Identification of Histidine 45 as the Axial Heme Iron Ligand of Heme Oxygenase-2*

Kazunobu IshikawaDagger , Kathryn Mansfield Matera§, Hong ZhouDagger par , Hiroshi Fujii**, Michihiko SatoDagger Dagger , Tetsuhiko Yoshimura**, Masao Ikeda-Saito§, and Tadashi YoshidaDagger §§

From the Dagger  Department of Biochemistry and Dagger Dagger  Central Laboratory for Research and Education, Yamagata University School of Medicine, Yamagata, 990-23, Japan, the § Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, the ** Institute for the Life Support Technology, Yamagata Techonopolis Foundation, Yamagata, 990, Japan

    ABSTRACT
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A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (Delta HHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (Delta H45A) and His-152 to Ala (Delta H152A) mutants have been prepared using this expression system. Delta H45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-Delta H45A complex. The Delta H152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified Delta H152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. Biophys. 302, 402-408; McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.

    INTRODUCTION
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Heme oxygenase (HO),1 a microsomal protein, catalyzes the regiospecific oxidative degradation of iron protoporphyrin IX (heme hereafter) to biliverdin, CO, and Fe in the presence of NADPH-cytochrome P-450 reductase, which functions as an electron donor (1-3). In the catalytic cycle of HO, the enzyme first binds one equivalent of heme to form a heme-enzyme complex, which exhibits spectroscopic properties similar to those of myoglobins and hemoglobins (4-7). The first electron donated from the reductase reduces the ferric heme iron to the ferrous state, and a molecule of oxygen subsequently binds to form a metastable oxy form (8). Subsequent electron donation to the oxy form initiates the three stepwise oxygenase reactions during which CO and an iron-biliverdin complex are produced. Heme, therefore, participates both as the prosthetic group for oxygen activation and as the substrate for the enzyme catalysis, a property unique to HO (9).

The presence of two isoforms of heme oxygenase has been established: 33-kDa heme oxygenase isoform-1, HO-1, which is inducible and highly expressed in the liver and spleen tissues; and 36-kDa heme oxygenase isoform-2, HO-2, which is constitutive and distributed throughout the body (3). The two isoforms are products of two distinctly different genes (10, 11). Amino acid sequence similarity between the two isoforms is only about 40% with an extra N-terminal 20-amino acid sequence present in HO-2; however, there are two stretches of highly conserved sequences with matched predicted secondary structure (11). Both HO-1 and HO-2 have hydrophobic sequences at their C-terminal ends which have been proposed to be involved in binding to microsomal membranes (12-14).

A recent development in the bacterial expression of a 30-kDa soluble form of rat HO-1, which lacks the hydrophobic C-terminal domain, has made it possible to prepare HO-1 in the quantities required for structural studies (15, 16). By combination of optical absorption, EPR, resonance Raman scattering, and site-directed mutagenesis, we have established that the axial heme ligand in the heme·HO-1 complex is a neutral form of the imidazole of His-25 (6, 7, 15). Similar conclusions were drawn in an independent study by Sun et al. (17). Our spectroscopic and enzymatic examinations of the 28-kDa tryptic HO-2 fragment-heme complex has shown that both isoforms display the same enzymatic activity and that the two isoforms have homologous active site structures: the proximal ligand of the heme HO-2 complex is also a neutral imidazole of the histidine residue (18). Hence, the molecular mechanism of the enzyme action is presumed to be similar between the two isoforms. Sequence comparison predicts that His-45 in the HO-2 sequence corresponds to His-25 of HO-1, and thus, His-45 appears to be a primary candidate for the heme proximal ligand in the heme-HO-2 complex. However, Maines and co-workers (19, 20) reported that His-152 in HO-2, which corresponds to His-132 in the HO-1 sequence, was the essential His residue in the HO activity. His-132 in HO-1 does not play a role in the enzyme catalysis but is an important residue for folding the HO-1 protein (15, 16). Identification of the proximal heme ligand in HO-2 is yet to be established. HO-2 has attracted attention recently since the CO produced by HO-2 has been proposed to serve as a physiological messenger molecule in neurotransmission and vascular tone regulation (21-24). In light of the physiological significance of HO-2, we think it important to determine the roles of the conserved histidine residues and the heme axial ligand in the heme·HO-2 complex.

