Báo cáo khoa học: Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase pot

In general, HO-1 is respon-sible for the excretion of aged⁄ disused heme as well as recycling of iron [7,8] and HO-2 is associated with sig-nal transduction through the production of CO,

Synechocystis sp PCC 6803 is a true heme oxygenase

Properties of the heme and enzyme complex

Xuhong Zhang1, Catharina T Migita2, Michihiko Sato3, Masanao Sasahara1and Tadashi Yoshida1

1 Department of Biochemistry, Yamagata University School of Medicine, Japan

2 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan

3 Central Laboratory for Research and Education, Yamagata University School of Medicine, Japan

Heme oxygenase (HO) was first reported in

mamma-lian systems as a microsomal enzyme [1], in which the

hydrophobic 26 amino acid residues at the C-terminus

anchor the protein to the membrane [2,3] HO

cata-lyzes the regiospecific oxidative degradation of heme

to biliverdin IXa, iron, and CO via

a-meso-hydroxy-hemin, verdoheme, and the iron(III)–biliverdin IXa

complex at the expense of three molecules of oxygen

and seven electrons (Scheme 1) [4,5] The electrons are supplied from NADPH through microsomal NADPH-cytochrome P450 reductase (CPR) Two isozymes of

HO, denoted as HO-1 and HO-2, have been identified

in mammalian systems [6] In general, HO-1 is respon-sible for the excretion of aged⁄ disused heme as well as recycling of iron [7,8] and HO-2 is associated with sig-nal transduction through the production of CO, where

Keywords

biliverdin; cyanobacterium heme oxygenase;

EPR; ferredoxin; heme oxygenase

Correspondence

T Yoshida, Department of Biochemistry,

Yamagata University School of Medicine,

Iida-nishi 2-2-2, Yamagata 990-9585, Japan

Fax: +81 23 6285225

Tel: +81 23 6285222

E-mail: tyoshida@med.id.yamagata-u.ac.jp

(Received 25 August 2004, revised 10

December 2004, accepted 17 December

2004)

doi:10.1111/j.1742-4658.2004.04535.x

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity

in amino acid sequence have been identified in the cyanobacterium Synechocystissp PCC 6803 Isoform-1, Syn HO-1, has been characterized, while isoform-2, Syn HO-2, has not In this study, a full-length ho2 gene was cloned using synthetic DNA and Syn HO-2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein Like Syn HO-1, the purified Syn HO-2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXa, CO and iron in the presence of reducing systems such as NADPH⁄ ferredoxin reductase⁄ ferredoxin and sodium ascorbate The activity of Syn HO-2 was found to be comparable to that of Syn HO-1 by measuring the amount of bilirubin formed In the reaction with hydrogen peroxide, Syn HO-2 con-verted heme to verdoheme This shows that during the conversion of hemin

to a-meso-hydroxyhemin, hydroperoxo species is the activated oxygen spe-cies as in other heme oxygenase reactions The absorption spectrum of the hemin–Syn HO-2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin-Syn HO-1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa-coordinate low spin species Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components These observations suggest that the structure

of the heme pocket of the Syn HO-2 is different from that of Syn HO-1

Abbreviations

CPR, cytochrome P450 reductase; HO, heme oxygenase; Fd, ferredoxin; FNR, ferredoxin reductase; KPB, potassium phosphate buffer; rHO-1, heme oxygenase-1 of Rattus norvegicus; Syn HO-1, heme oxygenase-1 of Synechocistis sp PCC 6803; Syn HO-2, heme oxygenase-2

of Synechocystis sp PCC 6803; EPR, electron paramagnetic resonance.

CO stimulates the formation of cGMP as a possible

physiological messenger akin to NO [9] Additionally,

HO plays an important role in the defense against

oxi-dative stress, as lipophilic bilirubin IXa, the reduced

form of biliverdin IXa, works as a potent endogenous

antioxidant similar to vitamin E [10,11]

HO is also found in some pathogenic bacteria,

where it is essential for the heme-based iron acquisition

needed to survive and produce proteinaceous poisons

[12–14] In contrast to mammalian HO, bacterial HO

is soluble owing to the lack of a C-terminal

hydropho-bic region The reaction mechanisms of bacterial HOs

are essentially similar to those of mammalian HOs

Prokaryotic plant heme oxygenase activity was first

found in a red alga, Cyanidium caldarium, and then in

cyanobacteria, Synechocystis sp PCC 6701 and PCC

6803, which have now been studied for 20 years [15–

20] The HO of cyanobacteria and prokaryotic red

algae is responsible for the biosynthesis of

photorecep-tive bilins such as phycocyanobilin and

phycoerythro-bilin, as these bilins are synthesized from biliverdin

IXa, a product of the HO reaction [15]

