Fax/Tel: + 81 83 933 5863, E-mail: ctmigita@po.cc.yamaguchi-u.ac.jp Abbreviations: hemin, ferric protoporphyrin IX; heme, iron proto-porphyrin IX either ferrous or ferric form; hydroxyh
Trang 1Expression and characterization of cyanobacterium heme oxygenase,
a key enzyme in the phycobilin synthesis
Properties of the heme complex of recombinant active enzyme
Catharina T Migita1, Xuhong Zhang2and Tadashi Yoshida2
1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan; 2 Department of Biochemistry, Yamagata University School of Medicine, Japan
An efficient bacterial expression system of cyanobacterium
Synechocystis sp PCC 6803 heme oxygenase gene, ho-1,
has been constructed, using a synthetic gene A soluble
protein was expressedat high levels andwas highly
purified, for the first time The protein binds equimolar
free hemin to catabolize the boundhemin to
ferric-bili-verdin IXa in the presence of oxygen andreducing
equivalents, showing the heme oxygenase activity During
the reaction, verdoheme intermediate is formed with the
evolution of carbon monoxide Though both ascorbate
andNADPH-cytochrome P450 reductase serve as an
electron donor, the heme catabolism assisted by ascorbate
is considerably slow and the reaction with
NADPH-cytochrome P450 reductase is greatly retarded after the
oxy-heme complex formation The optical absorption
spectra of the heme-enzyme complexes are similar to those
of the known heme oxygenase complexes but have
some distinct features, exhibiting the Soret band slightly
blue-shiftedandrelatively strong CT bands of the high-spin component in the ferric form spectrum The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated EPR of the nitrosyl heme complex has establishedthe nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex The spectroscopic characters as well
as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, Hmu O
Keywords: cyanobacterium heme oxygenase isoform-1; EPR; heme complex; protein expression; spectroscopy
Photoreceptor chromophores in the plant kingdom are
categorizedinto two groups of chlorophyll andphycobilin
Chlorophyll, which is containedin all plants including
cyanobacteria andprotoflorideophyceae, is synthesized
from protoporphyrin IX, a precursor of heme Phycobilins
of open-chain tetrapyrroles are produced from biliverdin
that is a product of heme degradation Accordingly, the
chlorophyll andphycobilin syntheses share the pathway of
protoporohyrin synthesis from d-aminolevulinic acid[1,2]
Phycobilins work as the main photoreceptor of
photosyn-thesis in procaryophyta, cyanobacteria andother primitive eucaryotic algae Phycobilin synthesis branches from chlo-rophyll synthesis at the iron insertion to protoporphyrin IX
to form heme that is catalyzedby ferrochelatase Then, heme is convertedto biliverdin IXaby an enzyme named heme oxygenase (HO) Biliverdin IXais further reduced and isomerizedto produce phycobilins such as phycoerythro-bilin andphycocyanophycoerythro-bilin [3–5] The enzymes catalyzing these reactions, phycobilin synthase(s), have not been identified yet In higher plants, phytochromobilins are also supposedto be synthesizedfrom biliverdin IXa [6] The phytochromobilins are precursors of the chromophore of phytochromes, which are photo-reversible light signal-transducing biliproteins andhave closely relatedstructure with phycobilins [7]
HO was first establishedin mammalian systems as a membrane-boundmicrosomal enzyme that catalyzedthe regiospecific oxidative degradation of heme [8] The enzymatic reaction requires three molecules of oxygen andsix electrons to convert ferric heme to the ferric-biliverdin complex and CO [9–13] NADPH coupled with cytochrome P450 reductase supplies the electrons in mammalian systems The mammalian HOs (inducible isoform-1 andconservedisoform-2 are known) andtheir heme complexes have been characterizedrelatively well
Correspondences to C T Migita, the Department of Biological
Chemistry, Faculty of Agriculture, Yamaguchi University,
1677–1 Yoshida, Yamaguchi 753–8515, Japan.
Fax/Tel: + 81 83 933 5863,
E-mail: ctmigita@po.cc.yamaguchi-u.ac.jp
Abbreviations: hemin, ferric protoporphyrin IX; heme, iron
proto-porphyrin IX either ferrous or ferric form; hydroxyheme, iron
meso-hydroxyl protoporphyrin IX; HO, heme oxygenase;
Syn HO-1, Synechocystis heme oxygenase isoform 1.
Enzymes: heme oxygenase (EC 1.14.99.3); NADPH cytochrome P450
reductase (EC 1.6.2.4).
