During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme–heme oxygenase or CO–verdoheme–heme oxyge-nase complexes was observed and
Trang 1oxygenase isoform-1 from Glycine max (soybean) )
coordination structure of the heme complex and
catabolism of heme
Tomohiko Gohya1, Xuhong Zhang2, Tadashi Yoshida2and Catharina T Migita1
1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan
2 Department of Biochemistry, Yamagata University School of Medicine, Japan
Heme oxygenase (HO, EC 1.14.99.3) catalyzes the
con-version of heme to biliverdin IXa, CO and free iron
through successive reduction and oxygenation
reac-tions in the presence of molecular oxygen and
elec-trons supplied by NADPH Studies on the structure and function of HO have been conducted mostly in mammalian enzymes, as HO was first identified in mammals [1–3] During the last decade, the HO genes
Keywords
ferredoxin; heme catabolism; heme
complex; higher-plant heme oxygenase;
spectroscopic characterization
Correspondence
C T Migita, Department of Biological
Chemistry, Faculty of Agriculture,
Yamaguchi University, 1677-1 Yoshida,
Yamaguchi 753-8515, Japan
Fax ⁄ Tel: +81 83 9335863
E-mail: ctmigita@yamaguchi-u.ac.jp
T Yoshida, Department of Biochemistry,
Yamagata University School of Medicine,
Iidanishi 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 9 August 2006, revised 1 October
2006, accepted 9 October 2006)
doi:10.1111/j.1742-4658.2006.05531.x
Heme oxygenase converts heme into biliverdin, CO, and free iron In plants, as well as in cyanobacteria, heme oxygenase plays a particular role
in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IXa as a direct synthetic resource In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soy-bean), was prepared to evaluate the molecular features of its heme com-plex, the enzymatic activity, and the mechanism of heme conversion The similarity in the amino acid sequence between GmHO-1 and heme oxygen-ases from other biological species is low, and GmHO-1 binds heme with
1 : 1 stoichiometry at His30; this position does not correspond to the prox-imal histidine of other heme oxygenases in their sequence alignments The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid– base transition and is converted to biliverdin IXa in the presence of NADPH⁄ ferredoxin reductase ⁄ ferredoxin, or ascorbate During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme–heme oxygenase or CO–verdoheme–heme oxyge-nase complexes was observed and was extracted as a bis-imidazole com-plex; it was identified as verdoheme A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence
of NADPH⁄ ferredoxin reductase ⁄ ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system
Abbreviations
AtHO-1, heme oxygenase isoform 1 of Arabidopsis thaliana; BVR, biliverdin reductase; CPR, cytochrome P450 reductase; Fd, plant
ferredoxin; FNR, ferredoxin:NADP + reductase; GmHO-1, heme oxygenase isoform 1 of Glycine max; heme, iron protoporphyrin IX, either ferrous or ferric forms; hemin, ferric protoporphyrin IX; HO, heme oxygenase; hydroxyheme, iron meso-hydroxyl protoporphyrin IX; KPB, potassium phosphate buffer; rHO-1, heme oxygenase isoform 1 of Rattus norvegicus; SynHO-1, heme oxygenase isoform 1 of
Synechocystis sp PCC 6803.
