Detection of a Tryptophan Radical as an Intermediate Species in
the Reaction of Horseradish Peroxidase Mutant (Phe-221
Trp) and
Hydrogen Peroxide*
Atsushi
Morimoto
,
Motomasa
Tanaka
§,
Satoshi
Takahashi
,
Koichiro
Ishimori
,
Hiroshi
Hori¶, and
Isao
Morishima
From the
Department of Molecular Engineering,
Graduate School of Engineering, Kyoto University, Kyoto 606-8501 and
the ¶ Division of Biophysical Engineering, Graduate School of
Engineering Science, Osaka University,
Toyonaka Osaka 560-8531, Japan
 |
ABSTRACT |
The crucial reaction intermediate in the reaction
of peroxidase with hydrogen peroxide
(H2O2), compound I, contains a porphyrin
-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the
tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome
c. To investigate the roles of the amino acid residue near
the heme active center in discriminating the function of the
peroxidases in these two enzymes, we prepared a CcP-like
HRP mutant, F221W (Phe-221
Trp). Although the rapid spectral
scanning and stopped-flow experiments confirmed that the F221W mutant
reacts with H2O2 to form the porphyrin
-cation radical at the same rate as for the wild-type enzyme, the
characteristic spectral features of the porphyrin
-cation radical
disappeared rapidly, and were converted to the compound II-type
spectrum. The EPR spectrum of the resultant species produced by
reduction of the porphyrin
-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from
a Trp radical as found for CcP. The sequential radical
formation from the porphyrin ring to the Trp residue implies that the
proximal Trp is a key residue in the process of the radical transfer
from the porphyrin ring, which differentiates the function of
peroxidases.
 |
INTRODUCTION |
Peroxidases catalyze one-electron oxidation of various substrates
by using H2O2 as an oxidant (1). In the first
step of the catalytic cycle, horseradish peroxidase
(HRP),1 one of the typical
peroxidases, yields an intermediate called compound I, in which the
ferric enzyme (FeIII) undergoes 2 equivalent oxidation to
yield an oxyferryl porphyrin
-cation radical (2). Compound I in HRP
effects one-electron oxidation of the substrates, small organic or
inorganic molecules, producing the second intermediate, compound II.
The remaining oxidized site, ferryl iron (FeIV), again
oxidizes substrates to be reduced back to the resting state. On the
other hand, the compound I of another typical peroxidase, cytochrome
c peroxidase (CcP), can catalyze the redox
reaction with cytochrome c (Cyt c), a sole
substrate of CcP (3, 4). Although the resting state of
CcP reacts with H2O2 to form the oxidized intermediate, compound I, as does HRP, CcP compound
I has a UV-visible spectrum similar to that of HRP compound II (5, 6).
Several spectroscopic studies combined with site-directed mutagenesis
(7-9) have revealed that CcP compound I has a stable indolyl cation radical at Trp-191 as one of the oxidized site. The
formation of the Trp radical would facilitate one electron transfer
from Cyt c to the porphyrin, inasmuch as Cyt c
cannot be directly accessible to the CcP heme active center
(10, 11). Thus, the position of the radical center in compound I is
closely related to function of peroxidase and amino acid residues that control the radical center can be considered to be one of key factors
to discriminate the functions of peroxidases.
On the basis of sequence alignment between HRP and CcP, an
amino acid residue corresponding to Trp-191 in CcP is a Phe
residue (Phe-221) in HRP (12). To investigate the control mechanism for
the position of the radical center in the reaction intermediates, some
mutagenic studies focused on Trp-191 in CcP have been
carried out and shown that a "HRP-type" CcP mutant, in
which the proximal Trp was replaced with Phe (Trp-191
Phe), yields
an oxyferryl porphyrin
-cation radical intermediate similar to that
of HRP compound I, suggesting that the proximal Trp controls the
position of the radical center (8, 9, 13). However, the radical transfer from the porphyrin ring to the proximal Trp has not yet been
confirmed, which is one of the most crucial processes in the formation
of CcP compound I.
In this paper, we have prepared a complementary HRP mutant enzyme to
the CcP mutant, a "CcP-type" HRP mutant
having a tryptophan residue at the position of Phe-221 (F221W) (Fig.
1), in order to elucidate the regulation
mechanism of radical transfer in peroxidase reaction intermediates.
NMR, EPR, and ICP emission spectroscopies were used in this study to
characterize the heme environmental structure of the mutant. These
spectroscopies, demonstrating formation of the Trp radical during the
oxidation of the mutant by hydrogen peroxide after the porphyrin
-cation radical formation, provide strong evidence that the radical
center on the porphyrin ring can be transferred to the Trp residue near
the heme in peroxidase, indicating that the Trp residue is the key
amino acid residue to differentiate the function of peroxidases.

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Fig. 1.
The heme environmental structure of HRP
(12). In this view, Phe-221 in HRP corresponds to Trp-191 in
CcP.
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EXPERIMENTAL PROCEDURES |
Materials--
General molecular biology supplies were obtained
from Takara and Perkin-Elmer. All buffer materials and other chemicals
were purchased from Wako Pure Chemical Industries, Ltd. and Nacalai Tesque, Inc. as the highest quality available.
