy nucleic acids Therefore, insects are thought to have several mechanisms for sequestering and controlling heme For example, it can be conjugated with such proteins as the heme binding protein Correspondence to T Yoshida, Department of Biochemistry, Yamagata University School of Medicine, Yamagata, Japan Fax: + 81 23 628 5225, Tel.: + 81 23 628 5222, E-mail: tyoshida@med.id.yamagata-u.ac.jp Abbreviations: HO, heme oxygenase; CPR, NADPH/cytochrome P450 reductase; DmHO, heme oxygenase of D melanogaster; DmDHO, truncated form of D melanogaster heme oxygenase; DmCPR, NADPH/cytochrome P450 reductase of D melanogaster; DmDCPR, truncated form of D melanogaster NADPH/cytochrome P450 reductase; Syn HO-1, heme oxygenase-1 of Synechocistis sp PCC 6803 Enzymes: heme oxygenase (EC 1.14.99.3); NADPH/cytochrome P450 reductase (EC 1.6.2.4) (Received 25 December 2003, revised March 2004, accepted March 2004) Keywords: biliverdin; Drosophila melanogaster; heme oxygenase; insect; NADPH/cytochrome P450 reductase Ó FEBS 2004 1714 X Zhang et al (Eur J Biochem 271) Scheme Heme degradation pathway Heme to biliverdin IXa catalyzed by HO and biliverdin IXa to bilirubin IXa catalyzed by biliverdin reductase isolated from the bug Rhodnius prolixus, to form complexes that serve as a source of heme and prevent cells from heme-induced oxidative injury [25] Although heme biosynthesis has been reported in insects, very little is known about its degradation In fact, no HO has been isolated and characterized from any insect species so far Interestingly, biliverdin IXc is present in larval integument and hemolymph in some species of Lepidoptera, such as Pieris brassica [26], Manduca sexta [27], and Samia cynthia ricini [28] These insects possibly possess a HO that selectively cleaves the c-meso site from heme However, biliverdin IXa, the isomer formed in humans, occurs in the hemolymph and integument of other insects [29] Throughout the last century, the fruit fly has been the workhorse for genetic studies in eukaryotes The recent decoding of the complete genome sequence of Drosophila melanogaster has provided us with the opportunity to identify all fruit fly genes, including those involved in heme metabolism [30] In the present study, we found a putative HO gene in D melanogaster by homology searching in FlyBase, a database of genetic and molecular data for the fruit fly The D melanogaster HO gene without the sequence coding for the last 21 amino acids was cloned and further expressed in Escherichia coli The truncated enzyme was obtained in high yield as a soluble, catalytically active protein, making it available for the first time for detailed mechanistic studies Japan) The synthesized cDNA was subjected to PCR amplification to generate the coding region of the putative D melanogaster HO (DmHO) A sense primer, DmHOF1 (5¢-GCGCAAAAGACATATGTCAGCGAGCGAAG-3¢) and an antisense primer, DmHOR1 (3¢-CGAGAGTTC ATTCTTTTCGAACTTTATG-5¢) were used to amplify the full length DmHO consisting of 296 amino acid residues The underlined nucleotide sequence of 5¢-CATATG-3¢ represents the NdeI recognition site involving an initiation codon The underlined nucleotide sequences of 3¢-ATT-5¢, and 3¢-TTCGAA-5¢ are the complementary sequences of a stop codon, and the HindIII recognition site, respectively Another primer set with DmHOF1 and antisense primer DmHOR2 (3¢-GCACGGTTAGAAATCTTCGAACGG GAGCGT-5¢) were used to prepare a truncated form of DmHO (DmDHO) which lacks a C-terminal hydrophobic domain consisting of 21 amino acid residues The underlined nucleotide sequence of 3¢-ATC-5¢ is the complementary sequences of a stop codon PCR amplification was carried out with AmpliTaq Gold (Applied Biosystems) for 30 cycles The PCR products were digested with NdeI and HindIII and then cloned into the NdeI and HindIII sites of the pMW172 expression vector The constructs encoding the full length and C-terminally truncated DmHO were named pMWDmHO and pMWDmDHO, respectively Both constructs were sequenced using the dye terminator cycle sequencing method Purification of recombinant DmDHO Experimental procedures cDNA cloning and expression of putative DmHO FlyBase shows the existence of a nucleotide sequence encoding a protein homologous to both human and rat HOs RT-PCR was used to prepare cDNA encoding the putative HO of D melanogaster Briefly, first-strand cDNA synthesis was performed at 42 °C for 60 using adult D melanogaster polyA-rich RNA (Clontech) as a template, oligo(dT) primer (Genset, Proligo Japan, Kyoto, Japan), and reverse transcriptase (ReverTra Ace; Toyobo, Osaka, E coli strain BL21 (DE3) was transformed with pMWDmDHO A single colony was picked up and precultured in mL Luria–Bertani medium containing 50 lgỈmL)1 ampicillin and 1% glucose at 37 °C overnight Then 200 lL of the preculture was added to 500 mL of the same medium