Trang 2have been identified in a wide range of biological
species, especially in pathogenic bacteria, and some of
them have been expressed and characterized [4–6]
Higher-plant HOs, however, have not been
investi-gated on a molecular basis by applying multiple
spect-roscopic methods to the purified protein, and are now
the least studied HOs
HO in plants is one of the plastid enzymes
partici-pating in phytochromobilin synthesis This enzyme
catalyzes the cleavage of heme into biliverdin IXa,
which is then reduced and isomerized to form
(3E)-phytochromobilin, a chromophore of the
photorecep-tor protein of the phytochrome family, which plays
critical roles in mediating photomorphogenesis, by
sensing far-red and red light [7] HO genes of higher
plants have been identified in a few moss plants,
several angiosperms (tobacco, tomato, pea, soybean,
rice plant, sorghum, Arabidopsis thaliana), and a
gym-nosperm (loblolly pine) [8] So far, the HY1 gene and
the HO3 and HO4 genes of Arabidopsis have been
expressed in Escherichia coli, and Cd-induced
expres-sion of HO-1 in soybean leaves has also been reported
[8–11] In the latter study, it was suggested that plant
HOs also play a role in protection against oxidative
cell damage [11] More recently, the PsHO1 gene of pea was expressed, and the HO activity of the protein product was examined [12] These studies have shown that the obtained proteins bind heme to generate a
1 : 1 complex, and CO and biliverdin IXa are gener-ated through heme catabolism, thereby confirming HO activity However, characterization of the heme com-plexes on a molecular basis and determination of the kinetics of heme catabolism have not been performed yet
The amino acid sequences reported for higher-plant HOs are highly homologous to each other; for example, soybean (Glycine max) HO isoform-1 (GmHO-1) has 71.7% homology to A thaliana HO-1 (AtHO-1), and HOs from other plant species have similar levels of homology On the contrary, the homology in amino acid sequences between plant HOs and HOs from other biological species is quite low, e.g 21% to cyanobacte-rial HO-1 (Synechocystis sp PCC 6803) (SynHO-1), 22% to rat HO-1 (rHO-1), 23% to corynebacterial HmuO, or 21% to neisserial HemO Comparison of the sequence alignment reveals that the catalytically pivotal residues, Gly139 and Asp140, of human HO-1 (and also rHO-1) are replaced by Ala and His, respectively,
Fig 1 Amino acid sequence of GmHO-1 as compared with the sequences of Arabidopsis, Synechocystis and rat HO-1s The lightly shaded letters indicate residues with sequence identity, and heavily shaded histidine residues are proximal heme ligands Bars below the alignments show a-helical parts (AH) in the crystal structures of heme–SynHO-1 and heme–rHO-1 and those presumed for heme–GmHO-1.
Trang 3in GmHO-1 (Fig 1), although the residues comprising
the distal F-helix part are relatively well conserved in
the whole sequence of GmHO-1 In mammalian HO-1,
Gly139 directly contacts with heme, and Asp140 is
known to be a key residue for enzymatic activity [3,13]
In addition, the proximal residue for the substrate
heme binding, His25 in rHO-1 (also in human HO-1),
is replaced by Lys, and only one His in the
correspond-ing distal A-helix part occupies a position 13 residues
away from the N-terminus Moreover, the Arg183
resi-due of mammalian HO-1, which participates in
a-meso-specific heme decomposition, is not conserved (Leu in
GmHO-1) [14] Thus, our first concern in examining
plant HOs is to determine whether the plant HO
lack-ing the residues critical for HO activity catalyzes heme
degradation in a similar fashion to the mammalian
enzymes
The next concern is to establish the mechanism of
electron transfer from NADPH to GmHO-1 Muramoto
et al reported that the AtHO-1 reaction required
addi-tional reductant besides ferredoxin reductase (FNR;
ferredoxin:NADP+ reductase; EC 1.27.1.2)⁄ ferredoxin
(Fd) and NADPH [9] On the other hand, we
have clarified that cyanobacterial SynHO-1 (and also
HO-2) shows full activity when coupled with
NADPH⁄ FNR ⁄ Fd, without a secondary reductant,
which had been suggested to be necessary for the HO
activity of cyanobacterial proteins [15–17] Then, we
wanted to determine whether the NADPH⁄ FNR ⁄ Fd
reducing system works fully in the heme conversion
into biliverdin by GmHO-1 as in the SynHO-1
reaction
To investigate these phenomena, we purified the
recombinant mature form of GmHO-1 protein,
exclu-ding the plastid transit peptides, based on the reported
amino acid sequence [8] by constructing a bacterial
expression system Spectroscopic analyses of the
molecular features of the heme–GmHO-1 complex and
of the mechanism of heme degradation were
per-formed, and the results were compared with those for
the heme complexes of SynHO-1 and rHO-1 We