Phytochromo-bilin, one of the photo-sensing bilins required for the

photomorphogenesis of higher plants, is also

consid-ered to come from biliverdin IXa[15,21–23] Like

bac-terial HOs, the plant HOs are soluble and supposed to

need ferredoxin (Fd) as an electron donor In 1996,

the entire genome sequence of Synechocystis sp PCC

6803 was published and two different HO genes, ho1

and ho2, were identified [24] Next, the molecular

clo-ning of the HY1 gene of Arabidopsis was performed

and the product of this gene expressed in Escherichia

colishowed heme oxygenase activity [25] More recent

success in decoding the genomes of plants such as

tomato, soybean and pea also suggests the presence of

HOs in these higher plants [26] Cornejo et al first

reported a bacterial expression and purification system for a protein (HO-1) encoded by the ho1 gene [27] and

we also established an efficient E coli expression sys-tem to obtain highly purified protein (Syn HO-1) [28] This success has allowed for molecular-based studies

on Syn HO-1 [28,29]

Bacterial expression of the ho2 gene of Synechocystis

sp PCC 6803 was also carried out by Cornejo et al and yielded a small amount of soluble fraction which did not show heme oxygenase activity [27] Neverthe-less, the expected product of the ho2 gene, Syn HO-2,

is highly homologous in amino acid sequence to Syn HO-1 (51%) and most of the residues critical for heme oxygenase activity in mammalian HO-1 are conserved [30–34] (Fig 1), so that Syn HO-2 is strongly sugges-ted to be an active enzyme To obtain the active form

of Syn HO-2 and clarify the enzymatic properties of this protein, we have constructed a bacterial expression system for the ho2 gene and successfully obtained puri-fied Syn HO-2 protein Accordingly, it has been estab-lished that Syn HO-2 binds hemin stoichiometrically and converts it into biliverdin IXa, CO and iron in the presence of oxygen and electrons, demonstrating that Syn HO-2 is a true heme oxygenase This is the first report of the characterization of the cyanobacterial HO-2 protein and its heme complex

Results and Discussion

Expression and purification of Syn HO-2 Culturing the cells at two temperatures, first at 37
C and then at 20
C, we could avoid the accumulation of inclusion bodies of Syn HO-2 The harvested cells were pale green, indicative of the expression of a catalyti-cally active Syn HO-2 We purified the Syn HO-2 from the soluble fraction by ammonium sulfate fractionation and subsequent column chromatography on Sephadex G-75, DE-52, and hydroxyapatite The final prepar-ation after chromatography on a hydroxyapatite col-umn was clear and colorless and gave a single band of

29 kDa with about 97% purity on SDS⁄ PAGE (Fig 2), the size expected from the deduced Syn HO-2 amino acid sequence (28.5 kDa) About 10 mg of pro-tein was obtained from 1 L of culture

Catalytic activity of Syn HO-2 First we measured the catalytic activities of Syn HO-2 and compared them with those of Syn HO-1 We used ferredoxin reductase (FNR)⁄ Fd equivalent to seven-tenths of Syn HO-2 or sodium ascorbate as reducing reagents We added desferrioxamine and biliverdin

Scheme 1 Heme degradation pathway Conversion of heme to

bili-verdin IXa catalyzed by HO.

reductase to the reaction mixture to facilitate the release of iron and biliverdin from the enzyme and to reduce the biliverdin to bilirubin, respectively Table 1 indicates that Syn HO-2 is enzymatically active The specific activity for heme breakdown was comparable

to that of Syn HO-1 in the presence of each of the reducing systems, although the ascorbate-supported activity of Syn HO-2 was stronger than that of Syn HO-1 The activity levels of both enzymes supported

by NADPH⁄ FNR ⁄ Fd were higher than those with ascorbate NADPH⁄ CPR supported activities of both HOs were considerably low Our recent study on Syn HO-1 crystals indicated that the positively charged sur-face interacting with an electron donor was narrower than that of mammalian HO-1 [29] Then, the elec-trons from CPR might not be transferred efficiently to Syn HO-1 or Syn HO-2

Properties of the heme-Syn HO-2 complex All HOs studied to date bind heme stoichiometrically

to form a substrate–enzyme complex Like other HOs, Syn HO-2 also binds hemin, with a dissociation constant of about 8.87 ± 2.1 lm to form a 1 : 1 stoi-chiometric complex (Fig 3, inset) The complex was stable and purified as described in Experimental pro-cedures

Fig 1 Amino acid sequence of Syn HO-2 as compared with the sequences of Syn HO-1, rHO-1 and rHO-2 The shaded letters indicate resi-dues with sequence identity.

