(Received for publication, March 28, 1995; and in revised form, June 6, 1995)
From the
A gene coding for the F172Y mutant of horseradish peroxidase
isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperda (SF-9) and Trichoplusia ni egg
cell homogenate (HighFive
Hemoprotein peroxidases catalyze the
H The first step of the normal catalytic sequence is reaction of the
heme prosthetic group of the peroxidase with H A high
resolution crystal structure is not available for HRP but such
structures are available for CcP(13) ,
LiP(14, 15) , and the peroxidase from Arthromyces
rhamosus (or Coprinus cinereus) (16, 17) . However, alignment of the highly conserved
sequences of HRP and the three crystalline peroxidases suggests that
the structure of HRP is very similar to that of the other
peroxidases(13, 14, 15, 16, 17, 18, 19) .
These alignments have tentatively identified the proximal
(His We have expressed a
synthetic HRP gene in a baculovirus/insect cell system and are carrying
out site-specific mutagenesis studies of the structure and function of
this enzyme(24, 25, 26) . We demonstrate here
that reaction of H
An equal molar mixture of oligonucleotides (55 pmol) in ligation
buffer (66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl
The media was harvested 4-6 days
post-infection. Following removal of the cells by centrifugation, the
media was concentrated to
Alignment of the HRP amino acid sequence with the sequences
of three peroxidases for which crystal structures are available
indicates that Phe
Figure 1:
Superposition of key active
site residues in the crystal structures of CcP and LiP. The catalytic
histidine (CcP, His
The protein was purified to homogeneity from
baculovirus infected HighFive
Figure 2:
SDS-polyacrylamide gel electrophoresis
(12% gel) analysis of the F172Y HRP mutant purified as reported in Table 1. Lane 1, molecular mass standards in kDa; lane 2, medium after dialysis and centrifugation; lane
3, eluate from S-Fast Flow column; lane 4, eluate from
Q-Fast Flow column; lane 5, native HRP
standard.
Figure 3:
pH
profile for the oxidation of guaiacol by wild type and F172Y HRP.
Slightly different protein concentrations were used in these assays to
clearly differentiate the two sets of data.
Figure 4:
Spectra of wild type F172Y HRP at
ambient temperature before addition of H
The rate of Compound I formation for F172Y
as determined by stopped flow methods was k In contrast to native HRP which has a
stable Compound I, (
Figure 5:
Rate of decay of the ferryl porphyrin
radical cation (Compound I) of F172Y HRP as a function of pH as
determined by stopped flow absorption spectroscopy. The buffer used to
obtain the individual values is indicated: acetate buffer (▪),
phosphate buffer (▴), and Tris buffer (
The same value for the rate is obtained in the presence or
absence of 5.0 mM Ca
Figure 6:
EPR spectra at 10 K of F172Y HRP (A) and wild type, recombinant HRP (B) which were
frozen 5 min after addition of 1.3 equivalents of
H
The narrow signal observed at 6 K for F172Y is consistent
with an isolated S = 1/2 radical on a tyrosine residue.
The lineshape appears isotropic with a g value near 2 as
expected for an isolated S = 1/2 spin, and does not show
the characteristic broadening that is observed for the porphyrin
Figure 7:
EPR
spectra at 6 K of F172Y HRP (A) and recombinant wild type HRP (C) 30 s after the addition of 1.3 equivalents of
H
At 2.3 K, the porphyrin
radical cation of a conventional Compound I structure is observed with
both native and F172Y HRP (Fig. 8). The signals are broad, weak,
and very temperature dependent and are therefore difficult to
quantitate accurately. Nevertheless, the porphyrin radical cation
signals are of approximately equal intensity for both native and F172Y
HRP, but the porphyrin radical cation of the F172Y mutant appears to
decay more rapidly. Very little residual ferric HRP or F172Y HRP is
detected by EPR, showing that 1.3 equivalents of H
Figure 8:
EPR spectra at 2.3 K of the porphyrin
radical cations obtained from native HRP (A) and F172Y HRP (B) 30 s after reaction with 1.3 equivalents of
H
A gene coding for the F172Y mutant of HRP has been expressed
via a baculovirus vector in Spodoptera frugiperda and T.
ni cells. The spectra of the mutant protein purified from the
incubation medium in the ferric and ferric cyano states are essentially
identical to those of the corresponding spectra of either native HRP or
recombinant, wild type HRP. The catalytic activity of the F172Y mutant
toward guaiacol peroxidation, as revealed by K Striking differences are
observed in the activated states of the F172Y mutant despite its high
degree of structural identity with the active site of native HRP.
