From the Department of Protein Chemistry, Institute of Molecular
Biology, University of Copenhagen, Øster Farimagsgade 2A,
DK-1353 Copenhagen K, Denmark
Transient-state kinetic analysis of compound I
formation for barley grain peroxidase (BP 1) has revealed properties
that are highly unusual for a heme peroxidase but which may be relevant to its biological function. The enzyme shows very little reaction with
H2O2 at pH > 5 and exhibited
saturation kinetics at higher H2O2
concentrations (kcatapp increases from 1.1 s
1 at pH 4.5 to 4.5 s
1 at pH 3.1 with an
enzyme-linked pKa < 3.7 (Rasmussen, C. B.,
Bakovic, M., Welinder, K. G., and Dunford, H. B. (1993) FEBS Lett. 321, 102-105)). In the present paper, it is
shown that the presence of Ca2+ gives rise to biphasic
kinetics for compound I formation, with a slow phase as described above
and a fast phase that exhibits a second order rate constant more
typical of a classical peroxidase (k1app = 1.5 × 107 M
1
s
1, which is pH-independent between 3.3 and 5.0). The
amount of enzyme reacting in the fast phase increases with
Ca2+ concentration (Kd = 4 ± 1 mM at pH 4.0), although it is also moderately inhibited by
Cl
. The absorption spectrum of BP 1, which appears to be
a five-coordinate high spin ferric in the resting state changes
insignificantly in the presence of Ca2+. In the presence of
Cl
, it becomes six-coordinate high spin
(Kd approximately 60 mM at pH 4.0) but
only if Ca2+ is also present. Fluoride binds to BP 1 with
monophasic kinetics in the presence of 0-5 mM
Ca2+. The activating effect of Ca2+ can be
mimicked only by replacing it with Sr2+ and
Ba2+ ions. Comparing these data with the crystal structure
of the inactive neutral form of BP 1 (Henriksen, A., Welinder, K. G., and Gajhede, M. (1997) J. Biol. Chem. 273, 2241-2248) and similar data for wild-type and mutant peroxidases of
plant and fungal origin suggests (i) a proton-induced conformational
change from an inactive BP 1 at neutral pH to a low activity BP 1 form
with a functional distal histidine and (ii) a Ca2+-induced
slow conformational change (at least compared with compound I
formation) of this low activity form to a high activity BP 1 with a
typical peroxidase reactivity. BP 1 is the first example of a plant
peroxidase whose activity can be reversibly controlled at the enzyme
level by pH- and Ca2+-induced conformational changes.
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INTRODUCTION |
Homologous heme-containing peroxidases are found in plants, fungi,
and bacteria. Plants use these enzymes in intracellular peroxide
scavenging and in extracellular protective processes and hormonal
signaling. Plants encode a small family (an estimated 10 genes) of
soluble and membrane-bound ascorbate peroxidases found in chloroplast,
cytosol, and microbodies (1-3) and a large family (more than 50 genes)
of peroxidases targeted for the secretory pathway (4). Interestingly,
ascorbate peroxidases have a prokaryotic origin (class I), since they
are more closely related in structure to mitochondrial (exemplified by
yeast cytochrome c peroxidase; CCP)1 and bacterial
peroxidases than to the secretory peroxidases of plants (class III) or
of fungi (class II) (5).
Horseradish peroxidase (HRP C) is the most studied and applied class
III peroxidase and catalyzes the oxidation of a broad range of organic
and inorganic substrates (AH) by peroxide (ROOH) via the following
three-step mechanism, which applies to the majority of heme peroxidases
(6).
The intermediates cpd I and II are very potent oxidants and have
redox potentials near 900 mV. Products are often radicals (A·)
that may continue in nonenzymatic reactions depending on their chemical
reactivity and the environment.
We wanted to understand how plants use and regulate the activity of
such a great number of rather unspecific peroxidase enzymes and chose
to study the structure-function relationships of barley grain
peroxidase (BP 1), since it showed only 1% of the specific activity of
HRP C under standard assay conditions at pH 5.0 (7). First, a
transient-state kinetics study revealed that BP 1 cpd I formation has a
pH optimum near 3.4 and that this reaction is rather slow and increases
in rate as the pH is decreased, saturating with increasing hydrogen
peroxide concentration, in complete contrast to HRP C (8). Second, a
steady-state kinetic study at pH 4.0 with ferulic acid, caffeic acid,
and coniferyl alcohol was interpreted assuming that the rate constant
k1 of BP 1 cpd I formation was enhanced in the
presence of reducing substrate, although the presence of 1 mM CaCl2 also contributed (3-fold in the case
of caffeic acid) (9). In the present study, we wanted to clarify the
role of Ca2+ and other ions in the kinetics of BP 1 cpd I
formation. The binding of fluoride was also studied to further probe
the nature of the active site and the mechanism of this activation
phenomenon, since it appears that the fluoride complex of peroxidases
can mimic aspects of cpd I formation and structure, such as the
dependence on an unprotonated distal histidine for the binding (see
Ref. 10 and references therein; Refs. 6, 11, and 12), hydrogen bonding
of the ligand to distal arginine (13-15), and formation of the
porphyrin
cation radical (10).