for incubation at 37 °C After the A600 of the culture reached about 1.0, the incubation was continued at 20 °C for 24 h The harvested cells were washed with 20 mM potassium phosphate buffer, pH 7.4, containing 134 mM KCl, resuspended in vols (9 mL per g E coli cells) 50 mM Tris/HCl buffer (pH 7.4) containing Ó FEBS 2004 mM EDTA, and lysed by lysozyme (final concentration 0.2 mgỈmL)1) for 30 at °C The lysed cells were briefly sonicated and centrifuged at 100 000 g for 60 min; the resulting supernatant was used as the soluble fraction For the purification, the soluble fraction was first subjected to ammonium sulfate fractionation The precipitate obtained at 33–55% saturation was collected by centrifugation, dissolved in 20 mM potassium phosphate buffer (pH 7.4) in a final volume of mL, and applied to a column (3.6 · 50 cm) of Sephadex G-75, pre-equilibrated with the same buffer Fractions with an intense 32 kDa band on SDS/PAGE were collected and applied to a DEAE-cellulose DE-52 column (2.6 · 30 cm) After the column had been washed with 50 mL 20 mM potassium phosphate buffer (pH 7.4) containing 100 mM KCl, the protein was eluted with 400 mL 20 mM potassium phosphate buffer (pH 7.4) with a linear gradient of 100– 400 mM KCl The fractions containing 32 kDa protein were then fractionated with a hydroxyapatite column (2.6 · 20 cm), using a linear gradient between 200 mL each 20 and 300 mM potassium phosphate buffer (pH 7.4) All fractions containing the 32 kDa protein were pooled and concentrated The buffer solution was changed to 50 mM potassium phosphate buffer (pH 7.4) by Sephadex G-25 column chromatography All procedures were conducted at °C, and the final products were stored at )80 °C Construction of DmDCPR expression plasmid A truncated form of NADPH/cytochrome P450 reductase of D melanogaster (DmDCPR) expression vector was constructed by the same method as described above An NdeI/HindIII cDNA fragment encoding amino acids 46–679 of DmCPR was amplified by RT-PCR using the primers DmCPRF1 (5¢-CTTCCTGCGTACGCA TATGAAGGAGGAGGA-3¢) and DmCPRR1 (3¢-CA GACCTCGATTCGAATAGGTTTTCGGTTG-5¢) The first 45 amino acids of DmCPR were deleted because this sequence involves a membrane-bound region One NdeI restriction site inside the target sequence was reduced by site-directed mutagenesis without changing any amino acid residues The PCR product was digested and inserted into the NdeI and HindIII restriction sites of pMW172 to form pMWDmDCPR Preparation and assay of DmDCPR Conditions for expressing DmDCPR and preparing a soluble fraction were similar to those for DmDHO described above The precipitate obtained from the soluble fraction at 40–65% ammonium sulfate saturation was suspended in 15 vols (15 mL per g E coli cells) 20 mM potassium phosphate buffer (pH 7.4) The suspension was applied to a DE-52 column and the protein was eluted with a 400 mL linear gradient of 100–400 mM KCl in 20 mM potassium phosphate buffer (pH 7.4) Yellow fractions with an intense 72 kDa band on SDS/PAGE were pooled and then loaded on a column of 2¢5¢-ADP Sepharose 4B (Amersham Pharmacia Biotech) The column was washed with 50 mL 0.1 M potassium phosphate buffer (pH 7.4), and DmDCPR was eluted with 20 mL 0.1 M potassium phosphate buffer D melanogaster heme oxygenase (Eur J Biochem 271) 1715 (pH 7.4) containing mgặmL)1 2Â(3Â)-AMP Finally, the 2¢(3¢)-AMP in the eluate was removed by passage through a Sephadex G-25 column The final products were stored in 50% glycerol at )80 °C The ability of DmDCPR to catalyze reduction of 2,6dichloroindophenol was assayed using 21 mM)1Ỉcm)1 as the absorption coefficient of the dye at 600 nm [31] Heme binding study Heme binding of DmDHO was tested by adding hemin to 12 lM DmDHO in mL 50 mM potassium phosphate buffer (pH 7.4) The reference cuvette contained mL 50 mM potassium phosphate buffer (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 Assay of DmDHO by measuring bilirubin formation The catalytic activity of DmDHO was determined after conversion of biliverdin IXa, produced by the enzyme, into bilirubin by biliverdin IXa reductase The NADPH/ DmDCPR reaction mixture contained in a final volume of 1.5 mL: 50 mM potassium phosphate buffer (pH 7.4), 26 lM hemin, lM DmDHO, 0.22 lM DmDCPR, 300 lM NADPH, and lM biliverdin reductase [32] NADPH was omitted from the control system When necessary, mM desferrioxamine was added to both the reaction and control systems The reaction was started by the addition of NADPH after preincubation at 37 °C, and monitored at 468 nm for 10 The value of 43.5 mM)1Ỉcm)1 was used as the absorption coefficient for bilirubin at 468 nm [33] The ascorbate system contained in a final volume of 1.5 mL: 50 mM potassium phosphate buffer (pH 7.4), 26 lM hemin, lM DmDHO, 50 mM sodium ascorbate, 60 lM NADPH, and lM biliverdin reductase Ascorbate was omitted