Reaction of the F172Y mutant with one equivalent of
H One possible
explanation for these data is that there are two populations of F172Y
that differ in extent of saturation of the calcium-binding sites on the
protein (32, 44) and that insufficient calcium
saturation of one of the two populations makes it less stable and gives
rise to the observed instability of Compound I. This possibility is
ruled out by the demonstration that the rates of Compound I formation
and decay, and the amplitude of the protein EPR signal, are insensitive
to the presence or absence of 5 mM Ca The results clearly show that oxidation of F172Y
HRP by H The
present results indicate that the presence or absence of a highly
oxidizable residue vicinal to the heme group is a major factor in
determining whether the second oxidation equivalent required for the
peroxidase-catalyzed reduction of H The
balance between porphyrin and protein oxidation is physiologically
relevant and can be observed in intact peroxidases. Thus, oxidation of
lactoperoxidase yields a porphyrin radical cation species that is
transformed with time into a CcP-like protein
radical(46, 47, 48) . Evidence for this
transformation includes the demonstration by titration with
ferrocyanide that two oxidizing equivalents are retained in the second
species despite its Compound II-like spectrum and the observation that
the initial species catalyzes the iodination of tyrosine and the
dimerization of iodotyrosine, whereas the second Compound I species
only catalyzed the dimerization reaction.
) cells. Homology modeling
with respect to three peroxidases for which crystal structures are
available places Phe
on the proximal side of the heme in
the vicinity of porphyrin pyrrole ring C. The pH optimum and
spectroscopic properties of the F172Y mutant are essentially identical
to those of wild type HRP. V
values show that
the mutant protein retains most of the guaiacol oxidizing activity.
Stopped flow studies indicate that Compound I is formed with
H
O
at the same rate (k
= 1.6
10
M
s
) at both pH 6.0 and 8.0 as it is with the
wild type enzyme. This Compound I species decays rapidly at a rate k
= 1.01 s
, pH 7.0, to a
second two-electron oxidized species that retains the ferryl (Fe
= O) absorption. EPR studies establish that a ferryl
porphyrin radical cation is present in the initial Compound I, but
electron transfer from the protein results in formation of a second
Compound I species with an unpaired electron on the protein (presumably
on Tyr
). The presence or absence of oxidizable amino
acids adjacent to the heme is thus a key determinant of whether the
second oxidation equivalent in Compound I is found as a porphyrin or
protein radical cation.
O
-dependent one-electron oxidation of proteins
and/or small organic or inorganic molecules(1, 2) .
Horseradish peroxidase isozyme C (HRP) (
)catalyzes the
oxidation of small substrates to free radical products, most notably
the conversion of phenols to phenoxy radicals(3) . In contrast,
cytochrome c peroxidase (CcP) only slowly oxidizes small
molecules but very efficiently oxidizes cytochrome c, a 12-kDa
protein that is its natural substrate(4, 5) . The
catalytic mechanisms of these two prototypical peroxidases have been
extensively studied, and the mechanistic understanding thus obtained
provides the foundation for current views on the structure and function
of all hemoprotein peroxidases, including myeloperoxidase(6) ,
eosinophil peroxidase(7) , and thyroid peroxidase(8) .
O
to give a two-electron oxidized species known as Compound
I(2, 3) . Compound I is reduced to Compound II by a
substrate-derived electron and to the resting ferric state by a second
electron. All known hemoprotein peroxidases have a common Compound II
structure in which the single oxidation equivalent is stored as a
ferryl (Fe
= O) species(1, 2) .
The same ferryl moiety is present in Compound I, but there are two
possible sites for the location of the second oxidation equivalent.