The data have been interpreted in structural terms based on the
crystallographic information for BP 1 presented in the accompanying paper (16) and on studies of other wild-type and mutant peroxidases. The crystal structures of BP 1 at pH 5.5, 7.5, and 8.5 show an inactive
peroxidase with the catalytic histidine turned away from the distal
cavity in response to the dislocation of a 21-residue segment usually
packing against the highly conserved distal catalytic residues and
calcium ion-binding site (16). The crystal structure of active BP 1 is
unknown, but it is assumed to resemble the active peanut and
horseradish peroxidases, which are rather similar (17, 18).
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EXPERIMENTAL PROCEDURES |
Materials--
BP 1 was purified from barley grain (19) and
stored at 4 °C as an ammonium sulfate precipitate or in freeze-dried
form. Precipitated protein was desalted on a 5-ml Sephadex G-25 column in pH 4.0 buffer, and freeze-dried BP 1 was dissolved in buffer and
centrifuged before use. The results were unaffected by the method of
storage. The RZ value
(ASoret max/A280) of the enzyme was 2.9, depending on pH and buffer (19). An extinction coefficient of 100 mM
1 cm
1 at
402 nm, pH 4.0, was used to determine the concentration of BP 1. Hydrogen peroxide concentration was determined and checked regularly
using an extinction coefficient of 43.6 M
1
cm
1 at 240 nm. All chemicals were of analytical grade,
and water was drawn from a Milli Q system.
Equipment--
Absorption measurements were performed by a
Beckman DU-70 or a Hewlett Packard 8452A diode-array spectrophotometer.
Absorption spectra of BP 1 in 1 and 100 mM
CaCl2 (Fig. 4, inset) were measured on a Cary 5 spectrophotometer (by Drs. B. Howes and G. Smulevich, University of
Florence). Rapid mixing stopped-flow experiments were carried out on an
Applied Photophysics SX-18MV stopped-flow spectrometer running in
standard absorption nonsequential mixing mode with a 1-cm light path
and controlled by an Acorn A5000 computer running dedicated software. A
diode-array detector was attached for fast recording of spectra (Fig.
3A). The temperature was controlled at 25 ± 1 °C by
a Heto DT water bath with a heater/cooler. Experiments were repeated at
least five times for each set of conditions. Kinetic data were fitted
either as single A(t) = A·exp(
kt) + P or double
A(t) = Aa·exp(
kat) + Ab·exp(
kbt) + P exponential functions, to obtain the end point
P, the rate constants k, and amplitudes
A for phases a and b, using the curve fitting facility
(modernized version of the Marquardt algorithm) of the instrument
software. The software Fig.P (Biosoft) was used for graphical
representation and calculation of apparent second order rate constants
and data error (S.E.).
Ionic Effects on BP 1 Reactions--
The transient-state
kinetics of the reaction between BP 1 and hydrogen peroxide was
examined using 1 µM BP 1 and 5-50 µM of hydrogen peroxide (final concentrations). The ionic strength of the
buffers was routinely adjusted by the addition of
K2SO4. The change in absorbance was followed at
398 nm, an isosbestic point between cpd I and II of BP 1 (Fig.
3B).
The effects of calcium and chloride ions on the reactions of BP 1 and
hydrogen peroxide or fluoride were examined in 50 mM sodium
citrate buffer without CaCl2 (with and without EDTA) and with CaCl2. CaCO3 and NaCl were used to
distinguish the specific effects of these two ions. CaCO3
was dissolved in citric acid, and the pH was adjusted with NaOH.
Buffers were freshly made, and precipitation was not observed. The
Kd for the binding of Ca2+ was
calculated from the kinetic data (amplitudes of fast phase as a
percentage of the total) for the reaction with hydrogen peroxide at pH
3.97 using the equation of Smith et al. (20), assuming Ca2+ binding to a single site,
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(Eq. 1)
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where A is the change observed,
A
is the maximum change, L is the
total ligand concentration, and P is the enzyme
concentration. Titration experiments of BP 1 with NaCl in 0, 1, 10, and
50 mM CaCO3 and titration with
CaCl2 in citrate buffer were performed by adding aliquots
of buffered NaCl or CaCl2 to the enzyme and monitored by
absorption spectrometry. The dissociation constant for
Cl
binding to the BP 1·Ca2+
complex was estimated by fitting the change in
A406
A310
versus Cl
concentration to a single
exponential function, assuming that the Kd for
Cl
binding is much higher than for Ca2+
binding and therefore did not significantly affect the concentration of
the BP 1·Ca2+ complex.
