The reactivity of a-hydroxyhaem and verdohaem boundto haem oxygenase-1 to dioxygen and sodium dithionite Hiroshi Sakamoto1, Yoshiaki Omata1, Shunsuke Hayashi1, Saori Harada1, Graham Palm
Trang 1The reactivity of a-hydroxyhaem and verdohaem bound
to haem oxygenase-1 to dioxygen and sodium dithionite
Hiroshi Sakamoto1, Yoshiaki Omata1, Shunsuke Hayashi1, Saori Harada1, Graham Palmer2
and Masato Noguchi1
1
Department of Medical Biochemistry, Kurume University School of Medicine, Japan;2Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USA
Recently we have shown that ferric a-hydroxyhaem bound
to haem oxygenase-1 can be converted to ferrous verdohaem
by approximately an equimolar amount of O2in the absence
of exogenous electrons [Sakamoto,H.,Omata,Y.,Palmer,
G.,and Noguchi,M (1999) J Biol Chem 274,18196–
18200] Contrary to those results,other studies have claimed
that the conversion requires both O2and an electron More
recently,Migita et al have reported that the major reaction
product of ferric a-hydroxyhaem with O2 is a ferric
porphyrin cation radical that can be converted to ferrous
a-hydroxyhaem with sodium dithionite [Migita,C T.,Fujii,
H.,Matera,K M.,Takahashi,S.,Zhou,H.,and Yoshida,
T (1999) Biochim Biophys Acta 1432,203–213] To clarify
the reason(s) for the discrepancy,we compared the reactions;
i.e a-hydroxyhaem to verdohaem and verdohaem to
bili-verdin,under various conditions as well as according to the
procedures of Migita We find that complex formation of a-hydroxyhaem with haem oxygenase may be small and a substantial amount of free a-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead
to a misinterpretation of the experimental results We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2 Finally,we find that dithionite seems to be inap-propriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase Keywords: haem oxygenase; a-hydroxyhaem; verdohaem; sodium dithionite; NADPH-cytochrome P450 reductase
Microsomal haem oxygenase (HO,EC 1.14.99.3) catalyses
the O2-dependent physiological degradation of haem using
reducing equivalents from NADPH-cytochrome P450
reductase,and produces biologically active molecules:
biliverdin,CO,and iron,which display both beneficial
and deleterious effects,depending on the circumstances
[1,2] Two isoforms of HO, HO-1 and HO-2, exist and are
different gene products HO-1 is highly expressed in the
spleen and liver,and is inducible not only by haem itself but
also by a variety of agents causing oxidative stress HO-1 is
also known as heat shock protein 32 and may protect cells
from oxidative damage through antioxidant activity of
bilirubin produced through the subsequent reduction of
biliverdin by biliverdin reductase HO-2,on the other hand,
is constitutively expressed in the brain,testes and vascular
systems Evidence accumulated in the past decade suggests
that the principal role of HO-2 is the production of CO as a
signal mediator (reviewed in [2–5]) However,this role of
CO as a signaling agent remains controversial [6]
The HO reaction consists of three sequential oxidation steps [7,8] Haem bound to the enzyme is first hydroxylated
at the a-meso-carbon,yielding a-hydroxyhaem The second step is the conversion of a-hydroxyhaem to verdohaem with the concomitant release of the a-meso-carbon as CO [9] Finally,the oxygen bridge of verdohaem is cleaved to produce iron and biliverdin Recently,the crystal structures
of both human [10] and rat [11] HO-1 in complex with haem have been determined and provide important information on the selective a-meso-hydroxylation The haem is sandwiched between two helices: the proximal helix bears His-25 as the ligand to the haem iron and the distal helix lies above the b-, c-,and d-meso-carbon atoms
of the porphyrin macrocycle These structural features probably direct the a-meso-accessibility of the ferric hydroperoxide that has been demonstrated as the porphy-rin-hydroxylating species [12–16] Moreover,no polar side chains at the distal side are in direct contact with the iron-bound ligand,i.e water or hydroxide,but only the backbone atoms of Gly139 and Gly143 are close enough
to hydrogen-bond to the distal ligand We have proposed that,if the amide nitrogen of Gly143 is the proton donor hydrogen-bonded to the iron-bound oxygen of the ferric hydroperoxide,it can stabilize the negatively charged oxygen and facilitate the electrophilic addition of the terminal oxygen to the a-methene bridge [11] The recent findings by Liu et al [17] that Gly143 mutants,all of which have lost the water ligand,have no HO activity support this proposal
Correspondence toM Noguchi,Department of Medical Biochemistry,
Kurume University School of Medicine,67 Asahi-machi,
Kurume 830-0011,Japan.
