Peroxidases are enzymes catalyzing the oxidation of a variety of
organic and inorganic compounds by hydrogen peroxide or related
compounds. Horseradish peroxidase (HRP) (
)has a carbohydrate
content of 18% (1) and consists of more than 30
multiforms(2) . The predominant form is isoenzyme C (HRPC) that
is a monomeric glycoprotein of 308 residues with eight oligosaccharide
side chains. It has a calculated protein M
of
33,922 (3) and has been shown to contain up to 2 mol of
calcium ions/mol of protein(4) . Peroxidase action involves
2e
oxidation of the enzyme by hydrogen peroxide to
give a species known as compound I. This then reverts to the resting
state via two successive 1e
reactions with reducing
substrate molecules, the first yielding a second enzyme intermediate,
compound II(1) . Reduction of compound II to native enzyme is
often rate-limiting in the peroxidase catalytic cycle. However,
operating with limiting concentrations of H
O
and a large excess of reducing substrate it is possible to make
compound I formation the rate-controlling step but it is impossible to
make the conversion of compound I to compound II rate-limiting for
native HRPC(1) .
Poulos and Kraut (5) proposed a
mechanism for the peroxidase-catalyzed heterolytic cleavage of the
RO-OH bond based on the crystal structure of cytochrome c peroxidase. Two essential features of this mechanism were
acid-base catalysis by a distal histidine (His-42 in HRP) and charge
stabilization of a precursor enzyme-substrate complex by the conserved
distal arginine (Arg-38). Hence, compound I formation is proposed to be
a two-step mechanism. In the first step there is a pre-equilibrium of
reactants to form at least a precursor complex,
HRP-H
O
, and in the second step the O-O bond is
cleaved producing compound I and water:

Kinetic evidence for this mechanism in HRPC (6, 7) and microperoxidase (8) has been
obtained using stopped-flow cryoenzymology. The spectrum of a new
intermediate, proposed to be an hyperporphyrin species (compound 0) was
obtained at [H
O
] > K
. It was proposed that this new
intermediate was formed from an HRP-H
O
complex
after an oxidation step involving electron transfer between
HO
and the ferric heme group. Mechanism 1
has also been proposed for cytochrome c peroxidase following
studies with cytochrome c peroxidase variants and hydrogen
peroxide(9, 10) .
The proposed role of Arg-38 in
the formation of compound I suggests that changing this polar residue
to a neutral one should significantly decrease the stabilization of the
precursor enzyme-substrate complex and inhibit the subsequent
heterolytic cleavage of the oxygen-oxygen bond. In an attempt to
observe the formation and accumulation of compound 0 we have
constructed a HRP mutant in which Arg-38 has been replaced by Leu using
site-directed mutagenesis. This HRPC variant has been characterized
with respect to its reaction with hydrogen peroxide and to the
reactivity of its compound I to different reducing substrates. Nuclear
Overhauser (11) and NMR relaxation(12, 13) experiments suggest that phenol and other aromatic
substrates bind distal to the heme and close to the heme C18 methyl
with the aromatic protons 6-10 Å from the heme
group(14) . Different binding sites for guaiacol, thioanisol,
and iodide have been proposed based on studies of HRP modification with
-meso substituents(15, 16) . However, the exact
nature of the reducing substrate binding site of HRPC is still not
known and the role of distal heme residues such as histidine 42 and
arginine 38 not resolved(14) . Substitution of arginine 38 by
leucine in HRPC gave us the opportunity to study the role of this
residue in modulating the binding and activity of reducing substrates
with the enzyme.
MATERIALS AND METHODS
Reagents
HRP (EC 1.11.1.7) isoenzyme C (Type VI)
was purchased from Sigma and used without further purification.
HRPC
was obtained as described by Smith et al.(17) . The concentrations of HRPC and HRPC
were determined spectrophotometrically by using 
= 102 mM
cm
(18) and 
= 92
mM
cm
, respectively.
Reagent grade H
O
(30% v/v) was obtained from
BDH and its concentration was calculated by iodide titration with
HRPC(19) . ABTS, guaiacol, and p-cresol were purchased
from Sigma and ascorbic acid from Merck. Stock solutions of the
reducing substrates were prepared in 0.15 mM phosphoric acid
to prevent autoxidation. All other chemicals were of analytical grade
and supplied by Merck.
