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 w
Trang 1Unique features of recombinant heme oxygenase of Drosophila
oxygenases studied
Xuhong Zhang1, Michihiko Sato2, Masanao Sasahara1, Catharina T Migita3and Tadashi Yoshida1
1
Department of Biochemistry and2Central 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-stepoxidation 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: tyoshida@med.id.yamagata-u.ac.jp
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,
accep ted 9 March 2004)
Trang 2isolated 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 42C for 60 min using adult
D melanogasterpolyA-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 melanogasterHO (DmHO) A sense primer, DmHOF1 (5¢-GCGCAAAAGACATATGTCAGCGAGCGAAG-3¢) and an antisense primer, DmHOR1 (3¢-CGAGAGTTC ATTCTTTTCGAACTTTATG-5¢) were used to amp lify 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 stopcodon, and the HindIII recognition site, respectively Another primer set with DmHOF1 and antisense primer DmHOR2 (3¢-GCACGGTTAGAAATCTTCGAACGG 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 stopcodon PCR amplification was carried out with AmpliTaq Gold (Applied Biosystems) for 30 cycles The PCR products were digested with NdeI and HindIII and then cloned into the NdeI and HindIII 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)1ampicillin and 1% glucose at 37C overnight Then 200 lL of the preculture was added to 500 mL of the same medium for incubation at 37C After the A600
of the culture reached about 1.0, the incubation was continued at 20C for 24 h The harvested cells were washed with 20 mMpotassium phosphate buffer, pH 7.4, containing 134 mMKCl, resuspended in 9 vols (9 mL per g
E coli cells) 50 m Tris/HCl buffer (pH 7.4) containing
Scheme 1 Heme degradation pathway Heme
to biliverdin IXa catalyzed by HO and verdin IXa to bilirubin IXa catalyzed by bili-verdin reductase.
Trang 32 mM EDTA, and lysed by lysozyme (final concentration
0.2 mgÆmL)1) for 30 min at 4C 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 mM 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 mM
potas-sium phosphate buffer (pH 7.4) containing 100 mM KCl,
the protein was eluted with 400 mL 20 mM potassium
phosphate buffer (pH 7.4) with a linear gradient of 100–
400 mM 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 mM potassium phosphate buffer
(pH 7.4) All fractions containing the 32 kDa protein
were pooled and concentrated The buffer solution was
changed to 50 mM potassium phosphate buffer (pH 7.4)
by Sephadex G-25 column chromatography All
proce-dures were conducted at 4C, 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¢-CTTCCTGCGTACGCA
TATGAAGGAGGAGGA-3¢) and DmCPRR1 (3¢-CA
GACCTCGATTCGAATAGGTTTTCGGTTG-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 NdeI and HindIII 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 mM 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 mM KCl in 20 mM 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
0.1Mpotassium phosphate buffer (pH 7.4), and DmDCPR
was eluted with 20 mL 0.1Mpotassium 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 mM )1Æcm)1as the absorption coefficient of the dye at 600 nm [31]
Heme binding study Heme binding of DmDHO was tested by adding hemin to
12 lM DmDHO in 2 mL 50 mM potassium phosphate buffer (pH 7.4) The reference cuvette contained 2 mL
50 mM potassium phosphate buffer (pH 7.4) alone A solution of 1 mMhemin was added in 4 lL aliquots to both test and reference cuvettes with 5 min equilibration between additions at 25C 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 mM potassium phosphate buffer (pH 7.4),
26 lMhemin, 1 lMDmDHO, 0.22 lMDmDCPR, 300 lM NADPH, and 6 lM biliverdin reductase [32] NADPH was omitted from the control system When necessary,
1 mMdesferrioxamine was added to both the reaction and control systems The reaction was started by the addition
of NADPH after 3 min preincubation at 37C, and monitored at 468 nm for 10 min The value of 43.5 mM )1Æcm)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 mMpotassium phosphate buffer (pH 7.4), 26 lM hemin, 1 lMDmDHO,
50 mM sodium ascorbate, 60 lM NADPH, and 6 lM 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 30C between 350 and
750 nm We used three electron donor systems, NADPH/ DmDCPR, ascorbate, and H2O2 The standard reaction mixture for the NADPH/DmDCPR system consisted of
10 lM DmDHO–hemin complex, 0.22 lM DmDCPR and
1 mMdesferrioxamine in a final volume of 1.5 mL 50 mM potassium phosphate buffer (pH 7.4) After 3 min preincu-bation, the reaction was started by the addition of 20 lL
10 mMNADPH (final concentration, 0.13 lM) The ascor-bate reaction mixture contained 10 lM DmDHO–hemin complex and 1 mM desferrioxamine in a final volume of 1.5 mL 50 mMpotassium phosphate buffer (pH 7.4) After
3 min preincubation, the reaction was initiated by the addi-tion of 20 lL 1M sodium ascorbate (final concentration,
13 lM) The HO system consisted of 10 lM DmDHO–
