Tài liệu Báo cáo khoa học: Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied docx

Unique features of recombinant heme oxygenase of
Drosophila
melanogaster
compared with those of other heme
oxygenases studied
Xuhong Zhang
1
, Michihiko Sato
2
, Masanao Sasahara
1
, Catharina T. Migita
3
and Tadashi Yoshida
1
1
Department of Biochemistry and
2
Central Laboratory for Research and Education, Yamagata University School of Medicine, Japan;
3
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan
We cloned a cDNA for a Drosophila melanogaster homo-
logue of mammalian heme oxygenase (HO) and constructed
a bacterial expression system of a truncated, soluble form
of D. melanogaster HO (DmDHO). The purified DmDHO
degraded hemin to biliverdin, CO and iron in the presence
of reducing systems such as NADPH/cytochrome P450
reductase and sodium ascorbate, although the reaction rate
was slower than that of mammalian HOs. Some properties
of DmHO, however, are quite different from other known
HOs. Thus DmDHO bound hemin stoichiometrically to

form a hemin–enzyme complex like other HOs, but this
complex did not show an absorption spectrum of hexa-
coordinated heme protein. The absorption spectrum of the
ferric complex was not influenced by changing the pH of the
solution. Interestingly, an EPR study revealed that the iron
of heme was not involved in binding heme to the enzyme.
Hydrogen peroxide failed to convert it into verdoheme. A
spectrum of the ferrous–CO form of verdoheme was not
detected during the reaction from hemin under oxygen and
CO. Degradation of hemin catalyzed by DmDHO yielded
three isomers of biliverdin, of which biliverdin IXa and two
other isomers (IXb and IXd) accounted for 75% and 25%,
respectively. Taken together, we conclude that, although
DmHO acts as a real HO in D. melanogaster, its active-site
structure is quite different from those of other known HOs.
Keywords: biliverdin; Drosophila melanogaster; heme oxy-
genase; insect; NADPH/cytochrome P450 reductase.
Heme oxygenase (HO, EC 1.14.99.3) was first characterized
in mammals as a microsomal enzyme that catalyzes the
three-step oxidation of hemin to biliverdin IXa,CO,and
free iron, via a-meso-hydroxyhemin, verdoheme, and ferric
iron–biliverdin complex [1–3] (Scheme 1). To date two
mammalian isozymes of HO have been identified [4]: HO-1,
an inducible enzyme that is highly expressed in the spleen
and liver; HO-2, a constitutive enzyme found abundantly in
the brain and testes. The two isozymes have about 43%
similarity at amino acid level, and both have a C-terminal
hydrophobic domain that is involved in binding to micro-
somal membrane. Both HO-1 and HO-2 have been
demonstrated to play important roles in physiological iron

homeostasis [5,6], antioxidant defense [7,8], and possibly the
cGMP signaling pathway [9,10]. Although HO-3 was once
reported as an isozyme of HO, its function is not yet well
defined [11].
HO has also been found and characterized in bacteria
[12–14] and plants [15–18] and other species such as
Rhodophyta [19]. In contrast with mammalian HO,
these HOs are water-soluble enzymes because they lack
a membrane-anchoring domain at the C-termini of their
sequences. In pathogenic bacteria, HO is thought to help
bacteria to acquire iron from heme-containing proteins
found in their host cells for survival and toxin production.
In plants, biliverdin is used for the biosynthesis of photo-
responsive bilins such as phycobilins and phytochromobi-
lins [15–19]. Although the HOs have been characterized
structurally and functionally in most species, very little is
known about HO in insects.
Heme is extremely important in insects. It is a vital
nutrient for most, if not all, insects for their embryonic
development [20], although they do not use it as a transport
vehicle or storage vessel for oxygen. Heme also serves as the
prosthetic moiety of hemoproteins, such as hemoglobin
[21,22], catalase [23] and nitric oxide synthase [24], which are
essential for biological function. However, heme is poten-
tially toxic to insects, particularly blood-sucking insects such
as mosquitoes, because it catalyzes oxidative reactions that
can damage membranes and destroy nucleic acids. There-
fore, insects are thought to have several mechanisms for
sequestering and controlling heme. For example, it can be
conjugated with such proteins as the heme binding protein

Correspondence to T. Yoshida, Department of Biochemistry,
Yamagata University School of Medicine, Yamagata, Japan.
Fax: + 81 23 628 5225, Tel.: + 81 23 628 5222,
E-mail:
Abbreviations: HO, heme oxygenase; CPR, NADPH/cytochrome
P450 reductase; DmHO, heme oxygenase of D. melanogaster;
DmDHO, truncated form of D. melanogaster heme oxygenase;
DmCPR, NADPH/cytochrome P450 reductase of D. melanogaster;
DmDCPR, truncated form of D. melanogaster NADPH/cytochrome
P450 reductase; Syn HO-1, heme oxygenase-1 of Synechocistis sp.
PCC 6803.
Enzymes: heme oxygenase (EC 1.14.99.3); NADPH/cytochrome P450
reductase (EC 1.6.2.4).
(Received 25 December 2003, revised 2 March 2004,
accepted9March2004)
Eur. J. Biochem. 271, 1713–1724 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04077.x
isolated from the bug Rhodnius prolixus, to form complexes
that serve as a source of heme and prevent cells from
heme-induced oxidative injury [25].
Although heme biosynthesis has been reported in
insects, very little is known about its degradation. In fact,
no HO has been isolated and characterized from any
insect species so far. Interestingly, biliverdin IXc is present
in larval integument and hemolymph in some species of
Lepidoptera, such as Pieris brassica [26], Manduca sexta
[27], and Samia cynthia ricini [28]. These insects possibly
possess a HO that selectively cleaves the c-meso site from
heme. However, biliverdin IXa, the isomer formed in
humans, occurs in the hemolymph and integument of
other insects [29].