Diverse spectroscopic and physical measurements, including magnetic
circular dichroism, electron nuclear double resonance, magnetic
susceptibility, and resonance Raman spectroscopy, clearly establish
that the second oxidation equivalent in Compound I of HRP is stored as
a porphyrin radical cation(2) . The HRP Compound I structure
therefore can be represented as
[Por
Fe
= O]. In
contrast, the second oxidation equivalent in the case of CcP is stored
as an unpaired electron on a tryptophan residue (Trp
)
located on the proximal side of the heme(9) . The CcP Compound
I structure therefore can be represented as
[Prot
Fe
= O]. Some
of the structural features that promote the formation of a protein
radical in CcP have been
determined(10, 11, 12) , but the factors that
stabilize a porphyrin radical cation in HRP and other peroxidases with
respect to oxidation of nearby residues remain obscure.
) and distal (His
) histidines and the
arginine (Arg
) of HRP suggested by the structures of the
crystalline peroxidases (13, 14, 15, 16, 17) to be
involved in the formation and stabilization of Compounds I and II.
Sequence alignments have also been used, in conjunction with NMR data,
to tentatively identify residues in the vicinity of the heme group. An
aromatic residue adjacent to the 1- and 8-methyl groups of the heme was
initially identified by such arguments as
Tyr
(20, 21) , but more recent NMR
studies suggest that the residue is a phenylalanine rather than a
tyrosine(22, 23) . (
)
O
with the F172Y mutant of
HRP, in which a tyrosine has been introduced into the structure near
the heme group, initially yields a normal Compound I intermediate. The
porphyrin radical cation of this Compound I, however, oxidizes the
protein to give a CcP-like Compound I in which the second oxidation
equivalent is present as a protein radical. The results demonstrate
that one of the parameters that determine whether the second oxidation
equivalent is on the porphyrin or the protein is the presence or
absence of oxidizable residues near the heme.
Materials
Guaiacol was purchased from Sigma. HRP
(grade 1) was from Boehringer Mannheim. Restriction enzymes were
purchased from New England Biolabs (Beverley, MA) or Boehringer
Mannheim. S Fast Flow and Q Fast Flow resins were from Pharmacia.
Oligonucleotides were synthesized at the University of California, San
Francisco, Biomolecular Resource Center using an Applied Biosystems
380B DNA synthesizer. Heat-inactivated fetal calf serum, penicillin,
streptomycin, and Escherichia coli strain DH5 were
obtained from the University of California, San Francisco, Cell Culture
Facility. DNA miniprep columns were from Promega (Madison, WI). All
insect cell culture media contained 100 µg/ml streptomycin sulfate
and 100 units/ml penicillin ``G.'' Guaiacol peroxidation and
thioanisole sulfoxidation activities were assayed as reported elsewhere (26) .
Construction of the F172Y HRP Gene
The F172Y
mutant gene was constructed by cassette mutagenesis between the BspEI and BstEII restriction sites of a
synthetic HRP gene (British Biotechnologies, Ltd.). The cassette
sequence was identical to the gene sequence except it contained the
tyrosine codon TAC at amino acid position 172:
, 10 mM dithiothreitol, and 0.4 mM ATP) was annealed by heating at 100 °C for 3 min and cooling
slowly to room temperature over a period of 1 h. The plasmid pUC-HRP
containing the synthetic gene cloned into pUC19 was digested with BspEI and BstEII, gel purified, and ligated
with the annealed cassette. The ligation mixture was used to transform E. coli strain DH5
to ampicillin resistance. Positive
clones were screened by restriction digests and verified by DNA
sequencing. A 762-base pair SacI/NcoI fragment of the
HRP gene containing the mutation was then exchanged into the
baculovirus vector pVLHRP2 (24) which contains the same HRP
gene as well as the HRP 5`-leader sequence. Positive clones were
identified by restriction digestion. The purified plasmid DNA from one
clone (pVLHRPF172Y) was used for HRP gene incorporation into
baculovirus.
Production of Recombinant Virus
Recombinant virus
was generated in Sf9 cells growing in Hink's TNM-FH insect tissue
culture media (JRH Biosciences, Lenexa, KA) containing 10%
heat-inactivated fetal calf serum using the BaculoGold transfection kit (Pharmingen, San Diego). Plaque-purified virus
was used to infect cell monolayers in 3 ml of media. After 3 days the
medium was removed and used as first passage virus stock (pass 1
stock). Genomic viral DNA from 0.75 ml of this media was screened for
the presence of HRP gene insertion using polymerase chain reaction
procedures and primers from Invitrogen. HRP gene insertion was verified
by sequencing the polymerase chain reaction-amplified fragment. A
400-µl aliquot of pass 1 stock was then used to infect 1
10
cells in a 100-ml culture. After 4 days of growth, the
medium was harvested and used for large scale expression after the
viral titer had been determined.
