©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heme Oxygenase-2
PROPERTIES OF THE HEME COMPLEX OF THE PURIFIED TRYPTIC FRAGMENT OF RECOMBINANT HUMAN HEME OXYGENASE-2 (*)

(Received for publication, December 27, 1994)

Kazunobu Ishikawa (1) Noriko Takeuchi (3) Satoshi Takahashi (4) Kathryn Mansfield Matera (3) Michihiko Sato (2) Shigeki Shibahara (5) Denis L. Rousseau (4) Masao Ikeda-Saito (3) Tadashi Yoshida (1)(§)

From the  (1)Department of Biochemistry and (2)Central Laboratory for Research and Education, Yamagata University School of Medicine, 990-23, Japan, the (3)Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, (4)AT& Bell Laboratories, Murray Hill, New Jersey 07974, and the (5)Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, Sendai, 980, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS and DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recombinant human microsomal heme oxygenase-2 was expressed in Escherichia coli. Tryptic digestion of the membrane fraction, in which the wild-type enzyme was localized, yielded a soluble tryptic peptide of 28 kDa, which retained the ability to accept electrons from NADPH-cytochrome P-450 reductase and the enzymatic activity for conversion of heme to biliverdin. The tryptic fragment, when purified to apparent homogeneity, bound one equivalent of heme to form a substrateenzyme complex that had spectroscopic properties characteristic of heme proteins, such as myoglobin and hemoglobin. Optical absorption, Raman scattering, and EPR studies of the heme-tryptic fragment complex revealed that the ferric heme was six coordinate high spin at neutral pH and six coordinate low spin at alkaline pH, with a pK value of 8.5. EPR and Raman scattering studies indicated that a neutral imidazole of a histidine residue served as the proximal ligand in the heme-heme oxygenase-2 fragment complex. The reaction with hydrogen peroxide converted the heme of the heme oxygenase-2 fragment complex into a verdoheme-like intermediate, while the reaction with m-chloroperbenzoic acid yielded a oxoferryl species. These spectroscopic properties are similar to those obtained for heme oxygenase-1, and thus the catalytic mechanism of heme oxygenase-2 appears to be similar to that of heme oxygenase-1.


INTRODUCTION

Microsomal heme oxygenase (HO) (^1)is the rate-limiting enzyme in the mammalian heme degradation pathway. The enzyme converts iron-protoporphyrin IX (heme) into biliverdin IXalpha with the accompanying production of CO and release of free iron through successive mono-oxygenase reactions, which require three O(2) molecules and electrons donated by NADPH-cytochrome P-450 reductase (Tenhunen et al., 1969). HO has two isozymes, referred to as HO-1 and HO-2 (Maines et al., 1986). HO-1, an inducible form, is mainly distributed in reticuloendothelial cell-rich tissues, such as spleen and liver (Tenhunen et al., 1970). HO-1, with a molecular mass of 33 kDa, was first purified from microsomes of pig spleen in 1978 (Yoshida and Kikuchi, 1978a), then from rat liver in 1979 (Yoshida and Kikuchi, 1979). The purified enzyme itself is not a hemoprotein, but the substrate-enzyme complex formed by 1:1 binding with heme exhibits light absorption spectral properties similar to hemoproteins such as myoglobin and hemoglobin (Yoshida and Kikuchi, 1978a; 1979). The heme degradation mechanism catalyzed by HO-1 is unique; heme serves both as the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O(2) (Yoshida and Kikuchi, 1978b). Recent developments in the bacterial expression of a 30-kDa soluble form of rat HO-1 have made it possible to prepare HO-1 in the large quantities required for spectroscopic studies (Ishikawa et al., 1992). Using optical absorption, EPR, and resonance Raman scattering, we have recently established that the axial ligand in the ferrous heme-HO-1 complex is a neutral imidazole of His-25 (Takahashi et al., 1994a, 1994b; Ito-Maki et al., 1995). Similar conclusions were drawn in independent studies by Sun et al.(1993, 1994). Recent NMR studies suggested an open heme pocket of the heme-HO-1 complex (Hernández et al., 1994).