Construction of an (R38L)HRPC
Gene
A polymerase chain reaction-based technique with the
HRPC synthetic gene (17) as template was used to generate
insert DNA bearing the Arg-38 mutation. The construction of the
(R38L)HRPC
gene was carried out as described by Smith et al.(20) for the (F41V)HRPC
mutant,
except that oligonucleotide N1 replaced V1. N1 was identical to the
wild-type synthetic gene sequence between nucleotide positions
111-147, except that it contained the Leu codon TTA at position
126 (5`-GCTGCTTCAATATTATTACTGCACTTCCATGAC-3`).Amplified DNA
fragments (404 base pairs) bearing this mutation were cloned in-frame
into the wild-type gene at the unique SspI and XhoI
sites in the HRP expression vector pAS5(20) . Double-stranded
DNA sequence analysis by the dideoxy chain termination method, using
Sequenase version 2 (21) and oligonucleotides S1 and N1 (20) as sequencing primer, confirmed the expected sequence
change.
Preparation of (R38L)HRPC
Growth and
induction of Escherichia coli strains producing the
recombinant peroxidase variant were as described
previously(20) . Folding and activation of (R38L)HRPC
recovered from E. coli inclusion bodies were achieved
essentially by the method of Smith et al.(17) with
the modifications subsequently described by Smith et
al.(20) . Purified enzyme was desalted into 10 mM sodium MOPS, pH 7.0, and stored in liquid nitrogen until use. The
concentration of the enzyme was determined using the Soret extinction
coefficient determined by the pyridine hemeochrome method (22) .
Pre-Steady State Kinetics
Transient kinetics were
monitored in a stopped-flow spectrophotometer (model SF-51, Hi-Tech
Scientific, Salisbury, UK) in 10 mM sodium phosphate buffer,
pH 7.0. Data were recorded through an RS232 interface with a
microcomputer. Compound I formation was monitored at 401 nm, isosbestic
for compound I and compound III (see ``Results'').
(R38L)HRPC
compound I was generated by mixing enzyme (2.0
µM) with 2 volumes of hydrogen peroxide (4.0
µM) in a simple flow-mixing device and was used within 5
min of preparation. Temperature was controlled at 25 or 10 °C,
using a Techne C-400 circulating bath with a heater-cooler.
Spectrophotometry
Stopped-flow rapid-scan
spectrophotometry was carried out with the same stopped-flow
spectrophotometer described above equipped with an MG 6000 diode array
system. Ultraviolet/visible absorption spectra were recorded in quartz
cuvettes (1 cm) on a Shimadzu UV-2101PC spectrophotometer with a
spectral bandwidth of 1 nm and a scan speed of 120 nm/min. Steady-state
assays were carried out with the same instrument by measuring the
appearance of products. Tetraguaiacol formation was followed at 470 nm
(
= 26.6 mM
cm
), 4-methyl-o-benzoquinone at 400
nm (
= 1.14 mM
cm
), and ABTS radical at 414 nm (
= 31.1 mM
cm
). The assay medium was 10 mM sodium/phosphate buffer, pH 7.0. Other conditions and reagents are
detailed in the text and in the legends to figures and tables.
Spectrophotometric Determination of Substrate Binding
Constants
Difference spectra of the Soret region (350-480
nm) of the ferric (R38L)HRPC
with reducing substrates minus
ferric (R38L)HRPC
were recorded in 1-cm quartz
microcuvettes in 10 mM sodium MOPS, pH 7.0. The reference and
the sample cuvettes contained 0.4 ml of enzyme solution (10
µM) for base line recording. Increasing amounts of
guaiacol (in the same buffer as the enzyme) or p-cresol (in
ethanol) were then added to the sample cuvette. The same volume of
solvent was added to the reference cuvette. The contents were stirred
with a plastic rod before recording the spectrum. Equilibrium
dissociation constants (K
) for the complex
formation were calculated using the following expression(12) :

where
A is the change in absorbance, S is the
substrate concentration, and
A
is the
change in absorbance at saturating concentration of the substrate.
Similar titrations were carried out with plant and recombinant HRPC. In
all cases the K
values were 3 orders of magnitude
greater than the enzyme concentration and the free concentration of
substrate was assumed to be equal to its initial concentration.