Fig 2 SDS ⁄ PAGE of the purified Syn HO-2 protein Lane 1,

molecular mass marker; lane 2, 10 lg of purified protein.

Absorption spectra of the complexes of heme and

HOs resemble the spectrum of myoglobin with one

exception, the HO of Drosophila melanogaster In the

HO of fruit fly, the iron of heme was not involved in

binding to the enzyme, resulting in a different

spec-trum from the other HOs [35] Figure 3 exhibits

opti-cal absorption spectra of the iron(III), iron(II), oxy,

and CO-bound forms of the heme-Syn HO-2 complex

at pH 7.4 Interestingly, the spectrum of the iron(III)

complex has two peaks at 576 and 540 nm besides the

Soret band at 412 nm in the visible region, which

resembles that of the complex of iron(III) heme and

Syn HO-1 at alkaline pH [28], suggesting that the

major species of the iron(III) heme iron of Syn HO-2

at neutral pH is a hexa-coordinate low spin species

This is quite different from other known HOs The

Soret peak of the iron(III) complex was slightly

red-shifted at pH 8.0 with a slight decrease in its

absorb-ance and slightly blue-shifted at pH 6.0 with a slight

increase in its absorbance However, the dependence

on pH was not fully reversible because of denaturation

of the protein By the pyridine hemochrome method, the extinction coefficient at 412 nm for the iron(III) heme–Syn HO-2 complex is calculated to be

110 mm)1Æcm)1 The electron paramagnetic resonance (EPR) spec-trum of the iron(III) resting state of heme-Syn HO-2

in pH 7.4 solution reveals that the heme in Syn HO-2

is in an admixture of high-spin and low-spin states (Fig 4A) Though the high spin species exhibits an apparently axially symmetric type of spectrum with

g^¼ 6 and gII¼ 2, small rhombicity is observed at the perpendicular component of the spectrum, which is not seen in the spectrum of the high-spin heme Syn

Table 1 Activities of the purified Syn HO-2 The HO activity was

determined from the initial rate of bilirubin formation with the

NADPH ⁄ FNR ⁄ Fd, NADPH ⁄ CPR or sodium ascorbate system at

30
C, pH 7.4 All the measurements were done in triplicate.

Reaction system

Bilirubin formation (nmolÆmg protein)1Æh)1) Syn HO-2 Syn HO-1 NADPH ⁄ FNR ⁄ Fd 622 ± 21 603 ± 27

NADPH ⁄ CPR 81 ± 3 69 ± 8

Sodium ascorbate 424 ± 20 273 ± 23

Fig 3 Absorption spectra of various forms of the Syn HO-2–heme

complex The concentration of the complex was 7 lm The spectra

are the iron(III) (solid line), iron(II) (dotted line), iron(II)-CO (dashed

line), and oxy (dotted-dashed line) complexes Inset, titration of Syn

HO-2 (9 lm) with hemin as monitored by the increase in

absorb-ance at 412 nm ()) The increase in absorbabsorb-ance because of the

addition of hemin in the absence of Syn HO-2 is indicated (*).

Fig 4 EPR spectra of the heme–Syn HO-2 complexes (A) The iron(III) resting state complex in pH 7.4 solution (solid line) and in

pH 8.0 solution (dotted line) EPR conditions were microwave quency, 9.35 GHz, microwave power, 1 mW, field modulation fre-quencies, 100 kHz, field modulation amplitude, 10 G, and sample temperature, 10 K (B) The15NO bound iron(II) complex measured with microwave power, 0.2 mW, field modulation amplitude, 2 G,

at 25K, where other conditions are the same as those described

in (A).