Purification of F172Y HRP
Recombinant enzyme was
expressed using Trichoplusia ni egg homogenate
(HighFive) cells (Invitrogen) growing in shaker flasks
at 27 °C with Sf-900 II SFM serum-free insect cell media (Life
Technologies Inc.). Pass 2 stock was added to 300 ml of T. ni cells (
2
10
cells/ml) in a
Fernbach culture flask to give a multiplicity of infection of 5. After
1 h at 27 °C the volume was increased to 800 ml with serum-free
media. Two h later, 10 ml of a 500 µM hemin solution was
added to the expression flask. The hemin solution was made by
dissolving 3.3 mg of hemin in 10 ml of 10 mM NaOH. Immediately
after the hemin was dissolved, the solution was carefully neutralized
with a few drops of 0.5 N HCl. The hemin solution was filter
sterilized before addition to the expression flask. Three days
post-infection another 10 ml of hemin solution was added to the
expression flask.
50 ml using a spiral channel
ultrafiltration apparatus (YM-10 membrane, Amicon, Beverley, MA). The
crude concentrated media was dialyzed against 10 mM NaOAc, pH
5.0, and applied to a column of S Fast Flow (1.5
40 cm)
equilibrated with 10 mM NaOAc, pH 5.0, buffer. The column was
washed with the same buffer (50 ml) and then eluted with 20 mM NaCl in NaOAc buffer. The fractions containing peroxidase activity
were pooled and dialyzed against 10 mM Tris, 5 mM
CaCl
, pH 7.5, buffer. The protein was then washed through a
small column (1.5
2 cm) of Q Fast Flow. HRP does not bind to
anion-exchange media under these conditions, but the remaining
impurities bound tightly. The pure enzyme was stored at -70
°C and lyophilized when higher concentrations of enzyme were
desired.
Stopped Flow Kinetics
Stopped-flow experiments
were performed on an Applied Photophysics Sequential DX-17 MV
stopped-flow instrument. The sample handling unit was fitted with two
drive syringes mounted inside a thermostated-bath compartment to allow
for variable temperature experimentation. A 1.0-cm path-length cell was
employed. First- and second-order curve fitting and rate constants were
calculated from the average of at least five traces, using a Marquardt
algorithm based on a routine
``curfit''(27, 28) . The absorption spectra
at indicated time points were calculated with software provided by
Applied Photophysics. This consisted of slicing the appropriate time
points across a series of kinetic traces at different wavelengths and
then splining the points of a specific time group(29) . The
concentrations of HRP and the F172Y HRP mutant in these studies were
between 1 10
and 5
10
M. The concentrations were calculated assuming the molar
absorptivity for the F172Y mutant is the same as that of native HRP
(
= 1.02
10
M
s
). The concentration of H
O
was either 10-fold that of either HRP or F172Y HRP for
pseudo-first-order conditions (i.e.k
for
HRP
Compound I), or the concentration was matched 1:1 to
H
O
-enzyme for second-order experiments for k
and the subsequent measurement of k
(i.e. for the decay of Compound I). All
experiments involving the formation of Compound I were followed at 402
nm, the largest absorbance change for this transition. All experiments
involving the conversion of Compound I to final product were followed
at 420 nm (greatest
A). All solutions contained 100
mM KCl buffered at a concentration of 25 mM with
either acetate, <pH 6, phosphate, pH 6-8, or Tris-OH, >pH
8. To solutions at pH 5.47, 7.1, and 8.56, Ca
(5.0
mM) was added to ensure that the calcium sites were filled.
EPR Spectra
A 1.3-fold molar excess of
HO
was added to solutions of either HRP (313
µM) or F172Y HRP (329 µM) in 25 mM Tris buffer, pH 7.0, containing 100 mM KCl and, for some
experiments, 5 mM Ca
. Within less than 30 s,
the samples were transferred to EPR tubes and were quickly frozen to 77
K. After the EPR spectra were recorded, the samples were quickly thawed
to 298 K, allowed to stand for 5 min, and refrozen to 77 K before again
recording their EPR spectra. Finally, the samples were thawed to 298 K
for 30 min before refreezing and recording the final set of EPR
spectra. EPR spectra were recorded in 3 mm O.D. quartz tubes on a
Bruker ESP300 spectrometer at X-band using 100 KHz field modulation.
