Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean) ) coordination structure of the heme complex and catabolism of heme Tomohiko Gohya1, Xuhong Zhang2, Tadashi Yoshida2 and Catharina T Migita1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan Department of Biochemistry, Yamagata University School of Medicine, Japan Keywords ferredoxin; heme catabolism; heme complex; higher-plant heme oxygenase; spectroscopic characterization Correspondence C T Migita, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan Fax ⁄ Tel: +81 83 9335863 E-mail: ctmigita@yamaguchi-u.ac.jp T Yoshida, Department of Biochemistry, Yamagata University School of Medicine, Iidanishi 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 August 2006, revised October 2006, accepted October 2006) doi:10.1111/j.1742-4658.2006.05531.x Heme oxygenase converts heme into biliverdin, CO, and free iron In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IXa as a direct synthetic resource In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion The similarity in the amino acid sequence between GmHO-1 and heme oxygenases from other biological species is low, and GmHO-1 binds heme with : stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid– base transition and is converted to biliverdin IXa in the presence of NADPH ⁄ ferredoxin reductase ⁄ ferredoxin, or ascorbate During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme–heme oxygenase or CO–verdoheme–heme oxygenase complexes was observed and was extracted as a bis-imidazole complex; it was identified as verdoheme A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence of NADPH ⁄ ferredoxin reductase ⁄ ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system Heme oxygenase (HO, EC 1.14.99.3) catalyzes the conversion of heme to biliverdin IXa, CO and free iron through successive reduction and oxygenation reactions in the presence of molecular oxygen and elec- trons supplied by NADPH Studies on the structure and function of HO have been conducted mostly in mammalian enzymes, as HO was first identified in mammals [1–3] During the last decade, the HO genes Abbreviations AtHO-1, heme oxygenase isoform of Arabidopsis thaliana; BVR, biliverdin reductase; CPR, cytochrome P450 reductase; Fd, plant ferredoxin; FNR, ferredoxin:NADP+ reductase; GmHO-1, heme oxygenase isoform of Glycine max; heme, iron protoporphyrin IX, either ferrous or ferric forms; hemin, ferric protoporphyrin IX; HO, heme oxygenase; hydroxyheme, iron meso-hydroxyl protoporphyrin IX; KPB, potassium phosphate buffer; rHO-1, heme oxygenase isoform of Rattus norvegicus; SynHO-1, heme oxygenase isoform of Synechocystis sp PCC 6803 5384 FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al have been identified in a wide range of biological species, especially in pathogenic bacteria, and some of them have been expressed and characterized [4–6] Higher-plant HOs, however, have not been investigated on a molecular basis by applying multiple spectroscopic methods to the purified protein, and are now the least studied HOs HO in plants is one of the plastid enzymes participating in phytochromobilin synthesis This enzyme catalyzes the cleavage of heme into biliverdin IXa, which is then reduced and isomerized to form (3E)phytochromobilin, a chromophore of the photoreceptor protein of the phytochrome family, which plays critical roles in mediating photomorphogenesis, by sensing far-red and red light [7] HO genes of higher plants have been identified in a few moss plants, several angiosperms (tobacco, tomato, pea, soybean, rice plant, sorghum, Arabidopsis thaliana), and a gymnosperm (loblolly pine) [8] So far, the HY1 gene and the HO3 and HO4 genes of Arabidopsis have been expressed in Escherichia coli, and Cd-induced expression of HO-1 in soybean leaves has also been reported [8–11] In the latter study, it was suggested that plant HOs also play a role in protection against oxidative Heme catabolism by soybean heme oxygenase-1 cell damage [11] More recently, the PsHO1 gene of pea was expressed, and the HO activity of the protein product was examined [12] These studies have shown that the obtained proteins bind heme to generate a : complex, and CO and biliverdin IXa are generated through heme catabolism, thereby confirming HO activity However, characterization of the heme complexes on a molecular basis and determination of the kinetics of heme catabolism have not been performed yet The amino acid sequences reported for higher-plant HOs are highly homologous to each other; for example, soybean (Glycine max) HO isoform-1 (GmHO-1) has 71.7% homology to A thaliana HO-1 (AtHO-1), and HOs from other plant species have similar levels of homology On the contrary, the homology in amino acid sequences between plant HOs and HOs from other biological species is quite low, e.g 21% to cyanobacterial HO-1 (Synechocystis sp PCC 6803) (SynHO-1), 22% to rat HO-1 (rHO-1), 23% to corynebacterial HmuO, or 21% to neisserial HemO Comparison of the sequence alignment reveals that the catalytically pivotal residues, Gly139 and Asp140, of human HO-1 (and also rHO-1) are replaced by Ala and His, respectively, Fig Amino acid sequence of GmHO-1 as compared with the sequences of Arabidopsis, Synechocystis and rat HO-1s The lightly shaded letters indicate residues with sequence identity, and heavily shaded histidine residues are proximal heme ligands Bars below the alignments show a-helical parts (AH) in the crystal structures of heme–SynHO-1 and heme–rHO-1 and those presumed for heme–GmHO-1 FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS 5385 Heme catabolism by soybean heme oxygenase-1 T Gohya et al in GmHO-1 (Fig 1), although the residues comprising the distal F-helix part are relatively well conserved in the whole sequence of GmHO-1 In mammalian HO-1, Gly139 directly contacts with heme, and Asp140 is known to be a key residue for enzymatic activity [3,13] In addition, the proximal residue for the substrate heme binding, His25 in rHO-1 (also in human HO-1), is replaced by Lys, and only one His in the corresponding distal A-helix part occupies a position 13 residues away from the N-terminus Moreover, the Arg183 residue of mammalian HO-1, which participates in a-mesospecific heme decomposition, is not conserved (Leu in GmHO-1) [14] Thus, our first concern in examining plant HOs is to determine whether the plant HO lacking the residues critical for HO activity catalyzes heme degradation in a similar fashion to