found that, in spite of the low homology of the amino
acid sequence with those of known HOs, the heme–
GmHO-1 complex has similar spectroscopic
character-istics to those of the heme complexes of cyanobacterial,
mammalian or bacterial HOs [15,18,19] GmHO-1
con-verts combined heme into biliverdin IXa, retaining
a-regiospecificity, and releasing CO and free iron, in
the presence of oxygen and NADPH⁄ FNR ⁄ Fd,
with-out requiring additional reducing agents, albeit the
coordination structure of the verdoheme intermediate is
apparently different from that of the known
verdo-heme–HO complexes
Results
Expression and purification of GmHO-1
By culturing the cells at two temperatures, first at
37C and then at 25 C, we avoided the accumulation
of inclusion bodies of GmHO-1 The harvested cells were brown in color, unlike the cells expressing rHO-1
or SynHO-1, which were greenish due to the accumu-lated biliverdin; nevertheless, the E coli cells expressed active GmHO-1, as will be described later It has been reported that the E coli cells expressing the HY1 gene encoding AtHO-1 have a yellowish-brown tinge [9] We purified the GmHO-1 from the soluble fraction by ammonium sulfate fractionation and subsequent col-umn chromatography on Sephadex G-75 and DE-52 The ammonium sulfate fraction and active G-75 frac-tions were tinged with yellow We do not know the nature of this yellow substance(s) at present The final preparation after chromatography on a DE-52 column was clear and colorless, and gave a single band of
26 kDa with about 97% purity on SDS⁄ PAGE, the size expected from the deduced GmHO-1 amino acid sequence (26.1 kDa) About 100 mg of protein was obtained from 1 L of culture
Spectroscopic features of the heme–GmHO-1 complex
The optical absorption spectra of the heme–GmHO-1 complex in the ferric, ferrous, CO-bound and
O2-bound forms are typical of heme proteins and similar to those of the SynHO-1 or rHO-1 complexes (Fig 2) The stoichiometry of the heme binding was confirmed to be 1 : 1 by the titration plots shown in the inset The optical absorption data for heme– GmHO-1, together with those of SynHO-1 and rHO-1, are summarized in Table 1 The absorption maxima of the O2-bound and CO-bound forms of heme–GmHO-1 are slightly red-shifted compared with those of heme– SynHO-1 and heme–rHO-1
The EPR spectrum of heme–GmHO-1 at pH 7.0 shows the heme mostly in the rhombic ferric high-spin state, with gx¼ 5.95, gy¼ 5.68 and gz¼ 2.00 (Fig 3A) Here, anisotropy of the gxy component is apparently larger than that of heme–rHO-1, as shown in the partly expanded spectra (a-1 in Fig 3), indicating that in-plane anisotropy of heme is relatively large In addition, small amounts of low-spin species are also observed in the neutral solution, as distinctly seen in the partly expan-ded spectrum (a-2 in Fig 3) The EPR spectrum of the
15NO-bound GmHO-1 (nitrosylheme GmHO-1) is characteristic of six-coordinate heme proteins with the
Trang 4nitrogenous proximal ligand, indicating hyperfine
splitting due to a14N nucleus (nuclear spin 1, giving the
triplet splitting) in addition to a 15N nucleus (nuclear
spin 1⁄ 2, giving the doublet splitting) at the g2
compo-nent (Fig 3C) This strongly suggests coordination of a
histidinyl residue to the proximal site of the heme The
spectral features of15NO-heme–GmHO-1 are somewhat
different from those of the nitrosylheme complexes of
SynHO-1 (Fig 3D) and rHO-1 (Fig 3E), whereas those
of the latter two are very alike The EPR parameters
of the nitrosylheme–HO complexes as well as those of
the low-spin heme–HO complexes are listed in Table 2
Acid–base transition of heme–GmHO-1
The features of the optical absorption spectrum of
the ferric heme–GmHO-1 complex reversibly change,
depending on pH, between acidic (pH 6.5) and alka-line (pH 10.5) conditions The absorption maxima
of the alkaline form are listed in Table 1 The
pKa value of this acid–base transition was estimated
to be 8.2 by the method described in Experimental procedures
Such an acid–base transition of heme–GmHO-1 was also observed by EPR The EPR spectrum of heme– GmHO-1 at pH 8.7 exhibited a small high-spin signal
at gxy 6 and prominent peaks of the low-spin heme
Table 1 Optical absorption data of the heme–HO-1 complexes.
Types of
heme
Protein
GmHO-1
kmax(nm)
SynHO-1
kmax(nm)
rHO-1
kmax(nm) Soret Visible Soret Visible Soret Visible
CO-bound 420 539, 569 427 536, 566 419 535, 568
Alkaline 414 539, 577 427 537, 575 414 540, 575
Fig 2 Absorption spectra of the various forms of heme–GmHO-1.
Spectra are of the ferric (red), ferrous (blue), ferrous–CO (black)
and ferrous–oxy (green) forms Inset: titration plots of GmHO-1
(4.8 nmol) with hemin (0.4 m M ), monitored by the increase in
absorbance at 405 nm The pink off-line dots indicate the results
for the titration without protein.