Site-directed Mutagenesis--
Site-directed mutagenesis of
recombinant HRP at position 221 was carried out by the use of the
polymerase chain reaction-based technique with T7 HRP as a template
(14). To construct the insert DNA fragment with mutation, the synthetic
oligonucleotide 5'-TCCGCAGATCCCAGTCCACT-3' was used as a
primer to multiply mutated DNA fragment. The amplified DNA fragment
bearing the mutation was ligated into the T7 expression vector by using
the unique NdeI and NcoI restriction enzyme sites (15). The expression vector for the mutated HRP was transformed into
Escherichia coli strain BL 21 with ampicillin resistance. Positive clones were screened by SDS-polyacrylamide gel
electrophoresis, and introduction of the mutation was confirmed by
double-stranded DNA sequence analysis using the dideoxy chain
termination method with a model 373 DNA sequencer (Applied Biosystems).
No additional mutations in the mutated HRP gene were detected.
Expression, Reconstitution, and Purification of
HRP--
Wild-type and mutant HRPs were expressed in E. coli strain BL 21 and extracted from the inclusion bodies as
described previously by Nagano et al. (14). Reactivation of
apoHRP in the presence of calcium and heme, and purification of holoHRP
was followed by the method previously reported with some modifications
(16, 17). The RZ values of purified wild-type and F221W
mutant HRPs were 3.2 and 2.3, respectively. Peroxidase concentration
was estimated by using the Soret extinction coefficient in the pyridine
hemochrome form by monitoring at 556 nm (18). The extinction
coefficients for wild-type and F221W mutant HRPs were 103 (
= 403 nm) and 113 mM
1.cm
1 (
= 409 nm), respectively.
Proton Nuclear Magnetic Resonance (1H NMR)
Spectroscopy--
1H NMR spectra were recorded at 500 MHz
on a Bruker Advance DRX500 spectrometer. We used a LOSAT pulse sequence
with 64,000 data transforms of 150 kHz and 8.5-µs 90° pulse. The
probe temperature was controlled at 290.0 K by a temperature control
unit of the spectrometer. The volume of the NMR samples were 400 µl
and the concentrations used for the measurements were about 1.0 mM (heme basis) in 50 mM sodium phosphate
buffer, pH 7.0. Peak shifts were referenced to tetramethylsilane.
Reaction with Hydrogen Peroxide--
Spectra were obtained with
a rapid scanning spectrophotometer (Unisoku). Compounds I of wild-type
and F221W HRPs were generated by addition of 1 equivalent of
H2O2 to the resting state solution. The
formation of compound I was monitored by a Unisoku stopped-flow spectrophotometer at 25.0 °C in 50 mM sodium phosphate
buffer, pH 7.0. 2.0 µM HRP was used to determine the
elementary reaction rate constant. More than 10-fold excess of
H2O2 was used to ensure the pseudo-first-order
kinetics. The formation rate of compound I (k1)
was measured by following decrease of the absorbance at 395 nm, the
isosbestic point between compound I and compound II. For the F221W
mutant, the electron transfer rate from Trp-221 to the porphyrin
(kET) was measured at 412 nm, the isosbestic point between the ferric state and the oxyferryl species. The reduction
rate of compound II (k3) was obtained by almost
the same method as the formation rate of compound I. The
k3 values for the wild-type and mutant HRPs were
acquired by adding guaiacol or ferrocyanide 5 min after addition of a
slight excess of H2O2. The rate of formation of
compound II was followed at 424 nm for wild-type HRP and 419 nm for the
mutant.
Electron Paramagnetic Resonance (EPR) Spectroscopy--
EPR
spectra of HRPs were measured by a Varian E-12 spectrometer equipped
with an Oxford ESR-900 liquid helium cryostat. Measurements were
carried out at the X-band (9.22 GHz) microwave frequency at 5 and 77 K. The range of the microwave power is 0.01 mW to 100 mW. The sample
concentration was ~400 µM, and the volume was 50 µl.
The sample solution was frozen in EPR tube by submersion in liquid
nitrogen over 1 min after addition of a small excess of
H2O2 to ferric wild-type enzyme. To observe the
EPR signal from the unstable radical species in the F221W mutant, the
rapid mixing, freeze-quench technique was used (19). The photolysis experiments were performed with a tungsten light.
Quantitative Analysis for Calcium Ion Bound in HRP--
The
amount of calcium ion bound in the HRP samples in deionized water at pH
7.0 was determined by ICP emission spectroscopy (Jarrel Ash ICAP-500).
The samples analyzed here contained 1.2 mg of calcium/liter.
Peroxidase Activity--
Oxidation activities for guaiacol and
ABTS were measured by monitoring the increase of the absorbance at 470 and 405 nm, respectively on a Perkin-Elmer UV-visible spectrophotometer
(Lambda 19) at 25.0 °C. For the guaiacol oxidation, 1 mM
H2O2 was mixed with 5.1 mM guaiacol
in 50 mM sodium phosphate buffer at pH 7.0, after which 5 nM enzyme was added to initiate the reaction. For the ABTS
oxidation, 5 nM enzyme was added to the solution containing 1 mM H2O2 and 0.90 mM
ABTS in 50 mM sodium phosphate buffer, pH 7.