(Received17 September 2002, revised9 December 2002,
accepted10 December 2002)
Trang 2using recombinant proteins [14–16] The crystal structures
of the human HO-1 [17] andrat HO-1 [18] have been
published The reaction mechanism of heme degradation,
especially that of the first oxygenation step from heme to
a-hydroxyheme, has been clarified by recent works [19,20]
Recently, heme oxygenase has also been foundin some
pathogenic bacteria [21–28] They are water-soluble
pro-teins andmainly function to release iron from heme of the
host cells, which is necessary for the survival of bacteria
andfor causing diseases The crystal structure of the
bacterial HO (Hem O) has been reported[27] The
reaction mechanism is supposedto be analogous to those
of mammalian HOs [22,23,25]
Algal andcyanobacterial HOs have been studiedas a cell
extract for the last 20 years [29–33] The proteins possessing
heme oxygenase activity have been obtainedfrom redalga,
Cyanidium caldarium, andcyanobacterium, Synechocystis
sp PCC 6701 andPCC 6803 [33], to date The algal HO is a
soluble protein, localizedin the plastids The in vitro heme
oxygenase activity is supposedto needreducedferredoxin
andthe secondreductant such as ascorbate In 1993, the
HO gene of rhodophyta Porphyra purpurea (pbsA) was
isolated[34] andin 1996, the entire genome sequence of
Synechocystis sp PCC 6803 was determined, identifying
two different HO genes (ho1 and ho2) [35] Trials of cloning
andexpression of these genes in Escherichia coli yielded
single active protein, HO-1, from the ho1 gene [36] Another
cyanobacterial HO gene was identified in the complete
genomic sequence of Nostoc (Anabaena) sp PCC 7120, very
recently [37] On the other hand, recent research has
reportedthat, in higher plants, the Arabidopsis thaliana
hy1gene encords a protein related to HO [38–40] Thus, HO
seems to present ubiquitously in the plant kingdom, as a key
enzyme for the synthesis of photon-accepting
chromo-phores However, knowledge of the characteristics of plant
HO is limitedbecause large amounts of purifiedprotein
have not been available so far
In this study, we have constructed an efficient bacterial
expression system of the HO-1 protein, basedon the ho1
gene sequence of cyanobacteria, Synechocystis sp PCC
6803 [35] andhave succeededin obtaining highly purified
soluble protein, Syn HO-1, in a large scale This is the first
report of the characterization of the isolatedcyanobacterial
HO-1 protein andits heme complexes, applying the optical
absorption andelectron paramagnetic resonance (EPR)
spectroscopies
Experimental procedures
Construction ofSynechocystis heme oxygenase-1
expression plasmid, pMWSynHO1
Plasmidpurification, subcloning, andbacterial
transfor-mations were carriedout as previously described[23] A
T7 promoter-basedexpression vector, pMW172 (a gift
from K Nagai, MRC Laboratory of Molecular Biology,
Cambridge, UK) was used to make the expression
plasmidpMWSynHO1 for the recombinant Synechocystis
heme oxygenase-1 by incorporating a double-stranded
synthetic oligonucleotide with unique restriction enzyme
sites for SpeI, SacI, AvrII, ClaI, and MluI between the
NdeI and HindIII sites A 720-base pair synthetic gene
coding for the entire Syn HO-1 was synthesized from nine oligonucleotides and their complements constructed
by 55–99mer nucleotides Double strand DNAs, Oligo I
to Oligo IX, were ligatedstep by step into the restriction enzyme sites of the plasmid, by use of T4ligase Oligo I andOligo II were insertedbetween the sites of NdeI and SpeI; Oligo III, between the sites of SpeI and SacI; Oligo
IV andOligo V, between SacI and AvrII; Oligo VI, between AvrII and ClaI; Oligo VII andVIII, between ClaI and MluI; Oligo IX, between MluI and HindIII Escherichia coli strain JM109 was usedfor DNA manipulation The nucleotide sequence of the expression plasmid, pMWSynHO1, was determined by an Applied Biosystems 373A DNA sequencer
Protein expression and purification
A 10-mL inoculum in Luria–Bertani medium (+ 50 lgÆmL)1 ampicillin : 0.1% glucose) was preparedfrom plates of transformed E coli BL21 (DE3) cells carrying pMWSyn-HO1 Cultures (500 mL) were inoculatedwith 1 mL
of the inocula andgrown in Luria–Bertani medium (+ 200 lgÆmL)1 ampicillin) at 37C until D600 reached 0.8–1.0 The cells were grown for an additional 16 h at 25C, harvestedby centrifugation, andstoredat)80 C until use Typical yieldof cells from a 500-mL culture was 2 g The E coli cells (10 g), resuspended into 90 mL Tris buffer (pH 7.4, + 2 mM EDTA), were lyzed(2 mg lyso-zyme per g cells) with stirring at 4C for 30 min After sonication (Branson 450 Sonifire) andcentrifugation at
39 000 g for 1 h, the resulting supernatant was converted into 35–60% (NH4)2SO4 fraction andcentrifuged The subsequent precipitates, containing the Syn HO-1 protein, were dissolved in 20 mM potassium phosphate buffer (pH 7.4) andappliedto a Sephadex G75 column (3.6· 50 cm), pre-equilibratedwith the same buffer The protein fractions elutedin the potassium phosphate buffer, with the intense 27 kDa bandon SDS/PAGE, were gathered and directly loaded onto a DEAE-cellulose (DE-52) column (2.6· 28 cm) After washing the column with 50 mL of 20 mM potassium phosphate (pH
7.4)-50 mMKCl, the protein was elutedwith 400 mL of 20 mM potassium phosphate (pH 7.4) using a linear gradient, 50–250 mM KCl Collectedfractions with high protein content were further run through a hydroxylapatite column (2.6· 20 cm) The protein was elutedwith 400 mL of potassium phosphate (pH 7.4) using a linear gradient, 20–200 mM Only fractions with the single bandat 27 kDa