Fax: + 81 942 31 4377,Tel + 81 942 31 7544,
E-mail: mnoguchi@med.kurume-u.ac.jp
Abbreviations: HO,haem oxygenase; rHO-1,truncated rat haem
oxygenase-1; OEPO,octaethyloxophlorin;
OEOP,octa-ethyloxoporphyrin; OEB,octaethylbilindione.
Enzyme: microsomal haem oxygenase (HO,EC 1.14.99.3).
(Received 30 May 2002,revised 20 August 2002,
accepted 5 September 2002)
Trang 2Although major advances have been made in
under-standing the nature of the first hydroxylation of haem,the
mechanisms for the subsequent steps,especially the
conver-sion of a-hydroxyhaem to verdohaem,are still unclear On
the basis of spectroscopic studies of a-hydroxyhaem in
complex with chemical ligands [18–21] and with proteins
such as apomyoglobin [22] or HO [23,24], the ferric
a-hydroxyhaem is deprotonated and assumes the resonance
structure (Scheme 1),a high-spin ferric enolate (1a)-keto
(1b) tautomer and a low-spin ferrous p neutral radical (1c)
(reviewed in [15,25]) It is widely accepted that direct binding
of O2 to 1c at the haem edge triggers CO extrusion and
verdohaem formation However,the requirement for
redu-cing equivalents as well as the oxidation state of the
resultant verdohaem remains controversial
Thus,Matera et al [26],and subsequently Migita et al
[27],have reported that this conversion requires both O2and
an exogenous electron to yield the ferrous verdohaem
(Scheme 2A) They chemically synthesized
a-hydroxyhae-min and prepared its complex with a soluble rat HO-1
protein According to their results,most ( 70%) of ferric
a-hydroxyhaem bound to the enzyme was converted to a
ferric porphyrin cation radical upon exposure to O2,along
with generation of the ferrous verdohaem as a minor
product (less than 30%) The major product,the ESR-silent
porphyrin cation radical,can be returned to the ferrous
a-hydroxyhaem by addition of sodium dithionite However,
it was not clearly explained why partial formation of ferrous
verdohaem took place during the reaction of
a-hydroxy-haem with O2without the addition of an electron
By contrast,Liu et al [23] reported that in the anaerobic
reaction of the ferric haem-human HO-1 complex with 1
molar equivalent of H2O2,ferric a-hydroxyhaem was
produced,which upon exposure to air was converted to
ferric verdohaem They also directly obtained ferric
verdo-haem in the reaction of the verdo-haem-HO complex with H2O2in
air They concluded that the reaction only needs O2and that
an electron is required for conversion of ferric verdohaem to
the ferrous state (Scheme 2B) We have reported [24] that
under strictly anaerobic conditions employing synthetic
a-hydroxyhaem bound to rat HO-1 (rHO-1),this
conver-sion is achieved with an almost equimolar amount of O2in
the absence of reducing equivalents and that the resultant
verdohaem appeared to be in the ferrous state (Scheme 2C)
In contrast to the previous studies,the pathway we have
proposed does not require any reducing equivalents,and
instead postulates generation of an oxidizing equivalent
with the formal stoichiometry of (1/2) H2O2[28]
In the present study,we have attempted to clarify the
reason(s) for these discrepancies At first,we carefully
compared the absorption spectra of our ferric,ferrous and
CO-ferrous a-hydroxyhaem-HO complexes with those prepared by Matera et al [26] Next,we compared the conversion of a-hydroxyhaem to verdohaem and of verdo-haem to biliverdin under various conditions including those used by Migita et al [27]
E X P E R I M E N T A L P R O C E D U R E S
Materials
A truncated version of rHO-1 lacking the 22-amino acid C-terminal hydrophobic stretch was expressed in Escheri-chia coliand purified as described [29] The catalytic activity
of this rHO-1 was comparable to that of the wild type NADPH-cytochrome P450 reductase was purified from rat liver as previously described [30] Gases of high purity, argon (99.999%),O2(99.99%) and N2(99.99%) were used (Iwatani,Fukuoka,Japan) All other chemicals used were
of analytical grade
Formation of a-hydroxyhaem-rHO-1 complex Unless otherwise stated,the following manipulations were carried out anaerobically in a glove box filled with N2 at room temperature (25C) a-Hydroxyhaemin was synthes-ized as described [24] A concentrated anaerobic rHO-1
Scheme 1 The resonance structure of a-hydroxyhaem.