from the control system Reduction was initiated by the addition of ascorbate Other conditions were the same as those for the NADPH/DmDCPR system Reaction of hemin bound to DmDHO by NADPH/DmDCPR or sodium ascorbate in the presence of desferrioxamine Spectral changes were recorded at 30 °C between 350 and 750 nm We used three electron donor systems, NADPH/ DmDCPR, ascorbate, and H2O2 The standard reaction mixture for the NADPH/DmDCPR system consisted of 10 lM DmDHO–hemin complex, 0.22 lM DmDCPR and mM desferrioxamine in a final volume of 1.5 mL 50 mM potassium phosphate buffer (pH 7.4) After preincubation, the reaction was started by the addition of 20 lL 10 mM NADPH (final concentration, 0.13 lM) The ascorbate reaction mixture contained 10 lM DmDHO–hemin complex and mM desferrioxamine in a final volume of 1.5 mL 50 mM potassium phosphate buffer (pH 7.4) After preincubation, the reaction was initiated by the addition of 20 lL M sodium ascorbate (final concentration, 13 lM) The H2O2 system consisted of 10 lM DmDHO– Ó FEBS 2004 1716 X Zhang et al (Eur J Biochem 271) hemin complex in a final volume of 1.5 mL 50 mM potassium phosphate buffer (pH 7.4) After preincubation, the reaction was started by the addition of H2O2 in water (final concentration 36 lM or 300 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 absorption coefficient at 240 nm [34] EPR spectroscopy EPR measurements were performed using 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–DmDHO complex was prepared by adding dithionite to the argon-saturated protein solution, containing Na15NO2, in an EPR tube Detection of CO To detect CO produced during the DmDHO reaction supported by NADPH/DmDCPR, myoglobin (H64L), a mutant with high affinity for CO [35], was used The reaction solutions contained 16 lM hemin–DmDHO complex, 1.6 lM DmDCPR, and 300 lM NADPH in 1.5 mL 50 mM potassium phosphate buffer (pH 7.4) Myoglobin mutant H64L, at a final concentration of 7.5 lM, was included in the test solution After the addition of NADPH to both cuvettes, the spectrum was recorded at intervals between 350 and 750 nm HPLC analysis of DmDHO reaction products The DmDHO reaction products of either NADPH/ DmDCPR or ascorbate were directly subjected to a Supelclean LC-18 solid-phase extraction column, preconditioned with 400 lL acetonitrile followed by 400 lL 0.1 M Tris/HCl buffer (pH 7.4) The product of the DmDHO reaction with H2O2 was loaded on the same column after hydrolytic conversion into biliverdin The column was washed with acetonitrile/water (1 : 9, v/v), and green pigment was then eluted with acetonitrile/water (1 : 1, v/v) This was lyophilized, and the residue dissolved in 5% HCl/methanol for esterification at °C overnight Water was added to the esterified product, and green pigment was extracted with chloroform The chloroform solution was washed with water and then analyzed by HPLC on a column of Capcell Pak C18 (SG 120, 4.6 · 150 mm) preequilibrated with degassed acetonitrile/water (3 : 2, v/v) at a flow rate of mLỈmin)1 The eluate was monitored at 310 nm The biliverdin dimethyl ester standards were eluted in the order biliverdin IXa (18.2 min), IXd (19.7 min), IXb (21.1 min), and IXc (31.1 min) protein concentrations by the Lowry method using BSA as standard [38] Results and Discussion Characterization of DmHO deduced from the nucleotide sequence of cDNA Both the full length (DmHO) and truncated (DmDHO) enzymes were obtained from adult D melanogaster polyArich RNA by the RT-PCR method The deduced amino acid sequences were the same as reported originally in the SwissProt Database (Q9VGJ9) with one exception; position 50 was not isoleucine but phenylalanine We think that Phe50 is more likely to be correct because: (a) Phe50 was coded in our three DNA fragments obtained by PCR using different template cDNA which was independently synthesized with oligo(dT), random, and gene-specific primers, respectively; (b) Phe37 in mammalian HO-1, which corresponds to Phe50 in DmHO, has an important role in the interaction with the a-meso edge of heme [39,40] and is conserved at the corresponding position of most HOs isolated from other species Sequence comparisons by FASTA searching show that DmHO is 32.4% and 30.3% identical in amino acid sequence with rat HO-1 and rat HO-2, respectively (Fig 1) Sequence alignment analysis indicated that DmHO contains a large catalytic domain at the N-terminus and a small hydrophobic domain at the C-terminus This structure is similar to mammalian HOs but different from bacterial, algal, and cyanobacterial HOs which lack the hydrophobic domain Moreover, the Swiss-model project (first approach mode) suggests that the overall structure of DmHO is similar to that of mammalian HO-1 In rat HO-1, His25 works as the proximal ligand of heme iron The His39 residue of DmHO corresponds to His25 