the mammalian enzymes The next concern is to establish the mechanism of electron transfer from NADPH to GmHO-1 Muramoto et al reported that the AtHO-1 reaction required additional reductant besides ferredoxin reductase (FNR; ferredoxin:NADP+ reductase; EC 1.27.1.2) ⁄ ferredoxin (Fd) and NADPH [9] On the other hand, we have clarified that cyanobacterial SynHO-1 (and also HO-2) shows full activity when coupled with NADPH ⁄ FNR ⁄ Fd, without a secondary reductant, which had been suggested to be necessary for the HO activity of cyanobacterial proteins [15–17] Then, we wanted to determine whether the NADPH ⁄ FNR ⁄ Fd reducing system works fully in the heme conversion into biliverdin by GmHO-1 as in the SynHO-1 reaction To investigate these phenomena, we purified the recombinant mature form of GmHO-1 protein, excluding the plastid transit peptides, based on the reported amino acid sequence [8] by constructing a bacterial expression system Spectroscopic analyses of the molecular features of the heme–GmHO-1 complex and of the mechanism of heme degradation were performed, and the results were compared with those for the heme complexes of SynHO-1 and rHO-1 We found that, in spite of the low homology of the amino acid sequence with those of known HOs, the heme– GmHO-1 complex has similar spectroscopic characteristics to those of the heme complexes of cyanobacterial, mammalian or bacterial HOs [15,18,19] GmHO-1 converts combined heme into biliverdin IXa, retaining a-regiospecificity, and releasing CO and free iron, in the presence of oxygen and NADPH ⁄ FNR ⁄ Fd, without requiring additional reducing agents, albeit the coordination structure of the verdoheme intermediate is apparently different from that of the known verdoheme–HO complexes 5386 Results Expression and purification of GmHO-1 By culturing the cells at two temperatures, first at 37 °C and then at 25 °C, we avoided the accumulation of inclusion bodies of GmHO-1 The harvested cells were brown in color, unlike the cells expressing rHO-1 or SynHO-1, which were greenish due to the accumulated biliverdin; nevertheless, the E coli cells expressed active GmHO-1, as will be described later It has been reported that the E coli cells expressing the HY1 gene encoding AtHO-1 have a yellowish-brown tinge [9] We purified the GmHO-1 from the soluble fraction by ammonium sulfate fractionation and subsequent column chromatography on Sephadex G-75 and DE-52 The ammonium sulfate fraction and active G-75 fractions were tinged with yellow We not know the nature of this yellow substance(s) at present The final preparation after chromatography on a DE-52 column was clear and colorless, and gave a single band of 26 kDa with about 97% purity on SDS ⁄ PAGE, the size expected from the deduced GmHO-1 amino acid sequence (26.1 kDa) About 100 mg of protein was obtained from L of culture Spectroscopic features of the heme–GmHO-1 complex The optical absorption spectra of the heme–GmHO-1 complex in the ferric, ferrous, CO-bound and O2-bound forms are typical of heme proteins and similar to those of the SynHO-1 or rHO-1 complexes (Fig 2) The stoichiometry of the heme binding was confirmed to be : by the titration plots shown in the inset The optical absorption data for heme– GmHO-1, together with those of SynHO-1 and rHO-1, are summarized in Table The absorption maxima of the O2-bound and CO-bound forms of heme–GmHO-1 are slightly red-shifted compared with those of heme– SynHO-1 and heme–rHO-1 The EPR spectrum of heme–GmHO-1 at pH 7.0 shows the heme mostly in the rhombic ferric high-spin state, with gx ¼ 5.95, gy ¼ 5.68 and gz ¼ 2.00 (Fig 3A) Here, anisotropy of the gxy component is apparently larger than that of heme–rHO-1, as shown in the partly expanded spectra (a-1 in Fig 3), indicating that in-plane anisotropy of heme is relatively large In addition, small amounts of low-spin species are also observed in the neutral solution, as distinctly seen in the partly expanded spectrum (a-2 in Fig 3) The EPR spectrum of the 15 NO-bound GmHO-1 (nitrosylheme GmHO-1) is characteristic of six-coordinate heme proteins with the FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al Heme catabolism by soybean heme oxygenase-1 Fig Absorption spectra of the various forms of heme–GmHO-1 Spectra are of the ferric (red), ferrous (blue), ferrous–CO (black) and ferrous–oxy (green) forms Inset: titration plots of GmHO-1 (4.8 nmol) with hemin (0.4 mM), monitored by the increase in absorbance at 405 nm The pink off-line dots indicate the results for the titration without protein Table Optical absorption data of the heme–HO-1 complexes Protein Types of heme Ferric e (mM)1Ỉcm)1) Ferrous Oxy CO-bound Alkaline pKa GmHO-1 kmax (nm) SynHO-1 kmax (nm) rHO-1 kmax (nm) Soret Visible Soret Visible Soret Visible 405 127 428 415 420 414 8.2 500, 630 402 128 557 427 541, 578 410 539, 569 427 539, 577 427 8.9 498, 631 404 140 555 431 537, 574 410 536, 566 419 537, 575 414 7.6 500, 631 554 540, 575 535, 568 540, 575 nitrogenous proximal ligand, indicating hyperfine splitting due to a 14N nucleus (nuclear spin 1, giving the triplet splitting) in addition to a 15N nucleus (nuclear spin ⁄ 2, giving the doublet splitting) at the g2 component (Fig 3C) This strongly suggests coordination of a histidinyl residue to the proximal site of the heme The spectral features of 15NO-heme–GmHO-1 are somewhat different from those of the nitrosylheme complexes of SynHO-1 (Fig 3D) and rHO-1 (Fig 3E), whereas those of the latter two are very alike The EPR parameters of the nitrosylheme–HO complexes as well as those of the low-spin heme–HO complexes are listed in Table Acid–base transition of heme–GmHO-1 The features of the optical absorption spectrum of the ferric heme–GmHO-1 complex reversibly change, Fig EPR spectra of the ferric heme–GmHO-1 (A, B) and nitrosylheme–HO (C–E) complexes EPR conditions were: microwave frequency, 9.35 GHz; microwave power, mW for (A) and (B) and 0.2 mW for (C)–(E); field modulation frequencies, 100 kHz; field modulation amplitude, 10 G for (A) and (B) and G for (C)–(E); signal acquisition temperature, K for (A) and (B) and 25 K for (C)–(E) (A) The ferric heme complex of GmHO-1 at pH 7.0 (0.1 M, KPB) a-1, Expansion of the gxy region of (A) (solid line) and of the corresponding part of the spectrum of ferric heme–rHO-1 (dotted line) a-2, Expansion of the higher-field region of (A) (B) The ferric heme–GmHO-1 complex at pH 8.7 (50 mM, Tris ⁄ HCl) (C–E) The 15 N-nitrosylheme complexes