While the physiological role of HO-1 in heme catabolism has been well established (Kikuchi and Yoshida, 1983; Maines, 1988), recently, HO-2 has attracted attention since it was reported that the CO produced by HO-2 might be a neurotransmitter and activate guanylyl cyclase in a manner similar to nitric oxide (Brune and Ullrich, 1987; Verma et al., 1993). HO-2, with a molecular mass of 36 kDa, is not inducible and is mainly distributed in the brain and testis (Maines, 1988). The amino acid sequence similarity between HO-2 and HO-1 is about 40%, but there are several stretches of highly conserved sequences with matched predicted secondary structure including the putative proximal His sites, His-25 in HO-1 and His-45 in HO-2 (Rotenberg and Maines, 1991; McCoubrey et al., 1992, 1993). As both isoforms display the same enzymatic activity, the active site structure, and hence the molecular mechanism of the enzyme action, is assumed to be analogous between the two isoforms. However, knowledge of the HO-2 active site structure has been limited, in part due to the difficulty in obtaining the large amount of enzyme necessary for spectroscopic studies. To understand the molecular mechanism of the enzyme action, knowledge of the active site structure is essential. To this end, we have constructed a bacterial expression system for the HO-2 protein using the human HO-2 cDNA (Shibahara et al., 1993) and have established a purification methodology to prepare a 28-kDa HO-2 tryptic fragment that retains heme degradation activity. We have carried out spectroscopic characterization of the heme-HO-2 fragment complex and obtained evidence that the structure of the heme pocket of HO-2 and the mechanism of heme degradation by HO-2 are very similar to those observed in HO-1.


EXPERIMENTAL PROCEDURES

Chemicals and Materials

The sources of reagents were as follows: restriction endonuclease and T(4) polynucleotide kinase from Toyobo; T(4) DNA ligase from Boehringer; DyeDeoxy Terminator Cycle sequencing kit from Applied Biosystems; prokaryotic expression vector, pTV118N, and isopropyl-1-thio-beta-D-galactopyranoside from TaKaRa; Escherichia coli strain XL1-blue from Stratagene; ampicillin, hemin, and bovine serum albumin from Sigma; nitrocellulose membranes from MSI; Sephadex G-75 and a molecular mass determination kit from Pharmacia Biotech Inc.; DEAE-cellulose (DE-32) from Whatman; hydroxyapatite from Seikagaku Kogyo; peroxidase-conjugated sheep IgG against rabbit IgG from Cappel; and synthetic oligonucleotide from Sawady Technology. NADPH-cytochrome P-450 reductase was purified from rat liver by the method of Yasukochi and Masters(1976). 1 unit of reductase activity was defined as described by Yoshida and Kikuchi (1978a). Partially purified biliverdin reductase was obtained from the pig spleen soluble fraction (Noguchi et al., 1979).

Construction of the Human Heme Oxygenase-2 Expression Plasmid

The prokaryotic expression vector, pTV118N, was linearized by digestion with NcoI and KpnI. A cDNA clone encoding the human HO-2, pHHO2-1 (Shibahara et al., 1993), was cut with AccI and KpnI. A synthetic 33-base nucleotide (5`-CATCTCAGGGGAAGTGGAAACCTCAGAGGGGGT-3`) and a 31-base nucleotide (3`-AGTCCCCTTCACCTTTGGAGTCTCCCCCATC-5`) were phosphorylated with T(4) polynucleotide kinase and then annealed. The resulting fragment from cDNA (AccI-KpnI, 33-976) was inserted into the NcoI and KpnI site of pTV118N using a double-stranded synthetic oligonucleotide as a linker between the NcoI and AccI sites. The resulting recombinant plasmid, named pTVHHO2, was transfected into E. coli strain XL1-blue. The nucleotide sequence was determined by the cycle sequencing method with a Perkin-Elmer polymerase chain reaction system 9600 and an Applied Biosystems 373A DNA sequencer.