HO-1 complex This suggests that the in-plane

anisot-ropy of the ligand field to the heme iron in Syn HO-2

is relatively large The seating of the heme in the heme

pocket might be less homogeneous than in Syn HO-1

As the pH of the solution rises from 7.4 to 8.0, the

sig-nal intensity of the high-spin species decreases and

alternatively that of the low-spin species increases

without a change in the g-values The low-spin

compo-nent of the spectrum shows that there are two kinds of

low-spin species: the major species has g-values of

2.69, 2.20, and 1.79 and the minor species has more

anisotropic g-values (Fig 4A, asterisks) The g-values

of the major species (2.69, 2.20, 1.79) are very close to

those (2.68, 2.20, 1.80) of the minor alkaline

compo-nent of heme–Syn HO-1 as well as to the values (2.67,

2.21, 1.79) of the alkaline form of heme-rHO-1 [28]

The g-values of the minor component of heme–Syn

HO-2 seem to be similar to those of the major

low-spin component (2.78, 2.14, 1.74) of heme–Syn HO-1

in alkaline solution [28] The low-spin species of heme–

Syn HO-2 therefore probably is the same species as the

alkaline forms of known heme–HO The existence of

alkaline forms is evidence that a water molecule is

pos-sessed at the distal site of the heme iron and strongly

interacts with the distal helix main chain or its

disso-ciable residues The close similarity of the g-values

sug-gests that the heme pocket milieu of heme-Syn HO-2

in alkaline solution more resembles that of rHO-1 than

that of Syn HO-1

The 15NO-bound form of heme-Syn HO-2 shows a

typical six-coordinated nitrosyl heme spectrum

(Fig 4B) The hyperfine splitting pattern at the g2

component indicates that nitrogen nuclei both of15NO

and of 14N-proximal ligand cause the splitting The

heme proximal ligand is considered to be His16, which

corresponds to His17 of the proximal ligand of

heme-Syn HO-1 EPR parameters of 15NO-heme–Syn HO-2

were compared with those of the Syn HO-1 and

rHO-1 complexes (Table 2) Hyperfine coupling constants,

A(15N-O) and A(14N-His), of the Syn HO-2 complex

are closer to those of the rHO-1 complex than Syn

HO-1 complex, indicating that the axial ligand

coordi-nation structure of heme-Syn HO-2 rather resembles

that of heme-rHO-1 In conclusion, EPR reveals that the heme-Syn HO-2 complex is in dynamic equilibrium between high- and low-spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components Further, part of the heme pocket structure of Syn HO-2 more resembles that of rHO-1 than that of Syn HO-1

Degradation of hemin bound to Syn HO-2 to biliverdin by the NADPH/FNR/Fd or sodium ascorbate systems

To reduce FNR-mediated heme degradation leading to nonbiliverdin products, we used FNR⁄ Fd equivalent

to one-twenty fourth of Syn HO-2 in this study With the addition of NADPH to the reaction mixture, the absorption showing a peak at 412 nm decreased gradu-ally until fingradu-ally, broad absorption bands centered near

380 and 690 nm appeared, indicative of the conversion

of hemin to biliverdin (Fig 5A) The formation of biliverdin is supported by the decrease in absorbance around 690 nm and concomitant increase in absorb-ance around 450 nm after the addition of biliverdin reductase, reflecting the conversion of biliverdin to bilirubin Similar spectrophotometric changes were observed in Fig 5B, where sodium ascorbate was used

as a reductant The decrease in absorbance at 412 nm supported by FNR⁄ Fd was slower than that with ascorbate This is because a relatively small amount of FNR⁄ Fd compared to Syn HO-2 was added to the reaction system Again, the absorption due to biliver-din was converted to that of bilirubin by the addition

of biliverdin reductase and NADPH

A previous study on rHO-1 indicated that when ascorbate was used as a reductant, the final heme deg-radation product was not biliverdin but iron(III) bili-verdin bound to the enzyme [36] Then, we conducted similar experiments without desferrioxamine The rates

of heme degradation did not differ in the presence or absence of desferrioxamine However, the intensities

of the absorbance around 690 nm in the NADPH⁄ FNR⁄ Fd system (Fig 5C) and ascorbate system (Fig 5D) were about two-thirds and one-half of those observed in the presence of desferrioxamine, respect-ively These results suggest that the final product of the Syn HO-2 reaction supported by both reducing systems is biliverdin and that the release of iron(III)-biliverdin from Syn HO-2 is slow

In independent experiments, we analyzed the stereo-selectivity of the products of the Syn HO-2 reaction supported by the NADPH⁄ FNR ⁄ Fd system and ascorbate system using HPLC In both cases, only

Table 2 EPR parameters of the iron(II) 15 NO bound heme

com-plexes of Syn HO-2, Syn HO-1, and rat HO-1 Data from Syn HO-1

and rat HO-1 are taken from reference [28].