Throughout the last century, the fruit fly has been the
workhorse for genetic studies in eukaryotes. The recent
decoding of the complete genome sequence of Drosophila
melanogaster has provided us with the opportunity to
identify all fruit fly genes, including those involved in heme
metabolism [30]. In the present study, we found a putative
HO gene in D. melanogaster by homology searching in
FlyBase, a database of genetic and molecular data for the
fruit fly. The D. melanogaster HO gene without the sequence
coding for the last 21 amino acids was cloned and further
expressed in Escherichia coli. The truncated enzyme was
obtained in high yield as a soluble, catalytically active
protein, making it available for the first time for detailed
mechanistic studies.
Experimental procedures
cDNA cloning and expression of putative DmHO
FlyBase shows the existence of a nucleotide sequence
encoding a protein homologous to both human and rat
HOs. RT-PCR was used to prepare cDNA encoding the
putative HO of D. melanogaster. Briefly, first-strand cDNA
synthesis was performed at 42 °C for 60 min using adult
D. melanogaster polyA-rich RNA (Clontech) as a template,
oligo(dT) primer (Genset, Proligo Japan, Kyoto, Japan),
and reverse transcriptase (ReverTra Ace; Toyobo, Osaka,
Japan). The synthesized cDNA was subjected to PCR
amplification to generate the coding region of the putative
D. melanogaster HO (DmHO). A sense primer, DmHOF1
(5¢-GCGCAAAAGA
CATATGTCAGCGAGCGAAG-3¢)
and an antisense primer, DmHOR1 (3¢-CGAGAGTTC

ATTCTTTTCGAACTTTATG-5¢)wereusedtoamplify
the full length DmHO consisting of 296 amino acid residues.
The underlined nucleotide sequence of 5¢-CATATG-3¢
represents the NdeI recognition site involving an initiation
codon. The underlined nucleotide sequences of 3¢-ATT-5¢,
and 3¢-TTCGAA-5¢ are the complementary sequences of a
stop codon, and the HindIII recognition site, respectively.
Another primer set with DmHOF1 and antisense primer
DmHOR2 (3¢-GCACGGTTAGAA
ATCTTCGAACGG
GAGCGT-5¢) were used to prepare a truncated form of
DmHO (DmDHO) which lacks a C-terminal hydrophobic
domain consisting of 21 amino acid residues. The underlined
nucleotide sequence of 3¢-ATC-5¢ is the complementary
sequences of a stop codon. PCR amplification was carried
out with AmpliTaq Gold (Applied Biosystems) for 30 cycles.
The PCR products were digested with NdeIandHindIII
andthenclonedintotheNdeIandHindIII sites of the
pMW172 expression vector. The constructs encoding the full
length and C-terminally truncated DmHO were named
pMWDmHO and pMWDmDHO, respectively. Both con-
structs were sequenced using the dye terminator cycle
sequencing method.
Purification of recombinant DmDHO
E. coli strain BL21 (DE3) was transformed with
pMWDmDHO. A single colony was picked up and
precultured in 5 mL Luria–Bertani medium containing
50 lgÆmL
)1
ampicillin and 1% glucose at 37 °Covernight.

Then 200 lL of the preculture was added to 500 mL of
the same medium for incubation at 37 °C. After the A
600
of the culture reached about 1.0, the incubation was
continued at 20 °C for 24 h. The harvested cells were
washed with 20 m
M
potassium phosphate buffer, pH 7.4,
containing 134 m
M
KCl, resuspended in 9 vols (9 mL per g
E. coli cells) 50 m
M
Tris/HCl buffer (pH 7.4) containing
Scheme 1. Heme degradation pathway. Heme
to biliverdin IXa catalyzed by HO and bili-
verdin IXa to bilirubin IXa catalyzed by bili-
verdin reductase.
1714 X. Zhang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
2m
M
EDTA, and lysed by lysozyme (final concentration
0.2 mgÆmL
)1
)for30minat4°C. The lysed cells were
briefly sonicated and centrifuged at 100 000 g for 60 min;
the resulting supernatant was used as the soluble fraction.
For the purification, the soluble fraction was first
subjected to ammonium sulfate fractionation. The preci-
pitate obtained at 33–55% saturation was collected by

centrifugation, dissolved in 20 m
M
potassium phosphate
buffer (pH 7.4) in a final volume of 5 mL, and applied to
a column (3.6 · 50 cm) of Sephadex G-75, pre-equili-
brated with the same buffer. Fractions with an intense
32 kDa band on SDS/PAGE were collected and applied
to a DEAE-cellulose DE-52 column (2.6 · 30 cm). After
the column had been washed with 50 mL 20 m
M
potas-
sium phosphate buffer (pH 7.4) containing 100 m
M
KCl,
the protein was eluted with 400 mL 20 m
M
potassium
phosphate buffer (pH 7.4) with a linear gradient of 100–
400 m
M
KCl. The fractions containing 32 kDa protein
were then fractionated with a hydroxyapatite column
(2.6 · 20 cm), using a linear gradient between 200 mL
each 20 and 300 m
M
potassium phosphate buffer
(pH 7.4). All fractions containing the 32 kDa protein
were pooled and concentrated. The buffer solution was
changedto50m
M