Expression of Heme Oxygenase-2 and Preparation of Soluble and Membrane Fractions

Transformed E. coli cells were pre-cultured overnight at 37 °C in Luria-Bertani medium, and then 2.5 ml of the preculture was added to 250 ml of the same medium. When the turbidity of the culture reached 0.4 at 600 nm, isopropyl-1-thio-beta-D-galactopyranoside (1 mM final concentration) was added to induce the expression of HO-2. Cultivation was continued for 7 h at 37 °C. Harvested cells were washed twice with 20 mM potassium phosphate buffer (pH 7.4) containing 134 mM KCl, resuspended in 9 volumes of 50 mM Tris-HCl buffer (pH 7.4) containing 2 mM EDTA, and lysed by lysozyme (final concentration of 1 mg/ml) for 30 min at 4 °C. The lysed cells were briefly sonicated until they lost their viscosity and were then centrifuged at 105,000 times g for 1 h; the resulting supernatant was used as the soluble fraction. The precipitate was resuspended in the same buffer and used as the membrane fraction.

Preparation of a 28-kDa Tryptic Fragment of the Heme Oxygenase-2 Protein

The bacterial membrane fraction (1 g of protein) was digested with 5 mg of trypsin for 30 min at 4 °C in 100 ml of 20 mM sodium phosphate buffer (pH 7.4). The digestion was stopped by addition of 25 mg of soybean trypsin inhibitor; then, the solubilized fraction was obtained by centrifugation at 105,000 times g for 1 h. The solubilized fraction was first subjected to ammonium sulfate fractionation. The precipitate obtained at 50-80% saturation of ammonium sulfate was collected by centrifugation and dissolved in 20 mM potassium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride in a final volume of 4 ml. The subsequent purification procedures including gel filtration on Sephadex G-75, DEAE-cellulose chromatography, and hydroxyapatite chromatography were essentially the same as those employed for purification of the tryptic fragment of rat liver HO-1 (Yoshida et al., 1991), except that sodium phosphate and NaCl were replaced by potassium phosphate and KCl, respectively.

Preparation of the Human Heme Oxygenase-2 Antibody

The purified tryptic fragment of HO-2 was subjected to SDS-PAGE, stained with Coomassie Brilliant Blue, cut out from the gel, crushed, and emulsified with Freund's adjuvant. Human HO-2 antiserum was prepared from a female rabbit injected with the emulsion. The rabbit received four intradermal injections (0.1 mg of protein) at 2-week intervals. 2 weeks after the final injection, blood was withdrawn, and the antibody was prepared as previously described (Shibahara et al., 1979).

Spectroscopic Methods

Light absorption spectra were recorded on a Hitachi U-3210 spectrophotometer at 20 °C. X-band EPR spectra were obtained with a Bruker ESP-300 spectrometer with an Oxford liquid helium cryostat as previously described (Takahashi et al., 1994a). Resonance Raman spectra were obtained as previously described with a charged coupled device detector (Takahashi et al., 1994a, 1994b). The excitation wavelengths were selected to maximize the scattering intensity from the complex under study. For the ferric heme complexes, the excitation wavelength was 406.7 nm, and for the ligand-free ferrous complex, it was 441.6 nm.

Other Procedures

The N-terminal amino acid sequence was determined with an ABI model 470A protein sequencer. Heme oxygenase enzyme assays (Yoshida and Kikuchi, 1978a), immunoblot analyses (Yoshida et al., 1991), SDS-PAGE (Laemmli, 1970), and protein assays (Lowry et al., 1951) were performed according to published procedures.


RESULTS and DISCUSSION

cDNA Sequence of Human Heme Oxygenase-2

We determined the nucleotide sequence of the coding region of cDNA for human HO-2 (pHHO2-1) (Shibahara et al., 1993) and found that the deduced primary structure had several differences from that originally reported by McCoubrey et al.(1992). In Fig. 1, the two predicted amino acid sequences are aligned and compared with those of rat (Rotenberg and Maines, 1990) and rabbit HO-2 (Rotenberg and Maines, 1991). The amino acid sequence deduced from the present cDNA sequence (Human-I in Fig. 1) agrees well with the previously reported result (Human-M) except for the stretch of eight amino acids between positions 281 and 288 (underlined in Fig. 1) and for position 86. The possible origin of this discrepancy is not apparent at this moment. The present sequence is more similar to the other two species than that derived from the previously reported DNA sequence (McCoubrey et al., 1992), and we think that our sequence is thus more likely to be correct.