Scheme 2 Three reaction formulae proposed for the conversion of a-hydroxyhaem to verdohaem (A),(B) and (C) are formulated after Matera et al [26,27], Liu et al [23] and Sakamoto et al [24], respectively The porphyrin substituents are omitted for clarity.
Trang 3solution ( 0.5 mM) in 0.1Mpotassium phosphate buffer
(pH 7.4) was prepared in a Reacti-vial sealed with a
Mininert valve (Pierce,Rockford,IL,USA) by gentle
stirring of the solution for 3 h at 5C under flowing argon
gas moistened by passing through anaerobic water
Recon-stitution of the ferric a-hydroxyhaem-rHO-1 complex was
carried out as follows; a portion of the rHO-1 solution was
transferred to a custom-made anaerobic titrator [24],that
contained 1 mL of the buffer Then a-hydroxyhaemin
dissolved in a small amount of 0.1MNaOH saturated with
argon was added,with the molar ratio to rHO-1 being
0.9 The final concentration of rHO-1 was 70 lM The
amounts of a-hydroxyhaem added were determined as the
pyridine haemochrome of a-hydroxyhaemin dimethyl ester
using e422 nm¼ 153.6 mM )1Æcm)1[22] By this procedure,
i.e addition of the alkaline a-hydroxyhaem solution into the
rHO-1 protein in the potassium phosphate buffer (pH 7.4),
it was found that complex formation was easily
accom-plished (within 3 min) Hence,we adopted this procedure as
the routine method for preparing the
a-hydroxyhaem-rHO-1 complex
Formation of ferrous verdohaem-rHO-1 complex
The ferrous verdohaem-rHO-1 complex was prepared in
two different ways In the first method,the complex was
prepared from the a-hydroxyhaem-rHO-1 complex by
addition of an approximately equimolar amount of O2
[24] In the second method,the complex was prepared by
reconstitution from synthetic a-verdohaem and rHO-1 [31]
In each method,the concentration of verdohaem was
determined as pyridine verdohaemochrome using
e680 nm¼ 28.5 mM )1Æcm)1 in pyridine-H2O (1 : 2 v/v) or
e685 nm¼ 31.2 mM )1Æcm)1in pyridine-CHCl3(3 : 7 v/v) as
reported [31]
Titrations
Titration experiments were performed as previously
des-cribed [24] Sodium dithionite or NADPH was dissolved
into the potassium phosphate buffer (pH 7.4) in a
custom-made vessel that was based on a flask previously reported
[32] The concentration of sodium dithionite was determined
by reductive titration of oxidized lumiflavin 3-acetate (e446 nm¼ 11.3 mM )1Æcm)1) with the dithionite solution, assuming that 1 mol of dithionite reduces 1 mol of lumi-flavin 3-acetate by 2-electron reduction [32,33] The con-centration of NADPH was determined using e339 nm¼ 6.2 mM )1Æcm)1 The O2-saturated buffer (1.25 mM as O2) was prepared by bubbling O2 into the buffer solution for
3 h Each of these titrants (dithionite,NADPH and O2) was loaded into a separate syringe equipped with a needle and a screw-threaded plunger Exchange of the syringe containing different titrant was carried out under continu-ous argon flow through the top inlet of the titrator Exposure of a sample in the titrator to atmospheric O2was attained by bubbling air through a long needle into the solution phase
Instruments Optical absorption spectra were recorded on a Varian Cary
50 UV-visible spectrophotometer in a glove box filled with