To this end, we have developed a new bacterial expression system for a 33-kDa truncated, soluble human HO-2 protein lacking its C-terminal membrane anchoring segment. Using this expression system, we have prepared two mutants of HO-2, H45A and H152A. We report here that the His-152 to Ala mutation retains the heme oxygenase activity and that His-152 is not a significant residue for the enzyme function but an important residue for stability of the heme oxygenase protein. In contrast, the His-45 to Ala replacement abolishes the enzyme activity, and spectroscopic examination establishes that the imidazole group of His-45 is the axial ligand in the heme·HO-2 complex.

    EXPERIMENTAL PROCEDURES
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Chemicals and Materials-- The sources of reagents are as follows: T4 polynucleotide kinase and sequencing primer for pTV118N, P7 (3'-CAGCACTGACCCTTTTGGGACCGC-5'), from Toyobo; restriction endonucleases, from New England Biolabs; T4 DNA ligase from Boehringer Mannheim; DyeDeoxyTM Terminator Cycle Sequencing kit from Perkin-Elmer Corp.; Pfu polymerase from Stratagene; ampicillin, hemin, and bovine serum albumin from Sigma; nitrocellulose membranes from Micron Separations Inc.; Sephadex G-75 from Pharmacia; DE-32 from Whatman; peroxidase-conjugated sheep IgG against rabbit IgG from Cappel; synthetic oligonucleotides from Sawady Technology; Escherichia coli strain BL21 from Novagen. A T-7 promoter prokaryotic expression vector, pMW172, was a generous gift from Dr. K. Nagai (MRC Laboratory of Molecular Biology, Cambridge, U.K.). NADPH-cytochrome P450 reductase was solubilized from rat liver microsomes with Triton X-100 and purified by the method of Yasukochi and Masters (25). Partially purified biliverdin reductase was prepared from the cytosolic fraction of porcine spleen by the method of Noguchi et al. (26).

Construction of Expression Plasmids of Truncated Soluble Form of Human HO-2-- In our previous work on human HO-2, we constructed an expression vector for the full-length human HO-2 protein, pTVHHO2 (18). Trypsin digestion of the recombinant full-length HO-2 yielded an enzymatically active 28-kDa tryptic fragment of the enzyme encompassing from Met-30 to Lys-274. Because the "putative" key His residue at position 45 is very close to the N-terminal after the tryptic digestion (Met-30), we were concerned with possible effects from the removal of the N-terminal 29 amino acids on the HO function. In addition, expression and purification of a water-soluble form is expected to be easier than the tryptic digestion of the detergent-solubilized full-length protein during the purification process as manifested in the recombinant HO-1 protein preparation (15, 16). Therefore, in this work, we have constructed a new expression vector, pMWDelta HHO2, which contains a human HO-2 cDNA coding sequence covering the amino acid residues from 1 to 288, from the full-length HO-2 expression vector, pTVHHO2, which was used in our previous studies (18).

pTVHHO2 was digested with ApaI and KpnI, and the two DNA fragments were isolated. The larger DNA fragment includes the pTV118N expression vector and the 1 to 63 nucleotide HO-2 coding sequence (the A of the initiation codon ATG is defined as nucleotide residue 1). The smaller DNA fragment corresponds to the HO-2 cDNA sequence from 64 to 997. The smaller DNA fragment was further cleaved by BpmI, and the ApaI/BpmI fragment, which includes the HO-2 coding sequence from 64 to 850, was isolated. Synthetic nucleotides, 5'-TCCGAACAGCTATGTAGGTAC-3' and 3'-GAAGGCTTGTCGATACATC-5' were phosphorylated and annealed. The larger ApaI/KpnI fragment of pTVHHO2, the ApaI/BpmI fragment, and the double-stranded oligonucleotide were ligated to form the pTVDelta HHO2 plasmid, which includes the human HO-2 coding sequence from Met-1 to Met-288 but lacks the coding sequence for the human HO-2 C-terminal hydrophobic region (Ala-289 to Met-316). A polymerase chain reaction (PCR) was carried out with pTVDelta HHO2 as a template and a synthetic nucleotide X (5'-AACAGCATATGGAGCGCCCACAGCTCGA-3') and P7 as primers. The nucleotide X corresponds to the nucleotide sequence from positions -8 to +20 of pTVDelta HHO2, except that ACC at positions from -3 to -1 was changed to CAT so as to create a new NdeI restriction site. The target fragment was digested with NdeI and HindIII and cloned into pMW172 to construct pMWDelta HHO2.