Figure 1: Comparison of the HO-2 amino acid sequence deduced from the cDNA nucleotide sequence for human, rat, and rabbit enzymes. Human-I and -M represent the sequences predicted by the present paper and by McCoubrey et al.(1992), respectively. Colons indicate identical residues between the four sequences. In the rabbit and rat sequences, dashes are introduced to maximize the similarity between the three sequences. Asterisks denote deleted amino acids in the sequence of McCoubrey et al.(1992).



Expression of Heme Oxygenase-2 in E. coli and Its Intracellular Localization

The harvested cells carrying the HO-2 expression vector, pTVHHO2, were green, while control cells transformed with pTV118N were brown. This change in color is due to the conversion of heme to biliverdin in the bacterial cells as observed in the bacterial expression of the HO-1 protein (Ishikawa et al., 1991). The HO-2 protein expression in the sonicates and soluble and membrane fractions was assessed by SDS-PAGE analysis (Fig. 2A, lanes3, 5, and 7) and compared with samples prepared from control cells (lanes2, 4, and 6). Two extra bands with molecular masses of 36 and 33 kDa appeared in the sonicates of the cells harboring the expression vector (lane3). These two bands were obscured in the soluble fraction (lane5) by the background but became more striking in the membrane fraction (lane7). In addition to the intact 33-kDa HO-1 protein, the bacterial expression of rat HO-1 also produced a 30-kDa active HO-1 protein, which was shown to be the product of proteolytic digestion of the membrane-anchoring C-terminal portion of the HO-1 protein (Ishikawa et al., 1991). By analogy, we assume that the 36-kDa band is the full-length form of HO-2, and the 33-kDa band is a partially digested species.


Figure 2: Expression of HO-2 in E. coli and its intracellular localization. A, SDS-PAGE of cultured E. coli cells harboring the expression vector and comparison with control E. coli cells. Lane1, molecular mass markers; lanes2 and 3, sonicates of control and expressed cells, respectively; lanes4 and 5, soluble fractions of control and expressed cells, respectively; lanes6 and 7, membrane fractions of control and expressed cells, respectively. Each sample (50 mg of total protein) was loaded on a 10% gel, and after electrophoresis, the gel was stained with Coomassie Brilliant Blue. B, Western blot of cultured E. coli cells harboring the expression vector. Lane1, sonicates; lane2, soluble fraction; lane3, membrane fraction; lane4, membrane fraction after tryptic digestion. Each sample (50 mg of total protein) was subjected to SDS-PAGE with a 10% gel followed by immunoblot analysis.



Identity of the 36- and 33-kDa bands as HO-2 was confirmed by Western blots of the sonicates of the cells harboring the expression vector, which showed two bands of 36 and 33 kDa (Fig. 2B, lane1). Western blotting analysis also clarified the localization of the expressed HO-2 protein, as much stronger bands were observed in the membrane fraction. The 36-kDa protein was specifically localized in the membrane fraction (lane3), indicating that the full-length HO-2 spontaneously integrated into the bacterial membrane. The 33-kDa protein was found in both the membrane and soluble fractions (lanes2 and 3), and the 33-kDa protein apparently lacking its C-terminal region acquired water solubility.

The heme oxygenase activities of the soluble and membrane fractions were 17.4 and 140.7 nmol of bilirubin formed/mg of total protein/h, respectively, and the sonicate had an enzyme activity of 103 nmol/mg/h. The heme degrading activity, and hence the HO-2 protein, was localized primarily in the membrane fraction, consistent with the Western blotting results.

