(Received for publication, December 27, 1994)
From the
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.
Microsomal heme oxygenase (HO) ()is the rate-limiting
enzyme in the mammalian heme degradation pathway. The enzyme converts
iron-protoporphyrin IX (heme) into biliverdin IX
with the
accompanying production of CO and release of free iron through
successive mono-oxygenase reactions, which require three O
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
(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.
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).
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.
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.
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 (Takahashi et al., 1994a,
1994b). At low pH, lines at 1481 (
) and 1564
cm
(
) were obtained for the
heme-HO-2 complex and at 1482 (
) and 1563
cm
(
) for the heme-HO-1 complex,
and at high pH, lines at 1503 (
) and 1579
(
) cm
were present in the heme-HO-2
spectra compared with lines at 1503 (
) and 1581
(
) 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 value of acid-base
transition; the HO-2 pK
value of 8.5 is about 1 pH
unit higher than the pK
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
for the
acid-base transition in various hemoproteins has a wide range of
values, and the observed pK
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.
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.
Wilks and Ortiz de
Montellano(1993) found that the reaction of the heme-HO-1 complex with
HO
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
O
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
O
, the H
O
-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 HO
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
O
; 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D21243[GenBank].