on SDS/PAGE were finally collected The protein concen-trations were estimatedby Lowry’s methodusing crystalline bovine serum albumin as standard
Reconstitution of Syn HO-1 with hemin
An alkaline-hemin solution of 0.86 mMin 4.6 lL increments was added to the 10 lMsolution of Syn HO-1 in 2 mL of 0.1Mpotassium phosphate buffer (pH 7.0) Optical absor-bance at 402 nm was monitored for each addition of the hemin solution andplottedagainst the volume of added hemin solutions to construct titration curves The heme–Syn HO-1 complex was purifiedby Sephadex G-25 andDEAE-cellulose (DE-52) column chromatography
Trang 3Optical absorption spectroscopy
Optical absorption spectra were recorded on a Shimadzu
UV-2200 spectrophotometer at 25C The ferrous
heme-Syn HO-1 complex was preparedin a sealedcuvette by the
addition of dithionite to the 0.1M potassium phosphate
(pH 7.0) solution of hemin-Syn HO-1 that was previously
deoxygenated by use of oxygen absorber (Iuchi, A500–50S)
andsaturatedwith argon The CO complex of heme-Syn
HO-1 was preparedby displacing argon filledin the space of
a sealedcuvette containing the ferrous-Syn HO-1 solution
with CO The oxy complex was preparedby introducing air
into the anaerobic sample of ferrous heme-Syn HO-1
generatedby the reduction of the ferric complex with
NADPH/reductase pH titration of the hemin-Syn HO-1
complex was conducted with the 1M Tris solution from
pH 6.0–8.5 andwith the 1M NaOH solution from
pH 8.5–11.0 Determination of the pKa value was
per-formedby a curve fitting with the calculatedcurves of the
fraction of alkaline form vs pH for given pKavalues in the
Henderson–Hasselbalch equation
EPR spectroscopy
EPR spectra were measuredby a Bruker E500 spectrometer,
operating at 9.35–9.55 GHz, with an Oxfordliquidhelium
cryostat The15NO-boundheme–Syn HO-1 complex was
prepared by adding dithionite to the argon-saturated
solution of Syn HO-1 and15NaNO2in EPR tubes
Reaction of the heme–Syn HO-1 complex with ascorbic
acid and NADPH-cytochrome P450 reductase
Ascorbic acid(final concentration 10 mM) was added to an
optical cell containing heme–Syn HO-1 (8.4 lM) in 2 mL
of 0.1M potassium phosphate buffer (pH 7) at 25C
Spectral changes between 240 and900 nm were recorded
until the reaction was completedby monitoring the
maximum loss of the Soret band(A402) andthe formation
of biliverdin (A730) In the experiments using
NADPH-reductase, the 14 equivalent of NADPH was added to the
solution of heme–Syn HO (8.5 lM) containing 55 nM of
the reductase Soluble cytochrome P450 reductase is a
recombinant human enzyme, which lacks hydrophobic
region consisting of N-terminal 50 amino acidresidues
For construction of expression plasmid, we used the gene
giftedfrom F J Gonzalez of the National Institute of
Health (Bethesda, Maryland, USA), to be published
elsewhere
HPLC analysis of the heme–Syn HO-1 reaction products
For the ascorbate-assistedreaction, ascorbate (final
con-centration 100 mM) was added to a mixture of heme–Syn
HO-1 (20 lM) anddesferrioxamine (2 mM) in 2 mL of
0.1M Tris/HCl buffer (pH 7.4) For the
NADPH/reduc-tase supportedreaction, NADPH (final concentration,
0.5 mM) was added to the solution of heme–Syn HO-1
(20 lM) andreductase (55 nM) in 2 mL of 0.1MTris/HCl
buffer (pH 7.4) After 2 h, the reactants were hydrolyzed
with HCl to ensure the full conversion into free biliverdin
Each solution was subjectedto a Supelclean LC-18 solid
phase extraction column prewashedwith acetonitrile/water (1 : 9, v/v) andelutedwith acetonitrile/water (1 : 1, v/v) Lyophilization of the collectedfractions gave green pigment, which was then dissolved in 5% HCl/methanol andkept at 4C overnight The product was extracted with chloroform andanalyzedby a column of Capcell Pak C18 (SG 120, 4.6· 150 mm) pre-equilibratedwith degassed acetonitrile/water (3 : 2, v/v) at a flow rate of
1 mLÆmin)1 The biliverdin standards were eluted in the order a (21.5 min), d (23.0 min), b (24.7 min), and c (37.0 min)
Results
Expression and purification of Syn HO-1
We have successfully expresseda recombinant cyanobac-terial Syn HO-1 protein using a synthetic gene constructed from nine oligonucleotides Amino acid sequence of the Synechocystissp PCC 6803 heme oxygenase (Syn HO-1) has been comparedwith those of relatedmammalian and bacterial HOs in Fig 1 The harvestedBL21 cells carrying the Syn HO-1 expression vector, pMWSynHO1, were green, same as reportedfor the clonedprotein from Synechocystis sp PCC 6803 ORF sll1184 [36] andfor other mammalian andbacterial heme oxygenase proteins [23,25,28,42] Accumulation of a green pigment strongly suggests the production of biliverdin, so that the Syn HO-1 is supposedto be expressedas a catalytically active protein, which has been confirmedas describedlater Recombinant Syn HO-1 was obtainedas a soluble protein The purifiedprotein through a hydroxylapatite column showeda single bandat 27 kDa on the SDS/ PAGE (Fig 2, lane 2 and3) Three litters of cell-cultured solution ( 12 g of cell) yielded 30 mg of the purified protein
Formation of the heme–Syn HO-1 complex When an aliquot of the alkaline-hemin solution is added into the solution containing Syn HO-1, the resultant solution gives the optical absorption spectrum which has a Soret bandat 402 nm, that is apparently distinguishable from the Soret bandof free hemin Utilizing this difference, the stoichiometry of the heme binding reaction ratio to Syn HO-1 was examined The inset of Fig 3 illustrates obtainedtitration plots It clearly indicates that the Syn HO-1 protein (10 lM) is saturatedat a ratio of