of rat HO-1 and therefore is likely to be the proximal ligand The crystal structure of human HO-1 [39] shows that Thr21, Glu29 and Phe207 are on the proximal side of the heme In DmHO, Thr35 corresponding to Thr21 of HO-1 is conserved, but the other two amino acid residues are not The crystal structure of HO-1 also shows that the backbone atoms of the two glycine residues, Gly139 and Gly143, which are highly conserved among the known sequences of HOs, directly contact the heme [39,40] In the DmHO sequence, Gly143 is present, but Gly139 is replaced by alanine, as in the sequence of Arabidopsis HO [17] Site-directed mutagenesis studies revealed that Asp140 is involved in the oxygen activation mechanism in mammalian HO-1 [41,42], but this amino acid residue is not found in DmHO These features of DmHO suggest that, although the ternary structure of DmHO is similar to that of mammalian HO-1, the structure of the heme pocket is somewhat different Other procedures Sequence translation and sequence alignment were performed using the WISCONSIN PACKAGE from the Genetic Computer Group (Madison, WI, USA) and CLUSTAL W multiple sequence alignment program at the EBI (EMBLEBI) H64L protein, a mutant of myoglobin, was purified by published methods [36] Hemin concentrations were measured by the method of Paul & Theorell [37], and Expression and purification of DmHO and DmDHO To obtain the full length and truncated forms of recombinant DmHO, two expression plasmids, pMWDmHO and pMWDmDHO, were constructed E coli strain BL21(DE3) transformed with pMWDmHO expressed a protein in the membrane fraction which gives a strong 34 kDa band on SDS/PAGE In contrast, E coli harboring pMWDmDHO Ó FEBS 2004 D melanogaster heme oxygenase (Eur J Biochem 271) 1717 Fig Amino acid sequence of DmDHO compared with reported DmHO, rat HO-1 and rat HO-2 * indicates positions that have a single, fully conserved residue : indicates that one of the ÔstrongÕ groups is fully conserved indicates that one of the ÔweakerÕ groups is fully conserved expressed a soluble 32 kDa protein mainly in the soluble fraction Molecular sizes of 34 kDa and 32 kDa are in agreement with the calculated values of 34 112 Da for full length DmHO and 31 777 Da for DmDHO These observations indicate that the C-terminal hydrophobic sequence composed of 21 amino acid residues acts as an anchor to membranes, similar to rat HO-1 [43,44] The truncated forms of mammalian HOs, in which the hydrophobic membrane-binding domains are removed, fully retain hemedegrading activity [2,3] Therefore, we presumed that the truncated form of DmHO also retains its activity As described below, DmDHO is a soluble, catalytically active protein and therefore we used only DmDHO in this study The expression of DmDHO by culturing the transformed E coli cells at 37 °C resulted in an accumulation of the expressed protein mostly in inclusion bodies However, culturing the transformed bacteria at 37 °C then 20 °C as described in Experimental procedures increased significantly the yield of the recombinant protein in the soluble fraction Expression of DmDHO, however, did not turn the culture medium green This phenomenon is distinct from that observed for E coli cells expressing mammalian, bacterial, and cyanobacterial HOs and raises questions about the heme-degrading activity of DmDHO We purified the expressed DmDHO from the soluble fraction by ammonium sulfate fractionation and subsequent column chromatography on Sepahdex G-75, DE-52 and hydroxylapatite The purified DmDHO gave a 32 kDa band with  95% purity on SDS/PAGE (lane in Fig 2) About 25 mg protein was obtained from L culture Expression and purification of DmDCPR Cultured E coli cells transformed with pMWDmDCPR were light yellow caused by constitutive flavins of DmDCPR During purification, we used this color along with the 72 kDa band on SDS/PAGE for detecting Fig SDS/PAGE of the purified DmDHO and DmDCPR Lane 1, Molecular mass marker; lane 2, lg purified DmDHO; lane 3, lg purified DmDCPR DmDCPR The purification procedures involved ammonium sulfate fractionation and column chromatography on DE-52 and 2¢5¢-ADP Sepharose 4B The purified fraction showed a single band of 72 kDa (lane in Fig 2), similar to the calculated value of 71 740 Da The 2,6-dichloroindophenol-reducing activity of purified DmDCPR was similar Ó FEBS 2004 1718 X Zhang et al (Eur J Biochem 271) to that of purified rat CPR About 10 mg protein was obtained from L culture Catalytic activity of DmDHO As mentioned above, expressed DmDHO in E coli cells did not turn the color of host cells green, so that we measured the ability of DmDHO to catalyze the conversion of hemin into biliverdin in vitro We used NADPH/DmDCPR and ascorbate as reducing reagents and biliverdin IXa reductase to reduce the biliverdin IXa produced by DmDHO to bilirubin Table suggests that