Site-directed Mutagenesis for H45A and H152A Mutants-- Two independent PCRs for construction of pMWDelta H45A, in which His-45 was replaced by Ala, were performed with pMWDelta HHO2 as a template DNA. Primers for the 5'-side region amplification were a synthetic nucleotide X and a mutagenic primer (3'-GGTTCCTTCGTCGGCTGGCCCGTCT-5'). The underlined bases are complementary codon for Ala-45. Primers for the 3'-side region amplification were a flanking primer (5'-AAACACCCAGTTTGTCAAGGACTTC-3') and a synthetic nucleotide Y (3'-GGCTTTCCTTCGACTCAACCGACGACGGTG-5') which is the complementary nucleotide sequence from positions 2,461 to 2,490 of pMW172 plasmid. The fragments for the 5'-side region and the 3'-side region, obtained after PCRs, were phosphorylated with T4 polynucleotide kinase and then digested by AccI and HindIII, respectively. The resulting two fragments were cloned into the pMWDelta HHO2 predigested by AccI and HindIII. For construction of pMWDelta H152A, a similar procedure was employed except that oligonucleotides of 3'-ATGACCACCGGCGACGTATGTGGGC-5' (the underlined bases are complementary codon of Ala-152) and 5'-CTACATGGGGGATCTCTCGGGGGGC-3' were used as mutagenic and flanking primers, respectively. The entire coding sequences of these plasmids were determined by an Applied Biosystems 373A DNA sequencer.

Preparation of a Truncated, Soluble Form of Wild-Type HO-2-- A fresh single colony of E. coli strain BL21 transformed with pMWDelta HHO2 was precultured overnight at 37 °C in 10 ml of Luria-Bertani medium. Then, 100 µl of the preculture was used to inoculate 500-ml cultures in the same medium, which was incubated at 37 °C for 16 h. The harvested cells carrying the HO-2 expression vector, pMWDelta HHO2, were green, while control cells transformed with pMW172 were brown. The green color is due to the conversion of heme to biliverdin in the bacterial cells as observed in the bacterial expression of HO-1 protein (13, 16). The harvested cells were washed and lysed as described previously (15). SDS-PAGE analysis showed a major band, with a molecular mass of 33 kDa, in the soluble fraction of the cells harboring the expression vector but not in the precipitated fraction. In contrast, the 33-kDa band did not appear in the lysates of the control cells. The 33-kDa band cross-reacted with the human HO-2 antibody. These results indicate that the truncated HO-2 protein, Delta HHO-2, with a molecular mass of 33 kDa was expressed in the bacteria and present in the soluble fraction of the bacterial lysate. A minor band with a molecular mass of 31 kDa was also present in the soluble fraction. This band likely corresponds to a partially degraded form of Delta HHO-2 because this band is also reactive to antibodies against human HO-2.

The soluble fraction obtained after centrifugation was treated with solid ammonium sulfate, and the precipitate was obtained between 35 and 55% ammonium sulfate saturation. The precipitate was dissolved in 20 mM sodium phosphate buffer, pH 7.4, and the HO-2 protein was purified by the two-step column chromatography on Sephadex G-75 and DE-32, according to the procedures used for the truncated rat HO-1 (15), except that the buffer did not contain phenylmethylsulfonyl fluoride. The purified enzyme showed a single protein band of 33 kDa on SDS-PAGE stained with Coomassie Brilliant Blue. About 20 mg of pure Delta HHO-2 was obtained from a single 500-ml culture.

Preparation of Delta H45A Mutant-- E. coli BL21 transformed with pMWDelta H45A was cultured as described for the expression of wild-type. The harvested cells transformed with pMWDelta H45A were not green but brown. However, when the cell lysate was subjected to SDS-PAGE, we could detect a 33-kDa major band with a minor band at 31 kDa, as observed for the wild-type HO-2 expression. As is the case for the wild-type expression, these two bands were present in the soluble fraction but were absent in the precipitated fraction. They cross-reacted with the human HO-2 antibody. As the Delta H45A mutant was recovered in the soluble fraction, we purified this mutant by the same procedure as that used for the wild-type enzyme, except that Western blotting analysis was utilized to detect the mutant HO-2 protein.