1 : 1 hemin to protein, thereby establishing that Syn HO-1 binds equimolar hemin to form the hemin-enzyme complex, same as mammalian andbacterial HOs [14,23,25]
Spectroscopic characterization Figure 3 exhibits optical absorption spectra of the ferric-, ferrous-, oxy-, andCO-boundforms of heme–Syn HO-1 The Soret bands of the ferric and ferrous forms, having maxima at 402 and427 nm, respectively, are slightly blue-shiftedcomparedwith those of mammalian (404 and431 nm) andbacterial (404, 406 and434 nm) heme–
HO complexes By contrast, absorption maxima of the
Trang 4Soret andQ (a and b) bands of the oxy form and the
CO-bound form do not show specific differences from
those of mammalian or bacterial HOs The small band
at 634 nm seen in the spectrum of the CO-boundform is
thought to come from the verdoheme-CO complex
generatedby the reaction of ferrous Syn HO-1 with
oxygen contaminated Compared with the visible
spec-trum of the ferric form of heme-rat HO-1, the specspec-trum
of ferric heme–Syn HO-1 shows distinctively stronger CT
bands (498 and 630 nm) and weaker Q-bands (575 and
535 nm) in pH 7.0 potassium phosphate solutions (data
not shown) Optical absorption data of the heme-HO
complexes with different taxonomical origins are
sum-marizedin Table 1 The absorption coefficient at 402 nm
for the ferric heme–Syn HO-1 complex is determined to
be 128 mM )1Æcm)1 by the pyridine hemochrome method
[23], which is the smallest among the values reportedfor
the mammalian andbacterial HOs
EPR of the heme–Syn HO-1 complex at pH 7 exhibits
an axially symmetric high-spin spectrum originatedfrom
the ferric ion in approximately tetragonal ligandfield
(g¼ 6 and g ¼ 2, upper spectrum in Fig 4) The axially
symmetric spectra and g-values are similar to the ferric
high-spin state of mammalian andbacterial heme-HO
complexes [14,24]
pH dependence of the heme–Syn HO-1 complex The optical absorption spectrum of the ferric heme–Syn HO-1 complex varies depending on pH As pH increases from
6 to 10, the Soret peak shifts from 402 to 418 nm gradually andthe peaks at 498 and630 nm in the visible region are alternatedwith the peaks at 537 and575 nm, as shown in Fig 5, panel A The expanded visible region spectrum shows that the CT bands derived from the high-spin species remain
at pH 10 This pH-dependent alteration is reversible between
pH 6 and10 The pKavalue of this acid–base transition is estimatedbasedon the increase of absorbance at 418 nm as
pH increases Curve fitting of the fraction of the alkaline form
to the calculatedvalues using the Henderson–Hasselbalch equation yielded the best-fitted result with pKa¼ 8.9 (Fig 5B)
EPR spectra of the heme–Syn HO-1 complex also show the pH dependency As the pH increases from 7 to 10, intensity of the axially symmetric spectrum is reduced and instead, the low-spin signals newly appear This change is also reversible Apparently two types of low-spin signals are observed(Fig 4, the lower spectrum) The major species (denoted as A) with g1¼ 2.78, g2¼ 2.14, and g3¼ 1.74 shows larger anisotropy than the minor species (denoted as B) with g ¼ 2.68, g ¼ 2.20, and g ¼ 1.80 Two kinds of
Fig 1 Amino acid sequence alignment of Synechocystis, mammalian, and bacterial heme oxygenases The plus sign indicates similar, while the asterisk indicates identical amino acid residues Nostocho, cyanobacterial Nostoc sp PCC 7120 [37], Hmu O, C diphtheriae [23], andHem O,
N meningitidis A 2855 [25].
Trang 5alkaline forms have been also observedfor the heme
complexes of bacterial HO (Hmu O), while the single
alkaline species detected for the heme–rat HO-1 complex
[14,24] The g-values of the alkaline forms of heme-HO complexes are presentedin Table 2
EPR of the nitrosyl heme–Syn HO-1 complex The EPR spectrum of the15NO complex of ferrous heme– Syn HO-1 is representedin Fig 6 The rhombic spectrum typical of a six-coordinated nitrosyl heme complex exhibits a triplet of doublet splitting at the g2component, that comes from the interaction between nuclear spins of14N (I¼ 1) and
15N (I¼ ½) andan electron spin, respectively By compa-rison of the spectra of known nitrosyl heme–HO complexes [14,24,41], the doublet component with a hyperfine coupling constant of 31.1 gauss is reasonably assignedto the 15N nucleus of 15NO on the distal site of heme Similarly, the triplet component with the hyperfine splitting of 7.1 gauss is attributable to the14N nuclei of the axial ligand trans to the nitrosyl ligand This firmly establishes that the proximal ligandof the heme–Syn HO-1 complex is a nitrogenous base The close value of hyperfine coupling constant of the proximal 14N nuclei to those of establishedhistidyl axial ligandin heme–HOs strongly suggests that the nitrogenous proximal ligandof the heme–Syn HO-1 complex also has histidyl origin in the proximal site (Table 3)
Catalytic turnover of the heme–Syn HO-1 complex The time course spectra of the heme-conversion reaction in the presence of ascorbate are depicted in Fig 7, panel A Addition of ascorbate to the heme–Syn HO-1 complex commences the reaction, which is monitoredby the steady decrease of the Soret and 498 nm bands and the shift of the bandat 630 nm to the longer-wavelength direction At the same time, a broadbandwith the maximum at approxi-mately 690 nm appears andincreases with time The newly
Fig 2 SDS/PAGE of purified Syn HO-1 protein Lane 1, molecular
mass markers; lane 2, 2.4 lg of protein andlane 3, 24 lg of protein.