DmDHO does degrade hemin to biliverdin IXa in the presence of each of the reducing systems, indicating that DmHO of fruit fly is a real HO Interestingly, the specific activity of heme breakdown was very low, almost one-quarter that with ascorbic acid, although DmDCPR equivalent to one-fifth of DmDHO was used In the case of rat HO-1, despite using rat CPR equivalent to about one-thirtieth of rat HO-1, the activity of heme degradation was half that seen in the ascorbate system [45] This suggests that effective electron transfer does not occur from DmDCPR to DmDHO With rat HO-1, heme breakdown to biliverdin in the presence of ascorbate is accelerated by desferrioxamine, a ferric iron chelator, because in that system the final product is not biliverdin but its precursor, ferric biliverdin, bound to HO-1 protein [46] Therefore, we assayed DmDHO activity Table Activities of purified DmDHO HO activity was determined from the initial rate of bilirubin formation with NADPH/DmDCPR or sodium ascorbate systems in the absence/presence of desferrioxamine and the presence of biliverdin reductase All measurements were performed in triplicate Values are mean ± SD Bilirubin formation [nmolỈ(mg protein))1Ỉh)1] Reducing system –Desferrioxamine +Desferrioxamine NADPH/DmDCPR Sodium ascorbate 32 ± 1.2 138 ± 250 ± 543 ± 10 in converting heme into bilirubin via biliverdin IXa in the presence of desferrioxamine As a result, the conversion activities with addition of either NADPH/DmDCPR or ascorbate increased by about eightfold and fourfold, respectively This suggests that spontaneous iron release from the ferric biliverdin–DmDHO complex in both systems is slow The specific activity of DmDHO was highest in the ascorbate system in the presence of desferrioxamine but still only about one-quarter that of rat HO-1 As described below, HPLC analysis showed that 75% of the total biliverdin produced by DmDHO is the IXa isomer As biliverdin IXa reductase has a preference for the a-isomer as substrate [47], the total yield of biliverdin is significantly underestimated if measured as the amount of bilirubin IXa eventually formed Recently it was reported that coupled oxidation of myoglobin with ascorbic acid is mediated by exogenous peroxide generated by reaction of ascorbate with oxy-myoglobin, because the reaction is inhibited by catalase [48] In the case of DmDHO, inclusion of 10 lM catalase had no effect, clearly showing that the DmDHO reaction does not depend on exogenous peroxide Properties of the heme–DmDHO complex All HOs so far reported bind heme stoichiometrically to form stable complexes with absorption spectra resembling those of myoglobin Like other HOs, DmHO also binds hemin to form a : stoichiometric complex (inset of Fig 3) To isolate this complex, we added excess (twofold) hemin to DmDHO and chromatographed the mixture on DE-52 or hydroxylapatite However, we obtained only DmDHO without hemin, indicating weak binding of hemin to DmDHO In fact, from the hemin titration, we obtained a value of 27 ± lM for the heme dissociation constant (Kd) This value is significantly higher than those for HmuO (2.5 ± lM) [12] and human HO-1 (0.84 ± 0.2 lM) [49] Figure shows the optical absorption spectra of purified DmDHO titrated with molar equivalent of hemin The ferric form of the DmDHO–heme complex has a broad Soret band with a peak at 390 nm and a smaller peak at 602 nm (solid line) As previously reported, the ferric heme Fig Absorption spectra of various forms of the DmDHO–heme complex ––, oxidized form; ỈỈỈỈ, reduced form; - - -, CO-bound form Inset, difference titration of DmDHO with hemin Precise procedures are described in Experimental procedures The increments in absorbance as the difference at 412 nm were plotted, because the difference was maximum at this wavelength Ó FEBS 2004 iron in the rat HO-1–hemin complex at neutral pH is sixcoordinate, high spin, and the Soret maximum undergoes a red shift with increasing pH, having an apparent pKa value of 7.6 [50] In contrast, the Soret maximum of the DmDHO– hemin complex was not influenced by increasing the pH to 10.0 We assumed that the ferric heme iron of the complex was not in the six-coordinate state, presumably lacking the water molecule at the distal site To confirm this, we carried out an EPR study As shown in Fig 4, the EPR spectrum of the hemin–DmDHO complex exhibits a highly rhombic, highspin state of hemin, showing pronounced difference from that of the hemin complex of cyanobacterial heme oxygenase isoform-1, Syn HO-1, which was determined to be in a six-coordinate high-spin state with a distal water molecule and a proximal histidine [16] The lower field feature of the Fig EPR spectra of the ferric DmDHO and Syn HO-1 complexes at neutral pH Both spectra were obtained at K, applied field modulation frequencies, 100 kHz, field modulation