Extraction of Delta H152A Mutant from Inclusion Bodies and Its Renaturation-- E. coli BL21 transformed with pMWDelta H152A was cultured as described for the expression of wild-type. The harvested cells carrying pMWDelta H152A were brown indicating that endogenous heme was not converted to biliverdin. However, SDS-PAGE analysis of the lysate of the harvested cells clearly showed that a protein band with a molecular mass of 33 kDa was expressed. The 33-kDa protein cross-reacted with the HO-2 antibody, indicating that Delta H152A was indeed expressed at the same level as the wild-type enzyme but that the expressed enzyme did not exhibit enzyme activity in the E. coli. SDS-PAGE also demonstrated that Delta H152A was recovered exclusively in the precipitated fraction. These observations suggest that the expressed Delta H152A protein accumulates in the host cells as an inclusion body. Therefore, we purified Delta H152A from inclusion bodies as described below.

To 10 g of packed cells in 100 ml of 50 mM Tris-HCl (pH 7.2) containing 2 mM EDTA were added 10 mg of lysozyme and stirred for 1 h at 4 °C. The mixture was then sonicated 10 times at maximum power with a Branson Sonifier 250 for 30 s with a 4-min interval between sonication and centrifuged at 8,000 × g for 20 min. The precipitates were suspended with 100 ml of 0.5 M sucrose with a Hitachi HG 30 homogenizer, followed by centrifugation at 8,000 × g for 20 min. Precipitates were treated with 100 ml of 20 mM sodium phosphate buffer (pH 7.4) containing 1% Triton X-100 and 1% sodium deoxycholate. The mixture was left for 1 h and then centrifuged at 8,000 × g for 20 min. The resulting precipitates were solubilized by homogenization in 10 ml of 30 mM Tris-HCl buffer (pH 7.2) containing 30 mM NaCl, 1 mM dithiothreitol, 5 mM 2-mercaptoethanol, and 8 M urea, followed by dialysis for 24 h against 2 liters of 20 mM sodium phosphate buffer, pH 7.4, containing 1 mM dithiothreitol and 5 mM 2-mercaptoethanol; the buffer was replaced 3 times. No SH-protecting reagents were added to the final buffer. After dialysis, the sample was centrifuged at 100,500 × g for 30 min, and the supernatant was collected. The supernatant was judged as a pure soluble H152A protein by SDS-PAGE.

Heme Oxygenase Activity Assay-- The assay method was similar to that described previously (4) with minor modifications. The standard reaction mixture contained, in a final volume of 1.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, an appropriate amount of HO, 30 nmol of hemin, 0.15 mg of bovine serum albumin, partially purified biliverdin reductase (5 nmol/min), 1 µmol of NADPH, and 1 unit of NADPH-cytochrome P-450 reductase. The reaction was initiated by the addition of NADPH and was carried out for 10 min at 37 °C. The absorbance difference at 468 nm was recorded between the standard and control systems; the latter system contained all components except for NADPH. The value of 43.5 mM-1 cm-1 was used for the extinction coefficient of bilirubin at 468 nm (4). One unit of enzyme was defined as the amount of enzyme catalyzing the formation of 1 nmol of bilirubin/h under these conditions.

Analytical Methods-- Light absorption spectra were recorded by Hewlett-Packard 8453 and Hitachi U-3210 spectrophotometers at 20 °C. EPR spectra were obtained by a Bruker ESP-300 spectrometer operating at 9.45 GHz equipped with an Oxford liquid helium flow cryostat. Microwave frequency was monitored by a frequency counter (HP-5350), and an NMR gauss meter (Bruker ER-035 M) was used to determine the magnetic flux density. Far-UV CD spectra (260 to 160 nm) were measured as described previously (15). Immunoblotting with polyclonal HO-2 antibodies (27) and protein assay by Lowry's method (28) were performed according to the published procedures.