N2at room temperature (25C) X-band ESR spectra were recorded at 5 K using a JEOL ESR spectrometer (JES FE3X) equipped with a JEOL liquid helium cryostat (ES-LTR5X) The instrumental conditions were: modulation frequency,100 kHz; modulation amplitude,1 millitesla; microwave frequency,9.88 GHz; and microwave power,
10 lW The microwave frequency was calibrated with a microwave frequency counter (Advantest) and the magnetic field strength was determined with a JEOL NMR counter (JEOL ES-OC1)
R E S U L T S A N D D I S C U S S I O N
Formation of a-hydroxyhaem-rHO-1 complex The various forms of a-hydroxyhaem bound to HO-1 were first investigated by Matera et al [26]; the optical spectrum
of the ferric form exhibited a rather broad Soret band, whereas the Soret bands of the ferrous and ferrous-CO forms were narrow (Fig 1B) The ratios of the intensities of the Soret maximum of the ferric (spectrum a),and ferrous
Fig 1 Comparison of the absorption spectra of several a-hydroxyhaem-HO-1 complexes (A) The ferric (spectrum a,–),ferrous (spectrum b,ÆÆÆ),and CO-ferrous a-hydroxyhaem-HO-1 complex (spectrum c,- - -) prepared in this study The final concentrations of rHO-1 and a-hydroxyhaemin were
69 and 61 l M ,respectively See Experimental procedure for details (B) The ferric (spectrum a,–),ferrous (spectrum b, ÆÆÆ),and CO-ferrous a-hydroxyhaem-HO-1 complex (spectrum c,- - -) reported by Matera et al [26] Figure 1 from [26] is replotted in order to make the apparent heights of absorption maxima of CO-ferrous complex of both preparations the same.
Trang 4complexes (spectrum b) to that of the CO-ferrous complex
(spectrum c) were 0.55 and 0.78,respectively The optical
spectra of the three forms of our a-hydroxyhaem-rHO-1
complex are shown in Fig 1A; the Soret maxima at 407,
433,and 421 nm for the ferric (spectrum a),ferrous
(spectrum b),and CO-ferrous (spectrum c)
forms,respect-ively,agree with those reported by Matera et al [26]
However,the sharpness of the Soret band of our ferric form
was remarkable in that the ratios of intensities of the Soret
maximum of our ferric and ferrous forms to that of the
CO-ferrous form were 0.65,and 0.81,respectively
Com-parison of these ratios to those of Matera et al suggested
that formation of their complex between ferric
a-hydroxy-haem and HO was incomplete,and this led us to reexamine
the process of complex formation
Upon reversing the order of additions,i.e concentrated
rHO-1 solution was added to the neutral a-hydroxyhaem
solution in the same pH 7.4 buffer,complex formation took
over 6 h for completion (data not shown) We further noticed
that keeping a-hydroxyhaem for several minutes in the
neutral pH buffer before the addition of rHO-1,led to
minimal complex formation (Fig 2A) In both cases,we
observed some precipitation of brown-colored particles In Fig 2A,a slight decrease in absorbance between 300 and
600 nm (compare spectra a and b) was probably due to the precipitation of free a-hydroxyhaem These observations indicated that a-hydroxyhaem might be apt to aggregate during incubation at neutral pH,and consequently,complex formation may become markedly slow or nonexistent An additional interesting finding was that subsequent addition