Fig 3 EPR spectra of the ferric heme–GmHO-1 (A, B) and nitro-sylheme–HO (C–E) complexes EPR conditions were: microwave frequency, 9.35 GHz; microwave power, 1 mW for (A) and (B) and 0.2 mW for (C)–(E); field modulation frequencies, 100 kHz; field modulation amplitude, 10 G for (A) and (B) and 2 G for (C)–(E); sig-nal acquisition temperature, 8 K for (A) and (B) and 25 K for (C)–(E) (A) The ferric heme complex of GmHO-1 at pH 7.0 (0.1 M , KPB) a-1, Expansion of the g xy region of (A) (solid line) and of the corres-ponding part of the spectrum of ferric heme–rHO-1 (dotted line) a-2, Expansion of the higher-field region of (A) (B) The ferric heme–GmHO-1 complex at pH 8.7 (50 m M , Tris ⁄ HCl) (C–E) The
15 N-nitrosylheme complexes of GmHO-1, SynHO-1, and rHO-1, respectively.
Trang 5at the lower magnetic fields (Fig 3B) The g-values of
this low-spin species were the same as those of the
low-spin species observed at neutral pH (denoted as
gak in Fig 3A, expanded lower-field spectrum)
Accordingly, this species was determined to be the
alkaline form of heme–GmHO-1, which coordinates a
hydroxyl anion at the distal site of heme Another
low-spin species, denoted as g* in Fig 3A, seems to be
a denatured form of heme–GmHO-1, because similar
low-spin species were sometimes observed for other
heme–HO complexes (data not shown)
Single species of the alkaline form were observed for
heme–GmHO-1, the same as for rHO-1, but distinct
from SynHO-1, which exhibits two kinds of alkaline
forms (Table 2) The anisotropy in g-values, gak
1–gak
3,
of the alkaline form of heme–GmHO-1 is somewhat
smaller than that of the other two HO complexes This
might indicate that the axial ligand field is relatively
strong in GmHO-1, due to steric control imposed by
the distal helix, in accord with the observation that
the in-plane anisotropy is large in the ferric state of
heme
Determination of the proximal ligand
Based on the EPR results for nitrosylheme–GmHO-1,
candidates for the proximal ligand of heme were
searched for in the amino acid sequence of GmHO-1,
around the position corresponding to the proximal His
of other HOs (Fig 1) His30 was found to be only
nitrogenous ligand capable of coordinating to the
fer-ric heme, so the GmHO-1 mutant H30G was prepared,
and the optical absorption and EPR spectra of its
heme complex were determined The H30G mutant
also accommodated heme with 1 : 1 stoichiometry, like
other HO mutants that lack the proximal histidine,
but the optical spectrum of the heme–H30G complex exhibited an asymmetrically broadened Soret band with the blue-shifted maximum at around 390 nm, compared with the Soret-band features of the wild-type complex, and no other characteristic bands at the vis-ible region (data not shown) Such features of the spec-trum are commonly seen in the heme complexes of
HO mutants lacking the proximal His [20,21] The heme–H30G complex did not decompose the bound heme enzymatically in the presence of either ascorbate
or NADPH⁄ FNR ⁄ Fd (data not shown) EPR meas-urements on the heme–H30G complex provided critical evidence for the lack of coordination of a protein resi-due at the proximal site, yielding the deformed spec-trum of high-spin heme (Fig 4A), which means that the heme is in the multiple configuration in the heme pocket, and the spectrum of nitrosylheme is typical of the 15NO coordination without the sixth ligand (Fig 4B) Accordingly, it has been established that the proximal ligand of heme–GmHO-1 is His30
Exogenous ligand binding
To investigate the nature of the heme pocket in GmHO-1, the apparent equilibrium constant for bind-ing of nitrogenous ligands, imidazole and azide, to heme–GmHO-1 was evaluated As imidazole was added to the solution of heme–GmHO-1, the Soret
Table 2 EPR parameters of the low-spin and 15 NO-bound forms of
heme–HO-1 complexes.
Protein
Alkaline Neutral Alkaline-1 Alkaline-2 Alkaline
Low spin
15
NO
Fig 4 EPR spectra of (A) ferric heme and (B) 15N-nitrosylheme complexes of the H30G mutant of GmHO-1 EPR conditions were the same as those described in Fig 3.