of GmHO-1, SynHO-1, and rHO-1, respectively depending on pH, between acidic (pH 6.5) and alkaline (pH 10.5) conditions The absorption maxima of the alkaline form are listed in Table The pKa value of this acid–base transition was estimated to be 8.2 by the method described in Experimental procedures Such an acid–base transition of heme–GmHO-1 was also observed by EPR The EPR spectrum of heme– GmHO-1 at pH 8.7 exhibited a small high-spin signal at gxy  and prominent peaks of the low-spin heme FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS 5387 Heme catabolism by soybean heme oxygenase-1 Table EPR parameters of the low-spin and heme–HO-1 complexes T Gohya et al 15 NO-bound forms of Protein GmHO-1 SynHO-1 rHO-1 Alkaline Low spin g1 g2 g3 15 NO a (15N), G a (14N), G g1 g2 g3 Neutral Alkaline-1 Alkaline-2 2.63 2.21 1.82 2.86 2.29 1.59 2.78 2.14 1.74 2.68 2.20 1.80 27 7.6 2.09 2.01 1.96 31 7.1 2.08 2.00 1.96 Alkaline 2.67 2.21 1.79 26 7.5 2.08 2.01 1.97 at the lower magnetic fields (Fig 3B) The g-values of this low-spin species were the same as those of the low-spin species observed at neutral pH (denoted as gak in Fig 3A, expanded lower-field spectrum) Accordingly, this species was determined to be the alkaline form of heme–GmHO-1, which coordinates a hydroxyl anion at the distal site of heme Another low-spin species, denoted as g* in Fig 3A, seems to be a denatured form of heme–GmHO-1, because similar low-spin species were sometimes observed for other heme–HO complexes (data not shown) Single species of the alkaline form were observed for heme–GmHO-1, the same as for rHO-1, but distinct from SynHO-1, which exhibits two kinds of alkaline forms (Table 2) The anisotropy in g-values, g ak –g ak , of the alkaline form of heme–GmHO-1 is somewhat smaller than that of the other two HO complexes This might indicate that the axial ligand field is relatively strong in GmHO-1, due to steric control imposed by the distal helix, in accord with the observation that the in-plane anisotropy is large in the ferric state of heme Determination of the proximal ligand Based on the EPR results for nitrosylheme–GmHO-1, candidates for the proximal ligand of heme were searched for in the amino acid sequence of GmHO-1, around the position corresponding to the proximal His of other HOs (Fig 1) His30 was found to be only nitrogenous ligand capable of coordinating to the ferric heme, so the GmHO-1 mutant H30G was prepared, and the optical absorption and EPR spectra of its heme complex were determined The H30G mutant also accommodated heme with : stoichiometry, like other HO mutants that lack the proximal histidine, 5388 Fig EPR spectra of (A) ferric heme and (B) 15N-nitrosylheme complexes of the H30G mutant of GmHO-1 EPR conditions were the same as those described in Fig but the optical spectrum of the heme–H30G complex exhibited an asymmetrically broadened Soret band with the blue-shifted maximum at around 390 nm, compared with the Soret-band features of the wild-type complex, and no other characteristic bands at the visible region (data not shown) Such features of the spectrum are commonly seen in the heme complexes of HO mutants lacking the proximal His [20,21] The heme–H30G complex did not decompose the bound heme enzymatically in the presence of either ascorbate or NADPH ⁄ FNR ⁄ Fd (data not shown) EPR measurements on the heme–H30G complex provided critical evidence for the lack of coordination of a protein residue at the proximal site, yielding the deformed spectrum of high-spin heme (Fig 4A), which means that the heme is in the multiple configuration in the heme pocket, and the spectrum of nitrosylheme is typical of the 15NO coordination without the sixth ligand (Fig 4B) Accordingly, it has been established that the proximal ligand of heme–GmHO-1 is His30 Exogenous ligand binding To investigate the nature of the heme pocket in GmHO-1, the apparent equilibrium constant for binding of nitrogenous ligands, imidazole and azide, to heme–GmHO-1 was evaluated As imidazole was added to the solution of heme–GmHO-1, the Soret FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al Heme catabolism by soybean heme oxygenase-1 Table Equilibrium constants for imidazole and azide ion binding to the heme–HO-1 complexes Numbers in parentheses indicate relative values normalized to the values of GmHO-1 Protein GmHO-1 )1 Kimidazole (M ) Kazide (· 102 M)1) SynHO-1 rHO-1 35 (1) 43 (1) 210 (6) 17 (0.4) 1400 (40) 200 (4.7) band maximum of the complex shifted from 405 to 410 nm and decreased in intensity, without showing a distinct isosbestic point At the same time, the heights of the absorption peaks in the visible region (500 and 630 nm) also decreased, and then new peaks appeared at 531 and 568 nm with increasing intensity, indicating formation of imidazole-bound heme–GmHO-1 (data not shown) The association constant was estimated on the basis of the changes in absorbance at 402 nm (imidazole-free form) and at 422 nm (imidazole-bound form) in the difference spectra of imidazole-free minus imidazole-titrated forms, as described in Experimental procedures In the same way, azide binding to heme–GmHO-1 was also examined In this case, a clear isosbestic point was observed at 412 nm, between the Soret band maxima of the azide-free form at 405 nm and of the azide-bound form at 421 nm, so the relative amounts of the nonbound and azide-bound forms were estimated directly from the values of absorbance at these maxima (data not shown) The estimated association constants for imidazole and azide binding to heme–GmHO-1 are summarized and compared with those for heme–SynHO-1 and heme–rHO-1 in Table Heme degradation by GmHO-1 Heme degradation by HOs can be monitored through the changes in optical absorption spectra, because ferric heme, oxyheme and verdoheme intermediate complexes of HO and free biliverdin, products of the HO reaction, all exhibit characteristic absorption bands (Scheme 1) As shown in Fig 5A, addition of ascorbate to the solution of heme–GmHO-1 initiates the reaction, as revealed by gradual diminution of the Soret band After several minutes, a broad band appears at around 660–675 nm; this increases in intensity with time, indicating the formation of biliverdin In this case, neither bands of the oxy form (at 541 and 578 nm) nor bands of verdoheme and CO–verdoheme (at  690 and at  640 nm, respectively) are observed, implying that the first step of heme conversion is ratelimiting The apparent initial rate of