Protein g 3 g 2 g 1 A([15N]NO) ⁄ G A([14N]His) ⁄ G

Syn HO-2 2.082 2.006 1.965 27.2 7.4

Syn HO-1 2.079 2.003 1.962 31.1 7.1

rat HO-1 2.086 2.008 1.986 26.0 7.4

the a-isomer of biliverdin was detected (data not

shown), indicating a-specificity of the Syn HO-2

reaction

Reaction of hemin bound to Syn HO-2 with

hydrogen peroxide

In the case of mammalian HO, an iron(III)

hydroper-oxide species was speculated to be an intermediate in

the first oxygenation step, as hydrogen peroxide

con-verted hemin to iron(III) verdoheme via

a-meso-hydroxyhemin (Scheme 1) [37] This was confirmed

experimentally [38–40] Then, we reacted the hemin–

Syn HO-2 complex with hydrogen peroxide and found

that a iron(III) verdoheme–Syn HO-2 complex was

formed (data not shown) This result shows that a

iron(III) hydroperoxide species must be an active

inter-mediate in the first oxygenation step from hemin to

a-meso-hydroxyhemin in the Syn HO-2 reaction, like for other HOs studied

Verdoheme formation during the course

of heme degradation With rHO-1, hemin bound to the enzyme was conver-ted into iron(II)-CO form of verdoheme under O2 and

CO, and the reaction was arrested at this stage because

CO inhibits the further conversion of verdoheme to iron(III)-biliverdin (Scheme 1) [41] To detect the iron(II)–CO form of the verdoheme–Syn HO-2 com-plex, we carried out similar experiments in an atmo-sphere of approximately 5% CO, 5% O2 and 90% N2 (v⁄ v ⁄ v) The spectrum (Fig 6, dotted line) recorded

2 min after the start of the reaction has four peaks at

415, 540, 570, and 637 nm in the visible region The first peak is attributable to the iron(III)–Syn HO-2

Fig 5 Reaction of hemin bound to Syn HO-2 with the NADPH ⁄ FNR ⁄ Fd system or sodium ascorbate in the presence or absence of desfer-rioxamine (DFO) Spectrum of the hemin–Syn HO-2 complex (solid line); 10 min after the start of the reaction depicted between 350 and

450 nm (dotted line); 50 min after the start of the reaction (dashed line); after the addition of biliverdin reductase (dotted-dashed line) In the ascorbate system (B and D), NADPH was added together with biliverdin reductase Inset, five-fold enlarged spectra between 500 and

750 nm.

complex and the peaks at 540 and 570 nm are due to

an admixture of the iron(III) and the iron(II)-CO

complexes The peak at 637 nm is attributable to

the CO-bound verdoheme–Syn HO-2 complex The

absorption at 637 nm increased with time and reached

a maximum 2 min after the start of the reaction The

broken line, a spectrum recorded a further 3 min later,

has a decreased absorbance at 637 nm and increased

absorbance around 690 nm, indicative of the

conver-sion of verdoheme to biliverdin The spectral changes

depicted in Fig 6 together with the result described in

the preceding section indicate that verdoheme is an

intermediate of the Syn HO-2 reaction, like other HO

reactions

Detection of CO during the Syn HO-2 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⁄ FNR ⁄ Fd-supported reaction The Soret band

of myoglobin was monitored at 1-min intervals after

the addition of NADPH to both the sample and the

reference cuvette As depicted in Fig 7, the myoglobin

Soret band shifted from 393 to 425 nm with the

appearance of a⁄ b bands at 568 and 538 nm and

absorbance at 425 nm increased These results indicate

that the iron(III) form of myoglobin was reduced to

the iron(II) form by the NADPH⁄ FNR ⁄ Fd system

and this was followed by CO binding to yield the

iron(II)-CO form, the authentic absorption spectrum

of which is depicted in the inset of Fig 7 This

experi-ment clearly demonstrates the formation of CO during

the degradation of heme by Syn HO-2 in line with the mechanism shown in Scheme 1

Conclusive remarks Although a previous effort to express the ho2 gene

in E coli cells by Cornejo et al resulted in mostly insoluble protein [27], we fortunately obtained soluble Syn HO-2 protein on a large scale As the conditions

of cell culture seemed to be similar in both cases, the difference in the plasmid used might be the cause