Kinetic Data Analysis
Pre-steady state kinetic
data were analyzed by fitting the absorbance time curves to exponential
functions using a least-squares minimization procedure. The values of K
and V
for HRPC
and (R38L)HRPC
on varying H
O
at fixed saturating concentrations of reducing substrate were
calculated by triplicate measurements of
at each
[H
O
] concentration. The same
procedure was used to determine these kinetic constants for reducing
substrates using a saturating concentration of
H
O
. The reciprocal of the variances of
were used as weighting factors in the non-linear regression fitting of
versus [substrate] data to the Michaelis-Menten
equation(23) . The fitting was carried out by using a
Gauss-Newton algorithm (24) implemented in a BASIC program.
Initial estimates of K
and V
were obtained from the Lineweaver-Burk equation.
RESULTS
Properties of (R38L)HRPC
The enzyme,
which was purified from the folding medium by FPLC using
cation-exchange chromatography on a Mono-S HR5/5 column (Pharmacia),
eluted at the same salt concentration as HRPC
. Preparations
were judged to be homogeneous by the observation of a single band on a
Coomassie Blue-stained reducing SDS-polyacrylamide gel electrophoresis
gel. The spectrum of (R38L)HRPC
at pH 7.0 is shown in Fig. 1. This enzyme has a similar spectrum to that reported for
the same variant in cytochrome c peroxidase (9) but
with a 3-nm displacement of the Soret band to 399 nm in the HRP mutant.
Moreover, the spectrum of (R38L)HRPC
has peaks at 500 and
646 nm and a shoulder at 383 nm. The extinction coefficient for the
Soret maximum (399 nm) calculated using the pyridine hemeochrome method
was 86 mM
cm
. The
preparations used had ratios of absorbance at 398/280 nm (Rz value) of
3.2. Heme incorporation data indicated that the preparations were not
contaminated with inactive heme-free enzyme (100% heme incorporation).
The electronic absorption spectrum of (R38L)HRPC* in the Soret region
is unusually broad and although the spectrum appears consistent with
pentacoordination of the heme iron at pH 7.0 more conclusive resonance
Raman data are required for a precise determination of the heme
coordination state of this mutant(25) .
Figure 1:
Spectrum of (R38L)HRPC
(solid line) and (R38L)HRPC
compound I (dashed line) in 10 mM Na
-phosphate
buffer, pH 7.0. Compound I was formed as was described under
``Materials and Methods.''
Reaction of (R38L)HRPC
with Hydrogen
Peroxide
(R38L)HRPC
reacted with a 10-fold
excess of hydrogen peroxide to slowly yield a stable compound I species (Fig. 2). The spectrum for this intermediate was exactly the
same as that described for native HRPC compound I (18) with an
extinction coefficient of 57 mM
cm
in the Soret region. (R38L)HRPC
compound I was stable in the reaction media for more than 30 min.
After this time, compound I slowly converted to the ferric state with
no detectable accumulation of compound II (data not shown). In contrast
when the same experiment was carried out with wild-type HRPC, a very
unstable compound I was formed which converted spontaneously to
compound II with an isosbestic point at 395 nm. Compound II was
subsequently reduced to resting ferric enzyme.
Figure 2:
Rapid-scan stopped-flow of the reaction of
(R38L)HRPC
(1.6 µM) with a 10-fold excess of
hydrogen peroxide (16 µM) in 10 mM Na
-phosphate buffer, pH 7.0, 25 °C. The first
scan was taken 4.4 ms after the flow stopped, and the subsequent scans
were at 0.5-s intervals. The arrows show the direction of
absorbance change with time.