of sodium dithionite to the mixture of rHO-1 and a-hydroxyhaem in which almost no complexes had yet been formed (Fig 2A),caused the smooth formation of the ferrous complex with a Soret maximum at 433 nm This process was complete within 2 h,and the sample solution became clear (Fig 2B) We concluded that the ferrous complex was mainly formed through reduction of free a-hydroxyhaem by dithionite followed by its direct binding
to the enzyme,rather than by reduction of the enzyme-bound ferric a-hydroxyhaem,as no complex formation was seen when the cytochrome P450 reductase system was employed
in place of sodium dithionite (data not shown) The finding that ferric a-hydroxyhaem is only slowly incorporated into the enzyme at neutral pH would explain the apparent broadness in the Soret band of the ferric complex
It is well known that ferric a-hydroxyhaem,prepared in aqueous solvent [20] or in a noncoordinating organic solvent such as chloroform [34],readily dimerizes This dimer formation is observed only with ferric a-hydroxy-haem [20,34] As reduction of the a-hydroxy-haem iron allows the meso-oxygen atom to be protonated [26], the dimeric structure could not be maintained in the ferrous state Hence,we suggest that in these experiments the free a-hydroxyhaem rapidly dimerized and subsequently aggre-gated and precipitated and that reduction of ferric a-hydroxyhaem by sodium dithionite reversed this aggre-gation and consequently facilitated complex formation with the enzyme This in turn explains why the spectra of both our and Matera’s ferrous and CO-ferrous complexes are almost identical (Fig 1)
Reaction of ferric a-hydroxyhaem with excess O2 Migita et al [27] reported that upon exposure of ferric a-hydroxyhaem bound to HO-1 to air,it was oxidized to a ferric porphyrin cation radical However,we have demon-strated that the anaerobic addition of an equimolar amount
of O2 converts the ferric a-hydroxyhaem to the ferrous verdohaem [24] To investigate the effect of excess O2 on ferric a-hydroxyhaem,we again titrated the a-hydroxy-haem-rHO-1 complex with O2 under strictly anaerobic conditions (Fig 3A) As previously reported [24],the addition of an approximately equimolar amount of O2to the a-hydroxyhaem complex yielded a mixture of ferrous CO-verdohaem (640 nm) and CO-free ferrous verdohaem (690 nm) (Fig 3A,spectrum a) The absorbances at 400 and 690 nm were almost unaffected by further addition of
up to 3 equivalents of O2 Further addition of O2led to a decrease in absorbance (Fig 3A,panel) Spectrum b obtained with five equivalents of O2 still showed the absorption maxima characteristic of verdohaem but the absorbance was reduced over the entire spectrum,suggest-ing that the ferrous verdohaem underwent a further reaction with the excess O2 We found that the dark green color of the ferrous verdohaem decreased during the O titration
Fig 2 Formation of the a-hydroxyhaem-rHO-1 complex in the ferric
and ferrous states (A) a-Hydroxyhaemin (38 nmol) was dissolved in
0.75 mL of potassium phosphate buffer (pH 7.4) and allowed to stand
for 30 min,prior to addition of 0.25 mL of the rHO-1 solution
(103 nmol) The final concentrations of rHO-1 and a-hydroxyhaemin
were 103 and 38 l M ,respectively Spectra were recorded immediately
(spectrum a,–) and after 1-h incubation (spectrum b,- - -) (B) Spectra
were recorded immediately (–),and at 30 min,1 h (ÆÆÆ), and 2 h (- - -)
after addition of 1.4 eq of sodium dithionite (54 nmol) to the sample of
spectrum b.