Low temperature control was achieved by an Air Products LTR-3 Helitran
liquid helium transfer line and cryostat. Temperatures below 4 K were
achieved by maintaining a measured vacuum above the helium flow.
Background spectra on blank samples collected under identical
conditions were subtracted from each spectrum. EPR simulations were
performed using the program qpowder (30) on a 486/66 DX2
personal computer.
corresponds to Leu
in
CcP, Val
in LiP, and Leu
in the A. rhamosus peroxidase (31) . These residues are located
in all three crystal structures on the proximal side of the heme close
to pyrrole ring C (Fig. 1)(13, 14, 15, 16, 17) .
The sequence and structural relationships between HRP and the three
crystalline peroxidases suggest that Phe
occupies a
similar site in the HRP structure. We have therefore chosen to replace
Phe
with a tyrosine residue to determine the consequences
of placing a structurally similar but more oxidizable residue close to
the heme group. It should be noted that the residue equivalent to
Phe
in the peroxidase crystal structures does not
interact directly with the proximal histidine iron ligand or with its
hydrogen bonding network, so that a mutation of Phe
should not directly influence the axial ligand-heme
interaction(13, 14, 15, 16, 17) .
; LiP, His
) and arginine
(CcP, Arg
; LiP, Arg
) and the adjacent
aromatic residue (CcP, Trp
; LiP, Phe
) are on
the distal side above the heme plane. The proximal histidine iron
ligands (CcP, His
; LiP, His
) are below the
heme plane, as are Leu
of CcP and Val
of
LiP, the residues that correspond to Phe
of
HRP(14) . These latter residues are off the edge of, and below,
heme pyrrole ring C.
Expression and Kinetic Characterization of the F172Y HRP
Mutant
A gene coding for the F172Y HRP mutant was constructed by
cassette mutagenesis between the BspEI and BstEII restriction sites of a commercially available HRP gene.
The cassette sequence was identical to the gene sequence except for
substitution of the tyrosine codon TAC for the phenylalanine codon at
position 172. A 762-base pair SacI/NcoI fragment of
the HRP gene encompassing the mutated region was then placed into the
previously constructed baculovirus vector pVLHRP2(24) . This
vector contains the HRP gene and a 5`-leader sequence that directs
excretion of the protein into the medium. The mutated vector was
incorporated into baculovirus and was then used to express the F172Y
HRP protein. Viral stocks were obtained by expressing the mutant
baculovirus in Sf9 cells, but large scale protein expression was done
in shaker flasks with HighFive cells. The use of
serum-free media in the expression system simplified protein
purification.
cell culture media by ion
exchange chromatography (Table 1). The concentrated culture
medium was applied to a long thin column of the cation-exchanger S Fast
Flow at pH 5.0. Although binding of the protein to the column is not
optimal at this pH, F172Y HRP denatures at lower pH values. Following
elution with a small amount of NaCl (20 mM), the active
fractions were combined, equilibrated into pH 7.0 Tris buffer, and
passed through a plug of the anion-exchanger Q Fast Flow. HRP does not
bind to anion exchangers at neutral pH, but the remaining impurities
bind strongly and are easily removed. Ca
was added to
the neutral Tris buffer to help stabilize the protein(32) . The
F172Y HRP mutant was thus obtained as a pure protein, as judged by
SDS-polyacrylamide gel electrophoresis, in a yield of 15 mg/liter of
culture (Fig. 2). The R
value of
the purified protein was 2.9, and its specific activity, determined by
guaiacol oxidation, was 86 µmol min
mg
. The R
value
suggests that a small fraction of the protein does not contain a heme
prosthetic group because the R
value for
pure, native HRP is between 3 and 3.4 (3) . The kinetic
parameters for the oxidation of guaiacol by recombinant wild type HRP
and the F172Y mutant are similar: HRP, K
= 7.63 mM, V
=
0.71 nmol s
pmol
; F172Y, K
= 2.62 mM, V
= 0.41 nmol s
pmol
. The protein concentrations used for
these experiments were based on the Soret absorbance of the
catalytically active enzyme. The V
for the
oxidation of guaiacol by the F172Y mutant is thus approximately half
that for the wild type enzyme.
pH Profile for Guaiacol Oxidation
Mutations within
the active site of HRP may affect the pH optimum for guaiacol
oxidation. The pH profile for guaiacol oxidation was therefore examined
by determining the activity of the enzyme in buffers of different pH
values adjusted to an ionic strength of 0.1 with NaCl (Fig. 3)(33, 34) . No difference in pH profile
was found between wild type HRP and the F172Y mutant. The pH optimum is
approximately 6.4 for both wild type and F172Y HRP.