In order to determine
the pseudo-first order rate constant for compound I formation, k
, the concentration of H
O
was increased through the range 20 µM to 50
mM. At high peroxide concentrations (>0.1 mM),
compound I was formed over a period of seconds followed by compound III
formation with an isosbestic point at 401 nm but without detectable
accumulation of compound II (Fig. 3). This wavelength was used
for the determination of k
. The time dependences
of the absorbance change at 401 nm can be fitted to a simple
exponential function. Under pseudo-first order conditions, with
hydrogen peroxide in large excess, the dependence of k
was linear at low hydrogen peroxide concentration (
0.5
mM) (Fig. 4A), whereas at high hydrogen
peroxide concentrations (up to 50 mM), k
approaches a limiting value (Fig. 4B). These
dependences can be explained if the reaction between (R38L)HRPC
and H
O
follows the mechanism described in . If the first step in this mechanism equilibrates rapidly (k
[H
O
] + k
k
) the
expression for k
is:

Figure 3:
Rapid-scan stopped-flow of the formation
of (R38L)HRPC
compound I and its conversion to compound III
in 10 mM Na
-phosphate buffer, pH 7.0, 25
°C. The reaction was started by mixing 2 µM enzyme and
0.5 mM hydrogen peroxide. The first scan was taken 1.23 ms
after the flow stopped, and the subsequent scans were at 1-s intervals.
The arrows indicate the direction of absorbance change with
time.
Figure 4:
Dependence of k
, the pseudo-first order rate constant
for (R38L)HRPC
compound I formation, on the hydrogen
peroxide concentration at pH 7.0 and 25 °C. A,
20-500 µM hydrogen peroxide. B,
0.02-50 mM hydrogen
peroxide.
where k
= k
and K
= (k
+ k
)/k
(26) . Therefore, k
is directly calculated from the limiting value (Fig. 4B), whereas k
can be
determined assuming that k
k
from the slope of the dependence of k
versus [H
O
] at low
concentrations of hydrogen peroxide (Fig. 4A). The
values for the elementary rate constants for (R38L)HRPC
compound I formation are k
= 1.1
± 0.1
10
M
s
and k
= 142
± 10 s
. The value of k
is about 3 orders of magnitude lower than that for native HRPC
(1.7 ± 0.1
10
M
s
) and non-glycosylated recombinant HRPC
(1.6 ± 0.1
10
M
s
).
Direct Observation of the HRP-H
O
Intermediate
The saturation kinetics observed with
(R38L)HRPC
indicate that at high
[H
O
] a reaction intermediate is
formed in a rapid pre-equilibrium step prior to compound I formation.
Simulation of mechanism 1 using the measured rate constants for the
(R38L)HRPC
mutant shows that at 50 mM hydrogen
peroxide the time required for the maximum accumulation of the
intermediate is 2.5 ms. In order to minimize any possible stopped-flow
artifacts occurring in the first few milliseconds of the reaction, the
detection of this intermediate was carried out at a lower temperature.
At 10 °C, the values for k
and k
were 6.3
10
M
s
and 33
s
, respectively, and the maximum concentration of
the intermediate, 78% of the initial enzyme, was calculated to be
formed at 10 ms. Under these experimental conditions, the resting
enzyme converts to compound I but without the appearance of an
isosbestic point, suggesting the accumulation of at least one
intermediate, possibly the complex HRP-H
O
. The
time course at 374 nm, isosbestic between ferric enzyme and the
transient intermediate, shows a lag period of about 6 ms (Fig. 5A). The solid circles are simulated
data points calculated using and the above values of k
and k
. The time course of
the reaction (monitored at 357 nm isosbestic between ferric enzyme and
compound I) exhibits a maximum absorbance at about 10 ms after which
time the formation of an intermediate species is essentially complete
and its conversion to compound I is in progress (Fig. 5B). Again the simulated data points shown in Fig. 5B lie on the observed absorbance time curve. The
spectrum for this intermediate, shown in Fig. 6, resembles that
of resting enzyme but with peaks at 397, 487, and 580 nm and a shoulder
at 530 nm. This spectrum does not resemble that of the hyperporphyrin
published by Baeck and Van Wart(6, 7) . We suggest
that this intermediate is the HRP-H
O
complex.
However, these data do not exclude the possibility of the subsequent
formation of compound 0, although we were not able to detect directly
this species under our conditions.