Trang 6band maximum of the complex shifted from 405 to
410 nm and decreased in intensity, without showing a
distinct isosbestic point At the same time, the heights
of the absorption peaks in the visible region (500 and
630 nm) also decreased, and then new peaks appeared
at 531 and 568 nm with increasing intensity,
indica-ting formation of imidazole-bound heme–GmHO-1
(data not shown) The association constant was
esti-mated on the basis of the changes in absorbance at
402 nm (imidazole-free form) and at 422 nm
(ole-bound form) in the difference spectra of
imidaz-ole-free minus imidazole-titrated forms, as described
in Experimental procedures In the same way, azide
binding to heme–GmHO-1 was also examined In this
case, a clear isosbestic point was observed at 412 nm,
between the Soret band maxima of the azide-free
form at 405 nm and of the azide-bound form at
421 nm, so the relative amounts of the nonbound and
azide-bound forms were estimated directly from the
values of absorbance at these maxima (data not
shown) The estimated association constants for
imi-dazole and azide binding to heme–GmHO-1 are
sum-marized and compared with those for heme–SynHO-1
and heme–rHO-1 in Table 3
Heme degradation by GmHO-1
Heme degradation by HOs can be monitored through
the changes in optical absorption spectra, because
ferric heme, oxyheme and verdoheme intermediate complexes of HO and free biliverdin, products of the
HO reaction, all exhibit characteristic absorption bands (Scheme 1) As shown in Fig 5A, addition of ascorbate to the solution of heme–GmHO-1 initiates the reaction, as revealed by gradual diminution of the Soret band After several minutes, a broad band appears at around 660–675 nm; this increases in inten-sity with time, indicating the formation of biliverdin
In this case, neither bands of the oxy form (at 541 and
578 nm) nor bands of verdoheme and CO–verdoheme (at 690 and at 640 nm, respectively) are observed, implying that the first step of heme conversion is rate-limiting The apparent initial rate of heme degradation
by GmHO-1 is about three times higher than that of degradation by SynHO-1, but nearly four times lower than that of degradation by rHO-1 in the presence of
1200 equivalents of ascorbate (Table 4)
To establish the physiologic electron-donating system
in the higher-plant HO reaction, the heme–GmHO-1 reaction was carried out in the presence of NADPH coupled with FNR⁄ Fd (Fig 5B) After addition of NADPH, the Soret band maximum of heme–GmHO-1 immediately shifted from 405 to 415 nm, and at the same time, distinct absorption bands of oxyheme appeared at 540 and 579 nm Then, a broad band appeared at around 660 nm, and was maximal 9–12 min after initiation of the reaction The spectral features of the final reaction mixture were analogous, but not iden-tical, to those of the ascorbate reaction, which was mainly due to free biliverdin, probably because of the overlapping of absorption bands of the intermediate complexes Product analysis by HPLC revealed that only the a-isomer of biliverdin IX was produced in both the NADPH⁄ FNR ⁄ Fd-supported and ascorbate-assis-ted GmHO-1 reactions (data not shown) Catalase did not affect the heme–GmHO-1 reaction in the presence
of either ascorbate or NADPH⁄ FNR ⁄ Fd The apparent initial heme degradation rate in the presence of
Table 3 Equilibrium constants for imidazole and azide ion binding
to the heme–HO-1 complexes Numbers in parentheses indicate
relative values normalized to the values of GmHO-1.
Protein
Kimidazole( M )1) 35 (1) 210 (6) 1400 (40)
K azide (· 10 2
Scheme 1 Pathways of heme degradation by heme oxygenase (HO), elucidated for the mammalian HO-1 Most HOs other than those of mammalian origin are also known to cleave heme at the a-meso position selectively to produce biliverdin IXa.