Thioanisole Sulfoxidation
Stereoselective
thioanisole sulfoxidation is catalyzed by native HRP, but the rate and
stereoselectivity of the reaction are increased in the F42V
mutant(25, 26) . The stereospecificity of thioanisole
sulfoxidation by F172Y HRP was therefore examined to determine if the
mutation alters the topology in the vicinity of the ferryl oxygen.
However, the enantiomeric excess obtained with F172Y HRP (ee 76%) is
similar to that observed with the wild type enzyme (ee 70%). These
results suggest that the distal active site pocket is not significantly
altered by the F172Y mutation.Spectroscopic Properties of the F172Y HRP
Mutant
The spectrum of ferric F172Y HRP is identical to those of
the native enzyme and the recombinant wild type protein. The same
spectroscopic identity characterizes the ferric cyano complexes of
native ( 362, 422, 538), recombinant wild type
(
362, 422, 538), and F172Y HRP (
362, 422, 540).
Formation and Decay of Compound I
Reaction of the
F172Y HRP mutant with one equivalent of HO
initially gives a Compound I spectrum that changes with time to a
Compound II-like spectrum (Fig. 4). Similar treatment of native
HRP gives a Compound I spectrum that does not decay significantly
within 1 h(42) .
O
(A), immediately after addition of one equivalent of
H
O
(B), and 5 min after the addition
of one equivalent of H
O
(C).
= 1.6
10
M
s
at both pH 6 (100 mM phosphate
buffer) and pH 8 (50 mM Tris buffer, 100 mM KCl).
This value is the same as those reported in the literature for native
HRP (k
= 1.0 to 2.0
10
M
s
)(35, 36, 37) .
This was determined using the equation rate
= k
[HRP] where k
= k
[H
O
] under
pseudo-first-order conditions.
)Compound I formed from F172Y decays and
the decay exhibits a modest pH dependence (Fig. 5). At pH 7, the
rate of decay in the absence of added substrate is 1.01 s
([F172Y] = 5.0
10
M) and, as shown by a linear dependence of rate
on the concentration of F172Y (not shown), follows the equation
rate
= k
[F172Y Compound
I].
). Some values
(
) were obtained in buffer used for the other point at the same pH
but in the presence of 5 mM Ca
.
. This suggests either
that the calcium site on the protein is filled even in the absence of
added calcium, or that the degree of saturation of this site does not
influence the rate of decay of Compound I.
EPR Studies
The decay of the Compound I
chromophore in the absence of an external electron donor suggests that
the electron required to reduce the ferryl species is provided by the
protein. EPR studies have therefore been carried out to determine
whether the decay of compound I is linked to the formation of a protein
radical. Indeed, a protein radical is detected when
HO
is added to F172Y HRP, and the sample is
then rapidly frozen to 10 K (Fig. 6A). Control
experiments show that an analogous protein radical signal is not
generated when native or wild type recombinant HRP is treated with
H
O
under the same conditions (Fig. 6B). The protein radical was quantitated by spin
integration with respect to a known Cu
standard under
conditions that showed no evidence of saturation for either sample. The
signal observed 30 s after the addition of H
O
corresponded to 11% of the F172Y mutant protein in the solution.
If the sample was warmed to ambient temperature for 5 min and then
refrozen, the signal intensity decreased to 6% of the total protein,
and decreased slightly to 5% of the protein if the sample was held at
ambient temperature for 30 min. To determine whether incomplete
saturation of the Ca
sites in the recombinant protein
contributed to radical formation and decay, the same experiment was run
in the presence of 5 mM Ca
. Essentially
identical results were obtained with the protein radical observed
corresponding to 11, 8, and 6% of the total F172Y HRP protein in the
sample.