Figure 5:
Time course at 375 nm (A) and 357
nm (B) of the reaction of 2 µM (R38L)HRPC
with 50 mM H
O
in 10 mM Na
-phosphate buffer, pH 7.0, 10 °C. The filled circles are simulated data points using a computer
program KSIM (supplied by Dr. N. Millar). The rate constants used for
the simulation, assuming mechanism 1, were k
= 6.3
10
M
s
; k
= 0.07
s
and k
= 33
s
, and the extinction coefficients used were: A, 
=


Figure 6:
Rapid-scan optical spectra of the reaction
of 2 µM (R38L)HRPC
with 50 mM H
O
in 10 mM Na
-phosphate buffer, pH 7.0, 10 °C. Curve
a is the spectrum of (R38L)HRPC
. Curves b and c are 10-ms and 0.1-s scans, respectively, initiated at the
start of mixing, which show the formation of the
HRP-H
O
complex and its conversion to compound
I.
Binding of Reducing Substrates to Ferric
(R38L)HRPC
Spectrophotometric titrations have shown
that substrates can be divided into two classes on the basis of the
difference spectra obtained when they bind to
HRP(16, 27) . A type I spectrum is induced by p-cresol while guaiacol gives a type II spectrum. The
difference spectrum obtained when guaiacol binds to plant HRPC (data
not shown), and the K
value (Table 1),
determined from the substrate concentration dependence of the amplitude
of the difference spectra, were essentially identical to those
previously reported(27) . Similar results were obtained when
guaiacol bound to recombinant HRPC (Fig. 7; Table 1).
However, the difference spectrum caused by binding of guaiacol to
(R38L)HRPC
was quite different (Fig. 7) and
resembles the type I spectra reported for the binding of phenol,
hydroquinone, and p-cresol to ferric HRPC(27) . The
spectrum had a trough with a minimum at 395 nm, a peak at 416 nm, and
an isosbestic point at 406 nm (Fig. 7). The value of K
for the binding of guaiacol to (R38L)HRPC
showed that it bound 2.3 times weaker than it did to both plant
and recombinant HRPC (Table 1).
Figure 7:
Difference spectrum for the binding of
guaiacol (A) and p-cresol (B) to
HRPC
, and guaiacol (C) and p-cresol (D) to (R38L)HRPC
.
Spectrophotometric studies of
the binding of p-cresol to (R38L)HRPC
also showed
that the mutation modified the binding of this substrate to the enzyme.
Both HRPC (28) and HRPC
(Fig. 7) gave a type
I spectrum and the calculated values for K
(Table 1) were very similar to those reported for the
native enzyme(28) . Binding of p-cresol to ferric
(R38L)HRPC
induced a spectrum with peaks at 385 and 413 nm (Fig. 7). The value of K
for the binding of p-cresol to ferric mutant enzyme was about 2.5 times higher
than for the binding of this substrate to both wild-type enzymes (Table 1). Therefore, substitution of the distal arginine 38 by
leucine in HRPC differentially modifies both the equilibrium constant
for the binding of aromatic electron donors to the ferric state of the
enzyme and the coordination state of the complex formed between ferric
HRP and aromatic substrates.
Reaction of (R38L)HRPC
Compound I with
Reducing Substrates
When (R38L)HRPC
compound I was
kinetically titrated with p-cresol or ascorbate, it was
apparently reduced directly to ferric enzyme with no accumulation of
compound II. It is well known that compound II exists in two
pH-dependent forms (pK = 8.6), the protonated form
being more reactive than the unprotonated
form(1, 29) . Titration experiments with
(R38L)HRPC
compound I at pH 10, designed to stabilize
compound II, again failed to detect any compound II (Fig. 8).
This was similar to the behavior reported for
(R38K)HRPC
(32) . Three different explanations are
possible for this observation: (a) a novel reaction pathway
exists; (b) compounds I and II are spectroscopically
indistinguishable (cf. compounds I and II in cytochrome c peroxidase); or (c) the reduction of compound II to
resting ferric enzyme is very much faster that the reduction of
compound I at both high and low pH values.
Figure 8:
The reduction of (R38L)HRPC
compound I at pH 10 (10 mM Na
borate
buffer). Spectra, a, 2 µM (R38L)HRPC
; b, 20 µM H
O
added to a; c, 1 µM ascorbic acid added to b; d, another 1 µM ascorbic acid added
to c.