Redox Potential of Fe2+/Fe3+
Couple--
The redox potentials of HRPs were monitored at 435 nm with
platinum electrode on a Perkin-Elmer UV-visible spectrophotometer (Lambda 19). The mediator was a mixture of safranine T, phenosafranine, benzylviologen, and
-hydroxyphenazine. The ferric enzymes were photoreduced at 25.0 °C in 50 mM sodium phosphate buffer
containing 50 mM EDTA, pH 7.0. The reductive titration of
HRP was carried out with a short irradiation with white light. This
measurement was repeated until HRP could not be reduced further by the
irradiation.
The midpoint potential of HRP (E0) was
determined by the plot of the monitored electrode potential
(Eh) against the percentage of reduced HRP
estimated from the absorbance change at 435 nM by using the
following Nernst equation.
|
(Eq. 1)
|
The
and F correspond to a number of electrons
involved in the redox reaction and Faraday constant, respectively. The
midpoint potential value of HRP was corrected by that of a
phenosafranine (
252 mV).
 |
RESULTS |
Reaction with Hydrogen Peroxide--
The resting state of
wild-type HRP reacts with H2O2 to form the
oxidized intermediate, compound I. This oxidized species, the porphyrin
-cation radical is characterized by a diminished absorbance in the
Soret region (9) as shown in Fig.
2a. Compound I was then
gradually one electron-reduced to compound II with shift of the Soret
peak from 402 to 420 nm and finally back to the resting state (20). In
the F221W mutant, the addition of H2O2 also
decreased the absorbance at the Soret band, which is characteristic of
the porphyrin
-cation radical formation (Fig. 2b).
However, the absorbance of the Soret band increased again faster than
in the course of the wild-type enzyme, and the resultant spectrum
(
max = 419 nm) corresponds to that of HRP compound II (
max = 420 nm). The rapid restoration of the absorbance
at the Soret band indicates that the porphyrin
-cation radical in
the F221W mutant is highly unstable and easily reduced. Even for the parent enzyme in the absence of the substrates, compound I is converted
to compound II with the donation of one electron from impurities in the
solution (21). However, the life time of the porphyrin
-cation
radical in the wild-type without the substrate is typically about 30 min (21), which is much longer than that of the porphyrin
-cation
radical in the F221W compound I (~5 s). Because the spectral pattern
in the Soret band for CcP compound I (oxyferryl-Trp radical)
is quite similar to that of compound II (oxyferryl) in HRP and were
quite similar in this mutant (22), we could not confirm a radical
formation on the newly introduced Trp residue in the second reaction
intermediate (
max = 419 nm) for the HRP mutant by the
absorption spectrum.

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Fig. 2.
Rapid spectral scanning experiments for
wild-type (a) and F221W mutant HRPs (b) after
the addition of 1 eq of H2O2. The
repetitive scans were recorded at (a) 0 (dotted
line), 21, 42, 84, 1029, 4179, 6279, 10479 ms and (b) 0 (dotted line), 21, 42, 63, 336, 1239 ms after addition of
H2O2.
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EPR Spectrum of the Intermediate Species--
In order to follow
the radical center formed by the reaction of the F221W mutant with
hydrogen peroxide, we used the EPR spectroscopy at 5 and 77 K in the
presence of an equimolar amount of H2O2. In the
parent enzyme, a broad EPR signal characteristic of compound I,
oxyferryl
-cation radical (Fig.
3a), was gradually diminished with the same rate as restoration of the absorbance of the Soret band,
which corresponds to reduction of the porphyrin
-cation radical and
formation of compound II, oxoferryl species, inasmuch as compound II is
EPR silent. In contrast to that of the parent enzyme, the F221W mutant
exhibited a rather sharp EPR signal at g
= 2.036 and g
= 2.007 (Fig. 3, b and
c), characteristic of the Trp radical in CcP
compound I (g
= 2.037 and
g
= 2.005) (23-25). Although we have not yet
succeeded in the quantitative analysis for the EPR measurements after
mixing the mutant with hydrogen peroxide, the second reaction
intermediate in which the Soret peak appeared at 419 nm seems to afford
these EPR signals characteristic of the Trp radical. These signals lost
their intensity with time and the radical center was quenched within 1 min after mixing with hydrogen peroxide (data not shown). On the other
hand, the absorption spectra of the mutant in 1 min after the mixing had the Soret peak at 419 nm (results not shown), indicating that the
heme iron is still in the oxyferryl state as found for native HRP (1).
These results indicate that the mutant HRP has another radical center
such as the Trp radical in CcP compound I.

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Fig. 3.
EPR spectra of wild-type compound I
(a) and F221W compound I (b-d). These
spectra were taken at 5 K (a-c) and 77 K (d)
over a range of 400 G. Microwave power was approximately 5 mW for
a, 1 mW for b, and 0.1 mW for c and
d. Each spectrum was from four accumulations of 60 s
duration.