    RESULTS AND DISCUSSION
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Properties of Truncated Soluble HO-2-- The specific activity of the purified Delta HHO-2 was about 1,100 nmol of bilirubin formed/mg of protein/h when assayed with NADPH-cytochrome P-450 reductase system. Purified Delta HHO-2 binds hemin at 1:1 molar ratio, and the optical absorption spectra of the Delta HHO2-heme complex in ferric, ferrous, and ferrous-CO forms were identical to those of the 28-kDa tryptic HO-2 fragment-heme complex (18). We designate the 33-kDa water soluble form, Delta HHO-2, as wild-type for the purpose of discussion.

Properties of Delta H45A-- Purified Delta H45A had no heme degradation activity when assayed by NADPH-cytochrome P-450 reducatase. Thus, His-45 of HO-2 is clearly essential for the heme degradation activity. One might claim that the loss of the enzyme activity in Delta H45A is due to the unfolding of the protein induced by the mutation. In a recent mutant Mb study, Hargrove et al. (29) found that mutations in the heme pocket can drastically alter the stability of apoMb, and some heme-pocket mutants of apoMb assume unfolded structures. To examine this possibility, we measured the CD spectra of wild-type and Delta H45A between 260 and 180 nm. As shown in Fig. 1A, the CD spectrum for Delta H45A is indistinguishable from that of the wild-type spectrum, thus the His-45 to Ala mutation does not appear to alter the global protein structure of the enzyme. This implies that the abolished enzyme activity of Delta H45A is not due to mutation-induced protein unfolding but to the specific effect of His-45 to Ala replacement.


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Fig. 1.   CD and optical absorption spectra of wild-type HO-2 and the Delta H45A mutant in 0.1 M phosphate buffer, pH 7.0, at 20 °C. A, top panel, CD spectra of wild-type and Delta H45A. Protein concentration was about 1 mg/ml, and an optical cuvette with a path length of 0.01 mm was used. B, bottom panel, light absorption spectra of the complex with heme and wild-type (solid line) and the Delta H45A mutant (dotted line). Inset, titration of Delta H45A (3.5 µM) by hemin as monitored by absorption increase at 400 nm (closed symbols). The data shown by open symbols are the absorbance increase at the same wavelength in the absence of Delta H45A protein.

To examine whether or not His-45 is the heme-linked residue of the heme-HO complex, we characterized the heme·Delta H45A complex spectroscopically. Fig. 1B compares the optical absorption spectra of the ferric form of an equivalent mixture of heme and Delta H45A with that of wild-type. The heme·wild-type complex exhibits a 6-coordinate ferric high spin spectrum (18), whereas a significantly different spectrum is observed for the heme·Delta H45A complex. The Soret maximum of the mutant complex is blue shifted to 400 nm, and its intensity is reduced with respect to that of the wild-type spectrum; the heme iron in the Delta H45A complex is also in a high spin state, but the spectral change upon the His-45 to Ala mutation is an indication of an altered coordination structure in the mutant complex. The optical absorption spectrum is similar to that of the heme complex of HO-1 Delta H25A, a proximal His to Ala Delta HO-1 mutant (15). Since the Soret region optical absorption spectrum of the heme-Delta H45A complex is different from that of free heme in pH 7 buffer, spectrophotometric titration of Delta H45A with hemin was carried out utilizing this difference. As shown in the inset of Fig. 1B, the titration curve of the combination of Delta H45A with hemin shows a well defined inflection point from which the molar stoichiometry of their binding was estimated to be 1:1. Therefore, we conclude that Delta H45A can form a stoichiometric complex with hemin just as wild-type does. The heme-Delta H45A complex exhibited an EPR spectrum of a ferric high spin hemoprotein at 6 K (data not shown). As seen for the spectrum of the heme complex of HO-1 Delta H25A (15), the gperp signal of the H45A spectrum is broader than that of the wild-type spectrum, indicating that the electronic structure of the heme iron in Delta H45A is different from that of wild-type. The spectral changes upon His-45 to Ala mutation indicate possible changes in the coordination state of the heme iron.