potassium phosphate buffer (pH 7.4)
by Sephadex G-25 column chromatography. All proce-
dures were conducted at 4 °C, and the final products were
stored at )80 °C.
Construction of DmDCPR expression plasmid
A truncated form of NADPH/cytochrome P450 reductase
of D. melanogaster (DmDCPR) expression vector was
constructed by the same method as described above. An
NdeI/HindIII cDNA fragment encoding amino acids
46–679 of DmCPR was amplified by RT-PCR using
the primers DmCPRF1 (5¢-CTTCCTGCGTACG
CA
TATGAAGGAGGAGGA-3¢)andDmCPRR1(3¢-CA
GACCTCG
ATTCGAATAGGTTTTCGGTTG-5¢). The
first 45 amino acids of DmCPR were deleted because this
sequence involves a membrane-bound region. One NdeI
restriction site inside the target sequence was reduced by
site-directed mutagenesis without changing any amino acid
residues. The PCR product was digested and inserted into
the NdeIandHindIII restriction sites of pMW172 to form
pMWDmDCPR.
Preparation and assay of DmDCPR
Conditions for expressing DmDCPR and preparing a
soluble fraction were similar to those for DmDHO described
above. The precipitate obtained from the soluble fraction
at 40–65% ammonium sulfate saturation was suspended
in 15 vols (15 mL per g E. coli cells) 20 m
M
potassium

phosphate buffer (pH 7.4). The suspension was applied to a
DE-52 column and the protein was eluted with a 400 mL
linear gradient of 100–400 m
M
KCl in 20 m
M
potassium
phosphate buffer (pH 7.4). Yellow fractions with an intense
72 kDa band on SDS/PAGE were pooled and then loaded
on a column of 2¢5¢-ADP Sepharose 4B (Amersham
Pharmacia Biotech). The column was washed with 50 mL
0.1
M
potassium phosphate buffer (pH 7.4), and DmDCPR
was eluted with 20 mL 0.1
M
potassium phosphate buffer
(pH 7.4) containing 7 mgÆmL
)1
2¢(3¢)-AMP. Finally, the
2¢(3¢)-AMP in the eluate was removed by passage through a
Sephadex G-25 column. The final products were stored in
50% glycerol at )80 °C.
The ability of DmDCPR to catalyze reduction of 2,6-
dichloroindophenol was assayed using 21 m
M
)1
Æcm
)1
as the

absorption coefficient of the dye at 600 nm [31].
Heme binding study
Heme binding of DmDHO was tested by adding hemin to
12 l
M
DmDHO in 2mL 50m
M
potassium phosphate
buffer (pH 7.4). The reference cuvette contained 2 mL
50 m
M
potassium phosphate buffer (pH 7.4) alone. A
solution of 1 m
M
hemin was added in 4 lL aliquots to both
test and reference cuvettes with 5 min equilibration between
additions at 25 °C. The absorbance between 350 and
750 nm was measured on a Beckman DU7400 single-beam
spectrophotometer.
Assay of DmDHO by measuring bilirubin formation
The catalytic activity of DmDHO was determined after
conversion of biliverdin IXa, produced by the enzyme,
into bilirubin by biliverdin IXa reductase. The NADPH/
DmDCPR reaction mixture contained in a final volume
of 1.5 mL: 50 m
M
potassium phosphate buffer (pH 7.4),
26 l
M
hemin, 1 l

M
DmDHO, 0.22 l
M
DmDCPR, 300 l
M
NADPH, and 6 l
M
biliverdin reductase [32]. NADPH
was omitted from the control system. When necessary,
1m
M
desferrioxamine was added to both the reaction and
control systems. The reaction was started by the addition
of NADPH after 3 min preincubation at 37 °C, and
monitored at 468 nm for 10 min. The value of
43.5 m
M
)1
Æcm
)1
was used as the absorption coefficient
for bilirubin at 468 nm [33]. The ascorbate system
contained in a final volume of 1.5 mL: 50 m
M
potassium
phosphate buffer (pH 7.4), 26 l
M
hemin, 1 l
M
DmDHO,

50 m
M
sodium ascorbate, 60 l
M
NADPH, and 6 l
M
biliverdin reductase. Ascorbate was omitted from the
control system. Reduction was initiated by the addition of
ascorbate. Other conditions were the same as those for the
NADPH/DmDCPR system.
Reaction of hemin bound to DmDHO by NADPH/DmDCPR
or sodium ascorbate in the presence of desferrioxamine
Spectral changes were recorded at 30 °C between 350 and
750 nm. We used three electron donor systems, NADPH/
DmDCPR, ascorbate, and H
2
O
2
. The standard reaction
mixture for the NADPH/DmDCPR system consisted of
10 l
M
DmDHO–hemin complex, 0.22 l
M
DmDCPR and
1m
M
desferrioxamine in a final volume of 1.5 mL 50 m
M
potassium phosphate buffer (pH 7.4). After 3 min preincu-

bation, the reaction was started by the addition of 20 lL
10 m
M
NADPH (final concentration, 0.13 l
M
). The ascor-
bate reaction mixture contained 10 l
M
DmDHO–hemin
complex and 1 m
M
desferrioxamine in a final volume of
1.5 mL 50 m
M
potassium phosphate buffer (pH 7.4). After
3 min preincubation, the reaction was initiated by the addi-
tion of 20 lL1
M
sodium ascorbate (final concentration,
13 l
M
). The H
2
O
2
system consisted of 10 l
M
DmDHO–
Ó FEBS 2004 D. melanogaster heme oxygenase (Eur. J. Biochem. 271) 1715
hemin complex in a final volume of 1.5 mL 50 m