Trang 5Spectrum b (Fig 3A) resembles the spectrum that Migita
et al assigned to the ferric a-hydroxyhaem p cation radical,
and is different from that of the ferric verdohaem-rHO-1
complex [24,31] The ferric verdohaem complex exhibits a
rhombic ESR spectrum with g-values of 2.54,2.14,and 1.88
that are typical of low-spin haemproteins possessing
hydroxide as the sixth ligand [24,31], but the product giving
spectrum b gave a poorly resolved axial signal at g¼ 2.02
and 1.98 (data not shown) The reaction product(s) of the
ferrous verdohaem with excess O2(hereafter referred to as
the O2-oxidized verdohaem) has not yet been fully clarified
Figure 3B shows the sequential reactions of the
O2-oxidized verdohaem with sodium dithionite and then
with CO and O2,according to the procedures reported by
Migita et al [27] The anaerobic addition of sodium
dithi-onite yielded spectrum c showing an asymmetric Soret band
with shoulders around 400 and 430 nm; subsequent addition
of CO produced spectrum d having a relatively sharp Soret
band at 420 nm The Soret maxima at 430 and 420 nm
indicated that the ferrous and CO-ferrous a-hydroxyhaem
bound to HO were the result of the additions of dithionite
and CO,but their yield was clearly small compared to that
obtained by Migita et al [27] Hence,it seems unlikely that
formation of the ferrous a-hydroxyhaem-rHO-1 complex
implied the recovery of the a-hydroxyhaem species from the ferric porphyrin cation radical that was their major product Rather we consider that the ferrous complex was newly formed upon reduction of the residual unbound ferric a-hydroxyhaem by sodium dithionite Exposure to air generated spectrum e with the characteristic absorption maxima of the CO-ferrous verdohaem complex at 407 and
636 nm Again,the absorption intensity at 636 nm was too small to regard CO-ferrous verdohaem as the major product Electrochemical studies have shown that the bis-cpyridine complex of iron(III) octaethyloxophlorin {(py)2FeIII (OEPO)} undergoes two reversible one-electron transfer processes as follows [25]:
fðpyÞ2FeIIðOEPOHÞ e ; Hþ
!
þe ; HþfðpyÞ2FeIII ðOEPOÞg
e
!
þe ðpyÞ2FeIIIðOEPOÞþ
ð1Þ
The solution of {(py)2FeIII(OEPO)} is air-sensitive and exposure of the solution to dioxygen results in the direct formation of octaethylverdohaem [(py)2FeII(OEOP)]Cl and the iron complex of octaethylbilindione {(py)2FeIII(OEB)} The role of [(py)2FeIII(OEPO)]+,corresponding to porphy-rin p cation radical of the protohaem system,in this model system for haem destruction remains to be determined [35]
If porphyrin p cation radical was produced by exposure of a-hydroxyhaem to dioxygen,as claimed by Migita et al [27],it is conceivable that unbound a-hydroxyhaem under-went the parallel process shown in Eqn 1
Degradation of verdohaem with sodium dithionite
or with the NADPH-cytochrome P450 reductase system
We next explored the degradation of the ferrous verdohaem bound to rHO-1 that was obtained from the reaction of the ferric a-hydroxyhaem complex with an equimolar amount of
O2,and compared the behavior of two reducing systems, namely: sodium dithionite and NADPH-cytochrome P450 reductase When sodium dithionite was added gradually to the verdohaem complex under anaerobic conditions,decrea-ses in absorbance at 400,535 and 690 nm took place and broad bands appeared at 431 and 795 nm (Fig 4A,spectrum a) The absorption around 795 nm initially increased and then decreased during the addition of dithionite The spectral changes appeared to be completed with about 4 eq of sodium dithionite The possibility of that the production of hydrogen peroxide caused degradation of verdohaem was ruled out because the dithionite solution was made anaerobically and was used anaerobically These findings thus suggested that the ferrous verdohaem had been converted to a further reduced form by the dithionite,because no similar spectra have been previously observed in the physiological degrada-tion of haem by HO (vide infra) The subsequent addidegrada-tion of
CO yielded an asymmetric Soret band with shoulders at 421 and 435 nm (Fig 4B,spectrum b) As described above, absorption at 421 nm implies that a small amount of the CO-ferrous a-hydroxyhaem complex was produced by reduction
of unbound ferric a-hydroxyhaem The additional absorp-tion at 435 nm may be related to a CO-adduct of the dithionite-reduced verdohaem Exposure to air led to a decrease in the Soret band,resulting in the production of a
Fig 3 Spectral changes in the reaction of the ferric
a-hydroxyhaem-rHO-1 complex with O 2 (A) Spectra were recorded after additions of
1.3 eq (spectrum a,–) and 5.0 eq (spectrum b,- - -) of O 2 Inset:
absorbances at 400 (s) and 690 (d) nm were plotted during additions
of 0–5 of O 2 (B) Spectra were recorded after addition of sodium
dithionite (spectrum c,–) to the sample of spectrum b,after addition of
CO (spectrum d,- - -) to the sample of spectrum c,and after exposure
to air of the sample of spectrum d (spectrum e, ÆÆÆ).