Steady-State Kinetics of (R38L)HRPC
with
Reducing Substrates
The kinetic parameters for
(R38L)HRPC
acting on ABTS, guaiacol, and p-cresol
and a comparison with the values obtained for the native plant and
recombinant HRPC enzymes are given in Table 2. The Arg-38
mutation differentially increased the K
for
hydrogen peroxide in the presence of these three substrates. The
difference in the apparent value for K
at pH
5.0 between HRPC and HRPC
was previously
reported(20) . It was proposed that this difference could be
related to the lack of glycosylation of the recombinant enzyme,
allowing readier access of the bulky charged substrate ABTS to its
binding site. The failure to observe the same effect on the value of K
for small substrates such as guaiacol and p-cresol supports this hypothesis. Therefore, comparing the
value of K
for ABTS and guaiacol for the two
non-glycosylated enzymes, HRPC
and (R38L)HRPC
,
it is apparent that the effect of the mutation on the K
for both electron donors is essentially the same (Table 2).
However, the effect of this mutation on the apparent K
for p-cresol was higher. The catalytic constant for the
oxidation of ABTS by the recombinant wild-type enzyme is drastically
decreased at pH 7.0 compared to the value at pH 5.0. This results in
guaiacol and p-cresol being better reducing substrates at pH
7.0. However, the R38L mutation reverses this trend, with ABTS being a
better substrate than guaiacol and p-cresol at pH 7.0. These
results show that arginine 38 not only modulates the formation of
compound I (increase in the
K

DISCUSSION
Mechanism of Compound I Formation: Effects of Arg-38
Mutation
A mechanism for compound I formation must be able to
explain the saturation kinetics observed at high hydrogen peroxide
concentration and be consistent with the formation of an
enzyme-substrate complex whose conversion to compound I is
rate-limiting for reactions in the absence of reducing substrates. The
simplest mechanism that is consistent with the data is mechanism 1. The
initial second-order step with implicit deprotonation of the peroxide
molecule is the binding of the anionic HO
ligand to the Fe(III) with proton transfer to a basic amino acid
residue. This is followed by a first-order step in which the
oxygen-oxygen bond is cleaved to yield compound I and water. However, a
more complex mechanism, needed to explain the formation of other
intermediates (i.e. compound 0; (6, 7, 8) ), is also compatible with our
data, although under our conditions we did not detect any intermediates
with a spectrum similar to that proposed for compound 0. Substitution of arginine 38 by Leu in HRP modifies both the
second-order rate constant of the formation of HRP-H
O
and the first-order rate constant for subsequent compound I
formation. The mutation decreases by 3 orders of magnitude the first
rate constant but it is not easy to estimate by how much it decreases
the second. It has been proposed that the O-O cleavage step is very
fast for wild-type peroxidases, probably of the order of 10
s
at room
temperature(9, 30) . If this value is true the
decrease induced by the arginine to leucine mutation would also be
about 3 orders of magnitude. However, we believe that this value is not
consistent with previous data. A value of 10
s
for k
would mean that the value of the
K

Smulevich et al.(25) have recently discussed the effect of changing Arg-38
Lys in HRP and Arg-48
Lys in cytochrome c peroxidase. The cytochrome c peroxidase variants R48K and
R48L react with hydrogen peroxide to form compound I factors of 2 and
200 times slower, respectively, than does native cytochrome c peroxidase(31) . In contrast, the R38K variant in
HRPC
showed a 500-fold decrease in the rate of compound I
formation(32) . Therefore, in cytochrome c peroxidase
a lysine residue is able to substitute quite well for arginine 48 with
respect to compound I formation, but it is not able to do so nearly as
efficiently in HRP. The data presented above show that the Arg
Leu mutation decreases the second-order rate constant for compound I
formation by 1,200-fold and k
by 10-100
times. This is not only consistent with the Poulos-Kraut mechanism (5) but also indicates that arginine 38 plays an important role
in the binding step of the peroxide molecule in the distal heme pocket
as previously suggested for an HRPC mutant in which arginine 38 had
been replaced by lysine(32) . Two possible functions of
arginine 38 in hydrogen peroxide binding are that the polar character
of this residue facilitates the access of the hydrogen peroxide
molecule to the heme and/or provides an electrostatic interaction with
the incoming peroxide which may also induce the deprotonation of
hydrogen peroxide at neutral pH.