M
potassium phosphate buffer (pH 7.4). After 3 min preincu-
bation, the reaction was started by the addition of H
2
O
2
in
water (final concentration 36 l
M
or 300 l
M
). The concen-
tration of H
2
O
2
in the original aqueous reagent solution was
determined spectroscopically using a value of 43.6
M
)1
Æcm
)1
for the absorption coefficient at 240 nm [34].
EPR spectroscopy
EPR measurements were performed using a Bruker E500
spectrometer, operating at 9.35–9.55 GHz, with an Oxford
ESR 900 liquid helium cryostat. The
15
NO-bound form of
the heme–DmDHO complex was prepared by adding

dithionite to the argon-saturated protein solution, contain-
ing Na
15
NO
2
,inanEPRtube.
Detection of CO
To detect CO produced during the DmDHO reaction
supported by NADPH/DmDCPR, myoglobin (H64L), a
mutant with high affinity for CO [35], was used. The
reaction solutions contained 16 l
M
hemin–DmDHO com-
plex, 1.6 l
M
DmDCPR, and 300 l
M
NADPH in 1.5 mL
50 m
M
potassium phosphate buffer (pH 7.4). Myoglobin
mutant H64L, at a final concentration of 7.5 l
M
,was
included in the test solution. After the addition of NADPH
to both cuvettes, the spectrum was recorded at 4 min
intervals between 350 and 750 nm.
HPLC analysis of DmDHO reaction products
The DmDHO reaction products of either NADPH/
DmDCPR or ascorbate were directly subjected to a

Supelclean LC-18 solid-phase extraction column, precondi-
tioned with 400 lL acetonitrile followed by 400 lL0.1
M
Tris/HCl buffer (pH 7.4). The product of the DmDHO
reaction with H
2
O
2
was loaded on the same column after
hydrolytic conversion into biliverdin. The column was
washed with acetonitrile/water (1 : 9, v/v), and green
pigment was then eluted with acetonitrile/water (1 : 1,
v/v). This was lyophilized, and the residue dissolved in 5%
HCl/methanol for esterification at 4 °C overnight. Water
was added to the esterified product, and green pigment was
extracted with chloroform. The chloroform solution was
washed with water and then analyzed by HPLC on a
column of Capcell Pak C18 (SG 120, 4.6 · 150 mm) pre-
equilibrated with degassed acetonitrile/water (3 : 2, v/v) at a
flow rate of 1 mLÆmin
)1
. The eluate was monitored at
310 nm. The biliverdin dimethyl ester standards were eluted
in the order biliverdin IXa (18.2 min), IXd (19.7 min), IXb
(21.1 min), and IXc (31.1 min).
Other procedures
Sequence translation and sequence alignment were per-
formed using the
WISCONSIN PACKAGE
from the Genetic

Computer Group (Madison, WI, USA) and
CLUSTAL W
multiple sequence alignment program at the EBI (EMBL-
EBI). H64L protein, a mutant of myoglobin, was purified
by published methods [36]. Hemin concentrations were
measured by the method of Paul & Theorell [37], and
protein concentrations by the Lowry method using BSA as
standard [38].
Results and Discussion
Characterization of DmHO deduced from the
nucleotide sequence of cDNA
Both the full length (DmHO) and truncated (DmDHO)
enzymes were obtained from adult D. melanogaster polyA-
rich RNA by the RT-PCR method. The deduced amino
acid sequences were the same as reported originally in the
SwissProt Database (Q9VGJ9) with one exception; position
50 was not isoleucine but phenylalanine. We think that
Phe50 is more likely to be correct because: (a) Phe50 was
coded in our three DNA fragments obtained by PCR using
different template cDNA which was independently synthes-
ized with oligo(dT), random, and gene-specific primers,
respectively; (b) Phe37 in mammalian HO-1, which corres-
ponds to Phe50 in DmHO, has an important role in the
interaction with the a-meso edge of heme [39,40] and is
conserved at the corresponding position of most HOs
isolated from other species.
Sequence comparisons by
FASTA
searching show that
DmHO is 32.4% and 30.3% identical in amino acid

sequence with rat HO-1 and rat HO-2, respectively (Fig. 1).
Sequence alignment analysis indicated that DmHO contains
a large catalytic domain at the N-terminus and a small
hydrophobic domain at the C-terminus. This structure is
similar to mammalian HOs but different from bacterial,
algal, and cyanobacterial HOs which lack the hydrophobic
domain. Moreover, the Swiss-model project (first approach
mode) suggests that the overall structure of DmHO is
similar to that of mammalian HO-1. In rat HO-1, His25
works as the proximal ligand of heme iron. The His39
residue of DmHO corresponds to His25 of rat HO-1 and
therefore is likely to be the proximal ligand. The crystal
structure of human HO-1 [39] shows that Thr21, Glu29 and
Phe207 are on the proximal side of the heme. In DmHO,
Thr35 corresponding to Thr21 of HO-1 is conserved, but
the other two amino acid residues are not. The crystal
structure of HO-1 also shows that the backbone atoms of
the two glycine residues, Gly139 and Gly143, which are
highly conserved among the known sequences of HOs,
directly contact the heme [39,40]. In the DmHO sequence,
Gly143 is present, but Gly139 is replaced by alanine, as in
the sequence of Arabidopsis HO [17]. Site-directed muta-
genesis studies revealed that Asp140 is involved in the
oxygen activation mechanism in mammalian HO-1 [41,42],
but this amino acid residue is not found in DmHO. These
features of DmHO suggest that, although the ternary
structure of DmHO is similar to that of mammalian HO-1,
the structure of the heme pocket is somewhat different.
Expression and purification of DmHO and DmDHO
To obtain the full length and truncated forms of recombin-