amplitude, 10 G, and microwave power, mW D melanogaster heme oxygenase (Eur J Biochem 271) 1719 Table Optical absorption data for the heme–DmDHO complex kmax (Soret) (emM)1Ỉcm)1) Ferric form Ferrous deoxy form CO form Oxy form kmax (visible) 390 428 420 410 602 559 538, 568 537, 575 (70) (80) (163) (72) spectrum further suggests that the ligand field around the hemin molecule is inhomogeneous, implying that orientation of hemin in the DmDHO heme pocket is unequal The highly rhombic feature of the ferric heme–HO complexes is common to the point-mutated HOs of proximal histidine (data not shown) and to the five-coordinated a-hydroxyhemin complex of HO-1 [2] Contrary to the sequencebased expectation, the spectrum of the ferrous 15NO-bound heme–DmDHO complex is typical of penta-coordinated 15 NO–heme complexes, differing from that of Syn HO-1, which is a hexa-coordinate 15NO–heme complex exhibiting the triplet hyperfine splitting due to the nuclear spin of one of the nitrogen nucleus of an imidazole of a histidine residue trans to the 15NO [16] (Fig 5) The hemin–DmDHO complex in alkaline solution (pH 10.0) does not show the spectrum of a typical hydroxide-coordinated low-spin form (data not shown) This is in accord with both the result of optical spectra and the revealed coordination structure of hemin without proximal histidine Accordingly, EPR results identify the hemin in the DmDHO complex to be uncoordinated by the protein residue, which is markedly different from other known hemin–HO complexes Reduction of the ferric heme with sodium dithionite under nitrogen gas yielded a ferrous form with a Soret band at 428 nm and a small peak at 559 nm (Fig 3, dotted line) After introduction of CO, the ferrous form changed to a ferrous–CO form with a Soret maximum at 420 nm and two small peaks at 538 and 568 nm in the visible region (Fig 3, broken line) To exchange the gas phase in the solution, the solution was quickly passed through a spin column of Sephadex G-25 in air The resulting solution exhibited a new spectrum with a Soret peak at 410 nm and two small peaks at 538 nm and 575 nm (data, not shown), indicating that the CO form was converted into the oxy form This oxy form was gradually turned into a ferric complex with an auto-oxidation rate (Kobs) of 3.5 · 10)3 s)1 This value is much higher than that of rat HO-1 (0.14 · 10)3 s)1) and comparable to those of some mutants, in which the hydrogen-bonding network to stabilize oxygen bound to iron is thought to be weak [42] Table shows optical absorption data for the heme– DmDHO complex Reaction of hemin bound to DmDHO by NADPH/DmDCPR or sodium ascorbate in the presence of desferrioxamine Fig EPR spectra of the ferrous 15NO-bound forms of the heme– DmDHO and Syn HO-1 complexes Both spectra were obtained at 20 K, applied field modulation frequencies, 100 kHz, field modulation amplitude, G, and microwave power, 0.2 mW As desferrioxamine increased biliverdin formation from hemin in both the NADPH/DmDCPR and ascorbate systems by facilitating the release of iron from the ferric biliverdin–DmDHO complex, we measured the degradation of hemin bound to DmDHO in the presence of desferrioxamine As depicted in Fig 6A, the addition of NADPH 1720 X Zhang et al (Eur J Biochem 271) Ó FEBS 2004 NADPH/DmDCPR, consistent with the results from the catalytic activity assay This spectrum also has small peaks around 537 and 575 nm attributable to the oxy complex The spectrum recorded 30 after the initiation of the reaction (broken line) shows two broad bands centered near 380 and 670 nm, indicative of slow biliverdin formation Again, after addition of biliverdin reductase and NADPH, a decrease in absorbance around 670 nm and concomitant increase near 460 nm were observed (solid line II) Comparison between the spectral intensities at 670 nm of the final product of both reducing systems suggests that about twice as much biliverdin is formed in the ascorbate system as in the NADPH/DmDCPR system We think that this is partly due to CPR-mediated heme degradation leading to nonbiliverdin products [51,52] In fact, we observed that about 40% of hemin was lost 120 after incubation of 2.6 lM hemin with 1.2 lM DmDCPR and 300 lM NADPH at 30 °C As the affinity of heme for DmDHO is low, some of the substrate may be degraded by DmDCPR, making it unavailable for the DmDHO reaction Reaction of the hemin bound to DmDHO by sodium ascorbate under O2 and CO Fig Reaction of hemin bound to DmDHO by NADPH/DmDCPR or sodium ascorbate in the presence of desferrioxamine (A) –– I, spectrum of the complex of hemin and DmDHO; ỈỈỈỈ, spectrum 10 after the addition of NADPH to start the reaction; - - -, 120 after the reaction; –– II, after the addition of biliverdin reductase Inset, enlarged spectra between 450 and 750 nm (B) –– I, spectrum of the complex of hemin and