The effects of Na+, K+, Mg2+,
Cd+2, Sr2+, Ba2+, Lu3+,
and Eu3+ on the reaction between BP 1 and hydrogen peroxide
were studied in 50 mM sodium citrate buffer, pH 3.97. The
effects of ionic strength were examined in 50 mM sodium
citrate buffer, pH 3.97, in the presence of 1 mM
CaCl2 at 0.04 M ionic strength and after adding K2SO4 or Na2SO4 to 0.5 M ionic strength. The ionic strength effects were also
analyzed at pH 3.25, 3.97, and 4.93 in the absence of Ca2+
at 0.04, 0.1, and 0.5 M ionic strengths obtained by adding
K2SO4 to 25 or 50 mM citrate
buffers with initial ionic strengths of 0.04 M. The effect
of the ionic strength on the BP 1 absorption spectra in the absence and
presence of 1 mM CaCl2 was examined at pH 3.25, 3.97, and 4.93 at 0.04, 0.1, and 0.5 M ionic strength using
the same buffers.
The spectral and kinetic effects of Ca2+ and
Cl
on fluoride binding to BP 1 were examined in a similar
way. Spectral titrations of BP 1 were performed at pH 3.10, 3.55, 3.97, 4.09, 4.35, 4.65, and 5.14 in duplicate or triplicate, adding 15-25
aliquots of concentrated NaF in each experiment, and calculating
Kd according to the Smith et al. (20)
equation (Equation 1). Experimental conditions are elaborated in the
figure and table legends.
 |
RESULTS |
Reaction of BP 1 with Hydrogen Peroxide
Effect of Calcium Ion--
The principal experimental observation
of this work is that the reaction of barley grain peroxidase BP 1 and
hydrogen peroxide can exhibit double exponential kinetics, indicating
that a slow and a fast reaction occur simultaneously. Fig.
1 illustrates that at pH 3.97 the
conversion to cpd I is slow and strictly monophasic in the absence of
Ca2+ (with or without EDTA), whereas it becomes biphasic in
the presence of Ca2+, with a fast phase approximately 100 times faster than the slow phase observed in absence of
Ca2+. The fast phase constitutes approximately 20% of the
total amplitude (total change of enzyme absorbance) in the presence of
1 mM Ca2+ and 90% in the presence of 50 mM Ca2+. We will refer to this rapid form of
the enzyme as the EA state (where A represents
"active").

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Fig. 1.
Biphasic kinetic traces of the reaction of BP
1 and H2O2 in response to calcium ion
concentration. The change in absorbance at an isosbestic point
between cpd I and II (Fig. 3B) is recorded. The reactions
were performed at 25 °C with 1 µM BP 1 and 5 µM H2O2, with no or 1 mM CaCO3 at ionic strength 0.1 M in
50 mM sodium citrate buffer, pH 3.97, and with 50 mM CaCO3 ionic strength > 0.1 M in 100 mM sodium citrate buffer, pH 3.97. The
data were fitted to a single (no calcium) or double exponential
function (calcium present).
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Analysis of the hydrogen peroxide dependence of the two phases shows
that they follow different mechanisms of reaction. The pseudo-first-order rates of the fast phase show linear dependence of
hydrogen peroxide (Fig. 2A)
and follow a typical peroxidase mechanism (6).
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(Eq. 2)
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The rates of the slow phase (slow state of the enzyme
ES) show saturation at high hydrogen peroxide
concentration (Fig. 2B), a mechanism perhaps involving one
or more reversible steps,
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(Eq. 3)
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following Michaelis-Menten type kinetics,
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(Eq. 4)
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or an initial slow conformational change of an inactive state
EI preceding cpd I formation,
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(Eq. 5)
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with the rates ki, k
i, and
k1s [H2O2] and
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(Eq. 6)
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could explain the observed kinetics. The kinetics of the slow
reaction have previously been extensively studied (8, 9) and
characterized by an apparent saturation rate constant
(kcatapp) and substrate
concentration at half-saturation
(Kmapp) at nine pH values pH
3.09-5.08 (8). The results obtained here are very similar. Table
I lists the saturation rate constants, whether kcatapp or
ki, and the initial slope or rate at low
[H2O2] for the slow phase. An enzyme-linked
pKa < 3.7 is found for kcatapp or ki.
At pH 3.97, the saturation rate is 2.0 s
1, and the
initial rate is 0.11 µM
1
s
1.

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Fig. 2.
Plots of kobs for the
fast (A) and slow (B) phase versus
hydrogen peroxide concentration at pH 3.33. The measurements were
performed in pH jump experiments in 50 mM sodium citrate buffer, 1 mM CaCO3, 0.1 M ionic
strength (final concentrations). Each data point represents the average
of at least five measurements ± S.E. The
kobs of the fast phase shows linear dependence
of hydrogen peroxide concentration with a slope of
k1app = 17.3 ± 0.7 µM 1 s 1, while
kobs of the slow phase shows saturation kinetics
with kcatapp = 5.5 ± 0.4 s 1.