ant DmHO, two expression plasmids, pMWDmHO and
pMWDmDHO, were constructed. E. coli strain BL21(DE3)
transformed with pMWDmHO expressed a protein in the
membrane fraction which gives a strong 34 kDa band on
SDS/PAGE. In contrast, E. coli harboring pMWDmDHO
1716 X. Zhang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
expressed a soluble 32 kDa protein mainly in the soluble
fraction. Molecular sizes of 34 kDa and 32 kDa are in
agreement with the calculated values of 34 112 Da for full
length DmHO and 31 777 Da for DmDHO. These obser-
vations indicate that the C-terminal hydrophobic sequence
composed of 21 amino acid residues acts as an anchor to
membranes, similar to rat HO-1 [43,44]. The truncated
forms of mammalian HOs, in which the hydrophobic
membrane-binding domains are removed, fully retain heme-
degrading activity [2,3]. Therefore, we presumed that the
truncated form of DmHO also retains its activity. As
described below, DmDHO is a soluble, catalytically active
protein and therefore we used only DmDHO in this study.
The expression of DmDHO by culturing the transformed
E. coli cells at 37 °C resulted in an accumulation of the
expressed protein mostly in inclusion bodies. However,
culturing the transformed bacteria at 37 °Cthen20°Cas
described in Experimental procedures increased significantly
the yield of the recombinant protein in the soluble fraction.
Expression of DmDHO, however, did not turn the culture
medium green. This phenomenon is distinct from that
observed for E. coli cells expressing mammalian, bacterial,
and cyanobacterial HOs and raises questions about the
heme-degrading activity of DmDHO.

We purified the expressed DmDHO from the soluble
fraction by ammonium sulfate fractionation and subse-
quent column chromatography on Sepahdex G-75,
DE-52 and hydroxylapatite. The purified DmDHO gave
a 32 kDa band with  95% purity on SDS/PAGE
(lane 2 in Fig. 2). About 25 mg protein was obtained
from 1 L culture.
Expression and purification of DmDCPR
Cultured E. coli cells transformed with pMWDmDCPR
were light yellow caused by constitutive flavins of
DmDCPR. During purification, we used this color along
with the 72 kDa band on SDS/PAGE for detecting
DmDCPR. The purification procedures involved ammo-
nium sulfate fractionation and column chromatography on
DE-52 and 2¢5¢-ADP Sepharose 4B. The purified fraction
showed a single band of 72 kDa (lane 3 in Fig. 2), similar to
the calculated value of 71 740 Da. The 2,6-dichloroindo-
phenol-reducing activity of purified DmDCPR was similar
Fig. 1. Amino acid sequence of DmDHO
compared with reported DmHO, rat HO-1 and
rat HO-2. * indicates positions that have a
single, fully conserved residue. : indicates that
one of the ÔstrongÕ groups is fully conserved.
. indicates that one of the ÔweakerÕ groups is
fully conserved.
Fig. 2. SDS/PAGE of the purified DmDHO and DmDCPR. Lane 1,
Molecularmassmarker;lane2,2lgpurifiedDmDHO;lane3,2lg
purified DmDCPR.
Ó FEBS 2004 D. melanogaster heme oxygenase (Eur. J. Biochem. 271) 1717
to that of purified rat CPR. About 10 mg protein was

obtained from 1 L culture.
Catalytic activity of DmDHO
As mentioned above, expressed DmDHO in E. coli cells did
not turn the color of host cells green, so that we measured
the ability of DmDHO to catalyze the conversion of hemin
into biliverdin in vitro.WeusedNADPH/DmDCPR and
ascorbate as reducing reagents and biliverdin IXa reductase
to reduce the biliverdin IXa produced by DmDHO to
bilirubin. Table 1 suggests that DmDHO does degrade
hemin to biliverdin IXa in the presence of each of the
reducing systems, indicating that DmHO of fruit fly is a real
HO. Interestingly, the specific activity of heme breakdown
was very low, almost one-quarter that with ascorbic acid,
although DmDCPR equivalent to one-fifth of DmDHO was
used. In the case of rat HO-1, despite using rat CPR
equivalent to about one-thirtieth of rat HO-1, the activity of
heme degradation was half that seen in the ascorbate system
[45]. This suggests that effective electron transfer does not
occur from DmDCPR to DmDHO.
With rat HO-1, heme breakdown to biliverdin in the
presence of ascorbate is accelerated by desferrioxamine, a
ferric iron chelator, because in that system the final product
is not biliverdin but its precursor, ferric biliverdin, bound to
HO-1 protein [46]. Therefore, we assayed DmDHO activity
in converting heme into bilirubin via biliverdin IXa in the
presence of desferrioxamine. As a result, the conversion
activities with addition of either NADPH/DmDCPR or
ascorbate increased by about eightfold and fourfold,
respectively. This suggests that spontaneous iron release
from the ferric biliverdin–DmDHO complex in both systems

is slow. The specific activity of DmDHO was highest in the
ascorbate system in the presence of desferrioxamine but still
only about one-quarter that of rat HO-1. As described
below, HPLC analysis showed that 75% of the total
biliverdin produced by DmDHO is the IXa isomer. As
biliverdin IXa reductase has a preference for the a-isomer as
substrate [47], the total yield of biliverdin is significantly
underestimated if measured as the amount of bilirubin IXa
eventually formed. Recently it was reported that coupled
oxidationofmyoglobinwithascorbicacidismediatedby
exogenous peroxide generated by reaction of ascorbate with
oxy-myoglobin, because the reaction is inhibited by catalase
[48]. In the case of DmDHO, inclusion of 10 l
M
catalase had
no effect, clearly showing that the DmDHO reaction does
not depend on exogenous peroxide.
Properties of the heme–DmDHO complex
All HOs so far reported bind heme stoichiometrically to
form stable complexes with absorption spectra resembling
those of myoglobin. Like other HOs, DmHO also binds
hemin to form a 1 : 1 stoichiometric complex (inset of
Fig. 3). To isolate this complex, we added excess (twofold)
hemintoDmDHO and chromatographed the mixture on
DE-52 or hydroxylapatite. However, we obtained only
DmDHO without hemin, indicating weak binding of hemin
to DmDHO. In fact, from the hemin titration, we obtained a
value of 27 ± 3 l
M
for the heme dissociation constant (K