DmDHO; ÆÆÆÆ, spectrum after the addition of ascorbate to start the reaction; - - -, 30 after the reaction; –– II, after the addition of biliverdin reductase and NADPH Inset, enlarged spectra between 450 and 750 nm to the reaction solution (solid line I) initiated heme degradation The spectrum recorded after 10 (dotted line) shows a red-shifted Soret maximum and two peaks at 538 and 575 nm in the visible region, indicating formation of an oxy form Formation of the oxy form is faster than its further degradation reaction, and loss of the Soret band was relatively slow The spectrum recorded after 120 (broken line) has a broad absorption centered at 670 nm, showing the conversion of hemin into biliverdin This is supported by a decrease in absorbance around 670 nm and concomitant increase near 460 nm due to bilirubin after addition of biliverdin reductase (solid line II) We also measured the spectral change in ascorbic acidsupported heme degradation in the presence of desferrioxamine The spectrum in Fig 6B recorded after the addition of sodium ascorbate (dotted line) shows that heme degradation proceeded faster than in the presence of With rat HO-1, hemin bound to enzyme is converted into ferrous–CO forms of verdoheme under O2 and CO, and the reaction stops at this stage because CO stops the further reaction of verdoheme to ferric-biliverdin [53] To detect the ferrous–CO forms of the verdoheme–DmDHO complex, we carried out similar experiments The spectrum (dotted line in Fig 7) recorded after the start of the reaction has three peaks at 538, 568, and 602 nm in the visible region; the former two peaks are due to the CO-bound form and the latter peak to the ferric form of the hemin–DmDHO complex However, we were unable to detect a peak around 640 nm attributable to the ferrous–CO form of verdoheme The broken line is a spectrum recorded 40 after the start of the reaction Again, absorption around 640 nm was not observed, but broad absorption in the red region increased, indicating biliverdin formation DmDHO shares several mechanistic features with other HOs, including CO formation, and therefore we believe that verdoheme is an intermediate in the DmDHO reaction We assume that verdoheme formation from the oxy form of the heme– DmDHO complex is slower than conversion of verdoheme into ferric biliverdin, which frustrates detection of the ferrous–CO form of verdoheme Reaction of the hemin–DmDHO complex with H2O2 In mammalian HO-1, a ferric hydroperoxy species is an active intermediate in the first oxygenation step [54–56] H2O2 hydroxylates heme at the a-meso position to form a-meso-hydroxyhemin, which is then converted into verdoheme in the presence of O2 [57] Therefore, we investigated whether H2O2 can support the conversion of hemin bound to DmDHO to verdoheme On addition of 3.6 molar equivalents of H2O2, the Soret band at 390 nm decreased gradually accompanied by a very small increase around 685 nm (data not shown) When 30 equivalents of H2O2 were used, a rapid decrease in the Soret band was observed However, the intensity of the absorption of the final reaction Ó FEBS 2004 D melanogaster heme oxygenase (Eur J Biochem 271) 1721 Fig Reaction of hemin bound to DmDHO by sodium ascorbate under O2 and CO ––, spectrum of the complex of hemin and DmDHO; ỈỈỈỈ, spectrum after the addition of ascorbate to start the reaction; - - -, 40 after the start of the reaction Inset, enlarged spectra between 500 and 800 nm product around 685 nm was almost the same as when 3.6 equivalents were used HPLC analysis showed that the amount of biliverdin formed was only 0.15% of that formed in the ascorbic acid/desferrioxamine-supported system (Fig 8C) These observations suggest that H2O2 oxidized DmDHObound heme to fragmentation products rather than to verdoheme Presumably, in the first stage of the DmDHO reaction, a hydroperoxy species is the active oxygen species, by analogy with mammalian HO-1, and this species is formed by binding of H2O2 to the ferric iron of the hemin– DmDHO complex We not know why verdohemochrome is not formed in the H2O2-supported DmDHO reaction Interestingly, a mutant of human HO-1, D140A, has similar properties [41] Detection of CO during the DmDHO 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/ DmDCPR-supported reaction The Soret band of myoglobin was monitored at 4-min intervals after the addition of NADPH to both the sample and reference cuvette As depicted in Fig 9, the myoglobin Soret band shifted from 393 to 425 nm with the appearance of a/b bands at 568 and 538 nm and A425 increased, indicating reduction of the ferric form of myoglobin to the ferrous form by the NADPH/DmDCPR system This was followed by CO binding to yield the ferrous–CO form, the authentic absorption spectrum of which is depicted in inset of Fig This experiment clearly demonstrates CO formation during