Purification of a Tryptic Fragment of Heme Oxygenase-2 Expressed in E. coli

Previously, we reported that the tryptic fragment of HO-1 obtained after solubilization of rat liver microsomes by mild trypsin treatment retained catalytic activity (Yoshida et al., 1991). Therefore, we tried to purify an active tryptic fragment of recombinant HO-2. After digestion of the membrane fraction, Western blotting analysis showed the presence of only one HO-2 species with a molecular mass of 28 kDa (Fig. 2, lane4). This fraction still retained heme degradation activity, and the 28-kDa enzyme was purified as described under ``Experimental Procedures.'' Table 1summarizes the purification procedure. The final preparation appeared to be homogenous as judged from SDS-PAGE (data not shown). The specific activity of the purified 28-kDa protein was 1,000 nmol/mg/h in the presence of 1 unit of P-450 reductase, about one-fourth that of native rat testis HO-2 (Trakshel et al., 1986). By this purification procedure, about 13 mg of the 28-kDa HO-2 tryptic fragment was purified from 1 g of the membrane fraction.



The N-terminal amino acid sequence of the 28-kDa fragment was determined as MADLSELLKEGTKEAHDRAE, which matches the deduced amino acid sequence from Met-30 to Glu-49 (Fig. 1), indicating that the N-terminal 29 amino acid residues were cut off by the trypsin digestion. Judging from the molecular size of the tryptic fragment, the C-terminal portion was also removed by digestion, perhaps by cleavage at Lys-274. The removal of both N- and C-terminal portions is likely to be responsible for the reduced enzyme activity.

Spectroscopic Properties of the Heme-Heme Oxygenase-2 Complex

The tryptic fragment readily bound heme to form an enzyme-substrate complex that titrated to a stoichiometric ratio of unity (data not shown). Hereafter in this paper, the heme complex of the 28-kDa HO-2 tryptic fragment complex will be designated as the heme-28-kDa fragment complex. Fig. 3illustrates the pH-dependent changes in the light absorption spectrum of the ferric heme-28-kDa fragment complex between pH 7.0 and 9.5. At pH 7.0, the complex has a Soret peak at 404 nm and bands at 500 and 631 nm in the visible region. The Soret absorption band is known to be sensitive to the coordination structure of ferric high spin hemoproteins (Giacometti et al., 1981; Morikis et al., 1990; Ikeda-Saito et al., 1992). On the basis of the Soret peak position, the ferric iron in the heme-28-kDa fragment complex at pH 7.0 is postulated to be six coordinate high spin. At alkaline pH, the high spin spectrum is replaced by a spectrum with bands at 413, 540, and 575 nm, similar to the low spin form of the hydroxide complex of methemoglobin. This pH-dependent spectral change is reversible between pH 7 and 10, and the pK(a) value of the change is estimated to be 8.5 as shown in the inset of Fig. 3.


Figure 3: Absorption spectra of the ferric heme-HO-2 complex between pH 7.0 and 9.5 at 20 °C. The 28-kDa fragment was used, and the pH values of the sample are listed in the figure. Inset, the fraction of the alkaline form calculated from the pH-dependent changes in the absorbance at 404 nm. The symbols are experimental values, and the curve is drawn by a least-squares fitting to the n = 1 Hendersen-Hasselbach equation.



The resonance Raman spectra of the ferric form of the heme-28-kDa fragment complex were measured at pH 7.0 and 9.4 (Fig. 4). Spectra were obtained, which were very analogous to those of the heme-HO-1 complex below and above its pK(a) (Takahashi et al., 1994a, 1994b). At low pH, lines at 1481 ((3)) and 1564 cm ((2)) were obtained for the heme-HO-2 complex and at 1482 ((3)) and 1563 cm ((2)) for the heme-HO-1 complex, and at high pH, lines at 1503 ((3)) and 1579 ((2)) cm were present in the heme-HO-2 spectra compared with lines at 1503 ((3)) and 1581 ((2)) cm in the heme-HO-1 complex. These lines are characteristic of a six coordinate high spin heme iron at pH 7.0 and of a six coordinate low spin state at pH 9.4.