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Despite the close similarity of the EPR signal between F221W HRP and
native CcP, some significant differences were also detected. The EPR spectrum of the Trp radical of CcP undergoes
substantial spectral changes on warming to 77 K. At this temperature,
the EPR signal from the Trp radical was broadened, which is
attributable to the multiple radical species corresponding to the
thermally excited states (25, 26). However, the EPR signal from the Trp
radical of the mutant at 77K (Fig. 3d) was almost same as that at 5 K under the low microwave power (Fig.
3c).2 The
thermally excited states for the Trp radical in CcP were also manifested in the photoillumination EPR measurements. By repeating
illumination, a new and much more intense EPR signal with complex
structures emerged and the characteristic EPR signal disappeared again
after the sample had been warmed to 193 K for 20 min and cooled to 77 K
(25). The reversible formation of the radical center on the Trp residue
via an intermediate species by illumination also suggests the possible
existence of several thermally excitable energy levels near the ground
state of the Trp radical in CcP (25). The EPR spectrum of
F221W HRP mutant, however, was almost insensitive to heating and
photoillumination (results not shown). Another distinct difference of
the EPR spectra between the two proteins is the power dependence of the
EPR signal. The EPR signal from the Trp radical of the F221W mutant was
significantly saturated at 0.1 mW (Fig. 3b), whereas more
microwave power was required to saturate the signal from
CcP.3 This is
interpreted by a longer spin lattice relaxation time (T1) (27) and weak interactions between the
radical and oxyferryl heme (28) in the HRP mutant.
1H NMR Spectroscopy--
Although prominent changes in
the reactivity toward hydrogen peroxide and formation of the another
radical center were induced by the mutation at Phe-221, the structural
alterations monitored by the hyperfine-shifted NMR of the heme
peripheral groups and amino acid residues near the heme iron were less
drastic (Fig. 4). For the ferric high
spin wild-type HRP, the resting state of peroxidases, the four peaks
from the heme peripheral methyl groups were observed at 84, 77, 73, and
56 ppm (5-, 1-, 8-, and 3-methyl groups, respectively) (29, 30). In the
spectrum for the F221W mutant, the corresponding peaks were slightly
shifted (80, 77, 70, and 55 ppm). A single resonance assignable to the N1H of the proximal His (His-170) in the mutant was also
observed with a slight shift at 99 ppm, compared with that (101 ppm) of wild-type HRP. These results suggest that the heme environmental structure of the F221W mutant is not altered largely by the
mutation.

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Fig. 4.
1H NMR spectra of ferric
wild-type HRP (a) and F221W mutant (b).
One-dimensional 1H NMR spectra of resting state wild-type
HRP (a) and resting state F221W HRP (b), showing
that the spectra were recorded at 500 MHz with solution condition of 50 mM sodium phosphate, pH 7.0 at 17.0 °C. TMS,
tetramethylsilane.
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Quantitative Analysis for Calcium Ion Bound in HRP--
It should
be also noted here that calcium ion bound near the heme in HRP is one
of the distinct structural difference between the two peroxidases, HRP
and CcP. HRP has one calcium ion at both of the proximal and
distal site, whereas none is contained in CcP. The bound
calcium ion in HRP stabilizes the protein structure and enhanced the
catalytic reactivity of HRP (31-34), whereas, in CcP, the
absence of the calcium ion is essential for the formation of the stable
radical center at the Trp residue (35, 36). Mutations near the heme
distal sites of HRP resulted in the loss of the bound calcium ion
(distal calcium) and the severe functional defects (37). In order to
confirm the binding of calcium ions in the mutant, we used ICP
measurement (38). The results from these experiments clearly showed
that the mutant can bind 2 mol of calcium ion/mol of mutant protein, as
found for wild-type HRP (Table I).
Peroxidase Activity--
In order to clarify the effect of the
mutation on catalytic oxidation activities for guaiacol and ABTS, their
activities under the steady-state condition were investigated. Relative
oxidation activities for guaiacol and ABTS were determined by observing the formation of the oxidation product of guaiacol and ABTS at 470 and
405 nm, respectively (14). Although the activities of the wild-type
enzyme were virtually the same as those reported previously (14),
significant depression of the activities (the remaining activities were
about 30% of the wild-type enzyme) were observed for both of the
substrates in the mutant as summarized in Table
II.
