From the Department of Biochemistry and
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
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ABSTRACT |
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A truncated, soluble, and enzymatically active
form of human heme oxygenase-2 (HHO2) was expressed in
Escherichia coli. To identify the axial heme ligand of
HO-2, His-45 to Ala (
H45A) and His-152 to Ala (
H152A) mutants
have been prepared using this expression system.
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-
H45A complex. The
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
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.
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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.
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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, pMWHHO2, 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).
Site-directed Mutagenesis for H45A and H152A Mutants--
Two
independent PCRs for construction of pMWH45A, in which His-45 was
replaced by Ala, were performed with pMW
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 pMW
HHO2 predigested by AccI and
HindIII. For construction of pMW
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 pMWHHO2 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, pMW
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,
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
HHO-2 because this band is also reactive to antibodies
against human HO-2.
Preparation of H45A Mutant--
E. coli BL21
transformed with pMW
H45A was cultured as described for the
expression of wild-type. The harvested cells transformed with
pMW
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
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 H152A Mutant from Inclusion Bodies and Its
Renaturation--
E. coli BL21 transformed with pMW
H152A
was cultured as described for the expression of wild-type. The
harvested cells carrying pMW
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
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
H152A was recovered
exclusively in the precipitated fraction. These observations suggest
that the expressed
H152A protein accumulates in the host cells as an
inclusion body. Therefore, we purified
H152A from inclusion bodies
as described below.
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 mM1
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.
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RESULTS AND DISCUSSION |
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Properties of Truncated Soluble HO-2--
The specific activity of
the purified HHO-2 was about 1,100 nmol of bilirubin formed/mg of
protein/h when assayed with NADPH-cytochrome P-450 reductase system.
Purified
HHO-2 binds hemin at 1:1 molar ratio, and the optical
absorption spectra of the
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,
HHO-2, as wild-type for the purpose of discussion.
Properties of H45A--
Purified
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
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
H45A between 260 and 180 nm. As shown in
Fig. 1A, the CD spectrum for
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
H45A is not due to mutation-induced protein unfolding
but to the specific effect of His-45 to Ala replacement.
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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 H152A mutant protein or His-152 to Ala
mutation reduces the HO enzyme activity. To assess the possibility of
the presence of nonfunctional
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
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
H152A, in the
following experiments.
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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 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
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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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