heme degradation by GmHO-1 is about three times higher than that of degradation by SynHO-1, but nearly four times lower than that of degradation by rHO-1 in the presence of 1200 equivalents of ascorbate (Table 4) To establish the physiologic electron-donating system in the higher-plant HO reaction, the heme–GmHO-1 reaction was carried out in the presence of NADPH coupled with FNR ⁄ Fd (Fig 5B) After addition of NADPH, the Soret band maximum of heme–GmHO-1 immediately shifted from 405 to 415 nm, and at the same time, distinct absorption bands of oxyheme appeared at 540 and 579 nm Then, a broad band appeared at around 660 nm, and was maximal 9–12 after initiation of the reaction The spectral features of the final reaction mixture were analogous, but not identical, to those of the ascorbate reaction, which was mainly due to free biliverdin, probably because of the overlapping of absorption bands of the intermediate complexes Product analysis by HPLC revealed that only the a-isomer of biliverdin IX was produced in both the NADPH ⁄ FNR ⁄ Fd-supported and ascorbate-assisted GmHO-1 reactions (data not shown) Catalase did not affect the heme–GmHO-1 reaction in the presence of either ascorbate or NADPH ⁄ FNR ⁄ Fd The apparent initial heme degradation rate in the presence of Scheme Pathways of heme degradation by heme oxygenase (HO), elucidated for the mammalian HO-1 Most HOs other than those of mammalian origin are also known to cleave heme at the a-meso position selectively to produce biliverdin IXa FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS 5389 Heme catabolism by soybean heme oxygenase-1 T Gohya et al Table Apparent rates of initial heme degradation (v) by GmHO-1, SynHO-1 and rHO-1 in the presence of ascorbate, NADPH ⁄ FNR ⁄ Fd (for GmHO-1 and SynHO-1), and NADPH ⁄ CPR (for rHO-1) The concentration of reactants in 0.1 M KPB (pH 7.0) are: sodium ascorbate, mM; heme–HO, lM; Fd, lM; FNR, 0.22 lM; CPR, 0.25 lM; and NADPH of indicated equivalent Protein GmHO-1 v (lMỈmin)1) Ascorbate (1200 eq.) NADPH (4 eq.) NADPH (8 eq.) SynHO-1 v (lMỈmin)1) rHO-1 v (lMỈmin)1) 0.17 0.26 0.41 0.051 0.35 0.39 0.63 0.52 – Bilirubin assay of the heme oxygenase activity of GmHO-1 In mammalian HO reactions, the end-product, biliverdin IXa, is further reduced to bilirubin by NADPH: biliverdin reductase (BVR; EC 1.3.1.24) To estimate the yield of free biliverdin produced by the GmHO-1 reaction, the bilirubin assay was carried out by use of rat BVR The overall yields of bilirubin after the heme conversion followed by biliverdin reduction under different conditions were estimated and compared with the bilirubin yields for SynHO-1 or rHO-1 reactions under comparable conditions (Table 5) Bilirubin yields were somewhat low for GmHO-1 and SynHO-1, but were comparable ( 50%) for the three HOs when desferrioxamine, a chelating agent of Fe3+, was applied to assist the extraction of Fe3+ from the ferric biliverdin– HO complexes Interestingly, when an excess amount of ascorbate was used as a reducing agent, the bilirubin yield in the GmHO-1 reaction was as high as 47% Detection of CO liberation and identification of reaction intermediates Fig Heme conversion by GmHO-1 (A) Spectra were recorded at the indicated time after addition of ascorbate (6 mM) to the solution of heme–GmHO-1 (5 lM in 0.1 M KPB, pH 7.0) (B) Spectra were recorded at the indicated times after addition of NADPH (40 lM) to the solution of heme–GmHO-1 (5 lM; 0.1 M KPB, pH 7.0), FNR (0.22 lM), and Fd (1 lM) NADPH ⁄ FNR ⁄ Fd of GmHO-1 was also compared with that of SynHO-1 under similar conditions, as well as that of rHO-1 in the presence of NADPH ⁄ cytochrome P450 reductase (CPR; NADPH:cytochrome P450 reductase; EC 1.6.2.4) (Table 4) This result suggests that the GmHO-1 reaction occurs as fast as the SynHO-1 reaction, supported by the NADPH ⁄ FNR ⁄ Fd reducing system, and as fast as the rHO-1 reaction supported by the NADPH ⁄ CPR reducing system under similar conditions 5390 Plausible verdoheme and CO–verdoheme intermediates were not detected in the course of heme degradation Table Bilirubin yields (%) in the heme conversion by HO coupled with the biliverdin conversion by BVR Proteins GmHO-1 NADPH (4 eq.) + NADPH (8 eq.) ⁄ BVRa NADPH (20 eq.) + desferrioxamine (1.1 mg) + BVRb Ascorbate (2400 eq.) SynHO-1 rHO-1 28 28 44 50 42 47 47 – – a Additional NADPH and BVR were supplied 45 after the first addition of NADPH (4 eq.) b Desferrioxamine and BVR were supplied 20 and 30 after addition of NADPH, respectively FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al Heme catabolism by soybean heme oxygenase-1 Fig Detection of CO produced during the GmHO-1 reaction by the H64L mutant of myoglobin (A, B) and detection of CO–verdoheme produced in the GmHO-1 reaction under CO (C, D) (A) Difference spectra of optical absorption spectra obtained for the reaction of heme– GmHO-1 (5 lM) in the presence of H64L (4 lM) minus those for the reaction of H64L (4 lM) alone, after addition of ascorbate (6 mM) at appropriate times (B) Difference spectra obtained for the reactions described in (A), except that NADPH (10 lM), FNR (0.22 lM) and Fd (1 lM) were used in place of ascorbate (C) Spectra obtained for the reaction of heme–GmHO-1 (5 lM) with ascorbate (6 mM) (D) Spectra obtained for the reaction of heme–GmHO-1 (5 lM) with FNR (0.22 lM), Fd (1 lM), and NADPH (40 lM) All solutions were in 0.1 M KPB (pH 7.0) by GmHO-1 by optical absorption spectrometry (Fig 5) To ascertain the liberation of CO, this reaction was performed in the presence of H64L, a myoglobin mutant with 40 times greater affinity for CO than the wild type [22] As shown in Fig 6A,B, the difference spectra, the spectra obtained in the presence minus those obtained in the absence of H64L, show an absorption peak at 423 nm, which is known to be specific for the CO-bound form of myoglobin, thereby proving liberation of CO in the heme conversion by GmHO-1 in the presence of either ascorbate (Fig 6A) or NADPH ⁄ FNR ⁄ Fd (Fig 6B) Next, to determine whether verdoheme is produced in the GmHO-1 reaction, this reaction was performed under a CO atmosphere The high partial pressure of CO should enhance coordination of CO to the verdoheme if it is produced As shown in Fig 6C, when ascorbate was added to the heme–GmHO-1 solution presaturated with CO in a sealed cuvette, the Soret band maximum shifted from 405 to 420 nm, and peaks FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS 5391 Heme catabolism