Trang 6biliverdin-iron chelate-like compound (Fig 4B,spectrum c)
[36] The absorption at 636 nm,which first increased and
then disappeared,indicates the formation and degradation of
a trace amount of the CO-ferrous verdohaem produced from
the free a-hydroxyhaem Acidification and extraction of the
product into chloroform gave biliverdin,but its yield was less
than 40% of that obtained from the reaction with
NADPH-cytochrome P450 reductase (data not shown) Thus,we
conclude that sodium dithionite is an inappropriate
reduc-tant for investigating normal haem degradation,such as that
seen with the NADPH-cytochrome P450 reductase system
In contrast to the reaction with dithionite,anaerobic
addition of up to 3.7 eq of NADPH in the presence of
NADPH-cytochrome P450 reductase caused no significant
changes in the spectrum of the ferrous verdohaem complex,
except for an increase in absorption at 340 nm due to the
added NADPH (Fig 5A) Subsequent addition of CO gave
a spectrum (Fig 5B,spectrum b) with absorption maxima
at 409 and 638 nm that are characteristic of the CO-ferrous verdohaem complex [31,37] Further exposure to air caused
a loss of the absorption maxima at 340,409 and 638 nm, and subsequent increases in the absorbances around 380 and 680 nm (Fig 5C,spectrum c) indicative of biliverdin formation [36] For the ring opening that occurs in the conversion of verdohaem to biliverdin,O2 and reducing equivalents are clearly necessary
In order to confirm the results obtained above,we further explored the degradation of the bound synthetic verdo-haem Additions of dithionite to the synthetic verdohaem-rHO-1 complex caused spectral changes (Fig 6A) similar to those shown in Fig 4A The addition of CO produced an absorption maximum at 438 nm (Fig 6B,spectrum b) that was considered to be a form of ferrous verdohaem further
Fig 5 Spectral change during the degradation of verdohaem bound to rHO-1 caused by the NADPH-cytochrome P450 reductase system (A) Spectra were recorded after additions of 0 (–),1.0,2.0,3.0 (ÆÆÆ),and 3.7 eq (spectrum a,- - -) of NADPH,in the presence of NADPH-cytochrome P450 reductase,to the verdohaem-rHO-1 complex obtained as in Fig 4A (B) The spectrum (–) is the same as spectrum a Spectrum b (- - -) was recorded after addition of CO (C) The spectrum (–) is the same as spectrum b Several spectra (ÆÆÆ) were recorded after exposure to air and spectrum c (- - -) is the final product.
Fig 4 Spectral change during the degradation of verdohaem bound to
rHO-1 caused by sodium dithionite (A) Spectra were recorded after
additions of 0 (–),0.9,1.1 (ÆÆÆ),and 4.6 eq (spectrum a,- - -) of sodium
dithionite to the verdohaem-rHO-1 complex obtained in the reaction
of the ferric a-hydroxyhaem-rHO-1 with an approximately equimolar
amount of O 2 (B) The spectrum (–) is the same as spectrum a
Spec-trum b (- - -) was recorded after addition of CO (C) The specSpec-trum (–)
is the same as spectrum b Several spectra (ÆÆÆ) were recorded after
exposure to air and spectrum c (broken line,- - -) is the final product.