Trang 7NADPH⁄ FNR ⁄ Fd of GmHO-1 was also compared
with that of SynHO-1 under similar conditions, as well
as that of rHO-1 in the presence of NADPH⁄
cyto-chrome P450 reductase (CPR; NADPH:cytocyto-chrome
P450 reductase; EC 1.6.2.4) (Table 4) This result
sug-gests that the GmHO-1 reaction occurs as fast as the
SynHO-1 reaction, supported by the NADPH⁄ FNR ⁄ Fd
reducing system, and as fast as the rHO-1 reaction
sup-ported by the NADPH⁄ CPR reducing system under
similar conditions
Bilirubin assay of the heme oxygenase activity
of GmHO-1
In mammalian HO reactions, the end-product, biliver-din IXa, is further reduced to bilirubin by NADPH: biliverdin reductase (BVR; EC 1.3.1.24) To estimate the yield of free biliverdin produced by the GmHO-1 reaction, the bilirubin assay was carried out by use of rat BVR The overall yields of bilirubin after the heme conversion followed by biliverdin reduction under dif-ferent conditions were estimated and compared with the bilirubin yields for SynHO-1 or rHO-1 reactions under comparable conditions (Table 5) Bilirubin yields were somewhat low for GmHO-1 and SynHO-1, but were comparable ( 50%) for the three HOs when des-ferrioxamine, a chelating agent of Fe3+, was applied to assist the extraction of Fe3+from the ferric biliverdin–
HO complexes Interestingly, when an excess amount
of ascorbate was used as a reducing agent, the bilirubin yield in the GmHO-1 reaction was as high as 47%
Detection of CO liberation and identification
of reaction intermediates Plausible verdoheme and CO–verdoheme intermediates were not detected in the course of heme degradation
Table 4 Apparent rates of initial heme degradation (v) by GmHO-1, SynHO-1 and rHO-1 in the presence of ascorbate, NADPH ⁄ FNR ⁄ Fd (for GmHO-1 and SynHO-1), and NADPH ⁄ CPR (for rHO-1) The con-centration of reactants in 0.1 M KPB (pH 7.0) are: sodium ascor-bate, 6 m M ; heme–HO, 5 l M ; Fd, 1 l M ; FNR, 0.22 l M ; CPR, 0.25 l M ; and NADPH of indicated equivalent.
Protein GmHO-1
v (l M Æmin)1)
SynHO-1
v (l M Æmin)1)
rHO-1
v (l M Æmin)1)
Table 5 Bilirubin yields (%) in the heme conversion by HO coupled with the biliverdin conversion by BVR.
Proteins
NADPH (4 eq.) + NADPH (8 eq.) ⁄ BVR a
NADPH (20 eq.) + desferrioxamine (1.1 mg) + BVR b
a Additional NADPH and BVR were supplied 45 min after the first addition of NADPH (4 eq.).bDesferrioxamine and BVR were sup-plied 20 min and 30 min after addition of NADPH, respectively.
Fig 5 Heme conversion by GmHO-1 (A) Spectra were recorded at
the indicated time after addition of ascorbate (6 m M ) to the solution
of heme–GmHO-1 (5 l M in 0.1 M KPB, pH 7.0) (B) Spectra were
recorded at the indicated times after addition of NADPH (40 l M ) to
the solution of heme–GmHO-1 (5 l M ; 0.1 M KPB, pH 7.0), FNR
(0.22 l M ), and Fd (1 l M ).
Trang 8by GmHO-1 by optical absorption spectrometry
(Fig 5) To ascertain the liberation of CO, this
reac-tion was performed in the presence of H64L, a
myo-globin mutant with 40 times greater affinity for CO
than the wild type [22] As shown in Fig 6A,B, the
difference spectra, the spectra obtained in the presence
minus those obtained in the absence of H64L, show an
absorption peak at 423 nm, which is known to be
spe-cific for the CO-bound form of myoglobin, thereby
proving liberation of CO in the heme conversion by
GmHO-1 in the presence of either ascorbate (Fig 6A)
or NADPH⁄ FNR ⁄ Fd (Fig 6B)
Next, to determine whether verdoheme is produced
in the GmHO-1 reaction, this reaction was performed under a CO atmosphere The high partial pressure of
CO should enhance coordination of CO to the verdo-heme if it is produced As shown in Fig 6C, when ascorbate was added to the heme–GmHO-1 solution presaturated with CO in a sealed cuvette, the Soret band maximum shifted from 405 to 420 nm, and peaks
Fig 6 Detection of CO produced during the GmHO-1 reaction by the H64L mutant of myoglobin (A, B) and detection of CO–verdoheme pro-duced in the GmHO-1 reaction under CO (C, D) (A) Difference spectra of optical absorption spectra obtained for the reaction of heme– GmHO-1 (5 l M ) in the presence of H64L (4 l M ) minus those for the reaction of H64L (4 l M ) alone, after addition of ascorbate (6 m M ) at appropriate times (B) Difference spectra obtained for the reactions described in (A), except that NADPH (10 l M ), FNR (0.22 l M ) and Fd (1 l M ) were used in place of ascorbate (C) Spectra obtained for the reaction of heme–GmHO-1 (5 l M ) with ascorbate (6 m M ) (D) Spectra obtained for the reaction of heme–GmHO-1 (5 l M ) with FNR (0.22 l M ), Fd (1 l M ), and NADPH (40 l M ) All solutions were in 0.1 M KPB (pH 7.0).