of the discrepancy In spite that an RNA blot ana-lysis of cyanobacteria grown in light suggested that ho2 is silent [27], the Syn HO-2 obtained in this study has shown heme oxygenase activity Similar to mammalian HO-1 and HO-2 as well as bacterial HOs (Hmu O, Hem O, Pig A) and Syn HO-1, Syn HO-2 binds a stoichiometric amount of heme to form a stable heme–HO complex Optical absorption and EPR spectroscopy reveal that heme–Syn HO-2 is mostly in the iron(III) low-spin state, which is a unique feature of this complex In spite of the low-spin resting state, the bound heme is converted

to biliverdin IXa, CO and free iron in the presence

of reducing equivalents such as ascorbate or NADPH⁄ FNR ⁄ Fd and oxygen The activity of Syn HO-2 for the catabolism of heme is comparable to that of Syn HO-1 as determined by the initial rate

Fig 6 Reaction of hemin bound to Syn HO-2 with the

NADPH ⁄ FNR ⁄ Fd system under O 2 and CO Spectrum of the

hemin–Syn HO-2 complex with FNR ⁄ Fd (solid line); 2 min after the

addition of NADPH to start the reaction (dotted line); 5 min after

the start of the reaction (dashed line) Inset, fivefold enlarged

spec-tra between 500 and 750 nm.

Fig 7 Detection of CO produced during the Syn HO-2 reaction The sample solution contained the hemin–Syn HO-2 complex, FNR ⁄ Fd and the H64L mutant of myoglobin Myoglobin was omitted from the reference solution The reaction was started by the addition of NADPH to both solutions and the difference spectrum was recor-ded Difference spectrum before the start of the reaction (dashed line); 0.5 min (dotted-dashed line); 1.5 min (dotted line); 4.5 min (solid line) after the start of the reaction Inset: Absorption spectra

of various forms of the H64L mutant of myoglobin CO-iron(II) form (solid line); iron(II) form (dotted line); iron(III) form (dashed line).

of bilirubin formation The a-selectivity of the

prod-uct is strictly retained The second intermediate of

the HO reaction, verdoheme, has also been detected

in the Syn HO-2 reaction and hydrogen peroxide has

been ascertained to substitute for O2 and electrons

in the conversion of hemin to verdoheme, thereby

implying that the chemistry of heme degradation by

Syn HO-2 is similar to that by other HOs

Accord-ingly, we conclude that Syn HO-2 is a true heme

oxygenase even though the physiological importance

of this isoform is unknown at this stage

It is noticeable that the EPR spectrum of nitrosyl

heme–Syn HO-2, in which the minute difference in the

ligand field around the heme is saliently represented, is

explicitly different from that of heme–Syn HO-1 As

85% of the amino acids composing of the distal-site

helix in Syn HO-1 are conserved in Syn HO-2, some

factor(s) other than the primary structure, may be

responsible for the difference in the heme-pocket

struc-ture We are now in the process of analyzing the

crys-tal structure of Syn HO-2

Experimental procedures

Construction of Synechocystis heme oxygenase-2

expression vector, pMWSynHO2

A 50 base pair double-stranded synthetic oligonucleotide

with unique sites for the restriction enzymes NdeI, Bsu36I,

NheI, EcoRI, XhoI, and HindIII was ligated between NdeI

and HindIII sites of a T7-promotor-based bacterial

expres-sion vector, pMW172, to make a plasmid tentatively

referred to as pMW-A Ten oligonucleotides and their

complements, 59–88 nucleotides in length, were synthesized

to construct a 752 base pair synthetic gene coding for the

entire Syn HO-2 from the ATG initiation codon to the

TAA stop codon Each nucleotide was phosphorylated

with T4 polynucleotide kinase, then annealed with its

com-plement to make a double-stranded DNA, e.g Oligo I to

Oligo X Oligo I was designed so that the 5¢ end could be

ligated to the NdeI site, whereas its 3¢ cohesive end was

complementary to the 5¢ end of Oligo II The 3¢ end of

Oligo II could be ligated to the Bsu36I site Similarly, the

5¢ ends of Oligos III, V, VII and IX were designed to

ligate to the Bsu36I, NheI, EcoRI, and XhoI sites,

respect-ively, and their 3¢ ends had sequences for ligation to the

5¢ ends of Oligo IV, VI, VIII and X The 3¢ end of Oligo

X had a sequence designed to ligate to the HindIII site

pMWSynHO2, double-stranded Oligo I to Oligo X were

ligated step by step into the restriction enzyme sites of

pMW-A The nucleotide sequence of the thus constructed

pMWSynHO2 was determined with an Applied Biosystems

373A DNA sequencer

Syn HO-2 expression and purification

A 5 mL inoculum in Luria–Bertani medium (+ 50 lgÆmL)1 ampicillin⁄ 0.1% glucose) was prepared from a plate of trans-formed E coli BL21 (DE3) cells carrying pMWSynHO2 Five-hundred milliliter cultures were inoculated with 300 lL