To gain further insights into the effect of the mutation on the
catalytic activity of HRP, we estimated the elementary reaction rates
in the catalytic cycle. The rate of formation of compound I was
determined by monitoring the decay of the absorption at 395 nM, an isosbestic point between compound I and compound II for the wild-type enzyme. The value found with the mutant was k1 = 1.0 × 107
M
1·s
1 at pH 7.0 and 25 °C,
which is virtually the same as that of the parent enzyme
(k1 = 1.2 × 107
M
1·s
1) (Table
III). The radical transfer rate from the
porphyrin ring (kET), which corresponds to the
electron transfer rate to the porphyrin ring, was monitored at 417 nm
(Fig. 5), the isosbestic point between
the ferric state and the oxyferryl species. The time course can be
fitted by a single exponential as shown in Fig. 5, and the rate was
estimated to be 65 s
1. The reduction of compound II to
the ferric state (k3 process) was examined in
the reaction of compound II with guaiacol or ferrocyanide which is an
electron donor to HRP. The k3 values for
guaiacol and ferrocyanide in the wild-type enzyme were 2.5 × 105 M
1·s
1 and
7.9 × 104
M
1·s
1, respectively,
corresponding to those in the native enzyme (39). As listed in Table
III, the k3 values for the reaction of the F221W mutant with both of the two substrates were depressed to about 30% of
that for the wild-type enzyme.

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Fig. 5.
Absorbance change at 417 nm, isosbestic
point, after F221W mutant and hydrogen peroxide are mixed. The
concentration of enzyme and hydrogen peroxide were 2 and 20 µM, respectively. The upper panel of the
figure shows the residuals from the best one-experimental fit to the
time course.
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Redox Potential of Fe2+/Fe3+
Couple--
Redox potential of Fe2+/Fe3+
couple is also one of the factors reflecting the formation of compounds
I and II in the peroxidases (40). The monitored electrode potentials
against the ratio of the reduced form to the total amount of enzyme
were fitted well by the theoretical Nernst equation (Equation 1) (data
not shown). The midpoint potentials of the wild-type enzyme (
261 mV)
was almost identical with that of HRP reported by Yamada et
al. (41). The redox potential of the F221W mutant (
178 mV) was
significantly higher than that of the parent enzyme and rather close to
that of native CcP (
182 mV).
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DISCUSSION |
Characterization of the Second Intermediate in the Reaction of the
F221W HRP Mutant with Hydrogen Peroxide--
The time course of the
absorbance during the reaction between HRP F221W and hydrogen peroxide
was characterized by the initial decrease in absorbance changes, which
was followed by the increase in absorbance at the Soret band. These
spectral changes were also encountered for native HRP, which
corresponds to the successive formation of the reaction intermediates,
compounds I and II. In the mutant, however, the life time for compound
I was extremely short and very rapid restoration was observed for the
absorbance of the Soret band. Another distinct difference between
wild-type and mutant HRP was found in their EPR spectra. In the
wild-type enzyme, a broad EPR signal characteristic of porphyrin
-cation radical appeared by addition of hydrogen peroxide (Fig.
3a) and gradually diminished by formation of compound II.
The F221W mutant, however, exhibited a completely different EPR signal
from that of the wild-type enzyme with the restoration of the
absorbance of the Soret band. Although the quantitative analysis of the
time course for the EPR spectrum of the F221W HRP mutant in the
presence of hydrogen peroxide has not been successful, the EPR signal
appeared concomitant with the restoration of the absorbance at the
Soret band, suggesting that the second reaction intermediate still has a radical center, as found for CcP (7-9). The shape and
g-values for the EPR signal of the mutant were also quite
similar to those of the Trp radical in CcP compound
I.4 A tyrosine residue, which
is also susceptible to the oxidation (19), would be another candidate
for the oxidized residue, but the EPR signal is quite different from
that observed for a CcP mutant, W191F (19). It is,
therefore, concluded that the second reaction intermediate in the
reaction of the F221W HRP mutant with hydrogen peroxide has a Trp
radical with FeIV as found for CcP compound
I.
The x-ray structure for wild-type HRP has revealed that HRP originally
has one Trp residue at position 117 between the D and D' helix (12).
However, the distance between Trp-117 and heme periphery is about 12 Å, and rapid mixing EPR experiment showed that this Trp residue never
formed a stable intermediate such as the Trp radical for CcP
compound I in catalytic cycle. It is highly unlikely that the large
structural changes are induced by the mutation we introduced in this
study and the distance between Trp-117 and heme periphery is
drastically shortened, inasmuch as the perturbations on the NMR
spectrum by the mutation were moderated.5 Moreover,
according to the x-ray structure for wild-type CcP, Trp-51
is also close to the heme active site (10). Although the Trp-51 residue
lies almost the same distance as the Trp-191 and parallel to the heme
periphery, a previous study has unambiguously identified that the
radical site can only be Trp-191, not Trp-51 (42). This finding in
CcP also supports the absence of the radical at Trp-117 in
HRP. It is, therefore, the Trp residue introduced by the mutation and
located near the heme periphery (5 Å) that is oxidized to form the Trp
radical as is Trp-191 in CcP.