Trang 9concomitantly appeared at 539 and 571 nm, indicating
formation of the CO-combined heme–GmHO-1
com-plex (Table 1) Injection of oxygen gas into this cuvette
decreased the Soret band so that it almost vanished
after 30 min, and instead, an absorption peak
appeared at 637 nm, suggesting the formation of
CO–verdoheme The 637 nm band disappeared
gradu-ally and was replaced by a new broad band with a
maximum at approximately 675 nm, identical to the
absorption band of free biliverdin The heme–GmHO-1
reaction under a CO atmosphere was also carried out
with the NADPH⁄ FNR ⁄ Fd reducing system (Fig 6D)
In this case, however, the 637 nm band was not
observed, but a broad band with a maximum at around
657 nm appeared Therefore, in the presence of
ascor-bate (Fig 6C), verdoheme is probably produced, but in
the presence of NADPH⁄ FNR ⁄ Fd, formation of the
verdoheme intermediate is still unclear because the
657 nm band is different from that of the well-known
verdoheme or CO–verdoheme bands at 688 and 637 nm,
respectively [23]
Heme degradation by HO is driven by hydrogen
peroxide, which substitutes for molecular oxygen and
probvides electrons to convert heme into verdoheme
[24] Therefore, heme–GmHO-1 was reacted with
H2O2 to ascertain whether verdoheme was actually
produced Soon after addition of H2O2 to the heme–
GmHO-1 solution, the Soret band intensity diminished
to nearly one-third and a relatively strong broad
band appeared in the visible region (kmax ¼
660 nm) (Fig 7) This 660 nm band is very similar to
that observed in heme degradation by GmHO-1 in the
presence of NADPH⁄ FNR ⁄ Fd (Figs 5B and 6D),
sug-gesting that the same intermediate, namely the 660 nm
species, is accumulated
To isolate and identify the 660 nm species, the heme–
GmHO-1 reaction was carried out with six equivalents
of H2O2under anaerobic conditions, to avoid the
degra-dation or successive conversion of the intermediate by
oxygen The green pigment was extracted from the
reac-tion product with acetone containing imidazole The
spectrum of the extract is shown in Fig 8B, exhibiting
peaks at 404, 534, 636 and 684 nm; the latter two differ
from the 660 nm band of the protein complex (Fig 8A)
When the solution of the extract was exposed to CO, the
684 nm band gradually shifted to 636 nm The reported
band maxima of bis-imidazole-coordinated verdoheme
are 400, 536 and 685 nm [23], so the spectra shown in
Fig 8B are considered to be a mixture of a
CO-coordi-nated monoimidazole complex and a bis-imidazole
com-plex of verdoheme Using the same methods, extracts
of the H2O2 reaction intermediates of heme–SynHO-1
and heme–rHO-1 were obtained The optical absorption
spectra of the acetone extracts (Fig 8C,D) showed similar features, indicating a common chromophore
In conclusion, it has been confirmed that the 660 nm
Fig 7 Heme degradation by GmHO-1 in the presence of H 2 O 2 Spectra were recorded at the indicated times after addition of H2O2 (40 l M in N2-saturated 0.1 M KPB) to the heme–GmHO-1 solution (5 l M in N 2 -saturated 0.1 M KPB).
Fig 8 Optical absorption spectra of the intermediates of heme–
HO reactions (A) Obtained from the reaction mixture of heme– GmHO-1 with H 2 O 2 (6 eq of the heme) under anaerobic condi-tions (B–D) Acetone extracts containing excess imidazole from the reaction mixtures of the heme–HO-1 complexes with H 2 O 2 (6 eq.)
in anaerobic conditions.