d
).
This value is significantly higher than those for HmuO
(2.5 ± 1 l
M
) [12] and human HO-1 (0.84 ± 0.2 l
M
)[49].
Figure 3 shows the optical absorption spectra of purified
DmDHO titrated with 1 molar equivalent of hemin. The
ferric form of the DmDHO–heme complex has a broad
Soret band with a peak at 390 nm and a smaller peak at
602 nm (solid line). As previously reported, the ferric heme
Table 1. Activities of purified DmDHO. HO activity was determined
from the initial rate of bilirubin formation with NADPH/DmDCPR or
sodium ascorbate systems in the absence/presence of desferrioxamine
and the presence of biliverdin reductase. All measurements were per-
formed in triplicate. Values are mean ± SD.
Reducing system
Bilirubin formation
[nmolÆ(mg protein)
)1
Æh
)1
]
–Desferrioxamine +Desferrioxamine
NADPH/DmDCPR 32 ± 1.2 250 ± 8
Sodium ascorbate 138 ± 5 543 ± 10
Fig. 3. Absorption spectra of various forms of
the DmDHO–heme complex. ––, oxidized

form; ÆÆÆÆ, reduced form; - - -, CO-bound form.
Inset, difference titration of DmDHO with
hemin. Precise procedures are described in
Experimental procedures. The increments in
absorbance as the difference at 412 nm were
plotted, because the difference was maximum
at this wavelength.
1718 X. Zhang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
iron in the rat HO-1–hemin complex at neutral pH is six-
coordinate, high spin, and the Soret maximum undergoes a
red shift with increasing pH, having an apparent pK
a
value
of 7.6 [50]. In contrast, the Soret maximum of the DmDHO–
hemin complex was not influenced by increasing the pH
to 10.0.
We assumed that the ferric heme iron of the complex was
not in the six-coordinate state, presumably lacking the water
molecule at the distal site. To confirm this, we carried out an
EPR study. As shown in Fig. 4, the EPR spectrum of the
hemin–DmDHO complex exhibits a highly rhombic, high-
spin state of hemin, showing pronounced difference from
that of the hemin complex of cyanobacterial heme oxy-
genase isoform-1, Syn HO-1, which was determined to be in
a six-coordinate high-spin state with a distal water molecule
and a proximal histidine [16]. The lower field feature of the
spectrum further suggests that the ligand field around the
hemin molecule is inhomogeneous, implying that orienta-
tion of hemin in the DmDHO heme pocket is unequal. The
highly rhombic feature of the ferric heme–HO complexes is

common to the point-mutated HOs of proximal histidine
(data not shown) and to the five-coordinated a-hydro-
xyhemin complex of HO-1 [2]. Contrary to the sequence-
based expectation, the spectrum of the ferrous
15
NO-bound
heme–DmDHO complex is typical of penta-coordinated
15
NO–heme complexes, differing from that of Syn HO-1,
which is a hexa-coordinate
15
NO–heme complex exhibiting
the triplet hyperfine splitting due to the nuclear spin of one
of the nitrogen nucleus of an imidazole of a histidine residue
trans to the
15
NO [16] (Fig. 5). The hemin–DmDHO
complex in alkaline solution (pH 10.0) does not show the
spectrum of a typical hydroxide-coordinated low-spin form
(data not shown). This is in accord with both the result of
optical spectra and the revealed coordination structure of
hemin without proximal histidine. Accordingly, EPR results
identify the hemin in the DmDHO complex to be uncoor-
dinated by the protein residue, which is markedly different
from other known hemin–HO complexes. Reduction of the
ferric heme with sodium dithionite under nitrogen gas
yielded a ferrous form with a Soret band at 428 nm and a
small peak at 559 nm (Fig. 3, dotted line). After introduc-
tion of CO, the ferrous form changed to a ferrous–CO form
with a Soret maximum at 420 nm and two small peaks at

538 and 568 nm in the visible region (Fig. 3, broken line).
To exchange the gas phase in the solution, the solution was
quickly passed through a spin column of Sephadex G-25 in
air. The resulting solution exhibited a new spectrum with a
Soret peak at 410 nm and two small peaks at 538 nm and
575 nm (data, not shown), indicating that the CO form was
converted into the oxy form. This oxy form was gradually
turned into a ferric complex with an auto-oxidation rate
(K
obs
)of3.5· 10
)3
s
)1
. This value is much higher than that
of rat HO-1 (0.14 · 10
)3
s
)1
) and comparable to those of
some mutants, in which the hydrogen-bonding network to
stabilize oxygen bound to iron is thought to be weak [42].
Table 2 shows optical absorption data for the heme–
DmDHO complex.
Reaction of hemin bound to DmDHO by
NADPH/DmDCPR or sodium ascorbate
in the presence of desferrioxamine
As desferrioxamine increased biliverdin formation from
hemin in both the NADPH/DmDCPR and ascorbate
systems by facilitating the release of iron from the ferric

biliverdin–DmDHO complex, we measured the degradation
of hemin bound to DmDHO in the presence of desferri-
oxamine. As depicted in Fig. 6A, the addition of NADPH
Fig. 4. EPR spectra of the ferric DmDHO and Syn HO-1 complexes at
neutral pH. Both spectra were obtained at 8 K, applied field modula-
tion frequencies, 100 kHz, field modulation amplitude, 10 G, and
microwave power, 1 mW.
Fig. 5. EPR spectra of the ferrous
15
NO-bound forms of the heme–
DmDHO and Syn HO-1 complexes. Both spectra were obtained at
20 K, applied field modulation frequencies, 100 kHz, field modulation
amplitude, 2 G, and microwave power, 0.2 mW.
Table 2. Optical absorption data for the heme–DmDHO complex.
k
max
(Soret) (em
M
)1
Æcm
)1
) k
max
(visible)
Ferric form 390 (70) 602
Ferrous deoxy form 428 (80) 559
CO form 420 (163) 538, 568
Oxy form 410 (72) 537, 575
Ó FEBS 2004 D. melanogaster heme oxygenase (Eur. J. Biochem. 271) 1719
to the reaction solution (solid line I) initiated heme