Fig 3 Optical absorption spectra of the heme–Syn HO-1 complexes The spectra are the ferric (–-–), ferrous (– –), ferrous-CO (––), andoxy (- - - -) complexes, respectively Inset, titration of Syn HO-1 (10 l M ) with hemin detected by the absorbance increase at 402 nm The background absorbance shown in without Syn HO-1 comes from added free hemin [Heme-Syn HO-1] ¼ 9.7 l M , in 0.1 M potassium phosphate (pH ¼ 7.0) The ferrous form was made by the addition of dithionite (150 l M ) under anaerobic condition and the spectrum was recorded after 15 min of incubation The oxy form was produced by the addition of air in the ferrous complex produced with NADPH (120 l M ) andthe spectrum was taken after 5 min of incubation.
Trang 6Table 1 Optical absorption data for the heme-heme oxygenase complexes with different taxonomical sources Syn HO-1, cyanobacterial Synechocystis
sp PCC 6803; rat HO-1, taken from refs [9] and[14]; Pig A, Pseudomonas aeruginosa, taken from ref [28]; Hmu O, Corynebacterium diphtheriae, taken from ref [23]; Hem O, Neisseriae meningitidis, taken from ref [25].
Ferric form
k max (Soret) (e (m M )1 )) 402 (128) 404 (140) 406 (129) 404 (150) 406 (179)
k max (visible) 630, 498 631, 500 632 630, 500
Ferrous deoxy form
Oxy form
k max (a, b) 574, 537 575, 539 570, 540 570, 540
CO form
k max (a, b) 566, 536 568, 535 567, 537 568, 538 568, 538 Alkaline form
k max (a, b) 575, 537 575, 540
Fig 4 EPR spectra of ferric heme–Syn HO-1 complexes in neutral and
basic solutions Measuring conditions: T ¼ 8 K, microwave frequency
9.55 GHz, field modulation 100 kHz, modulation amplitude 10 G,
microwave power 0.5 mW In the pH 10.6 spectrum, the low-spin
region is expanded fivefold The sample at pH ¼ 7.0, 100 lL of heme–
Syn HO-1 (400 l M ) in 0.1 M potassium phosphate; the sample at
pH ¼ 10.6, 120 lL of heme–Syn HO-1 (300 l M ) in 1 m M potassium
phosphate whose pH was adjusted with NaOH (1 ).
Fig 5 Determination of pK a for the heme–Syn HO-1 complex (A) Absorption difference spectra of the alkaline solutions, [heme-Syn HO-1] ¼ 7.9 l M , referring to the spectrum at pH 6.0 The visible-region is shown in the enlargedabsorption spectra (B) The fraction
of the alkaline form at given pH calculatedfor each value of pK a , 8.7 (–-–), 8.9 (––), 9.1(- - -), and9.3 (– –), basedon the Henderson–Has-selbalch equation The heavy dots are the fractions estimated from the experimentally obtainedabsorbance at 418 nm that is normalized against the value at pH 10.2.
Trang 7appearedvisible bandis suggestive of verdoheme or
biliverdin formation, or of their admixture Then, after
2 h, the reaction mixture was separately analyzedby HPLC,
confirming that the final product was biliverdin IXa(data
not shown) To examine the formation of the verdoheme
intermediate, this reaction was performed under the limited
oxygen condition As exhibited in Panel B in Fig 7, the
spectrum recorded after 2 h (the dashed-and-dotted line)
has peaks at 534, 637, and686 nm other than the Soret
peak The solid-line spectrum recorded after 4 h shows new
peaks at the Soret region (416 nm) andat 566 nm and
indicates the 686 nm band further increased The combined
double peaks at 600–750 nm are commonly observed in the
heme degradation by mammalian HO, which are markers
of verdoheme formation Peaks at 534 and 686 nm are
attributable to the ferrous–verdoheme complex and the
peak at 637 nm to the CO-bound verdoheme complex due
to the trapping of CO concomitantly produced The peaks
at 416 and566 nm are attributable to the CO-boundheme– Syn HO-1 complex (Table 1) Addition of CO transforms the solid-line spectrum into the broken-line spectrum, in which the peak at 637 nm is much enhancedandnew peaks
Table 2 g-Values and g-anisotropy of alkaline forms of heme–heme oxygenase complexes Data for Hmu O andrat HO-1 are taken from refs [24] and[14], respectively g-Anisotropy is defined as Dg ¼ g 1 ) g 3
Protein Species
Fig 6 EPR spectrum of the15N-nitrosyl heme–Syn HO-1 complex.