Trang 10species produced in the course of heme degradation by
GmHO-1 is verdoheme
Discussion
Homology and heme binding
Estimation of the secondary structure of GmHO-1
sug-gests that, in spite of low homology in the amino acid
sequence, GmHO-1 protein should consist of eight
a-helices common to other HOs whose crystal
struc-tures are known [3,25–29] (Fig 1) A recent modeling
study on pea HO-1 also suggested a similar structure
[12] In GmHO-1, however, a critical residue for HO
activity, the proximal His, which fixes heme to the
heme pocket of the enzyme and participates in the
activation of heme, is not at the position in which
it is present in SynHO-1 (His17) or rHO-1 (His25)
Instead, there is only one His (His30) in the
predic-ted proximal A-helix region (Fig 1) Experiments on
heme binding to GmHO-1 have demonstrated 1 : 1
stoichiometry, and the result of EPR investigations of
nitrosylheme–GmHO-1 indicate a nitrogenous
proxi-mal ligand of the heme, whereas the heme–H30G
complex shows neither a proximal protein ligand
(Fig 4) nor HO activity These findings have firmly
established that His30 of GmHO-1 is the proximal
heme ligand
Coordination structure of heme–GmHO-1
The optical absorption data for the GmHO-1 complex
in ferric, ferrous, oxy and CO-bound forms of heme
show that the coordination structure of the heme is
generally like that of SynHO-1 or rHO-1 (Fig 2 and
Table 1) The EPR spectrum of the ferric resting form
of heme–GmHO-1, however, shows that the rhombic
anisotropy is relatively large and close to that in the
ferric a-hydroxyheme complex of rHO-1 [30] This
means that the ligand field on the heme plane is
relat-ively anisotropic in GmHO-1 For this reason, it is
possible that the surrounding helices exert a greater
anisotropic ligand field effect on the heme plane than
the corresponding residues in SynHO-1 and rHO-1,
the amino acid sequences of which are considerably
different from that of GmHO-1
The observed acid–base transition strongly suggests
that the sixth, distal ligand of heme is a water
mole-cule, and the heme-bound water is supposed to be
con-nected to dissociable distal residue(s) through direct
or indirect hydrogen bonding The estimated pKavalue
of 8.2 is between the values of heme–rHO-1 (7.6) and
heme–SynHO-1 (8.9); in the latter two, the distal water
molecule interacts with respective Asp residues (Asp140 of rHO-1 and Asp131 of Syn HO-1) in the distal helix through hydrogen-bonding networks via water molecules in crystals [25,27] Unfortunately, the resolution of the reported crystal structures of heme– SynHO-1 and rHO-1 is not sufficiently high to allow accurate quantitative comparison of the length of the hydrogen-bonding network, so the reason for such a large difference in pKa values is unclear In GmHO-1, the corresponding residue to the Asp is His150, which
is also competent as a partner of the indirect hydrogen bonding with the heme-bound water This difference in the hydrogen-bonding counterpart would also affect the pKavalue
The EPR parameters of the nitrosylheme–HO com-plexes also give useful information on the heme pocket structure As shown in Table 2, the hyperfine splitting constants of the 15N nucleus of the distal NO and of the 14N nucleus of the proximal His of the GmHO-1 complex are closer to those of the rHO-1 complex than
to those of the SynHO-1 complex Thus, Fe–N(O) r-bonding in heme–GmHO-1 might be comparably strong to that in heme–rHO-1 [15] The strength of the Fe–N(His) bonding in the GmHO-1 and in rHO-1 complexes also appears to be the same, thereby imply-ing that the imidazole part of His30 is neutral and probably forms a hydrogen bond with Gln34, such that His25 of rHO-1 is stabilized by the hydrogen bonding with Glu29
Characterization of the heme pocket of GmHO-1
by exogenous ligand binding Azide, like imidazole, is a ligand with both r-donor and p-donor characteristics, but is a relatively stron-ger p-donor for stabilization of the higher oxidation states of metal ions In agreement with this, the bind-ing constants for the bindbind-ing of azide to ferric heme– HOs are one to two orders larger than those for imidazole (Table 3) Comparison of the Kazide values shows that heme–GmHO-1 and heme–SynHO-1 have smaller values than heme–rHO-1, suggesting weak relevance of the similarity in the amino acid sequences
of distal helices This difference does not necessarily mean that the ferric character of the former two is less than that of the latter, because polarity of the heme milieu as well as the steric conditions of the distal heme pocket could also affect Kazide Polar resi-dues might either stabilize the azide coordinated to heme or facilitate the access of anionic azide to the heme, and conversely, the steric effect of the distal residues might reduce the accessibility of azide The amino acid residues comprising the presumed distal