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Table I
The pH dependence of the reactions of the fast and slow forms of BP 1 and H2O2
The reactions were carried out at 25 °C with 1 µM BP 1 and 5-50 µM H2O2, in 50 mM
sodium citrate, 1 mM Ca2+ or in 25 mM
sodium citrate, 10 mM Ca2+, both at 0.1 M ionic strength, or in 50 or 100 mM sodium
citrate, 50 mM Ca2+, 0.2 M ionic
strength. Amplitudes were calculated from the lines fitted to the
stopped-flow data, and the apparent second order rate constants were
calculated from kobs (s 1)
versus [H2O2]. The fast phase
k1app seems to be independent of pH from 3.3 to 5.0 and is 15.7 ± 0.5 µM 1 s 1
(average of eight values ± S.E.).
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A dissociation constant (Kd) of 4 ± 1 mM for the binding of Ca2+ to BP 1 at pH 3.97 was calculated by fitting of the amplitudes of the fast phase
versus Ca2+ concentration (Table
II) as indicated under "Experimental
Procedures." The absorption spectrum of BP 1 changes very little upon
Ca2+ binding in the absence of Cl
(data not
shown). Consequently, the near heme environment is not changed directly
on Ca2+ binding.
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Table II
Effect of Ca2+ and Cl on the apparent second order
rate constant and amplitude of the fast phase of the reaction between
BP 1 and H2O2 at pH 3.97
Experimental conditions and calculations are as in Table I.
CaCl2 was used in the first eight experiments, whereas
combinations of CaCO3 and NaCl were applied to the last six
experiments.
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The binding of Ca2+ is strongly pH-dependent.
As shown in Table I, the amplitude of the fast phase in the presence of
1 mM Ca2+ is 60% at pH 3.33 and only 13% at
pH 4.23. At higher pH values, higher concentrations of Ca2+
are necessary to recruit more enzyme into the very active fast state.
At pH 5.52 and 5.98, very little activity and fast phase can be
observed. This pH dependence is the opposite of that expected for
normal Ca2+ binding sites in proteins, which usually
involve two unprotonated carboxylate side chains that become protonated
at lower pH and indicates that only the protonated
ES form can be activated by Ca2+.
The Ca2+ bound form (EA) has a
reactivity toward hydrogen peroxide typical of peroxidases. The
conformational change (ES to
EA; Scheme 1) is
slow compared with the rates of cpd I formation on the time scale of
these experiments, since two parallel reactions can be observed (Fig.
1).
At pH 3.33, if the experiments were not performed by a pH jump method,
the total amplitude (both phases) decreased to 80%, and the fast phase
decreased from 60% observed in pH jump experiments to 30% of the
total amplitude (Table I). The pH jump experiments required at least
10 s for completion of cpd I formation. Incomplete equilibration
among the three states of BP 1 is therefore unlikely to affect the
results. No heme bleaching was seen.
Spectra of Resting State, cpd I, and cpd II--
The time course
of the reaction of BP 1 and hydrogen peroxide at pH 3.97 is illustrated
by rapid scan diode-array absorption spectra (Fig.
3A). cpd I formation is seen
in a series of spectra taken over 5 s, and the decay to cpd II is
observed over 1000 s. The individual spectra of resting state BP
1, cpd I, and cpd II were calculated from these data by global
analysis. Absorbance maxima and well defined isosbestic points between
spectra were observed in the original spectra (Fig. 3A),
such as the isosbestic point between cpd I and II at 398 nm, which was
used throughout for monitoring of the kinetic studies of cpd I
formation. The reaction was carried out in 1 mM
CaCl2, which gave 80% free BP 1 (ES
form), 20% BP 1·Ca2+ complex (EA
form), and potentially <1% BP 1·Ca2+·Cl
complex (see below). The well defined isosbestic points of the spectra
of cpd I and II indicate that the ES and
EA forms generate the same intermediates as
subsequently found for the fluoride complex of these two forms.
Therefore, the amplitudes of the fast and slow phases are direct
measures of the amount of enzyme in the EA and
ES states, respectively, at a given
Ca2+ concentration and pH (Fig. 1; Table I).

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Fig. 3.
Time course of spectral changes during the
reaction of BP 1 and H2O2 and calculated
absorption spectra of intermediates. The reaction of 3 µM BP 1 and 50 µM
H2O2 in 50 mM sodium citrate buffer, 1 mM CaCl2, pH 3.97, 0.1 M
ionic strength, was recorded by rapid scan diode-array absorption
spectrometry for 10 and 1000 s. Panel A shows selected
spectra from each series. Panel B shows the calculated
spectra of resting state BP 1 ( ), cpd I (- - -), and cpd II
(- · -), isosbestic points and maxima. Panel C
shows residual absorbance between the experimental data and the
calculated spectra. Panel D shows the time course of
BP 1 conversion to cpd I, which slowly decays to cpd II.