Measuring conditions: T ¼ 30 K, microwave frequency 9.35 GHz,
fieldmodulation 100 kHz, microwave power 0.2 mW,
fieldmodula-tion amplitude 2G [heme–Syn HO-1] ¼ 430 l M , in 0.1 M potassium
phosphate (pH ¼7.0).
Table 3 EPR parameters of the ferrous15N-nitrosyl heme–heme
oxyg-enase complexes Data for Hmu O andrat HO-1 are taken from
refer-ence [24] and[14], respectively g-Anisotropy is defined as Dg ¼ g 3 – g 1
Protein Syn HO-1 Hmu O rat HO-1
A( 14 N-His) 7.1 G a 6.8 7.4
a
Value of A(14N-L).
Fig 7 Heme conversion by Syn HO-1 initiated by the addition of ascorbate (A) Spectra were recorded at the indicated time after the addition of ascorbate solution (10 m M ) to the heme–Syn HO-1 solu-tion (8.4 l M in 0.1 M potassium phosphate at pH 7.0) The Soret and
498 nm bands decrease with time, while the band at 680 nm appears andincreases The spectrum recordedafter 4 h indicates the formation
of mixture: free biliverdin, verdoheme, and verdoheme-CO (B) The reaction was conducted under argon atmosphere Spectra were recorded 2 h after the addition of ascorbate (–-–), 4 h after (––), and after the replacement of Ar in the space of the sealedcell with CO (– –).
Trang 8appears at 350 and541 nm while the peaks at 534 and
686 nm almost disappear The peaks at 350, 404, 541,
637 nm are very close to those reportedfor the CO bound
verdoheme–rat HO-1 complex [16] Accordingly, it can be
concluded that verdoheme is produced during the course of
heme degradation by Syn HO-1, accompanied by releasing
CO simultaneously The overall rate of the heme
degrada-tion by Syn HO-1 with ascorbate is roughly estimatedto be
one-fifth of that by rat HO-1 when the same amount of
enzyme andascorbate are used
Time course of the heme catabolic reaction by Syn HO-1
in the presence of NADPH cytochrome P450 reductase was
also examined As illustrated in Fig 8, the obtained spectra
are clearly discriminated from those of the
ascorbate-supported reaction Although addition of 14 equivalent of
NADPH to heme–Syn HO-1 in the presence of reductase
initiates the reaction, the reaction is almost at a standstill
from 6 to 15 min after addition of NADPH Shift of the
Soret maximum to 410 nm andappearance of the 534 and
573 nm bands in the visible region indicate that the oxy
complex of heme–Syn HO-1 is produced within 3 min and
accumulated Decomposition of the oxy complex appears
much slower than its formation anddoes not endeven after
210 min, exhibiting the bands of the remaining oxy
complex The 340 nm-bandof NADPH decreases in
proportion to the decrease of Soret band at 410 nm and
to the increases of broadbandspreading 600–700 nm The
latter bandwas confirmedto belong to biliverdin IXaby the
HPLC analysis (data not shown)
Discussion
Overall structure and heme binding
The primary structure of Syn HO-1 has very high identity
(38%) andsimilarity (67%) to that of human HO-1 [26]
Such resemblance is higher than the 57.4% homology of
cyanobacterial Nostoc sp PCC7120 [37] Other prokaryotic
HOs bear less resemblance to Syn HO-1: Hmu O, 31%
identity and 59% similarity; Hem O, 19% identity and 42%
similarity Then, the tertiary structure of Syn HO-1 protein
is expectedto resemble that of mammalian HO-1 Overall holdings of bacterial HO (Hem O) [27] and mammalian HO-1 [17,18] are known to be similar though their primary structure are less similar than that between Syn HO-1 and mammalian HO-1 Syn HO-1 binds equimolar hemin to form the stable heme–Syn HO-1 complex EPR of the nitrosyl heme–Syn HO-1 complex has establishedthat the proximal ligandof the heme–enzyme complex is a nitro-genous base The alignedsequence depictedin Fig 1 designates His17 as a potential candidate for the proximal ligandof heme–Syn HO-1, that corresponds to the estab-lishedproximal ligandof His25 in mammalian HO-1 and His20 in bacterial Hmu O [15,43]
Axial coordination structure of heme The cryogenic EPR has revealedthat the resting state
of heme–Syn HO-1 is in the axially symmetric ferric high-spin state at pH 7.0 At alkaline pH values, the high-high-spin state is partially convertedinto the low spin state This pH-dependent spin-state conversion is also observed at room temperatures (Fig 5) The alkaline forms of heme– Syn HO-1 have g-values that are close to those of the alkaline forms of heme–rat HO-1 andHmu O (Table 1), which are established to be the hydroxide-bound form generatedby deprotonation of the axially ligatedwater Therefore, the alkaline forms of heme–Syn HO-1 are also thought to be the hydroxide-bound forms, which are produced by the coordination of hydroxide originated from dissociation of the heme-bound or nearby water, correlating to the change of protonic equilibria of protic residues in the distal heme pocket As illustrated in Fig 5, the transition to the alkaline form is not completedeven at
pH 10
The determined pKavalue of 8.9 for the heme–Syn HO-1 complex is higher than that of rat heme–HO-1 andclose to that of bacterial heme–Hmu O (Table 1) Hence, it follows that the proton dissociation of distal water in Syn HO-1 is less favorable than that in rat HO-1 but is similar to that in Hmu O The amino acid residues constructing the distal helix in rat HO-1 (Leu129 to Met155) are almost all
Fig 8 Heme conversion by Syn HO-1 initi-ated by the addition of NADPH Spectra were recordedat the indicatedtime after the addi-tion of NADPH (final concentraaddi-tion 120 l M )
to the solution of heme–Syn HO-1 (8.5 l M ,
in 0.1 M potassium phosphate at pH 7.0) andreductase (55 n M ).