Trang 7reduced by sodium dithionite The absorption at 421 nm
that was seen in spectrum b of Fig 4B, was absent in
spectrum b because the synthetic verdohaem-rHO-1
com-plex contained no free a-hydroxyhaem Exposure to air
again produced a biliverdin-iron chelate-like compound
(Fig 6C,spectrum c),accompanied by a transient
appear-ance of absorption at 633 nm similar to that observed in the
experiments shown in Fig 4C As expected,with the
NADPH-cytochrome P450 reductase system,the synthetic
verdohaem was degraded to biliverdin (Fig 7A–C),in a
manner similar to that shown in Fig 5A–C
In an electrochemical study of bis-pyridine
octaethylver-dohaem [(py)2FeII(OEOP)]Cl,Ishizu and coworkers [38]
detected the one-electron reduced form of (py)2FeII(OEOP),
and formulated it as the p neutral radical {(py)2
FeII(OEOP)•} They also reported that,when the
electro-chemical reduction was carried out in the presence of
dioxygen,ring rupture took place and an open-chain poly
pyrrole iron complex was produced The absorption spectrum of the p neutral radical showing peaks at 439 and 740 nm closely resembles the dithionite-reduced form
of the verdohaem-HO-1 complex,which shows absorption peaks at 431 and 795 nm (Figs 4A and 6A) Exposure of the dithionite-reduced form to air gave a biliverdin-iron chelate-like compound Thus,the dithionite-reduced form is thought to correspond to the p neutral radical obtained in the (py)2FeII(OEOP) system Ishizu and coworkers pro-posed that the p neutral radical may be an intermediate in the haem decomposition process However it should be noted that such a species is never seen in the physiological haem degradation driven by cytochrome P450 reductase (Figs 5 and 7)
In conclusion,we find that,depending upon the recon-stitution conditions,the complex of ferric a-hydroxyhaem
Fig 7 Spectral change during the degradation of synthetic verdohaem bound to rHO-1 caused by the NADPH-cytochrome P450 reductase system (A) Spectra were recorded after additions of 0 (–),1.1,1.7,2.6 (ÆÆÆ),and 3.3 eq (spectrum a,- - -) of NADPH,in the presence of NADPH-cytochrome P450 reductase,to the synthetic verdohaem complexed with rHO-1 (B) The spectrum (–) is the same as spectrum a Spectrum b (- - -) was recorded after addition of CO (C) The spectrum (–) is the same as spectrum b Several spectra (ÆÆÆ) were recorded after exposure to air and spectrum c (- - -) is the final product.
Fig 6 Spectral change during the degradation of synthetic verdohaem
bound to rHO-1 caused by sodium dithionite (A) Spectra were recorded
after additions of 0 (–),0.4,0.6 (ÆÆÆ),and 3.8 eq (spectrum a,- - -) of
sodium dithionite to the synthetic verdohaem complexed with rHO-1.
(B) The spectrum (–) is the same as spectrum a Spectrum b (- - -) was
recorded after addition of CO (C) The spectrum (–) is the same as
spectrum b Several spectra (ÆÆÆ) were recorded after exposure to air and
the spectrum c (- - -) is the final product.
Trang 8with HO-1 can contain a substantial amount of free
a-hydroxyhaem and this can lead to an incorrect
interpret-ation of the nature of the enzyme-assisted conversion
of a-hydroxyhaem to verdohaem The reaction product of
a-hydroxyhaem complex with a stoichiometric amount of
O2 is ferrous verdohaem and is never a ferric porphyrin
cation radical Ferrous verdohaem bound to HO appears to
be relatively unstable and may be easily oxidized by excess
O2 These results confirm the reaction mechanism
previ-ously proposed [24],in which O2first attacks the ring carbon
adjacent to the a-oxy group of species 1c,resulting in a
dioxygen adduct that is then rearranged to produce CO and
ferrous verdohaem This intramolecular rearrangement is
accompanied by concomitant expulsion of an oxidizing
equivalent (Scheme 2C) At present we cannot explain the
fate of the oxidizing equivalent; some may react with HO
protein,the rest may be eliminated by continuous electron
flow from NADPH cytochrome P450 reductase The
dithionite-reduced form of ferrous verdohaem is considered
to be a p neutral radical species that cannot be an
intermediate in the physiological degradation of haem
Hence,the use of sodium dithionite should be avoided in the
study of the haem oxygenase reaction
A C K N O W L E D G E M E N T S
This work was supported in part by Grant-in-aid for Scientific
Research on Priority Areas (Biological Machinery (No 13033041))
from the Ministry of Education,Culture,Sports,Science and
Technology of Japan,Grant-in-aid for Scientific Research (C) (No.
12670125) from the Japan Society for the Promotion of Science,Grant
00K1100 from the Ichiro Kanehara Foundation,Grant GM 55807
from the National Institutes of Health,and Grant C636 from the Welch
Foundation.
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