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Fig. 3D demonstrates the formation of cpd I in 1 s and
its stability for 2-3 s. The slow form of BP 1 (complete absence of Ca2+) previously gave less stable (100 ms) and less
complete cpd I formation (about 60%) (8), which in comparison with the
present data (stable for 2 s; about 87% complete cpd I formation)
indicates greater stability of cpd I of the Ca2+-bound form
of BP 1. This was verified, since the lifetime of cpd I increases
considerably in the presence of 10 and 100 mM CaCl2 (not shown). However, cpd I of BP 1 is still very
unstable compared with other peroxidases. One reason for this could be the low pH of cpd I formation, which gives a higher redox potential for
cpd I and thus greater reactivity.
Effect of Chloride--
Experiments examining the effects of
calcium ion (see above) were first carried out with CaCl2.
The k1app for the fast phase,
however, showed a slight decline with increasing CaCl2
concentration (Table II), and spectral changes indicated a
Kd about 10-fold higher than the one derived from
the kinetic data. These data suggested that a strong calcium ion effect was overlaid by a weaker chloride effect, and further experiments were
carried out using CaCO3 and NaCl. Spectral analysis of BP 1 in 200 mM NaCl, in the absence of Ca2+, showed
that chloride does not bind to the EI or
ES forms of BP 1 (data not shown). In the
presence of Ca2+ ions, a Kd for the
binding of Cl
at pH 3.97 was calculated to be 62 ± 9 mM from spectral titration experiments using NaCl plus
CaCO3, and a similar estimate was obtained with
CaCl2 in citrate buffer (Fig.
4). Binding of Cl
to the
Ca2+-bound form of BP 1 (EA) changes
the Soret maximum from 401 to 406 nm and increases the extinction
coefficient. In the visible region, the
band at 498 nm shifts to
502 nm, and the CT1 band shifts from 640 to 637 nm (Fig. 4,
inset). An isosbestic point between the two spectra is
observed at 398 nm. The spectral changes suggest an increasing amount
of six-coordinate heme. Similar spectral changes were observed on
CCP-Cl
complex formation at pH 5.0 (the shoulder at 370 nm decreases, the Soret band increases in intensity and changes from
408 to 413 nm, the 505 band moves to 510 nm, and the CT1 band moves
from 645 to 640 nm) (12).

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Fig. 4.
Chloride binding to the BP
1·Ca2+ complex. Electronic absorption spectra of 3 µM BP 1 and 10 mM CaCO3 in 50 mM sodium citrate buffer, pH 3.97, ionic strength 0.1
M, and 0, 2.7, 15.9, 78.4, and 202.5 mM NaCl at
25 °C are shown. Arrows show the spectral change with
increasing chloride concentration. A dissociation constant
Kd = 62 ± 9 mM for chloride
release from the BP 1·Ca2+·Cl complex was
estimated from several titrations also using 50 mM CaCO3. The inset shows the spectra of BP 1 at 1 and 100 mM CaCl2.
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During cpd I formation, only the rate of the fast phase was affected by
Cl
, the rate decreasing with increasing Cl
concentration (Table II), strongly suggesting that Cl
binds only to the EA state. Neither the
amplitudes of the two phases nor the rate of the slow phase are
influenced by the presence of Cl
ions.
Effect of pH--
The pH dependence of the rate of the fast phase
(Table I) is different from that of the slow phase. In the pH range
from 3.3 to 5.0, the apparent second order rate constant is unchanged within experimental error, with an average of 15.7 ± 0.5 µM
1 s
1. At pH 5.52 at high
ionic strength, the second order rate constant seems to decrease to
7.1 ± 1.2 µM
1 s
1. At pH
5.98, fitting was not possible due to the small amplitude.
Other Ionic Effects--
Possible effects of a variety of cations
and anions and of ionic strength were analyzed. Only Sr2+
and Ba2+ gave rise to pseudo-first order rates for cpd I
formation and relative amplitudes of the fast phase comparable with
those found for experiments with Ca2+ (Table
III). Ionic strength adjustments, whether
done by Na2SO4 or
K2SO4, gave the same results. Consequently,
Mg2+, Na+, K+, Cd2+,
Lu3+, Eu3+, Ac
,
NO32
,
CO32
, and
SO42
show no strong specific ionic
effects on BP 1 cpd I formation.
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Table III
Effect of various ions on the apparent second order rate constant and
amplitude of the fast phase of the reaction between BP 1 and
H2O2
The reactions (at least five experiments deviating less than 10% from
the average) were carried out with 1 µM BP 1 and 5 µM H2O2 at 25 °C in 50 mM
sodium citrate, pH 3.97, with no Ca2+ present and no ionic
strength adjustment.
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Absorption spectra and the relative amplitudes of the two phases were
unaffected by ionic strengths of 0.04, 0.1, and 0.5 M in
citrate buffer containing 1 mM CaCl2 at pH
3.25, 3.97, and 4.93. The rates of the slow phase, however, increase
with ionic strength but less so at higher pH. If rates are 100% at 0.1 M ionic strength, then at pH 3.25 the rate is 30% at 0.04 M and 180% at 0.5 M ionic strength; at pH
3.97, 50 and 170%, respectively; and at pH 4.93, 100 and 125%,
respectively.