O
. Addition, mixing, and 5 min of incubation
were performed in 3-mm quartz EPR tubes at ambient temperature before
freezing to 77 K for storage. Instrument conditions were 100 microwatts
microwave power at 9.51 GHz, 1 Gauss field modulation at 100 KHz, 328
ms time constant, and 1
10
receiver
gain.
-cation radical of HRP Compound I or the Trp
radical
of CcP Compound I. These broad anisotropic signals arise from exchange
coupling of the radical species with the S = 1 ferryl
center (10) . In addition, the narrow signal for F172Y becomes
saturated above 50 microwatts at 6 K, indicating that it is not
efficiently relaxed by the ferryl center as is observed for the
radicals of wild type HRP and CcP. Finally, the narrow signal observed
for F172Y exhibits a complex hyperfine structure (Fig. 7A). No evidence for such a signal is observed
with samples of recombinant wild type HRP (Fig. 7C),
demonstrating that this signal is not an artifact of heterologous
protein expression. A simulation of this signal, using the g value and proton hyperfine tensors that have previously been
established for tyrosine radicals in
proteins(38, 39, 40) , is shown in Fig. 7B. Such radicals typically exhibit a small g anisotropy and an anisotropic hyperfine coupling to the C
and C
ring protons that are not expected to vary
significantly with sidechain conformation. The great variability in
observed tyrosine radical spectra arises from differences in
conformation of the aromatic ring about the
C
-C
dihedral angle, which dramatically
alters the proton hyperfine couplings to the two methylene protons on
the C
carbon. Spectra were simulated for all possible
values of this dihedral angle and at several values for
, the unpaired spin density on the C
ring
carbon
-orbital. The simulated spectrum in Fig. 7B used a value of
= 0.37, which is that
found previously for the Y
radical of photosystem
II(40) , the g tensor determined for the E. coli ribonucleotide reductase tyrosine radical (G
,
,
= 2.0091, 2.0046, 2.0023)(39) , and values
for the hyperfine coupling tensors to the C
and C
protons as determined for E. coli ribonucleotide
reductase(38) . The simulation implies a conformation for the
tyrosine of HRP F172Y in which the two methylene protons make angles,
= 20° and
=
140° with the tyrosine ring normal.
O
, acquired under conditions that show
hyperfine structure. Instrument conditions: 10 microwatts microwave
power at 9.51 GHz, 5.2 Gauss field modulation at 100 KHz, 164 ms time
constant, and 1.25
10
receiver gain. Shown in B is an EPR simulation using an anisotropic g tensor (g
= 2.0091, g
= 2.0046, g
=
2.0023), and anisotropic a tensors for the C
and
C
ring protons (a
=
-26.9, a
= -7.8, a
= -19.6 MHz) and using the
Euler angles,
= ±30,
= 0, and
= 0 relating the principal axes of the hyperfine and g tensors(37, 38, 39) . In addition, a
variable dihedral angle about the C
-C
bond was
assumed to result in isotropic hyperfine constants for the two
methylene protons of a
= B
cos
and a
= B
cos
,
where
+
= 120°
and
= 0.37 and B
= 162 MHz(37) . The simulation shown represents a
conformation with
= 20° and
= 140°.
O
are sufficient to completely oxidize the protein. No more than a
slight increase is observed in the ferric EPR signal of either protein
during the course of these experiments. The iron in both proteins thus
remains in the ferryl state during the 30-min course of the experiment
despite the decrease in the porphyrin and protein radical signals
observed during this time period. This is consistent with the fact that
the ferryl species is observed in the absorption spectrum for more than
2 h.
O
. Instrumental conditions were 20 milliwatts
microwave power at 9.51 GHz, 2.6 Gauss field modulation at 100 KHz,
1310 ms time constant, and 3.2
10
receiver gain.
Background spectra were collected under identical conditions for blank
samples and subtracted before display.