Although the present spectral data clearly showed that both of F221W
mutant HRP and CcP have a radical center on the Trp residue near the heme, the environment of the radical center is significantly different between the two peroxidases. A characteristic feature for the
Trp radical in the F221W HRP mutant is the rapid decay of the EPR
signal, which completely disappears within 60 s. On the other
hand, the Trp radical in CcP is quite stable and survives for more than 5 min in the absence of the substrate, cytochrome c. One of the structural factors responsible for the
unstable Trp radical in the HRP mutant would be the existence of a
cation near the Trp residue. A mutagenetic study on the CcP
has demonstrated that the introduction of the cation binding site into
the proximal site significantly enhanced electrostatic potential near
the Trp residue, resulting in the destabilization of the radical on the Trp residue (33). In the F221W mutant, the structural effects of the
mutation was localized near the mutation site and two calcium ions are
still bound as revealed by means of ICP measurement, implying the
electrostatic potential around the radical on the Trp is much higher in
the HRP mutant than that in CcP and the enhanced
electrostatic potential would destabilize the radical on the Trp
residue as found for the CcP
mutant.6
Another factor destabilizing the Trp radical would be the orientation
of Trp-221. On the basis of the x-ray crystal structure of
CcP (10), Trp-191 is in van der Waals contacts with Met-230 and Met-231 and is hydrogen-bonded with Asp-235 to fix the indole ring
at the optimum position for the radical formation. In fact, several
CcP mutants (M230L, M230I, M230Y, M231L, and D235N), in which these interactions are perturbed by the mutations (16, 18),
exhibited unstable Trp radicals. In the present work, the HRP mutant
would not form hydrogen bonds or van der Waals contacts to fix the Trp
residue at the optimal orientation as found for CcP, because
there are no amino acid residues to interact with the Phe-221 in HRP
(12). Therefore, Trp-221 in the HRP mutant is not fixed at the optimum
orientation as is Trp-191 in CcP, which eventually
destabilizes Trp-221 radical.
The temperature and power dependence values of the EPR signal from the
Trp radical in the HRP mutant are also indicative of the different
environment for the Trp radical from that in CcP. The long
spin-lattice relaxation time observed for the HRP mutant was also
encountered for the Tyr radical formed in the W191F CcP mutant (12). Because the Tyr residue trapping the radical in the mutant
CcP is located at more than 10 Å from the heme iron, the
weak electronic interaction results in the long spin-lattice relaxation. Although the distance from the heme iron to Trp-221 in the
mutant would be comparable to that from the heme to Trp-191 in
CcP, the configurational changes of the indole ring such as alteration of the orientation may affect the interaction with the heme.
The different environment around the Trp radical in the HRP mutant from
that in CcP is manifested in the EPR spectral changes upon
photo-illumination and heat up. As previously reported, the
photoillumination-dependent EPR spectrum of the Trp radical would correspond to several thermally excitable energy levels near the
ground state of the Trp radical in CcP and broadening at 77 K also supports the thermally excited multiple radical center (25, 26).
In a sharp contrast to the Trp radical in CcP,
photoillumination and heating did not significantly affect the EPR
spectrum of the HRP mutant,7
supporting the notion that the environmental structure and interactions with adjacent amino acid residues near the Trp radical are different in
the HRP mutant and CcP.
Radical Transfer from the Porphyrin Ring to the Trp
Residue--
As discussed in the previous section, the F221W HRP
mutant has a radical center in Trp-221 as the second reaction
intermediate in the reaction with hydrogen peroxide. Because the rapid
decrease of the absorbance at the Soret band immediately after addition of hydrogen peroxide unambiguously indicates the formation of the
oxyferryl-porphyrin
-cation radical at the first step of the
reaction of the mutant with hydrogen peroxide (43, 44), the radical
center would be transferred from the porphyrin ring to the Trp residues
to yield oxyferryl-Trp radical species as the second intermediate.
It is quite interesting to compare the present results with those from
the complimentary mutation in CcP, a HRP-like CcP
(9, 45). The HRP-like CcP has a phenylalanine residue at the
position of Trp-191 in which the radical center is located. Because a
phenylalanine residue is fairly resistant to the ring oxidation
compared with a tryptophan residue (12), the oxidation of Phe-191 was
not detected. Instead of the oxidation of the Phe residue, a transient porphyrin
-cation was observed and the radical center was
successively transferred to a tyrosine residue located 11 Å from the
heme iron, which affords a typical EPR signal of a tyrosine radical.
The results from the complimentary two mutants clearly showed that the
oxidation sensitive residue, Trp residue, near the heme periphery plays
crucial roles in the formation and transfer of the radical center
generated in the reaction of peroxidase with hydrogen peroxide.
It should be noted here that the radical transfer from the porphyrin
ring to the Trp residue would be also facilitated by the
destabilization of the
-cation radical on the porphyrin ring. As the
previous studies have proposed, the high valent porphyrin species such
as oxyferryl porphyrin
-cation radical are stabilized by the
electronic donation from the anionic axial ligands (46, 47). Because
the increase of the electronic donation has been considered to be
correlated with the decrease of the redox potential between ferric and
ferrous states (48), the redox potential for
Fe2+/Fe3+ couple serves as an indicator for the
stability of the high valent porphyrin species. In fact, heme enzymes
having porphyrin
-cation radical as the reaction intermediate such
as HRP (
250 mV) (41), catalase (
500 mV) (49). and P-450cam (
170
mV) (48), have their redox potentials much lower than that of human
myoglobin (+50 mV) (47) and sperm whale myoglobin (+55 mV) (50). For the F221W mutant, the redox potential of the
Fe2+/Fe3+ couple (
178 mV) was much higher
than that for the wild-type enzyme (
260 mV), rather close to that of
CcP (
182 mV). The higher redox potential corresponds to
the decreased electron donation from the axial ligand, which leads to
the destabilization of the high valent porphyrin species, oxyferryl
-cation radical. In the F221W mutant having the oxidation sensitive
Trp residue near the heme periphery, the destabilized porphyrin
-cation radical would readily be transferred to the Trp residue,
resulting in formation of the radical center on the Trp residue.