heme degradation by DmDHO HPLC analysis of the DmDHO reaction products HPLC analysis showed that the biliverdin formed in both the NADPH/DmDCPR/desferrioxamine and ascorbic acid/desferrioxamine systems contained three isomers, Fig HPLC analysis of the reaction products of hemin bound to DmDHO The product analysis with HPLC was described in Experimental procedures (S) Standard mixture of biliverdin IXa, IXb, IXc and IXd dimethyl esters; (A–C) esterified products from NADPH/ DmDCPR, sodium ascorbate, and H2O2 systems, respectively Ó FEBS 2004 1722 X Zhang et al (Eur J Biochem 271) Fig Detection of CO produced during DmDHO reaction The sample solution contained hemin–DmDHO complex, DmDCPR and H64L mutant of myoglobin Myoglobin was omitted from the reference solution The reaction was started by the addition of NADPH to both solutions The difference spectrum was recorded at 4-min intervals Inset: absorption spectra of various forms of H64L mutant of myoglobin ––, oxidized form; ỈỈỈỈ, reduced form; - - -, CO bound form IXa, IXb, and IXd, accounting for  75%, 16% and 8% of the total, respectively (Fig 8) This is unusual, because other HOs exclusively generate biliverdin IXa, except for Pig A of Pseudomonas aeruginosa, which forms both biliverdin IXb and IXd [14] The crystal structure of human HO-1 reveals a distal helix spanning the entire width of the heme, which sterically prevents access of the iron-bound hydroperoxy species to the b-meso, c-meso, and d-meso carbon atoms [39] Thus, the iron-bound hydroperoxy species can oxygenate only the a-mesocarbon of heme, leading to the exclusive a-meso-hydroxyheme formation The formation of three isomers of biliverdin by DmDHO implies that its heme pocket has a different structure from those of mammalian HO-1 and other a-specific HOs The EPR result suggesting the existence of several types of hemin conformation in the protein pocket is consistent with this non a-specific production of biliverdin We expected that DmDHO would produce the c-isomer of biliverdin because biliverdin IXc is present in some species of Lepidoptera However, we detected only trace amounts of the c-isomer in our in vitro studies of the soluble recombinant enzyme Concluding remarks We cloned a cDNA for D melanogaster homologous to mammalian HOs and constructed a bacterial expression plasmid of a truncated, soluble enzyme, DmDHO Purified recombinant DmDHO forms an enzyme–substrate complex with a stoichiometric amount of heme and catalyzes heme degradation to biliverdin isomers, CO and iron, although the specific activity was very low, in the presence of appropriate reducing systems These features are similar to those of other HOs, indicating that DmDHO is a true heme oxygenase in fruit fly Despite these similarities, DmDHO is distinctly different from other HOs (a) Unlike other HOs, the hemin– DmDHO complex is not in the six-coordinate high-spin state with a histidine residue as the proximal ligand and the iron of heme was not involved in forming the heme– DmHO complex (b) H2O2 does not support DmDHOdependent degradation of heme to verdoheme (c) CO– verdoheme cannot be detected during the catalytic reaction under oxygen and CO (d) In the final stage of the reaction, iron release from the ferric biliverdin–DmDHO complex is slow (e) The hemin catabolism of DmDHO is not a-specific and yields three isomers of biliverdin, IXa, IXb, and IXd Accordingly, we infer that the structure and hydrogen bonding of the DmHO active site is quite different from those of other HOs It is interesting to note that DmDHO was able to degrade heme to biliverdin, in spite of no direct binding of heme iron to the enzyme A similar but not identical observation was reported for a mutant of HmuO The H20A mutant in which His20 was replaced by Ala degraded hemin to verdoheme, a second intermediate of heme degradation [58] Further investigation of the structure to understand the mechanism of heme breakdown is needed Acknowledgements The bacterial expression vector pMW172 was a gift from Dr K Nagai, MRC Laboratory of Molecular Biology, Cambridge, UK The expression plasmid for the myoglobin mutant, H64L was a gift from Professor J S Olson, Rice University We thank Dr A F McDonagh, University of California, San Francisco, for helpful comments on the manuscript This work was supported in part by grants-in-aid from the 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structure from those of mammalian... peroxide Properties of the heme? ??DmDHO complex All HOs so far reported bind heme stoichiometrically to form stable complexes with absorption spectra resembling those of myoglobin Like other HOs, DmHO... spectrum of the hemin–DmDHO complex exhibits a highly rhombic, highspin state of hemin, showing pronounced difference from that of the hemin complex of cyanobacterial heme oxygenase isoform-1,