by soybean heme oxygenase-1 T Gohya et al concomitantly appeared at 539 and 571 nm, indicating formation of the CO-combined heme–GmHO-1 complex (Table 1) Injection of oxygen gas into this cuvette decreased the Soret band so that it almost vanished after 30 min, and instead, an absorption peak appeared at 637 nm, suggesting the formation of CO–verdoheme The 637 nm band disappeared gradually and was replaced by a new broad band with a maximum at approximately 675 nm, identical to the absorption band of free biliverdin The heme–GmHO-1 reaction under a CO atmosphere was also carried out with the NADPH ⁄ FNR ⁄ Fd reducing system (Fig 6D) In this case, however, the 637 nm band was not observed, but a broad band with a maximum at around 657 nm appeared Therefore, in the presence of ascorbate (Fig 6C), verdoheme is probably produced, but in the presence of NADPH ⁄ FNR ⁄ Fd, formation of the verdoheme intermediate is still unclear because the 657 nm band is different from that of the well-known verdoheme or CO–verdoheme bands at 688 and 637 nm, respectively [23] Heme degradation by HO is driven by hydrogen peroxide, which substitutes for molecular oxygen and probvides electrons to convert heme into verdoheme [24] Therefore, heme–GmHO-1 was reacted with H2O2 to ascertain whether verdoheme was actually produced Soon after addition of H2O2 to the heme– GmHO-1 solution, the Soret band intensity diminished to nearly one-third and a relatively strong broad band appeared in the visible region (kmax ¼ 660 nm) (Fig 7) This 660 nm band is very similar to that observed in heme degradation by GmHO-1 in the presence of NADPH ⁄ FNR ⁄ Fd (Figs 5B and 6D), suggesting that the same intermediate, namely the 660 nm species, is accumulated To isolate and identify the 660 nm species, the heme– GmHO-1 reaction was carried out with six equivalents of H2O2 under anaerobic conditions, to avoid the degradation or successive conversion of the intermediate by oxygen The green pigment was extracted from the reaction product with acetone containing imidazole The spectrum of the extract is shown in Fig 8B, exhibiting peaks at 404, 534, 636 and 684 nm; the latter two differ from the 660 nm band of the protein complex (Fig 8A) When the solution of the extract was exposed to CO, the 684 nm band gradually shifted to 636 nm The reported band maxima of bis-imidazole-coordinated verdoheme are 400, 536 and 685 nm [23], so the spectra shown in Fig 8B are considered to be a mixture of a CO-coordinated monoimidazole complex and a bis-imidazole complex of verdoheme Using the same methods, extracts of the H2O2 reaction intermediates of heme–SynHO-1 and heme–rHO-1 were obtained The optical absorption 5392 Fig Heme degradation by GmHO-1 in the presence of H2O2 Spectra were recorded at the indicated times after addition of H2O2 (40 lM in N2-saturated 0.1 M KPB) to the heme–GmHO-1 solution (5 lM in N2-saturated 0.1 M KPB) Fig Optical absorption spectra of the intermediates of heme– HO reactions (A) Obtained from the reaction mixture of heme– GmHO-1 with H2O2 (6 eq of the heme) under anaerobic conditions (B–D) Acetone extracts containing excess imidazole from the reaction mixtures of the heme–HO-1 complexes with H2O2 (6 eq.) in anaerobic conditions spectra of the acetone extracts (Fig 8C,D) showed similar features, indicating a common chromophore In conclusion, it has been confirmed that the 660 nm FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al species produced in the course of heme degradation by GmHO-1 is verdoheme Discussion Homology and heme binding Estimation of the secondary structure of GmHO-1 suggests that, in spite of low homology in the amino acid sequence, GmHO-1 protein should consist of eight a-helices common to other HOs whose crystal structures are known [3,25–29] (Fig 1) A recent modeling study on pea HO-1 also suggested a similar structure [12] In GmHO-1, however, a critical residue for HO activity, the proximal His, which fixes heme to the heme pocket of the enzyme and participates in the activation of heme, is not at the position in which it is present in SynHO-1 (His17) or rHO-1 (His25) Instead, there is only one His (His30) in the predicted proximal A-helix region (Fig 1) Experiments on heme binding to GmHO-1 have demonstrated : stoichiometry, and the result of EPR investigations of nitrosylheme–GmHO-1 indicate a nitrogenous proximal ligand of the heme, whereas the heme–H30G complex shows neither a proximal protein ligand (Fig 4) nor HO activity These findings have firmly established that His30 of GmHO-1 is the proximal heme ligand Heme catabolism by soybean heme oxygenase-1 molecule interacts with respective Asp residues (Asp140 of rHO-1 and Asp131 of Syn HO-1) in the distal helix through hydrogen-bonding networks via water molecules in crystals [25,27] Unfortunately, the resolution of the reported crystal structures of heme– SynHO-1 and rHO-1 is not sufficiently high to allow accurate quantitative comparison of the length of the hydrogen-bonding network, so the reason for such a large difference in pKa values is unclear In GmHO-1, the corresponding residue to the Asp is His150, which is also competent as a partner of the indirect hydrogen bonding with the heme-bound water This difference in the hydrogen-bonding counterpart would also affect the pKa value The EPR parameters of the nitrosylheme–HO complexes also give useful information on the heme pocket structure As shown in Table 2, the hyperfine splitting constants of the 15N nucleus of the distal NO and of the 14N nucleus of the proximal His of the GmHO-1 complex are closer to those of the rHO-1 complex than to those of the SynHO-1 complex Thus, Fe–N(O) r-bonding in heme–GmHO-1 might be comparably strong to that in heme–rHO-1 [15] The strength of the Fe–N(His) bonding in the GmHO-1 and in rHO-1 complexes also appears to be the same, thereby implying that the imidazole part of His30 is neutral and probably forms a hydrogen bond with Gln34, such that His25 of rHO-1 is stabilized by the hydrogen bonding with Glu29 Coordination structure of heme–GmHO-1 The optical absorption data for the GmHO-1 complex in ferric, ferrous, oxy and CO-bound forms of heme show that the coordination structure of the heme is generally like that of SynHO-1 or rHO-1 (Fig and Table 1) The EPR spectrum of the ferric resting form of heme–GmHO-1, however, shows that the rhombic anisotropy is relatively large and close to that in the ferric a-hydroxyheme complex of rHO-1 [30] This means that the ligand field on the heme plane is relatively anisotropic in GmHO-1 For this reason, it is possible that the surrounding helices exert a greater anisotropic ligand field effect on the heme plane than the corresponding residues in SynHO-1 and rHO-1, the amino acid sequences of which are considerably different from that of GmHO-1 The observed acid–base transition strongly suggests that the sixth, distal ligand of heme is a water molecule, and the heme-bound water is supposed to be connected to dissociable distal residue(s) through direct or indirect hydrogen bonding The estimated pKa value of 8.2 is between the values of heme–rHO-1 (7.6) and heme–SynHO-1 (8.9); in the latter two, the distal water Characterization of the heme pocket of GmHO-1 by exogenous ligand binding Azide, like imidazole, is a ligand with both r-donor and p-donor characteristics, but is a relatively stronger p-donor for stabilization of the higher oxidation states of metal ions In agreement with this, the binding constants for the binding of azide to ferric heme– HOs are one to two orders larger than those for imidazole (Table 3) Comparison of the Kazide values shows that heme–GmHO-1 and heme–SynHO-1 have smaller values than heme–rHO-1, suggesting weak relevance of the similarity in the amino acid sequences of distal helices This difference does not necessarily mean that the ferric character of the former two is less than that of the latter, because polarity of the heme milieu as well as the steric conditions of the distal heme pocket could also affect Kazide Polar residues might either stabilize the azide coordinated to heme or facilitate the access of anionic azide to the heme, and conversely, the steric effect of the distal residues might reduce the accessibility of azide The amino acid residues comprising the presumed distal FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS 5393 Heme catabolism by soybean heme oxygenase-1 T Gohya et al helix of GmHO-1 include several hydrophobic amino acids, and the distal side of the heme pocket of SynHO-1 has been reported to be less polar in total than that of rHO-1 [27] Thus, the less polar characteristics of the heme pocket of GmHO-1 might be associated with the smaller values of Kazide for GmHO-1 and SynHO-1, and with the ferric character of the heme The Kimidazole values of the three kinds of heme– HO-1 complexes are very different, and that of heme– rHO-1 is strikingly large The Kimidazole might reflect the magnitude of the vacancy in the distal side or the steric hindrance at the opening of heme pockets, due to the relatively large size of imidazole molecules, although flexibility of the distal pocket would also affect it Comparison of the opening side of heme pockets in crystal structures shows that Ile137 of SynHO-1 droops over the heme distal site, whereas the corresponding Val146 of rHO-1 is located relatively high above the distal side of heme (as is also true in human HO-1) [3,25,27] The difference in Kimidazole between heme–SynHO-1 and heme–rHO-1 may be attributable to this structural difference As shown in Fig 1, the presumed A-helix of GmHO-1 is 3–4 turns longer than the A-helices of SynHO-1 and rHO-1 Furthermore, the assumed F-helix, corresponding to the distal helices of SynHO-1 and rHO-1, is also relatively long, suggesting a unique structure of the opening of the heme pocket of GmHO-1 Such a structure might inhibit the approach of imidazole to the heme in GmHO-1, explaining the smallest Kimidazole value Mechanism of heme degradation by GmHO-1 Although some of the details are still unknown, the degradation of heme by HO follows the mechanism shown in Scheme Heme catabolism by mammalian HO proceeds sequentially by way of oxyheme, hydroxyheme, verdoheme and ferric biliverdin, and the presence of each intermediate is monitored by respective specific absorption bands Heme catabolism by HOs of mammals, pathogenic bacteria, cyanobacteria and probably insects is considered to have a similar mechanism, because the characteristic absorption bands of verdoheme and CO–verdoheme are observed or CO produced is detected [1,5,15,16,31,32] In heme degradation by GmHO-1, it has been confirmed that the final product is biliverdin IXa in the presence of either ascorbate or NADPH ⁄ FNR ⁄ Fd The oxyheme is apparently observed in the time-dependent spectra in Fig 5B, and CO excision from heme has been verified Furthermore, the intermediate with kmax ¼ 660 nm has been confirmed to be verdoheme (Figs and 8) Hydrogen peroxide also drives the con5394 version of heme–GmHO-1 to verdoheme–GmHO-1, as is the case for other heme–HO complexes (Fig 7) [24] Consequently, GmHO-1 has been established to be an HO that site-specifically oxygenates and cleaves heme into biliverdin IXa, CO and free iron in a manner similar to mammalian HO enzymes (Scheme 1) The initial heme degradation rate of GmHO-1, indicated in Table 4, is comparable to that of SynHO-1 in the presence of NADPH, FNR, and Fd, and also to that of rHO-1 in the presence of NADPH and CPR Here, NADPH ⁄ CPR has been established to be the physiologic electron-donating system for mammalian HO The yield of ferric biliverdin in the heme–GmHO1 reaction in the presence of NADPH ⁄ FNR ⁄ Fd is inferred to be also comparable to that in the heme– rHO-1 reaction, because chelating of the ferric iron enhances the bilirubin yield of the GmHO-1 and SynHO-1 reactions to a similar level as that with the rHO-1 reaction (Table 5) The lower yield of bilirubin in the GmHO-1 and SynHO-1 reactions in the presence of NADPH ⁄ FNR ⁄ Fd probably occurs due to reduction of ferric biliverdin to yield ferrous ion being less efficient with the limited number of electrons supplied from NADPH Accordingly, it can be concluded that GmHO-1 is fully active when electrons are supplied successively from NADPH by way of FNR and Fd to the combined heme in the presence of molecular oxygen This finding is inconsistent with previous reports that cyanobacterial or plant HO reactions require an additional reducing agent such as ascorbate [9,12,17] We propose that the NADPH ⁄ FNR ⁄ Fd reducing system could be the native electron-donating partner in the GmHO-1 reaction in plastids, as well as in the SynHO-1 reaction in the cytosol Although ascorbate, which is abundant in plant tissues (reported to be present at 1–20 mm [33]), probably contributes to some extent to the efficient reduction of the ferric ion and extraction of biliverdin from