Figure 4: Resonance Raman spectra of the ferric heme-HO-2 complex at pH 7.0 (A) and pH 9.4 (B). The samples (28-kDa fragment) at a concentration of 50 µM were dissolved in 0.1 M phosphate and 0.1 M CHES buffers, respectively. The excitation wavelength was 406.7 nm (6 milliwatt), and the spectra are the result of a 4-min total integration.



We have shown that a water and a hydroxide are the sixth ligand of the heme iron in the HO-1 complex at neutral and alkaline pH, respectively. The spectral similarity indicates that this is also the case for the heme-HO-2 complex, and suggests that the heme pocket structures of the two isozymes resemble each other. The major difference detected between the two isoforms is in the pK(a) value of acid-base transition; the HO-2 pK(a) value of 8.5 is about 1 pH unit higher than the pK(a) of 7.6 observed for the HO-1 complex (Takahashi et al., 1994a). Acid-base transitions observed in ferric hemoproteins with a water ligand are considered to be linked to the ionization of a distal amino acid residue that forms a hydrogen bond with the bound water ligand. The deprotonation of the distal residue, histidine in most cases, causes the ionization of the iron-bound water resulting in a predominantly low spin hydroxide form (Antonini and Brunori, 1971). The pK(a) for the acid-base transition in various hemoproteins has a wide range of values, and the observed pK(a) of 8.5 in the ferric heme-HO-2 fragment complex is similar to that in mammalian myoglobins (Antonini and Brunori, 1971). The possible difference between the two HO isoforms might be that the distal residue is different or that the distal residues are the same but the immediate environment is different.

Determination of the Proximal Heme Ligand

Fig. 5compares the EPR spectrum of the nitric oxide (NO) adduct of the ferrous heme-HO-2 28-kDa fragment complex with that of the HO-1 complex. The HO-2-nitric oxide complex exhibits an EPR spectrum with a rhombic symmetry (g(1) = 2.079, g(2) = 2.005, and g(3) = 1.986) similar to that of the HO-1 complex (Takahashi et al., 1994a). Experiments using ^14NO (data not shown) demonstrated that the doublet with a coupling constant of 3.2 millitesla associated with the g(2) signal in the HO-2 NO EPR spectrum is due to the I = 1/2 of the N. Thus, the triplet hyperfine splitting with a coupling constant of 0.69 millitesla is associated with the I = 1 ^14N nucleus of the axial ligand trans to the bound NO, as reported for the HO-1 complex (Takahashi et al., 1994a). This firmly establishes that the axial heme ligand of the heme-HO-2 complex is a nitrogenous base, likely an imidazole group of histidine as was shown for HO-1 (Takahashi et al., 1994a, 1994b). This assignment was refined by the resonance Raman spectrum of the ligand-free ferrous heme-28-kDa fragment complex (Fig. 6) in which the line at 215 cm is characteristic of an iron-histidine stretching mode in five coordinate ferrous hemoproteins (Kitagawa, 1988). This line is equivalent to that in the heme-HO-1 complex at 218 cm in the low pH form, which was proven to be the iron-histidine stretching mode (Takahashi et al., 1994b). Thus, we conclude that a neutral imidazole of a histidine residue serves as the iron proximal axial ligand in the HO-2 complex also.


Figure 5: EPR spectra of the NO complex of ferrous heme-heme oxygenase complexes recorded at 30 K with a microwave power of 0.2 milliwatts and 0.1 millitesla field modulation at 100 kHz. Top, HO-1 complex; bottom, HO-2 complex (28-kDa fragment).




Figure 6: Resonance Raman spectrum of the ferrous ligand-free heme-HO-2 complex. The sample (28-kDa fragment) at a concentration of 50 µM was dissolved in 0.1 M phosphate buffer. The excitation wavelength was 441.6 nm (6 milliwatts), and the spectrum is the result of a 16-min total integration.