The effect of Arg-38 mutations in
HRPC on the binding constant could explain the low activity of
metmyoglobin with respect to hydrogen peroxide (k
= 10
M
s
)(33) . In metmyoglobin the distal
histidine is still present in the active site, but Phe-43 takes the
place of the distal arginine(34) . The presence of a
phenylalanine residue in the active site could also have a profound
steric effect on the accessibility of hydrogen peroxide to the distal
pocket thereby decreasing the binding constant by up to 5 orders of
magnitude. However, the replacement of His-42 by Leu in HRPC decreases
the binding constant for hydrogen peroxide by 5 orders of magnitude. (
)This suggests that the principal feature in the formation
of an activated enzyme-HO
complex is the
transfer of a proton to the distal histidine to form an imidazolium
side chain thereby promoting the binding of HO
to the heme. It seems likely that both events take place in a
concerted manner involving both the catalytic arginine and histidine.
The effect of the Arg-38
Leu mutation on k
is most likely due to the proposed role of this residue in
stabilizing the transition state during O-O cleavage to form compound I
and water(5) .
Possible Structure for the ES Complex
The optical
spectrum of the newly detected intermediate provides some information
as to its likely structure. The spectrum of the intermediate is not
very different from that of the ferric enzyme. This is not surprising
since the (TMP)Fe(III)(t-BuO
) complex and
other ferric-oxyanion heme complexes have relatively normal Soret- and
Q-band spectra(35, 36) . The spectrum for this species
shows the loss of the 380 nm shoulder and small shifts in the 505- and
645-nm bands. Moreover, an increase in the Soret extinction coefficient
is also predicted. Having noted the similarity of these changes to
those observed when cytochrome c peroxidase ages(37) ,
we suggest that this new intermediate is a ferric hydroperoxy complex
in which one oxygen atom of HO
is
coordinated to the iron atom. Recently, a 2.2-Å crystal structure
of cytochrome c peroxidase oxyperoxidase has been obtained and
considered as a model for the transient ferric enzyme-peroxide
complex(38) . The authors support their proposal by arguing
that because oxyperoxidase and the enzyme-hydrogen peroxide complex
differ by only one electron and one proton their structures are likely
to be very similar. However, we did not detect any species with a
spectrum similar to that reported for oxyperoxidase in the reaction of
(R38L)HRPC
with hydrogen peroxide. On the other hand, it is
important to note that the compound I and compound II forms of both
plant and fungal peroxidases that differ by only one electron have
completely different absorption spectra.
The Aromatic Donor Molecule Binding Site of
HRP
The non-availability of a high resolution crystal structure
of HRP makes the identification of the binding site(s) for aromatic
donor molecules difficult. NMR data (12, 13) and
enzyme inactivation studies(15, 16) have located the
aromatic substrate binding site near the
-meso-heme edge. The
side chains of two phenylalanine residues are also implicated in the
binding process(14, 39) . The possibility of two
different binding sites, one for guaiacol or resorcinol, that induce
type II spectra when they bind to HRPC, and a second site for phenols
such as p-cresol, hydroquinone, and aniline, that yield a type
I spectrum, has been ruled out by NMR relaxation
studies(12, 13) . It has been concluded that the
UV/visible spectroscopic differences reflect different hydrogen bonding
interactions to residues that modulate the chromophore rather than
binding to different sites. The direct participation of distal heme
residues such as histidine 42 and arginine 38 in the binding of
aromatic electron donors remains unclear. Thus, histidine 42 has been
proposed to be involved in the binding of aromatic
substrates(14) , however, chemical modification of HRP with
diethyl pyrocarbonate, a histidine-specific reagent, did not modify the
dissociation constant for guaiacol binding(40) . However, it
has been reported that replacement of arginine 38 by lysine in
HRPC
greatly decreased the affinity for benzhydroxamic
acid(32) . Additional evidence for arginine 38 being directly
involved in the binding of aromatic substrates is presented in this
paper. Substitution of arginine 38 by leucine increases the
dissociation constants for the binding of guaiacol and p-cresol to the ferric enzyme, possibly by modifying the
hydrogen bond interactions in the complexes formed when they bind to
the enzyme (Fig. 7). This is consistent with the effect on the
binding of these reducing substrates to compound I and/or compound II
as indicated by the increase in the apparent K
values. These large differences in affinity cannot be
rationalized simply on the basis of a local perturbation resulting from
a single-site substitution. An arginine residue in a protein often
serves as a cationic site for the binding of a negatively charged group
in a substrate or cofactor(41) . Hence the simplest explanation
for the role of arginine 38 in substrate binding is a direct
electrostatic interaction between the positively charged guanidinium
group of arginine 38 and the partial negative charge developed on the
oxygen of the phenolic group of substrates such as p-cresol
and guaiacol, causing the orientation of the substrate in the active
site prior to electron transfer or hydrogen atom transfer in a similar
way to that proposed for the binding of benzhydroxamic
acid(32) . The polar character of arginine 38 may also
facilitate the access of the reducing substrate to its binding site on
compound I. These results, therefore, support models based largely on
NMR data that indicate key interactions between bound substrates and
the heme methyl C
H
, the conserved residue
arginine 38 and also possibly histidine
42(12, 13, 14) .