of the inocula and grown in Luria–Bertani medium (+ 200 lgÆmL)1ampicillin) at 37
C until the D600reached 0.8–1.0 The cells were grown for an additional 24 h at 20
C, harves-ted by centrifugation, and stored at)80
C prior to use The typical yield of cells from a 500 mL culture was 2 g

The E coli cells (10 g), resuspended in 90 mL of Tris⁄ HCl buffer (pH 7.4, +2 mm EDTA), were lysed (2 mg lysozyme per g cells) with stirring at 4
C for 30 min After sonication (Branson 450 Sonifire) and centrifugation

at 100 000 g for 1 h, the resulting supernatant was covered

with a 20–50% ammonium sulfate fraction and centrifuged The subsequent precipitates, containing the Syn HO-2 pro-tein, were dissolved in 20 mm potassium phosphate buffer (KPB) (pH 7.4) and applied to a Sephadex G-75 column (3.6· 50 cm), pre-equilibrated with the same buffer The protein fractions eluted in the KPB, with an intense 29 kDa band on SDS⁄ PAGE, were collected and directly loaded onto a column of DE-52 (2.6· 28 cm) The column was washed with 50 mL of 20 mm KPB (pH 7.4)-50 mm KCl, and the protein was eluted with 400 mL of 20 mm KPB (pH 7.4) using a linear gradient, 50–400 mm KCl Collected fractions with a high protein content were further run through a column of hydroxyapatite (2.6· 20 cm) Again the column was washed with 50 mL of 20 mm KPB (pH 7.4), and the protein was eluted with 200 mL of

50 mm KPB (pH 7.4) Only fractions with a single band at

29 kDa on SDS⁄ PAGE were gathered

Heme binding study

Heme binding of Syn HO-2 was tested by adding hemin to

9 lm Syn HO-2 in 2 mL of 50 mm KPB (pH 7.4) The ref-erence cuvette contained 2 mL of 50 mm KPB (pH 7.4) alone A solution of 1 mm hemin was added in 4 lL aliqu-ots to both test and reference cuvettes with 5 min equilibra-tion between addiequilibra-tions at 25
C The absorbance between

350 and 750 nm was measured on a Beckman DU7400 single-beam spectrophotometer

Preparation of a complex of Syn HO-2 and hemin

Syn HO-2 in 50 mm KPB (pH 7.4) was added to a 1.2 equivalent excess of hemin This solution was passed through a column of Sephadex G-25 equilibrated with

50 mm KPB (pH 7.4) Fractions colored brown were loaded onto a column of hydroxyapatite equilibrated with 50 mm KPB (pH 7.4) The passed fraction colored brown was collected and used as a complex of hemin and Syn HO-2 for spectrophotometric experiments

Optical absorption spectroscopy

Optical absorption spectra were recorded on a Beckmann,

DU7400 spectrophotometer at 25
C The iron(II) heme–

Syn HO-2 complex was prepared in a sealed cuvette by

the addition of dithionite to a 0.1 m KPB (pH 7.4)