Trang 4hemin complex in a final volume of 1.5 mL 50 mM
potassium phosphate buffer (pH 7.4) After 3 min
preincu-bation, the reaction was started by the addition of H2O2in
water (final concentration 36 lMor 300 lM) The
concen-tration of H2O2in the original aqueous reagent solution was
determined spectroscopically using a value of 43.6M )1Æcm)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 The15NO-bound form of
the heme–DmDHO complex was prepared by adding
dithionite to the argon-saturated protein solution,
contain-ing Na15NO2, in an EPR tube
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 lM hemin–DmDHO
com-plex, 1.6 lM DmDCPR, and 300 lM NADPH in 1.5 mL
50 mM potassium phosphate buffer (pH 7.4) Myoglobin
mutant H64L, at a final concentration of 7.5 lM, 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 lL 0.1M
Tris/HCl buffer (pH 7.4) The product of the DmDHO
reaction with H2O2was 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 4C 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
Trang 5expressed 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 37C resulted in an accumulation of the
expressed protein mostly in inclusion bodies However,
culturing the transformed bacteria at 37C then 20 C as
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 strong groups is fully conserved.
indicates that one of the weaker groups is
fully conserved.
Fig 2 SDS/PAGE of the purified DmDHO and DmDCPR Lane 1, Molecular mass marker; lane 2, 2 lg p urified DmDHO; lane 3, 2 lg purified DmDCPR.
Trang 6to 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 We used NADPH/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 oxidation of myoglobin with ascorbic acid is mediated by 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 lMcatalase 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) hemin to DmDHO 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 lMfor the heme dissociation constant (Kd) This value is significantly higher than those for HmuO (2.5 ± 1 lM) [12] and human HO-1 (0.84 ± 0.2 lM) [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))1Æh)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.
Trang 7iron 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 pKavalue
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 ferrous15NO-bound heme–DmDHO complex is typical of penta-coordinated
15NO–heme complexes, differing from that of Syn HO-1, which is a hexa-coordinate15NO–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 15NO [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 (Kobs) of 3.5· 10)3s)1 This value is much higher than that
of rat HO-1 (0.14· 10)3s)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 modulamodula-tion amplitude, 10 G, and
microwave power, 1 mW.
Fig 5 EPR spectra of the ferrous15NO-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
Trang 8to 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 lM hemin with 1.2 lM DmDCPR and
300 lM NADPH at 30C 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 O2and CO
With rat HO-1, hemin bound to enzyme is converted into ferrous–CO forms of verdoheme under O2and 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 H2O2
In mammalian HO-1, a ferric hydroperoxy species is an active intermediate in the first oxygenation step[54–56]
H2O2 hydroxylates heme at the a-meso position to form a-meso-hydroxyhemin, which is then converted into verdo-heme in the presence of O2[57] Therefore, we investigated whether H2O2can support the conversion of hemin bound
to DmDHO to verdoheme On addition of 3.6 molar equivalents of H2O2, the Soret band at 390 nm decreased gradually accompanied by a very small increase around
685 nm (data not shown) When 30 equivalents of H2O2 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.
Trang 9product 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 H2O2oxidized
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 H2O2to the ferric iron of the hemin–
DmDHO complex We do not know why
verdohemo-chrome is not formed in the H2O2-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 A425increased, 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 reacaddi-tion; - - -,
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 O systems, respectively.
Trang 10IXa, IXb, and IXd, 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) H2O2 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, and IXd 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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Fig 9 Detection of CO produced during DmDHO reaction The sample solution con-tained hemin–DmDHO complex, DmDCPR and H64L mutant of myoglobin Myoglobin was omitted from the reference solution The reaction was started by the addition of NADPH to both solutions The difference spectrum was recorded at 4-min intervals Inset: absorption spectra of various forms of H64L mutant of myoglobin ––, oxidized form; ÆÆÆÆ, reduced form; - - -, CO bound form.