degradation. The spectrum recorded after 10 min (dotted
line) shows a red-shifted Soret maximum and two peaks at
538 and 575 nm in the visible region, indicating formation
of an oxy form. Formation of the oxy form is faster than its
further degradation reaction, and loss of the Soret band was
relatively slow. The spectrum recorded after 120 min
(broken line) has a broad absorption centered at 670 nm,
showing the conversion of hemin into biliverdin. This is
supported by a decrease in absorbance around 670 nm and
concomitant increase near 460 nm due to bilirubin after
addition of biliverdin reductase (solid line II).
We also measured the spectral change in ascorbic acid-
supported heme degradation in the presence of desferri-
oxamine. The spectrum in Fig. 6B recorded 5 min after the
addition of sodium ascorbate (dotted line) shows that heme
degradation proceeded faster than in the presence of
NADPH/DmDCPR, consistent with the results from the
catalytic activity assay. This spectrum also has small peaks
around 537 and 575 nm attributable to the oxy complex.
The spectrum recorded 30 min after the initiation of the
reaction (broken line) shows two broad bands centered near
380 and 670 nm, indicative of slow biliverdin formation.
Again, after addition of biliverdin reductase and NADPH, a
decrease in absorbance around 670 nm and concomitant
increase near 460 nm were observed (solid line II).
Comparison between the spectral intensities at 670 nm of
the final product of both reducing systems suggests that
about twice as much biliverdin is formed in the ascorbate
system as in the NADPH/DmDCPR system. We think that
this is partly due to CPR-mediated heme degradation

leading to nonbiliverdin products [51,52]. In fact, we
observed that about 40% of hemin was lost 120 min after
incubation of 2.6 l
M
heminwith1.2l
M
DmDCPR and
300 l
M
NADPH at 30 °C. As the affinity of heme for
DmDHO is low, some of the substrate may be degraded by
DmDCPR, making it unavailable for the DmDHO reaction.
Reaction of the hemin bound to DmDHO by sodium
ascorbate under O
2
and CO
With rat HO-1, hemin bound to enzyme is converted into
ferrous–CO forms of verdoheme under O
2
and CO, and the
reaction stops at this stage because CO stops the further
reaction of verdoheme to ferric-biliverdin [53]. To detect the
ferrous–CO forms of the verdoheme–DmDHO complex, we
carried out similar experiments. The spectrum (dotted line in
Fig. 7) recorded 3 min after the start of the reaction has
three peaks at 538, 568, and 602 nm in the visible region; the
former two peaks are due to the CO-bound form and the
latter peak to the ferric form of the hemin–DmDHO
complex. However, we were unable to detect a peak around
640 nm attributable to the ferrous–CO form of verdoheme.

The broken line is a spectrum recorded 40 min after the
start of the reaction. Again, absorption around 640 nm was
not observed, but broad absorption in the red region
increased, indicating biliverdin formation. DmDHO shares
several mechanistic features with other HOs, including CO
formation, and therefore we believe that verdoheme is an
intermediate in the DmDHO reaction. We assume that
verdoheme formation from the oxy form of the heme–
DmDHO complex is slower than conversion of verdoheme
into ferric biliverdin, which frustrates detection of the
ferrous–CO form of verdoheme.
Reaction of the hemin–DmDHO complex with H
2
O
2
In mammalian HO-1, a ferric hydroperoxy species is an
active intermediate in the first oxygenation step [54–56].
H
2
O
2
hydroxylates heme at the a-meso position to form
a-meso-hydroxyhemin, which is then converted into verdo-
heme in the presence of O
2
[57]. Therefore, we investigated
whether H
2
O
2

can support the conversion of hemin bound
to DmDHO to verdoheme. On addition of 3.6 molar
equivalents of H
2
O
2
, the Soret band at 390 nm decreased
gradually accompanied by a very small increase around
685 nm (data not shown). When 30 equivalents of H
2
O
2
were used, a rapid decrease in the Soret band was observed.
However, the intensity of the absorption of the final reaction
Fig. 6. Reaction of hemin bound to DmDHO by NADPH/DmDCPR or
sodium ascorbate in the presence of desferrioxamine. (A) –– I, spectrum
of the complex of hemin and DmDHO; ÆÆÆÆ, spectrum 10 min after the
addition of NADPH to start the reaction; - - -, 120 min after the
reaction; –– II, after the addition of biliverdin reductase. Inset,
enlarged spectra between 450 and 750 nm. (B) –– I, spectrum of the
complex of hemin and DmDHO; ÆÆÆÆ, spectrum 5 min after the addition
of ascorbate to start the reaction; - - -, 30 min after the reaction; –– II,
after the addition of biliverdin reductase and NADPH. Inset, enlarged
spectra between 450 and 750 nm.
1720 X. Zhang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
product around 685 nm was almost the same as when 3.6
equivalents were used. HPLC analysis showed that the
amount of biliverdin formed was only 0.15% of that formed
in the ascorbic acid/desferrioxamine-supported system
(Fig. 8C).