Fig. 2 compares the EPR spectrum of the 15NO-bound ferrous heme-Delta H45A complex to that of the wild-type complex. The spectral shape of the Delta H45A mutant complex is typical of a five coordinate 15NO heme species (30), and the spectrum of the wild-type complex is 6 coordinate with an imidazole axial ligand as reported for the 28-kDa tryptic HO-2 fragment (18). This infers that, in the Delta H45A NO complex, the fifth coordination of the heme iron is vacant. On the basis of these observations, we conclude that His-45 is the proximal axial iron ligand in the heme·HO-2 complex and that the proximal His is essential for the HO-2 enzyme catalysis. This conclusion contradicts the proposal of Maines and co-workers who reported that His-152 might be the proximal heme ligand (19, 20). To address this discrepancy, we have prepared a His-152 to Ala mutant and examined its enzymatic and spectroscopic properties.


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Fig. 2.   EPR spectra of the ferrous 15NO compounds of the heme complexes of wild-type HO-2 (top) and the Delta H45A mutant (bottom). Measurements were carried at 30 K with an incident microwave power of 0.2 mW with a field modulation of 0.2 millitesla at 100 kHz.

Properties of H152A Mutant-- Specific heme degradation activity of H152A solubilized from inclusion bodies was only ~150 nmol of bilirubin formed/mg of protein/h under NADPH-cytochrome P-450 reductase system, about one-sixth of the wild-type enzyme activity. This indicates that either the soluble fraction contained a considerable amount of non-functional Delta H152A mutant protein or His-152 to Ala mutation reduces the HO enzyme activity. To assess the possibility of the presence of nonfunctional Delta H152A protein, we subjected the soluble fraction to gel-filtration column chromatography on Sephadex G-75. The elution profile of the G-75 column chromatography showed the presence of two protein peaks, one eluted at the void-volume and the other at the same elution volume as the wild-type protein. The latter fraction has a molecular mass of 33 kDa. Western blotting analysis showed both fractions cross-reacted with the native HO-2 antibody. The far-UV CD spectrum of the 33-kDa fraction was indistinguishable from that of the wild-type enzyme, indicating that the Delta H152A protein eluted as the 33-kDa fraction is properly folded. The void-volume fraction became turbid and its CD spectrum could not be obtained. The 33-kDa peak, which appeared to be homogenous as judged by the SDS-PAGE, had the same heme degradation activity as that of the wild-type enzyme. This is in contrast to the results of Maines and co-workers who claimed that His-152 is an essential His residue for the enzyme activity of HO-2 (19, 20). We used the second peak, 33 kDa HO-2 as Delta H152A, in the following experiments.

The optical absorption spectra of the heme-H152A in the ferrous, ferrous-CO, and ferrous-O2 forms were also the same as those of the wild-type complex (not shown). There was no difference detected in the auto-oxidation rate of the oxy form of the heme complex between Delta H152A and wild-type. In myoglobin, the hydrogen bond between the distal His and the bound oxygen plays a most crucial role in the inhibition of myoglobin heme iron auto-oxidation (31). If His-152 forms a hydrogen bond with the bound-oxygen, the rate of auto-oxidation rate of Delta H152A is expected to be different from that of the wild-type heme·HO complex. Fig. 3 illustrates the pH-dependent changes in the light absorption spectrum of the ferric heme·Delta H152A complex between pH 7 and 9.5. At pH 7, the heme·Delta H152A complex exhibits a typical hexacoordinate high spin spectrum with a Soret peak at 404 nm and bands at 500 and 631 nm in the visible region; the spectrum is indistinguishable from that of the wild-type heme complex shown in Fig. 1B. At alkaline pH, the high spin spectrum is replaced by a spectrum with bands at 413, 540, and 575 nm, which is identical to the wild-type spectrum in alkaline pH (18). This pH-dependent spectral change is reversible between pH 6 and 9.5, and the pKa value of the change is estimated to be 8.5 as shown in the inset of Fig. 3. These features, including spectral shape, peak positions, and the pKa value of the spectral transitions, are identical to those of the heme complex of the 28-kDa tryptic fragment of human HO-2 (18). If His-152 is linked with the coordinated water molecule in the ferric form, the pK value of the acid-base transition for the Delta H152A complex should be different from that of the wild-type complex.


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Fig. 3.   Light absorption spectra of the ferric heme·Delta H152A complex at pH 7.0, 8.5, and 9.0 at 20 °C. 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-Hasselback equation.