The Rate-limiting Step in the Peroxidase Cycle
One
possible explanation for the failure to observe compound II in the
reaction of (R38L)HRPC
compound I with reducing substrates
is that an increase in the stability of compound I with respect to
compound II is induced by the mutation. It is well established that the
reduction of compound II to native enzyme is usually the rate-limiting
reaction in the peroxidase cycle. The rates of reduction of compound I
and II often differ by factors in the range of
20-100(42) . However, Arthromyces ramosus peroxidase (43) forms a very stable compound I while
compound II is unusually unstable being rapidly reduced to ferric
enzyme. The instability of A. ramosus peroxidase compound II
was explained in terms of a higher reduction potential for this
species. A similar situation is observed in (R38L)HRPC
. The
drastically reduced reactivity of the compound I formed with a 10-fold
excess of hydrogen peroxide supports the hypothesis that the reduction
of compound II is not rate-limiting for this variant. However, the
failure to detect compound II at pH 10 causes us not to completely
exclude the possibility of novel reduction pathways for compound I
and/or compound II.
Mechanism of Compound I Reduction in HRP: Role of
Arg-38
The data presented above indicate that arginine 38 is not
only involved in compound I formation but also in other partial
reactions of the catalytic cycle. Substitution by leucine renders
compound II undetectable and modifies the kinetic constants for the
reactions with reducing substrates. As discussed above, the
modification of the apparent K
values for
guaiacol, p-cresol, and ABTS suggests that Arg-38 also has a
role in the binding/orientation of electron donors in the active site
of HRPC compound I. However, this effect alone cannot explain the
decrease in k
, since the rate equation for k
does not contain a binding constant term.
Other systems in which an arginine residue influences catalysis by
electrostatic effects include 2-keto-4-hydroxyglutarate aldolase (44) and transketolase(45) . We suggest that arginine
38 could have a catalytic role in the reduction of HRP compound I.
Interaction between the guanidinium group of arginine 38 and the ferryl
oxygen of HRP compound I would be expected to withdraw electron density
from the porphyrin radical cation and, thus facilitate the electron
transfer from the reducing substrate by increasing the redox potential.
This function of arginine 38 could explain both the decrease in the k
for electron donors and the different effect
of this substitution in the reactivity of cytochrome c peroxidase (9) where the radical cation is not located on
the porphyring ring, but on Trp-191 which is remote from the proposed
substrate binding site and arginine 48(46, 47) . The differential effect of the (R38L) mutation on HRP and
(R48L)cytochrome c peroxidase reactivities can, in part, be
explained in terms of the different polarity of their respective distal
heme pockets. Thus, the more polar pocket in cytochrome c peroxidase can still effectively promote charge separation in the
absence of the positively charged arginine. On the other hand, our data
clearly show that the removal of a positive charge in the active site
of HRP makes the reactivity for hydrogen peroxide similar to that
reported for metmyoglobin. In addition, we have also recently studied
by laser photolysis the reaction of the ferrous form of
(R38L)HRPC
with other heme ligands such as carbon monoxide
and cyanide ion(48) . Removal of arginine 38 from the active
center of HRP differentially modulates the kinetics of binding of CO
and HCN to the heme. The reassociation rates for both ligands approach
those previously reported for sperm whale myoglobin and human
hemeoglobin (49, 50) . Engineering proteins with
oxygen binding properties from peroxidases is now a real possibility.