These observations suggest that H
2
O
2
oxidized DmDHO-
bound heme to fragmentation products rather than to
verdoheme. Presumably, in the first stage of the DmDHO
reaction, a hydroperoxy species is the active oxygen species,
by analogy with mammalian HO-1, and this species is
formed by binding of H
2
O
2
to the ferric iron of the hemin–
DmDHO complex. We do not know why verdohemo-
chrome is not formed in the H
2
O
2
-supported DmDHO
reaction. Interestingly, a mutant of human HO-1, D140A,
has similar properties [41].
Detection of CO during the DmDHO reaction
Difference absorption spectroscopy in the presence of
mutated myoglobin, H64L, which has a high affinity for
CO, was used to detect CO formed during the NADPH/
DmDCPR-supported reaction. The Soret band of myo-
globin was monitored at 4-min intervals after the addition
of NADPH to both the sample and reference cuvette. As
depicted in Fig. 9, the myoglobin Soret band shifted from

393 to 425 nm with the appearance of a/b bands at 568 and
538 nm and A
425
increased, indicating reduction of the
ferric form of myoglobin to the ferrous form by the
NADPH/DmDCPR system. This was followed by CO
binding to yield the ferrous–CO form, the authentic
absorption spectrum of which is depicted in inset of
Fig. 9. This experiment clearly demonstrates CO formation
during heme degradation by DmDHO.
HPLC analysis of the DmDHO reaction products
HPLC analysis showed that the biliverdin formed in both
the NADPH/DmDCPR/desferrioxamine and ascorbic
acid/desferrioxamine systems contained three isomers,
Fig. 7. Reaction of hemin bound to DmDHO
by sodium ascorbate under O
2
and CO. ––,
spectrum of the complex of hemin and
DmDHO; ÆÆÆÆ, spectrum 3 min after the addi-
tion of ascorbate to start the reaction; - - -,
40 min after the start of the reaction. Inset,
enlarged spectra between 500 and 800 nm.
Fig. 8. HPLC analysis of the reaction products of hemin bound to
DmDHO. The product analysis with HPLC was described in Experi-
mental procedures. (S) Standard mixture of biliverdin IXa,IXb,IXc
and IXd dimethyl esters; (A–C) esterified products from NADPH/
DmDCPR, sodium ascorbate, and H
2
O

2
systems, respectively.
Ó FEBS 2004 D. melanogaster heme oxygenase (Eur. J. Biochem. 271) 1721
IXa,IXb,andIXd, accounting for  75%, 16% and 8%
of the total, respectively (Fig. 8). This is unusual, because
other HOs exclusively generate biliverdin IXa, except for
Pig A of Pseudomonas aeruginosa, which forms both
biliverdin IXb and IXd [14]. The crystal structure of
human HO-1 reveals a distal helix spanning the entire
width of the heme, which sterically prevents access of the
iron-bound hydroperoxy species to the b-meso, c-meso,
and d-meso carbon atoms [39]. Thus, the iron-bound
hydroperoxy species can oxygenate only the a-meso-
carbon of heme, leading to the exclusive a-meso-hydroxy-
heme formation. The formation of three isomers of
biliverdin by DmDHO implies that its heme pocket has a
different structure from those of mammalian HO-1 and
other a-specific HOs. The EPR result suggesting the
existence of several types of hemin conformation in the
protein pocket is consistent with this non a-specific
production of biliverdin. We expected that DmDHO
would produce the c-isomer of biliverdin because biliver-
din IXc is present in some species of Lepidoptera.
However, we detected only trace amounts of the
c-isomer in our in vitro studies of the soluble recombinant
enzyme.
Concluding remarks
We cloned a cDNA for D. melanogaster homologous to
mammalian HOs and constructed a bacterial expression
plasmid of a truncated, soluble enzyme, DmDHO. Purified

recombinant DmDHO forms an enzyme–substrate complex
with a stoichiometric amount of heme and catalyzes heme
degradation to biliverdin isomers, CO and iron, although
the specific activity was very low, in the presence of
appropriate reducing systems. These features are similar to
those of other HOs, indicating that DmDHO is a true heme
oxygenase in fruit fly.
Despite these similarities, DmDHO is distinctly different
from other HOs. (a) Unlike other HOs, the hemin–
DmDHO complex is not in the six-coordinate high-spin
state with a histidine residue as the proximal ligand and
the iron of heme was not involved in forming the heme–
DmHO complex. (b) H
2
O
2
does not support DmDHO-
dependent degradation of heme to verdoheme. (c) CO–
verdoheme cannot be detected during the catalytic reaction
under oxygen and CO. (d) In the final stage of the
reaction, iron release from the ferric biliverdin–DmDHO
complex is slow. (e) The hemin catabolism of DmDHO is
not a-specific and yields three isomers of biliverdin, IXa,
IXb,andIXd. Accordingly, we infer that the structure and
hydrogen bonding of the DmHO active site is quite
different from those of other HOs. It is interesting to note
that DmDHO was able to degrade heme to biliverdin, in
spite of no direct binding of heme iron to the enzyme. A
similar but not identical observation was reported for a
mutant of HmuO. The H20A mutant in which His20 was

replaced by Ala degraded hemin to verdoheme, a second
intermediate of heme degradation [58]. Further investiga-
tion of the structure to understand the mechanism of heme
breakdown is needed.
Acknowledgements
The bacterial expression vector pMW172 was a gift from Dr K. Nagai,
MRC Laboratory of Molecular Biology, Cambridge, UK. The
expression plasmid for the myoglobin mutant, H64L was a gift from
Professor J. S. Olson, Rice University. We thank Dr A. F. McDonagh,
University of California, San Francisco, for helpful comments on the
manuscript. This work was supported in part by grants-in-aid from
the Ministry of Education, Science, Sports, and Culture, Japan
(14580641).
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