the ferric biliverdin–enzyme complex, it is unlikely that ascorbate initiates the heme–GmHO-1 reaction as a physiologic electron donor Kinetic experiments show that the first step of heme degradation by GmHO-1 and SynHO-1 is obviously slower than that by rHO-1 in the presence of ascorbate (Table 4) This means that the first reduction of the ferric heme is harder to achieve in the GmHO-1 and SynHO-1 reactions than in the rHO-1 reaction (Scheme 1), and that the first step of heme conversion is rate-limiting in the ascorbate-supported GmHO-1 reaction (Fig 5A) Even for mammalian HO-1, the reducing ability of ascorbate is known to be thermodynamically insufficient to reduce ferric hemeHO-1 (EÂAsc ẳ + 80 mV vs FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al Eheme–human HO-1 ¼ ) 65 mV, at pH 7.4 and 25 °C) [34] We consider that this high resistance to the reduction of heme is disadvantageous to the plant HOs if they utilize ascorbate as the reducing agent for HO reactions Low CO affinity of heme–GmHO-1 It has been reported that heme catabolism by mammalian HOs under a CO atmosphere stops at the CO– verdoheme stage, due to the high affinity of CO for the verdoheme [35] In contrast to this, in the NADPH ⁄ FNR ⁄ Fd-assisted heme–GmHO-1 reaction performed under a CO atmosphere, CO-bound verdoheme was not detected (Fig 6D), and in the ascorbatesupported reaction, CO–verdoheme was observed only momentarily (Fig 6C) Thus, the affinity of verdoheme–GmHO-1 for CO appears to be very low The optical absorption spectrum of verdoheme–GmHO-1 is unique, having a broad absorption band with a maximum at 660 nm (Fig 7), not at the 686 nm of verdoheme–rHO-1 [23] Therefore, the electronic state of the verdoheme and accordingly the axial coordination structure of verdoheme in GmHO-1 seem to be unusual A resonance Raman study on verdoheme–rHO-1 suggested that verdoheme is in the six-coordinate state with the proposed ligand of hydroxide or water [23], so that the sixth ligand of verdoheme–GmHO-1 might be different from that of known HO complexes of verdoheme The stronger distal coordination should result in weaker affinity of CO for verdoheme In the ascorbate-supported reaction, such distal coordination onto verdoheme is not discernible, and instead, in a CO atmosphere, transient CO coordination is observed This is probably because the verdoheme is accumulated less, due to the limitation on verdoheme generation imposed by the first step of heme conversion Furthermore, a large excess of ascorbate might be advantageous for verdoheme degradation, by reducing verdoheme accumulation To address coordination of the distal ligand onto verdoheme–GmHO-1, site-directed mutations of possible distal residues are being studied Experimental procedures Construction of expression vector pMWGm HO-1 GmHO-1 expression vector pMWGmHO-1 was constructed in the same way as pMWSynHO-1 or pMWSynHO2, described previously [14,15] According to the reported amino acid sequence of GmHO-1 [8], we designed a nucleotide sequence encoding a mature-type enzyme from Ser26 Heme catabolism by soybean heme oxygenase-1 to Ser250 without the N-terminal transit region and with unique sites for the restriction enzymes NdeI, BstEII, BglII, EagI, NcoI and HindIII First, a 50-bp double-stranded synthetic oligonucleotide with unique sites for the aforementioned restriction enzymes was ligated between the 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, 44–91 nucleotides in length, were synthesized to construct a 681-bp synthetic gene coding for the maturetype GmHO-1 from the ATG initiation codon to the TAA stop codon Each nucleotide was phosphorylated with T4 polynucleotide kinase, and 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 BstEII site Similarly, the 5¢-ends of Oligos III, V, VII and IX were designed to ligate to the BstEII, BglII, EagI and NcoI 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 GmHO-1 expression vector pMWGmHO-1, doublestranded Oligo I to Oligo X were ligated step by step into the restriction enzyme sites of pMW-A To construct an expression plasmid for the H30G mutant of GmHO-1, PCR was used according to the method of Nelson and Long [36] The nucleotide sequence was determined with an Applied Biosystems (Foster City, CA, USA) 373A DNA sequencer Expression of GmHO-1 and purification A mL inoculum in LB medium (+ 50 lgỈmL)1 ampicillin ⁄ 0.1% glucose) was prepared from a plate of transformed E coli BL21 (DE3) cells carrying pMWGmHO-1 Five-hundred-milliliter cultures were inoculated with 200 lL of the inoculum and grown in LB medium (+ 200 lgỈmL)1 ampicillin) at 37 °C until the A600 nm reached 0.8–1.0 The cells were grown for an additional 24 h at 25 °C, harvested by centrifugation at 5000 g for 10 using a Kubota RA-3 rotor (Tokyo, Japan), and stored at ) 80 °C prior to use The typical yield of cells from a 500 mL culture was 1.7 g The E coli cells (10 g), resuspended in 90 mL of 50 mm Tris ⁄ HCl buffer (pH 7.4, + mm EDTA), were lysed [2 mg lysozym(g cells))1] with stirring at °C for 30 After sonication (Branson 450 Sonifire, Danbury, CT, USA) and centrifugation at 100 000 g for h using a Hitachi RP50T rotor (Tokyo, Japan), the resulting supernatant was covered with a 60–90% ammonium sulfate fraction and centrifuged at 12 000 g for 15 using a Kubota RA-3 rotor The subsequent precipitates, containing the GmHO-1 protein, were dissolved in 20 mm potassium FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS 5395 Heme catabolism by soybean heme oxygenase-1 T Gohya et al 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 26 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 of 50–250 mm KCl Only fractions with a single band at 26 kDa on SDS ⁄ PAGE were collected Other proteins The following proteins were expressed in E coli and purified to apparent homogeneity on SDS ⁄ PAGE according to the methods described in each reference: SynHO-1 [15], a truncated form of rat HO-1 [37], H64L mutant of myoglobin [38], maize ferredoxin type III [39], maize FNR [40], a truncated form of human CPR [41], and rat BVR [42] Heme binding and preparation of heme–GmHO-1 To determine the stoichiometry of heme binding to GmHO-1, 24 lL of a 200 lm protein solution (0.1 m KPB, pH 7.0) was titrated with each lL of a 400 lm hemin solution, prepared by diluting mm alkaline solution (NaOH, 10 mm) in 0.1 m KPB (pH 7.0) Absorbance at 405 nm was recorded for each addition of the hemin solution to construct titration curves vs the