The radical transfer from the porphyrin ring to the Trp residue would
shed light on the reaction mechanism of CcP. Despite extensive efforts to detect the radical transfer from the porphyrin ring to Trp-191, neither radical transfer nor porphyrin
-cation radical was confirmed in the wild-type enzyme. Although the formation of the porphyrin
-cation radical in CcP was supported by
use of the HRP-like mutant, W191F CcP (9, 45), the radical
transfer has not yet been shown in peroxidases. In this paper, we have demonstrated the radical transfer in HRP, of which structural features
are quite similar to that of CcP, strongly suggesting the
radical transfer from the porphyrin ring to the Trp residue in
CcP. As discussed in the previous section, however, the Trp radical in the HRP mutant is quite unstable, compared with that in
CcP. In other words, the radical transfer from the porphyrin ring to Trp-191 in CcP would be much faster than that in the
HRP mutant we obtained here, which has prevented us from directly observing the porphyrin
-cation radical and the radical transfer to
the Trp residue in CcP.
Reaction Mechanism of the F221W HRP Mutant with Hydrogen
Peroxide--
In the reaction of the F221W HRP mutant with hydrogen
peroxide, an additional intermediate having a radical center on the Trp
residue near the heme periphery was observed for the reaction cycle.
The reaction of the mutant with hydrogen peroxide, therefore, can be
explained by the mechanism shown by Scheme I.
The species denoted IA is an oxyferryl porphyrin
-cation radical as found for native HRP. The species IB
has a similar absorbance spectrum to that of compound II in native HRP,
but the EPR signal was characteristic of the Trp radical. The EPR
signal was diminished within a couple of minutes, whereas the
absorption spectrum is still the HRP compound II-type (species
IC). These results indicate that the species IB
is Trp radical and the species IC is HRP compound II. In
the presence of the substrate, the species IC was reduced back to the ferric resting state.
The kinetics for the steady-state and elementary reaction revealed the
rate constants for the formation of these intermediates. The value of
k1, the formation rate of the porphyrin
-cation radical for the mutant. is not affected by the mutation,
implying that both the deprotonation of hydrogen peroxide and the
cleavage of the O-O bond are not affected by the mutation. As our
previous studies have shown, the deprotonation and the O-O bond
cleavage depend on the basicity and orientation of the distal histidine (51), which is located at the opposite site of Phe-221. The NMR
spectrum of the F221W mutant also indicated only subtle structural perturbations around the heme moiety. It is therefore likely that the
structural perturbation by the mutation at Phe-221 is confined in the
mutation site at the proximal site, resulting in minor effects on the
k1 process.
The second step in the reaction of the mutant with hydrogen peroxide is
the formation of the Trp radical, which corresponds to the electron
transfer from the Trp residue to the porphyrin ring
(kET process). Although in the native
CcP the electron transfer rate from the Trp residue to the
porphyrin ring was not determined due to the extremely rapid oxidation
of Trp-191, a HRP-type CcP mutant (W191F) upon the reaction
with H2O2 showed the formation of a transient
porphyrin
-cation radical, followed by the formation of the tyrosine
radical (Tyr-236) (9, 45) and its formation rate was determined to be
54 s
1 by increase of the absorption at the Soret band. In
the mutant HRP, the formation rate of the Trp radical
(kET) determined by the absorption spectral
change was 65 s
1.
To estimate the value of k2, we have tried to
follow the time course of the EPR signal from the Trp radical, inasmuch
as the absorption spectrum was completely insensitive to the
disappearance of the radical. Unfortunately, however, the time-course
experiments by using EPR spectroscopy have not yet been successful due
to the limitation of the amount of the mutant protein and complexity in
the quantitative analysis for the EPR signal.
In reduction of compound II to the resting state,
k3 process, the reduction rate was decreased to
about 30% of that for the wild-type enzyme for both of the substrates,
guaiacol and ferrocyanide. Although the depression for the
k3 process was significant in the mutant, the
effects of the amino acid substitution at Phe-221 on the
k3 process would be rather small. Some mutations
around the heme active center severely inhibit reduction of compound II
and 10-30-fold decrease in k3 was observed
(14). In the reaction of the HRP mutant with hydrogen peroxide, the low
stability of compound I and successive intermediates prevent us from
confirming that the mutant quantitatively reacts with hydrogen peroxide
to form compound I and the absorption spectrum for compound II of the
mutant suggested that significant amount of the mutant enzyme was
inactivated during the reaction, probably due to the nonspecific radical transfer from the Trp radical. It is, therefore, likely that
the depression for the k3 process for the F221W
mutant can be attributed to the loss of the enzyme in the catalytic
reaction and the reactivity of the oxyferryl species in the mutant is
virtually the same as that of the wild-type enzyme. In the reaction of
the W191F CcP mutant with ferrocyanide, Kraut and co-workers
(8) also found a similar moderated depression for the
k3 process and concluded that the stability and
reactivity of the oxyferryl center in the CcP mutant would
be relatively unaffected by the mutation at Trp-191.