Trang 9conservedboth in Syn HO-1 andHmu O though the entire
primary structure of Syn HO-1 is much closer to that of rat
HO-1 than to that of Hmu O The crystal structure of the
hydroxide-bound heme–rat HO-1 showed that Gly143N is
located within hydrogen bonding distance (2.60 A˚) with the
heme-coordinating hydroxide [18] Recently, we have found
that the alanine mutation of residues on the distal helix of
rat HO-1 alters the pKavalue in order of 8.8 (S142A) > 8.6
(D140A) > 8.5 (R136A) > 8.0 (T135A) [44] It appears
that the closer is the mutatedresidue to G143, the higher is
the pKavalue, independent of the nature of the displaced
amino acid One possible explanation for the high pKavalue
of heme–Syn HO-1, andof Hmu O, is that the distal
ionizable group(s) that is responsible for the deprotonation
of the distal water is more distant from the heme axial site
than in rat HO-1
Multiple alkaline forms
The major component (species A) of the two alkaline
forms of heme–Syn HO-1 is present at an approximately
threefoldlarger quantity than the minor component
(species B) (Fig 4, Table 2) The bacterial heme-Hmu O
complex also forms two low-spin species, of which one is
far more predominant than the other (species B¢ andA¢ in
Table 2, respectively) [24] For the low-spin ferric heme
complexes in the groundelectronic states with dp spin
orbitals, the small but definite differences in the
coordina-tion circumstances are discriminated by g-values and
g-anisotropy [45] Species B from Syn HO-1, species B¢
from Hmu O, andthe species from rat HO-1 which has
only one alkaline form have very similar g-values and
g-anisotropy In these species, then, the distal hydroxide
protons are possibly fixedto the same direction relative to
the heme plain In rat HO-1, Gly143N resides in the
d-meso direction of heme, where the heme-coordinated
hydroxide least destabilizes dp orbitals of the heme iron,
resulting in smaller g-anisotropy Consistently,
g-aniso-tropy of these species is smaller than that of Species A (Syn
HO-1) andSpecies A¢ (Hmu O) In the latter species with
the larger g-anisotropy, the hydroxyl ligand might more
lean to the direction of the counter pyrrole Na–Naaxis,
where the dp orbitals are the most destabilized
Coordination structure of the nitrosyl heme complex
There are considerable numbers of studies aimed at
characterizing the coordination structure of the nitrosyl
heme complexes andheme proteins The rhombic type of
spectra obtainedfor the ferrous nitrosyl heme–HO
com-plexes is classifiedto type I andsupposedto contain a
bent Fe–N–O bondwith an angle of 120–150 [46] The
nitrosyl heme complex of Syn HO-1 has larger nitrogen
hyperfine coupling constant of the nitrosyl-nitrogen
nuc-leus, AN (15NO), andthe smaller one of the
proximal-ligandnitrogen nucleus, AN(14N-L), comparedwith those
of the nitrosyl heme–rat HO-1 complex (Table 3) In
addition, each of the g-values of the nitrosyl heme–Syn
HO-1 complex is smaller than that of the rat HO-1 or
Hmu O complexes The hyperfine interaction in the
nitrosyl heme complex arises from an unpairedelectron
that originally occupies the 2 pp* orbital of nitrogen oxide
andis delocalizedinto the metal d-orbitals through r- and p-interaction The larger AN(15NO) andthe smaller AN (14N-L) mean that the r-delocalization from the NO p* orbital to the iron d orbitals is reduced This phenomena can be interpretedon assumption that the Fe–N(O) distance is elongated due to the shortening of Fe–L bond Recent analysis of g-tensors of the six-coordinated nitrosyl iron(II) porphyrins with the imidazole ligand by density function theory describes that g-tensors of the type I complexes are sensitive to the Fe–N(Im) bondlength as well as to the orientation of the NO ligand(but not to the orientation of the imidazole ligand) [47] Changes in the Fe–N(Im) bondlength less than 0.5 A˚ is reflectedin deviations of the g-component up to 0.02, where the shorter are the distance, the smaller are the g-tensor components (g1, g2, and g3) Such small variation in the bondlengths is detectable only by ultra-high resolution X-ray crystallography [48] According to this theoretical estimation, our observation that all of the g1, g2, and g3 components of nitrosyl heme–Syn HO-1 are smaller than those of the rat HO-1 complex (Table 3) implies that the Fe–N(L) bondlength in nitrosylheme–Syn HO-1 is shorter than that in nitrosyl heme–rat HO-1, in accordance with the aforementioned assumption deduced from the consid-eration on AN The rhombic g-anisotropy thus reflects the difference of the heme pocket structures that perturb the coordination structure of the nitrosyl heme complexes In this meaning, the structure of either proximal or distal sites
of the heme pocket of heme–Syn HO-1 differs from that of heme–rat HO-1, andrather resembles that of bacterial heme–Hmu O
Protein modification of coordination geometry
in the heme–Syn HO-1 complex Among the heme complexes of Syn HO-1 andother wild type HOs reportedso far, the Syn HO-1 complex has distinctively small absorption maximum of the Soret bandwith a small absorption coefficient (Table 1) On the other hand, relative intensities of the 498 and 630 nm bands compared with those of the 575 and 535 nm bands in the visible region spectrum are larger than those observedin the spectrum of heme–rat HO-1 The former bands, referred as CT bands, are commonly distinctive in high-spin derivatives of ferric hemoproteins, while the latter bands (a- and b-bands) are usually weak in the high-spin derivatives but are distinctly observed in the low-spin derivatives [49] As the position and the intensity
of these bands are dependent not only on the spin state
or thermal spin-state equilibria but also on the nature of the sixth ligandandthe type of apoprotein, we couldnot attribute these features to one of the possible causes at present However, it can be mentionedthat the protein modification of coordination geometry in the heme–Syn HO-1 complex apparently differs from that in the known heme–HO complexes
Heme catabolism by Syn HO-1 The heme boundto Syn HO-1 is transformedinto biliverdin IXa regioselectively in the presence of oxygen andelectrons In the course of reaction, verdoheme
Trang 10intermediate is producedaccompaniedby CO release.