Comparison with the Results of Maines and Co-workers (19, 20), and with HO-1 H132A (16, 32)-- It is intriguing to consider why the same HO-2 mutant, H152A, used by Maines and co-workers (19, 20), had little enzyme activity while our mutant is fully active. We have been using human HO-2, while rat HO-2 was used by McCoubrey and Maines (19). The amino acid sequence around His-152 is quite similar between these two proteins; hence, it is highly unlikely that the difference in species is the cause of the discrepancies in the enzymatic properties. We used the purified Delta H152A protein for the spectroscopic and enzymatic studies, but Maines and co-workers did not purify the mutant enzyme. McCoubrey and Maines (19) measured heme oxygenase activity using a Triton-solubilized fraction of the precipitates of the bacterial cells in which H152A protein was expressed. It is possible that the preparations used by McCoubrey and Maines contained a significant amount of the unfunctional fraction of the mutant protein, since HO protein purification procedures required for removal of the unfunctional fraction were not employed in their work. It is also conceivable that the Triton-solubilized fraction used by McCoubrey and Maines might contain very small amounts of the H152A protein because Triton X-100 does not solubilize proteins from inclusion bodies (33). The diminished enzyme activity of H152A reported by McCoubrey and Maines (19) could be accounted for either by the presence of nonfunctional fractions and/or the absence of the mutant enzyme in their Triton X-100 bacterial extracts from the E. coli. Whatever the reason for the apparent discrepancies, the Delta H152A mutant enzyme prepared by our method exhibits spectroscopic and enzymatic properties essentially identical to those of the wild-type enzyme. Hence, we conclude that H152A is not an important residue for catalysis by this enzyme.

His-152 in HO-2 corresponds to His-132 in HO-1 so that it is also of interest to compare the properties of HO-2 Delta H152A to those of HO-1 Delta H132A. His-132 (HO-1) and His-152 (HO-2) are located in a span of 24 amino acids (residue 146 to 169 for HO-2 and 126 to 149 for HO-1) which is conserved in all forms of heme oxygenase with only one substitution: Leu-138 in HO-1 is replaced by Met in HO-2. HO-1 Delta H132A shares several key molecular properties with HO-2 Delta H152A. Both of them are expressed as inclusion bodies when the same expression system is used. The solubilized fraction of HO-1 Delta H132A from the inclusion body also includes both a functional enzyme and an inactive fraction that can be separated by gel filtration chromatography. As is the case for HO-2 Delta H152A, the purified Delta H132A has spectroscopic and enzymatic properties identical to those of wild-type (16). It is tempting to propose that HO-1 and HO-2 have the structural features in common at least near this 24-amino acid span.

The present results from HO-1 Delta H132A and HO-2 Delta H152A studies demonstrate the need for care in studying the roles of a specific amino acid by site-directed mutagenesis and bacterial expression of recombinant proteins. As is the case for both isoforms of heme oxygenase, certain mutant proteins, such as HO-1 Delta H132A and HO-2 Delta H152A, are expressed in inclusion bodies, while wild-type and other mutants, such as HO-1 Delta H25A and HO-2 Delta H45A, are expressed in soluble fractions (15, 16). In these cases, simple enzyme assays using bacterial extracts are not sufficient to conclude whether or not a particular residue is essential for enzyme activity. To avoid premature and erroneous conclusions as seen in the previous work on HO-1 His-132 (32) and HO-2 His-152 mutants (19, 20), possible formation of inclusion bodies must be assessed and careful purification of properly folded protein is necessary.

    FOOTNOTES

* This work was supported in part by Grants-in-aid for Scientific Research 06780575 (to K. I.), 08044240 (to T. Y.), 08249101 (to T. Y.), 08680675 (to T. Y.), 09235101 (to T. Y.), and 0948158 (to T. Y.) from the Ministry of Education, Science, Sports and Culture, Japan, and by Research Grant GM51588 (to M. I.-S.) from the National Institutes of Health. The purchase of the Bruker EPR instrument was in part supported by Grant RR05659 (to M. I.-S.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Chemistry, Baldwin-Wallace College, Berea, OH 44017.

par Present address: Harbin Preclinical College of Medicine of Medical University, Harbin, China.

§§ To whom correspondence should be addressed. Tel.: 81-236-28-5222; Fax: 81-236-28-5225.

1 The abbreviations used are: HO, heme oxygenase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Mb, myoglobin.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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