Fluoride Binding
Electronic Absorption Spectra--
Adding fluoride to BP 1 at pH
3.55 (Fig. 5) increases the intensity of
the Soret band significantly and shifts the maximum from 401 to 402 nm.
In the visible region, the 498-nm band shifts to 492 nm. The CT1 band
at 640 nm in the spectrum of the native enzyme is replaced by a rather
intense band at 611 nm in the fluoride-bound form. Well defined
isosbestic points are seen at 389, 555, and 635 nm. Similar changes are
observed in CCP·F
complex formation at pH 5.0 (12) and
in other wild type and mutant peroxidases and are consistent with the
formation of a six-coordinate high spin complex in which fluoride is
coordinated to the heme iron and strongly hydrogen-bonded to the distal
arginine (15).

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Fig. 5.
Complex formation of BP 1 and fluoride.
Electronic absorption spectra of 5.0 µM BP 1 and 0, 0.5, 2, 7, and 38 mM NaF in 50 mM sodium
citrate-phosphate buffer, 2 mM CaCl2, ionic
strength 0.1 M, pH 3.55, at 25 °C are shown.
Arrows show the spectral change with increasing fluoride
concentration. A dissociation constant Kd = 2.3 ± 0.2 mM for the BP1·fluoride complex was calculated from these data. The Kd values determined from
spectral titrations from pH 3.10 to 5.14 were all approximately 10 times smaller than those obtained from kinetic experiments (Table IV). The inset demonstrates monophasic kinetics of the formation
of the complex between 1 µM BP 1 and 10 mM
fluoride in 50 mM sodium citrate buffer, 2 mM
CaCl2, ionic strength 0.1 M, pH 3.97, 25 °C, despite the presence of Ca2+. The
kobs value of the example shown is 31.5 ± 0.08 s 1.
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Binding Kinetics--
Fluoride binding to BP 1 exhibits monophasic
reaction profiles unaffected by Ca2+ (Fig. 5,
inset), in contrast to the results for
H2O2 with Ca2+ present (Fig. 1).
The rate constants of association (kon) and dissociation (koff) and, thus, the kinetically
derived dissociation constant (Kd = koff/kon) are similarly
not affected by Ca2+ or Cl
at 2 mM CaCl2 (Table
IV). The association rate constant is
strongly dependent on pH and greatly favored at low pH, which indicates that the HF form is reacting (pKa = 3.14 (10)). The dissociation rate constant is largely independent of pH in the pH range
examined (Table IV). The dissociation constant, Kd, thus depends on pH in a similar way to kon,
i.e. rising sharply from pH 4.0 to 4.6.
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Table IV
The pH dependence of the binding of BP 1 and fluoride
Stopped-flow experiments were carried out at 25 °C using 1 µM BP 1 and 2.5-50 mM NaF in 50 mM sodium citrate, ionic strength 0.1 M, in the
presence or absence of 2 mM CaCl2. The rate of
association, kon, was determined from the slope of
kobs versus fluoride concentration, and
the rate of dissociation, koff, was determined from
the intercept at the y axis. The dissociation constant
Kd = koff/kon.
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The effects of Ca2+ and Cl
on the binding
kinetics of fluoride and BP 1 were examined more closely at pH 3.97 (Table V) but limited to a maximal
concentration of 5 mM CaCl2 due to low
solubility of CaF2. However, with Kd = 4 mM for the calcium binding, more than half of BP 1 will be
in the Ca2+-bound form, and biphasic kinetics should
therefore have been observed if the EA and
ES forms of BP 1 reacted differently. Therefore, it appears that fluoride binds to both forms, in contrast to
Cl
.
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Table V
Effect of Ca2+ and Cl on BP 1 and fluoride binding at
pH 3.95
Experimental conditions and calculations are as in Table III.
kon is independent of both ions and gives an average
value of 850 M 1 s 1.
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It is noteworthy that Kd determination by spectral
titrations as illustrated in Fig. 5 from pH 3.10 to 5.14 (Kd = 1.3 and 135 mM, respectively) gave
values 10 times smaller than those obtained from the kinetic
experiments. We speculate that the binding of fluoride
(kon) may include a spectroscopically silent
step 10 times faster than the change observed at 402 nm.
 |
DISCUSSION |
Kinetically Defined Forms of BP 1--
Scheme I gives a simple
overview of kinetically defined forms of BP 1 and the reaction
characteristics of these forms with hydrogen peroxide. BP 1 shows very
little activity (inactive form) at pH > 5, although 50 mM Ca2+ can induce measurable activity up to pH
6.0 (Table I). The kinetics suggest that formation of a slow reacting
BP 1 is triggered by the protonation of a group with
pKa < 3.7 (presumably a carboxylate).