Degradation of Heme Bound to Heme Oxygenase-2

A small amount of NADPH-cytochrome P-450 reductase (0.05 unit) was added to the complex of heme with the 28-kDa fragment of HO-2 in the presence of NADPH, and the changes in the absorption spectrum were followed. After the addition of the reductase, the heme bound to HO-2 was promptly and quantitatively degraded to biliverdin (data not shown), indicating that the substrate heme works as a prosthetic group for O(2) activation as is the case for HO-1 (Yoshida and Kikuchi, 1978b).

Wilks and Ortiz de Montellano(1993) found that the reaction of the heme-HO-1 complex with H(2)O(2) converted the bound heme into a verdoheme-like species but that the reaction with mCPBA formed only a stable oxoferryl species. This is also the case for the heme-HO-2 complex as shown in Fig. 7. Addition of H(2)O(2) to the heme-HO-2 28-kDa fragment complex (spectrumB) resulted in the formation of a verdoheme-like species with an absorption band at 688 nm, as observed for native and wild-type rat HO-1 (Yoshida et al., 1980, 1982; Yoshida and Noguchi, 1984; Wilks and Ortiz de Montellano, 1993). The reaction of the HO-2 complex with mCPBA yielded a species with an optical absorption spectrum (spectrumC) similar to that of Compound II (oxoferryl) of peroxidase enzymes, which was converted to the original spectrum by addition of ascorbic acid (data not shown). When mCPBA was added before H(2)O(2), the H(2)O(2)-dependent conversion to the verdoheme-like species was not observed. These features resemble those observed for the HO-1 complexes by Wilks and Ortiz de Montellano(1993) who suggested that the peroxo species is the active intermediate in HO-1, supporting the early proposal by Noguchi et al.(1983), but is different from the oxoferryl species of cytochrome P-450 or peroxidases (Dawson, 1988). The presence of a neutral proximal His in HO-1 further supports the peroxo-intermediate proposal (Takahashi et al., 1994a, 1994b), and the similar spectroscopic properties indicate that the molecular mechanism of action is analogous between the two isoforms.


Figure 7: Optical absorption spectra showing the reactions of the heme-HO-2 fragment complex with H(2)O(2) and mCPBA. A, spectrum of the complex of ferric heme and HO-2 (28-kDa fragment); B, spectrum recorded 5 min after the addition of 10 equivalent of H(2)O(2); C, spectrum recorded 5 min after the addition of 10 equivalent of mCPBA. All the spectra were recorded in 0.1 M phosphate buffer, pH 7.0, at 20 °C.



Conclusions

Re-examination of the cDNA sequence of human HO-2 rectifies the earlier results in the predicted amino acid sequence that matches those of the rat and rabbit enzyme more closely. The homogeneous 28-kDa tryptic fragment of HO-2 forms an enzyme-heme complex with a stoichiometric amount of heme and retains heme-degrading capability. The heme-HO-2 complex exhibits spectroscopic properties similar to those of the HO-1 complex, and both isoforms have the neutral form of a His imidazole ring as the proximal heme ligand. The major difference observed between the two isoforms is that the pK(a) of the distal residue, which is linked to the heme-bound water in the ferric form, is higher in HO-2 than in HO-1 by about 1 pH unit. Despite this difference, our results show that the molecular mechanism of the enzyme action of HO-2 is the same as that of HO-1 as far as can be discerned.


FOOTNOTES

*
This work was partially supported by Research Grants GM48714 (to D. L. R.) and GM39492 (to M. I.-S.) from the National Institute of General Medical Sciences and Grant 02680150 (to T. Y.) from the Ministry of Education, Science, and Culture of Japan. The purchase of the Bruker EPR spectrometer system was in part supported by Grant RR05659 (to M. I.-S.) from the National Center for Research Resources. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D21243[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-236-33-1114; Fax: 81-236-33-4020.

(^1)
The abbreviations used are: HO, heme oxygenase; PAGE, polyacrylamide gel electrophoresis; mCPBA, m-chloroperbenzoic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Masanao Sasahara for technical assistance and to Mariko Sato for help in preparation of the manuscript.


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