Compared with the reaction of native HRP with hydrogen peroxide (44),
the most prominent difference is the insertion of the radical transfer
process, kET process, to the reaction cycle in
the mutant. Although k2 has not been determined
in this study, the perturbations for the reaction rate constants of HRP
by the mutation were rather small. In other words, the activity of the HRP mutant with hydrogen peroxide is comparable to that of native HRP
and the mutation at Phe-221 to Trp can trap the radical from the
porphyrin ring, without affecting the reactivity of the heme iron.
These observations indicate that the Trp residue near the heme
periphery is crucial for the location of the radical center formed by
the reaction with hydrogen peroxide, which differentiates the function
of peroxidases; CcP uses the Trp radical for the effective
oxidation of the substrate protein, cytochrome c, and a
porphyrin
-cation radical would have some advantages in the oxidation of the aromatic substrates for HRP, because the aromatic substrates are small enough to interact with the porphyrin ring directly (1, 52-56).
Conclusions--
In summary, we have detected the Trp-221 radical
as the second intermediate species in the reaction of the F221W mutant
with hydrogen peroxide and demonstrated the radical transfer from the porphyrin ring to the Trp residue near the heme active center. This
result clearly indicates that the proximal Trp near the heme active
site is a key amino acid residue to control the location of the radical
center in peroxidase compound I and differentiate functions in
peroxidases. However, the Trp radical is unstable due to lack of the
interactions to fix the indole ring of the Trp residue at the optimal
orientation and presence of a cation (calcium ion) locating near the
Trp residue.
 |
ACKNOWLEDGEMENTS |
We are thankful to Dr. Kiyohiro Imai (Osaka
University) for assistance with the stopped-flow experiments. We are
also thankful to Dr. Shigeo Umetani (Kyoto University) for assistance
with the ICP experiments.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid 07309006 and 08249102 for Scientific Research (to I. M.) from the Ministry of Education, Science, Culture and Sports.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by research fellowships from the Japan Society for the
Promotion of Science for Young Scientists.
To whom correspondence should be addressed. Tel.:
81-75-753-5921; Fax: 81-75-751-7611; E-mail:
morisima{at}mds.moleng.kyoto-u.ac.jp.
1
The abbreviations used are: HRP, horseradish
peroxidase isozyme C; wild-type HRP, recombinant horseradish peroxidase
isozyme C expressed in E. coli; CcP, cytochrome
c peroxidase; Cyt c, cytochrome c;
ABTS, 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt; ICP, inductively coupled plasma; mW, milliwatt(s).
2
We measured the EPR spectrum of the F221W mutant
at 15 K, which is also similar to that at 5 and 77 K. The EPR pattern
was almost insensitive to temperature between 5 and 77 K, which is quite different from that of CcP (25).
3
We also examined the microwave power dependence
of the EPR signal from the Trp radical in the F221W mutant from 0.01 mW
to 10 mW. The saturation microwave power for the Trp radical in
CcP was much higher (H. Hori and T. Yonetani, unpublished
result).
4
The peroxy radical localized on tryptophan in
myoglobin also affords the similar EPR signal (57, 58). In this point,
we performed the same experiment under the condition which completely excluded the molecular oxygen by the reported method (58). The EPR
signal observed in anaerobic system is almost identical to the
characteristic signal of the Trp radical spectrum of the F221W HRP
mutant (g
= 2.036 and
g
= 2.007) in the aerobic system. Thus, the
EPR spectrum of the F221W HRP mutant is not due to a tryptophan peroxyl
radical as formed in myoglobin.
5
To confirm that the Trp newly introduced by the
mutation was oxidized by addition of hydrogen peroxide, we also
performed the amino acid analysis, because native HRP has one naturally occurring Trp. Unfortunately, however, the amino acid analysis for Trp
residue of the mutant HRP has not been successful, due to the low
sensitivity of Trp residue.
6
To examine the effect of the calcium ion binding
on the stability of the Trp radical, we have tried to remove the
calcium ion from the F221W mutant (26, 27). However, the mutant protein was so unstable to removal of the calcium ion that it was precipitated by denaturation.
7
However, the EPR signals from the Trp radical in
the F221W mutant showed the microwave power dependence (Fig. 3,
b and c). The signal broadening at
g
= 2.036 in the F221W mutant might correspond to the saturation broadening and/or multiple components of
the signals. Therefore, we cannot rule out the possibility that the Trp
radical in the HRP mutant has a multiple components under the high
power condition, but it is safely said that the radical character of
the HRP mutant is quite different from that of CcP.
 |
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