Protein Cross-linking Studies
The formation of a
tyrosine-free radical in the reaction of sperm whale myoglobin with
HO
leads to the formation of protein dimers due
to coupling of the tyrosine radicals from two protein molecules (41, 42) . Dimer formation accounts, in part, for
quenching of the protein radical generated in the reaction. To
determine if the tyrosine radical formed from the F172Y mutant gives
rise to similar protein-protein cross-linking, the protein was analyzed
by SDS-polyacrylamide gel electrophoresis after reaction with
H
O
. Intermolecular protein cross-linking was
not detected in these experiments (not shown). Tyr
is
thus located within the protein in a site that is sterically or
electrostatically protected from interaction with a reactive site in a
second protein molecule. This is reminiscent of the tyrosine radicals
formed with horse rather than sperm whale myoglobin, which are also
prevented by their protein environment from dimerizing to form
dityrosine links(41, 42) .
and V
measurements, is similar to
that of native HRP and recombinant, wild type HRP. Furthermore, the pH
profile for the oxidation of guaiacol by F172Y HRP is essentially
identical to that for the native protein. Finally, the absolute
stereochemistry of the sulfoxide produced from thioanisole, a sensitive
monitor of the active site structure and
topology(25, 26) , is not significantly changed by the
F172Y mutation. These results indicate that the F172Y HRP mutant folds
properly and has an active site that differs very little in structure
or topology from that of native HRP.
O
in the absence of an added substrate yields
initially a Compound I spectrum that decays with time to a Compound
II-like spectrum (Fig. 4). Reaction of native or recombinant
wild type HRP under the same conditions produces the normal Compound I
spectrum. Determination of the kinetic constants for the reactions of
these proteins with H
O
by stopped flow methods
shows that Compound I is formed at essentially the same pH-independent
rate by the F172Y mutant (k
= 1.6
10
M
s
) as
by native HRP (1.1-2.0
10
M
s
)(35, 36, 37) .
However, Compound I of F172Y HRP decays much more rapidly than that of
native HRP in the absence of added substrates. At pH 7, the decay rate
for F172Y HRP is k
= 1.01 s
(Fig. 5) whereas little decay is observed for Compound I
of native HRP within the time frame of these experiments(43) .
The basis for the decay of the F172Y HRP species with a Compound I
chromophore is unmasked by freeze-quench EPR studies which show that
the normal Compound I porphyrin radical cation is rapidly formed in the
reaction of both native and F172Y HRP with H
O
.
A protein radical, however, is simultaneously formed in the F172Y but
not native HRP reaction (Fig. 6). Spin quantitation indicates
that approximately 10% of the F172Y protein carries a protein radical
30 s after peroxide addition. The intensity of the protein radical
signal decays with time and accounts for 5% rather than 10% of the
protein after 30 min. Although it has not been possible from the EPR to
accurately determine the rate of decay of the porphyrin radical cation
due to the nature of the signal, qualitative comparison of the data
suggests that the porphyrin radical cation is more rapidly lost from
the F172Y mutant than from the native protein. This is supported by the
observation that the F172Y mutant gives a Compound II spectrum 2.5 h
after addition of one equivalent of H
O
whereas
native HRP gives the normal Compound I spectrum.
in the
incubation buffer.
O
produces a ferryl/porphyrin radical
cation Compound I structure analogous to that obtained with the native
protein. However, electron transfer from a protein residue gradually
reduces the porphyrin radical cation with concomitant formation of a
protein radical. The residue involved in this intramolecular electron
transfer reaction is presumably Tyr
because (a)
the corresponding reaction is not observed for native HRP within the
30-min time frame of the EPR experiments, and (b) the radical
signal can be adequately simulated with the parameters for a tyrosine
residue (Fig. 7). Quantitative conversion of the porphyrin
radical cation to the protein radical is not observed. Indeed, only
approximately 10% of the protein is found as the protein radical after
30 s, and the extent of protein radical formation decays as the
incubation time is lengthened. These results suggest that the protein
radical is in equilibrium with the porphyrin radical cation, and/or is
quenched by intra- or intermolecular reactions that occur at rates
competitive with its formation, so that only a steady state level of
protein radical is observed. The finding that reaction with
H
O
at ambient temperature produces a Compound I
species that decays to a species with a Compound II-like spectrum is
consistent with the hypothesis that the porphyrin radical cation
characteristic of Compound I is gradually quenched by oxidation of
Tyr
and that the Tyr
radical is quenched,
in turn, by reactions that do not produce stable radicals.
O
comes from
the porphyrin or the protein. These results concur with earlier work on
CcP which showed that a porphyrin cation radical is formed instead of
the usual protein radical when Trp
, the residue that is
normally oxidized, is replaced by a phenylalanine(45) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.