ES has a narrow pH range of activity (8) and a
maximal bimolecular rate constant of 6 × 105
M
1 s
1 at pH 3.47 at low
H2O2 concentration (Table I). In the presence of Ca2+, it appears that a conformational change, which is
slow compared with cpd I formation, converts ES
into the fast form, EA.
EA exerts typical peroxidase kinetics with
respect to cpd I formation and has an apparent second order rate
constant of 1.6 × 107 M
1
s
1 independent of pH in the range 3.3-5.0 where this
form predominates (Table I). The kinetic data suggest that the fast
EA form of BP 1 is a BP 1·Ca2+
complex with Kd = 4 mM at pH 4.0, assuming a single Ca2+ binding site involved in activation.
Among cations, only Sr2+ and Ba2+ were found to
substitute for Ca2+ (Table III). The slow and fast forms of
BP 1 were attributed to two independent phases in data from
stopped-flow studies of cpd I formation but were kinetically and
spectroscopically indistinguishable in reactions with fluoride;
i.e. the ES and
EA forms bind fluoride at similar rates
(kon; Tables IV and V). Fluoride binding,
however, is 2-4 orders of magnitude slower than cpd I formation by
ES and EA, respectively,
and could therefore include an ES to
EA type of rearrangement in the mechanism of
fluoride binding to ES. Common anions, excluding
those that are well known ligands of heme iron (F
,
CN
, N3
), showed no
specific effects on slow BP 1. The Cl
binds weakly
(Kd ~60 mM at pH 3.97) and only to the fast reacting BP 1-Ca2+ form, causing changes in the
absorption spectrum (see below) and inhibiting cpd I formation
moderately as indicated in Scheme I.
Structures of BP 1 Forms--
BP 1 is fairly stable at pH 3-11,
especially at neutral pH (19). The electronic absorption spectra of BP
1 at pH 3.95 and 8.30 are very similar (excepting the Soret peak shift
from 401 to 399 nm) and characteristic of a five-coordinate high spin
heme (19) with no low spin component to account for inactivity or reduced activity. The accompanying paper by Henriksen et al.
(16) on the crystal structures of BP 1 at pH 5.5, 7.5, and 8.5 in the absence of Ca2+ (EI form) confirms
this and shows a vacant and sterically hindered sixth coordination site
at heme Fe with distal Phe48 close by and parallel to the
heme at a C
-iron distance of 3.8 Å and the nearest
water 690-iron distance of 5.4 Å. The lack of activity of the
EI form, however, is undoubtedly due to the
distal His49 being turned away from the distal cavity and
unavailable for catalysis. A number of crystallographic, kinetic, and
spectroscopic studies have proven that the distal histidine in its
unprotonated form is essential to peroxidase cpd I formation and to the
binding of the majority of ligands to the heme iron and that it
contributes to the rate of cpd I formation by approximately
105 M
1 s
1 (21-24).
Since the apparent second order rate constants of both kinetic phases
at low H2O2 concentration are > 105 M
1 s
1, both the
ES and EA states of
active BP 1 seem to contain normally positioned unprotonated distal
histidine side chains. From the kinetic data, we therefore suggest that
protonation of a carboxylate side chain with pKa < 3.7 in inactive neutral BP 1 (EI) induces a
conformational change (the nature of this change and possible role of
Asp143 is discussed in the accompanying paper (16)), which
gives rise to a structural relationship between distal
His49 and heme iron typical of active peroxidases.
The kinetics defining the slow ES form of BP 1 show an abnormal peroxidase with kinetic characteristics comparable
with those of distal arginine mutants of CCP (25) and HRP C (24, 26). Thus, the arginine to leucine mutants of CCP and HRP C showed saturation kinetics at high hydrogen peroxide concentration and at
least 100-fold lower bimolecular rates. It was suggested that the
kinetics supported the reversible formation of a peroxidase-peroxide intermediate before heterolytic O-O bond cleavage (Equation 3) (23).
However, the distal arginine to lysine mutant of CCP (25) shows
saturation in the micromolar range of hydrogen peroxide concentration
like the slow ES state of BP 1 and may be a
better model, since it has charge and hydrogen-bonding properties more similar to those of arginine. A conformational gating mechanism involving the opening of the sterically hindered lysine mutant was
proposed to account for the cpd I formation kinetics in this mutant
(25). The rate constant at saturation was 200-300 s
1,
depending on the buffer used. We find a saturation rate constant of
only 2 s
1 at pH 3.97, which could then suggest a much
more extensive blockage of the active site and a conformational change,
such as EI to ES, prior
to cpd I formation (Equation 5). However, BP 1 seems so different from
other known peroxidases that only a structure of the
ES state observable in the absence of
Ca2+ at pH 3.1-4.0 can distinguish between these models
and provide an answer.