Therefore, the mechanism of heme conversion by Syn
HO-1 is foundto be fundamentally the same as that by
mammalian HOs, i.e heme is convertedto biliverdin
IXa, carbon monoxide, and iron through the three-step
reaction with the intermediates of a-meso-hydroxyheme
andverdoheme [13] In the heme–Syn HO-1 reaction,
the final product is free biliverdin even under the
ascorbate-supported reaction, differing from the product,
ferric-biliverdin IXa, in the ascorbate-supportedheme
catabolism by rat HO-1 andsimilar to that in the heme
catabolism of bacterial Hmu O under ascorbate [23]
Though both NADPH cytochrome P450 reductase and
ascorbate can support this reaction, the overall rate of heme
degradation is considerably slow in both systems compared
with that by mammalian HO, even slower than that by
bacterial Hmu O [23] Notably, the heme conversion with
NADPH cytochrome P450 reductase is retarded at the
oxy-complex, which has been observedfor the heme catabolism
neither by mammalian nor by bacterial HOs The collation of
time-course spectra of Panel A in Fig 7 with those of Fig 8
makes us realize that reduction of the ferric heme–Syn HO-1
complex followedby the oxy complex formation is
unfa-vorable in the ascorbate-supportedreaction as evidencedby
no accumulation of the oxy form By contrast, conversion of
the oxy complex (to the hydroxyheme complex) is extremely
slow in the NADPH-reductase supported reaction although
reduction of the ferric heme is sufficiently fast The slow
reduction rate of the ferric heme–Syn HO-1 complex by
ascorbate seems to imply the lower oxidation-reduction
potential of the heme iron in the Syn HO-1 complex
comparedwith that of the one in the rat HO-1 complex, that
might limit the overall reaction rate As for the retardation of
heme conversion under NADPH/reductase, the electron
transfer from NADPH cytochrome P450 reductase to the
oxy complex appears to be quite inefficient in the heme–Syn
HO-1 complex We have observedthat the presence of 30
equivalents of ascorbate together with reductase in the
reaction mixture avoids the retardation (data not shown)
This makes us speculate that the long-range electron
tunneling pathway through the Syn HO-1 protein, from
the binding site of reductase to the heme edge, is not the right
path The cyanobacterial cell must provide an effective and
successive electron-transfer system for Syn HO-1 Searches
for the inherent reducing system that works in the
physio-logical Syn HO-1 reaction are currently underway
Conclusive remarks
An effective bacterial expression system of cyanobacterial
Synechocystisheme oxygenase protein, was constructedfor
the first time andthe highly purifiedprotein, Syn HO-1, was
obtainedsuccessfully Syn HO-1 binds equimolar hemin to
form the heme–Syn HO-1 complex The resultant complex is
convertedto biliverdin IXaby the reaction with oxygen in the
presence of ascorbate or NADPH cytochrome P450
reduc-tase, forming detectable intermediates, the oxy-heme and
verdoheme complexes However, the overall reaction rate of
heme conversion is relatively slow Characteristics of the
heme–Syn HO-1 complex discriminate from those of
the other heme–HO complexes The resting state of the
heme–enzyme complex, which has a nitrogenous proximal
ligand, is in the ferric high-spin state The complex exhibits an acid–base transition with the pKavalue of 8.9, which is larger than that of the heme–rat HO-1 complex, suggesting that the proton dissociation of the distal water is less efficient The heme–enzyme complex generates two kinds of the alkaline form The nitrosyl heme–Syn HO-1 complex of type I is generated, which has a relatively large nitrosyl AN, small proximal ligandAN, and small g components with large anisotropy These characters strongly suggests that the heme pocket structure is different from that of mammalian HO andsomewhat resembles that of bacterial Hmu O, in spite of very high conservation of the amino acidresidues constitu-ting the heme pocket among these HOs
Acknowledgements
This work was supportedin part by a grant-in-aidfor Scientific Research 12680625 and14580641 (to T.Y.) from the Ministry of Education, Science, Sports and Culture of Japan.
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