volume of added hemin solution Separately, the absorbance at 405 nm of each free hemin solution was measured, and these were plotted together with that of the corresponding hemin– protein solutions The equivalent was determined at an inflection point of the titration curve Reconstitution of heme–GmHO-1 was carried out by adding a small excess of hemin to the protein solution, and then removing the excess hemin with a Sephadex G-25 column pre-equilibrated with 0.1 m KPB (pH 7.0) Optical absorption and EPR spectroscopy All of the optical absorption spectra were recorded on a Shimadzu (Tokyo, Japan) UV-2200 spectrophotometer at 25 °C Protein solutions were in 0.1 m KPB (pH 7.0), unless otherwise specified The ferrous form of heme–GmHO-1 was obtained by the addition of an appropriate amount of anaerobic dithionite solution (1 m) to heme–GmHO-1 (5 lm) in a UV cell (Aldrich, St Louis, MO, USA) with a screwtop open-cap tube furnished with a rubber septum, under anaerobic condition The CO-bound form was prepared by exposing the ferrous form to gaseous CO introduced into the anaerobic cuvette The oxy form of heme–GmHO-1 was obtained by reduction of the ferric 5396 form with NADPH (1 eq.) ⁄ FNR ⁄ Fd and exposing the reduced complex to small amount of oxygen gas The pH titration was performed using m Tris ⁄ HCl buffer for pH 6.5–8.3 and m NaOH solution for pH 8.3–11.4; aliquots were added to heme–GmHO-1 (8 lm) in 18 mm KPB (pH 6.00), and the pKa value was estimated according to the published method [14] Azide binding was carried out by addition of azide solutions (0.1 or m) to mL of heme–GmHO-1 solution (5 lm), followed by incubation for 10–15 on ice after each addition Optical absorption spectra were recorded after each addition of azide, and the absorbance values at 405 and 419 nm were monitored to estimate the mole fractions of azide-free and azide-bound forms of the complex, respectively Then both mole fractions were plotted against the concentration of azide of each solution The intersection point corresponding to the mole fraction of 0.5 of the respective forms gives the amount of azide necessary to attain the equilibrium, thereby giving the equilibrium constant for azide binding, Kazide Imidazole binding was also performed in a similar way with the use of m or 10 m imidazole stock solutions Kimidazole was estimated on the basis of the mole fractions of imidazole-free and imidazole-bound forms, which were estimated from the values of absorbance at 402 and 420 nm, respectively, in the difference spectra of imidazole-free minus imidazole bound forms EPR spectra were recorded on a Bruker (Karlsruhe, Germany) E500 spectrophotometer, operating at 9.35– 9.55 GHz, and at 6–8 K for ferric heme complexes or 20– 20 K for nitrosylheme complexes, with an Oxford liquid helium cryostat (ESR900) (Oxford, UK) The 15NO-bound form of heme–GmHO-1 was prepared by adding dithionite to the solution of heme–GmHO-1 containing Na15NO2 in an EPR tube made of extra-pure synthetic quartz Heme conversion and kinetic measurements Protein solutions were in 0.1 m KPB (pH 7.0), unless otherwise specified Heme conversion reactions in the presence of sodium ascorbate were started by adding appropriate amounts of sodium ascorbate solution (1 m) to the heme– GmHO-1 solution (5 lm) When NADPH ⁄ FNR ⁄ Fd was used as a reducing system, the reaction was started by addition of a given amount of NADPH (5–15 mm in 0.1 m KPB, pH 7.0) to the solution of heme–GmHO-1 (5 lm), Fd (1 lm), and FNR (0.22 lm), or to the solution of rHO-1 (5 lm) and CPR (0.22 lm) Optical absorption spectra of the reaction mixtures were recorded between 240 and 900 nm at appropriate time intervals until the HO reaction was completed The initial rate of heme degradation was estimated from the decreasing rate of the Soret band (A405, e ẳ 127 mm)1ặcm)1 for hemeGmHO-1, A402, e ẳ 128 mm)1ặcm)1 for hemeSynHO-1, and A404, e ¼ 140 mm)1Ỉcm)1 for heme–rHO-1, where the molar FEBS Journal 273 (2006) 5384–5399 ª 2006 The Authors Journal compilation ª 2006 FEBS T Gohya et al absorption coefficients were all determined by the pyridine hemochrome method, using A557, e ¼ 34.4 mm)1Ỉcm)1) Heme conversion by GmHO-1 in a CO atmosphere was carried out in the aforementioned anaerobic UV cell The reaction was initiated by the addition of O2 (1 mL) with a syringe Bilirubin assay Heme degradation by GmHO-1 or SynHO-1 (each lm in 0.1 m KPB, pH 7.0) was conducted in the presence of NADPH ⁄ FNR ⁄ Fd (20 lm, 0.22 lm, lm, respectively), and after the reaction was complete (approximately 45 after initiation of the reactions), BVR (1 lm) and additional NADPH (40 lm) were supplied In the rHO-1 (5 lm) reaction, CPR (0.2 lm) was used in place of FNR ⁄ Fd In the ascorbate-supported reactions, the heme–enzyme solution containing BVR (1 lm) and NADPH (40 lm) was reacted with ascorbate (12 mm) The end of each reaction was confirmed by the complete disappearance of the Soret band, and the quantity of bilirubin was determined from the A468 with e468 ẳ 55 mm)1ặcm)1 [43] Detection of CO To each solution of heme–GmHO-1 (5 lm) and FNR ⁄ Fd (0.22 and lm, respectively) or heme–GmHO-1 (5 lm) alone, H64L (4 lm) was added, and the heme conversion reaction was initiated by the addition of NADPH (10 lm) or ascorbate (6 mm), respectively Optical absorption spectra of the reaction mixtures were recorded at appropriate time intervals As a control experiment, the reaction of H64L with NADPH ⁄ FNR ⁄ Fd or ascorbate was carried out, and the difference spectra, in the presence minus in the absence of heme–GmHO-1, were obtained Extraction of the 660 nm intermediate Anaerobic H2O2 solution (480 lm) was injected into mL of a nitrogen-saturated solution of heme–GmHO-1 ( 80 lm in 0.1 m KPB, pH 7.0) in a capped UV cell filled with nitrogen gas Immediately, the color of the solution turned to green, indicating formation of the 660 nm species Then, a concentrated imidazole ⁄ acetone solution was added to the cell (1 : v ⁄ v), and the mixture was incubated for 30 on ice to complete the protein precipitation The supernatant of the solution was transferred to an eppendorf tube, followed by spinning down precipitates, and used for the optical absorption measurements Other 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absorption bands Heme catabolism by HOs of mammals, pathogenic bacteria, cyanobacteria and probably insects is considered to have a similar mechanism, because the characteristic absorption bands