solu-tion of hemin–Syn HO-2 saturated with argon The CO

complex of heme–Syn HO-2 was prepared by displacing

the argon in the space of a sealed cuvette containing the

iron(II)–Syn HO-2 solution with CO The oxy complex

was prepared by introducing air into the anaerobic sample

of iron(II) heme–Syn HO-2 generated by the reduction of

the iron(III) complex with a stoichiometric amount of

sodium dithionite

Assay of heme oxygenase activity by measuring

bilirubin formation

The catalytic activity of Syn HO-2 and Syn HO-1 was

determined after the conversion of biliverdin IXa produced

by the enzyme, to bilirubin by biliverdin reductase The

reaction mixture of the NADPH⁄ FNR ⁄ Fd system

con-tained in a final volume of 1.5 mL; 50 mm KPB (pH 7.4),

0.5 mg of bovine serum albumin, 20 lm hemin, 1 lm

enzyme, 0.7 lm maize FNR⁄ maize Fd III, 140 lm

NADPH, 1 mm desferrioxamine, and 6 lm recombinant rat

biliverdin reductase [42] NADPH was omitted in the

con-trol system The reaction was started by the addition of

NADPH after 3-min preincubation at 30
C, and

monit-ored at 468 nm for 10 min The value of 43.5 mm)1Æcm)1

was used as the extinction coefficient for bilirubin at

468 nm [43] Assay in the presence of NADPH⁄ CPR was

performed by the similar way except that 0.7 lm of rat liver

CPR was used instead of 0.7 lm of FNR⁄ Fd The

ascor-bate system contained in a final volume of 1.5 mL; 50 mm

KPB (pH 7.4), 0.5 mg of bovine serum albumin, 20 lm

hemin, 1 lm enzyme, 13.3 mm sodium ascorbate, 70 lm

NADPH, 1 mm desferrioxamine, and 6 lm biliverdin

reduc-tase Ascorbate was omitted from the control system

The reaction was initiated by the addition of ascorbate

Other conditions were the same as those for the

NADPH⁄ FNR ⁄ Fd system

Reaction of hemin bound to Syn HO-2 with

NADPH/FNR/Fd, sodium ascorbate, and

hydro-gen peroxide systems in the presence or absence

of desferrioxamine

Spectral changes were recorded at 30
C between 350

and 750 nm We used three electron donor systems,

NADPH⁄ FNR ⁄ Fd, ascorbate, and H2O2 The standard

reaction mixture for the NADPH⁄ FNR ⁄ Fd system

consis-ted of 8 lm Syn HO-2–hemin complex and 0.33 lm

FNR⁄ Fd in a final volume of 1.5 mL of 50 mm KPB

(pH 7.4) After 3 min preincubation, the reaction was started by the addition of 15 lL of 10 mm NADPH (final concentration, 0.1 mm) The reaction mixture for the ascor-bate system contained 8 lm Syn HO-2–hemin complex in a final volume of 1.5 mL of 50 mm KPB (pH 7.4) After

3 min preincubation, the reaction was initiated by the addition of 15 lL of 1 m sodium ascorbate (final concentra-tion, 10 mm) When desferrioxamine was added, a final concentration of 1 mm was used The H2O2system consis-ted of 8 lm Syn HO-2–hemin complex in a final volume of 1.5 mL of 50 mm KPB (pH 7.4) After 3 min preincuba-tion, the reaction was started by the addition of 15 lL of

1 mm H2O2 in water (final concentration, 10 lm) The concentration of H2O2in the original aqueous reagent solu-tion was determined spectroscopically using a value of 43.6 m)1Æcm)1for the extinction coefficient at 240 nm [44]

EPR spectroscopy

EPR measurements were performed with a Bruker E500 spectrometer, operating at 9.35–9.55 GHz, with an Oxford ESR 900 liquid helium cryostat The 15NO-bound form of the heme–Syn HO-2 complex was prepared by adding dithio-nite to the argon-saturated hemin–protein solution, con-taining Na15NO2in an EPR tube

Detection of carbon monoxide

To detect CO produced during the reaction supported by

a system of NADPH⁄ FNR ⁄ Fd, a myoglobin mutant, H64L, which has higher affinity for CO than the wild type [45], was included in the reaction mixture The reac-tion solureac-tions contained 8 lm hemin–Syn HO-2 complex,

(pH 7.4) H64L, at a final concentration of 6.5 lm, was included in the test solution After the addition of NADPH (final concentration, 0.1 mm) to both cuvettes, the spectrum was recorded 30 s after the start of the reaction and then recorded at 1-min intervals between

350 and 750 nm

Other procedures

Sequence translation and sequence alignment were per-formed using the Wisconsin Package from the Genetic Computer Group (Madison, WI, USA) and clustalw mul-tiple sequence alignment program at the EBI (EMBL-EBI) HPLC analysis of reaction products was done as previously described [35] Purified Syn HO-1 was obtained by a pub-lished method [28] Maize FNR [46], maize Fd III [47], and rat liver CPR [3] were purified to the single bands on SDS⁄ PAGE by published procedures The H64L mutant was purified according to published methods [48] Hemin concentrations were measured according to the method of

Paul and Theorell [49] Protein concentration was measured

by the Lowry method using bovine serum albumin as

standard [50]

Acknowledgements

This work was supported in part by grants-in-aid from

the Ministry of Education, Science, Sports, and

Cul-ture, Japan (14580641 and 16570108)

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 E coli expression

plas-mids for maize FNR and maize Fd III were gifts from

professor T Hase, Osaka University

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