Binding of fluoride mimics aspects of cpd I formation as summarized in
the Introduction. The results show that the rate of fluoride binding to
BP 1 is indistinguishable for the ES and
EA states (Table IV), indicating that heme iron
and distal histidine are equally accessible and functional in the two,
although the possibility cannot be excluded that the slow rate of
binding may also include the conversion between these forms. The
absorption spectrum of the complex is the same whether the calcium ion
is present or absent, which is also the case for the spectra of the resting state, cpd I and cpd II. The crystal structures of the fluoride
complex (13) and of cpd I of CCP (14) show that both the fluoride and
oxene ligand are stabilized by hydrogen bonding from N
of the distal arginine. The stabilizing effect of this interaction is
very strong and estimated to be 5.7 kcal/mol in CCP at pH 5.0 (Ref. 15
and erratum). By analogy, fluoride binding and cpd I formation by the
ES state of BP 1 seems, therefore, to be
followed by an adjustment making the distal Arg45 available
for stabilization of the ligand (23). However, the slow rate of
fluoride binding does not distinguish between a severely dislocated and
a typical distal arginine. It should also be noted that fluoride
binding to BP 1 is weaker (Kd = 4 or 25 mM at pH 4.0, determined by spectral titration and
kinetics, respectively, and 100 mM at pH 5.0, determined by
spectral titration) than to HRP C (Kd = 0.13 mM at pH 4.0 and 0.69 mM at pH 5.0 (11)) and to
CCP (Kd = 0.003 mM at pH 5.0; Ref. 12).
Accumulation of the EA form of BP 1 is clearly
the result of a rather slow conformational change either induced or
stabilized by increasing Ca2+ concentration at low pH (Fig.
1, Table II). The BP 1·Ca2+ complex behaves as an
entirely normal peroxidase in its reactions with hydrogen peroxide and
fluoride, and therefore, all structural properties, including those of
distal Arg45 and His49, must be highly similar
to those observed in the crystal structures of active cationic peanut
and horseradish peroxidases (17, 18).
Chloride binds exclusively to the EA state of BP
1 with a Kd = 60 mM at pH 4.0. This is
approximately 10-fold weaker than chloride binding to CCP (12).
Chloride binding to BP 1 and CCP gives very similar spectral changes
(see "Results"), which suggests similar binding sites. Yonetani and
Anni (12) demonstrated an increasing binding of chloride to CCP with
decreasing pH (Kd = 2.9, 2.4 × 10
2, and 1.0 × 10
2 M at
pH 6.0, 5.0, and 4.5, respectively), consistent with the binding of HCl
rather than Cl
(i.e. a mechanism similar to
that of fluoride binding involving distal histidine as the proton
acceptor (6, 10-12)). It appears, therefore, that both of these
ligands bind to the heme iron, despite the marked differences in their
resulting electronic absorption spectra (Figs. 4 and 5) (12), where the
chloride complex spectrum seems more similar to the spectrum of a
six-coordinate water-ligated peroxidase (27). The crystal structure of
a chloride-peroxidase complex is not known, however.
The lack of chloride binding to the slow ES
state of BP 1 indicates a distorted heme pocket that may be either
inaccessible to chloride or require an adjustment of side chains that
costs more in energy than can be gained on chloride binding. Fluoride binds to this state in a fashion that cannot be distinguished from the
binding to the EA state. The small HF can
therefore access heme iron in the ES state.
Furthermore, the binding of this ligand may provide sufficient energy
(1.6 kcal/mol more than chloride at pH 4.0, comparing their
Kd values determined by spectral titration) to
change the heme pocket structure from an
ES to an EA type in the
absence of the Ca2+ ion.
A Novel Ca2+ Binding Site?--
The crystal structures
of plant (class III) and fungal (class II) peroxidases targeted for the
secretory pathway show two conserved sites of Ca2+ binding,
one in each of the domains sandwiching the heme (17, 18, 28, 29). These
sites have been proposed to originate from an early gene duplication
event creating the present day two-domain peroxidase structures (3,
30). Amino acid sequence alignment demonstrated that the carboxylate
and hydroxyamino acid side chain ligands are conserved in all members
of these classes, including BP 1 (30). The refined crystal structure of
BP 1 at pH 7.5 (EI state) discloses a proximal
Ca2+ site that is similar to those of the known plant and
fungal peroxidases, but containing distorted distal domain with a
weakened metal binding site, proposed to hold a Na+ as
described in the accompanying paper (16). BP 1 preparation and
crystallization were performed in the absence of Ca2+, and
it can therefore be concluded that the proximal site binds Ca2+ more strongly than the distal site at pH 7.5. In
comparison, Ca2+ titration of cationic peanut peroxidase
complexed with cyanide monitored by NMR gave Kd = 0.1 µM for both the proximal and distal Ca2+
sites at pH 7.3 (31). However, it is unknown whether added Ca2+ would replace Na+ in the
EI form of BP 1. It is also noticeable that
Ca2+-depleted HRP C showed 80% of the rate of cpd I
formation by native HRP C (32) and not a 100-fold decrease as BP 1 shows in the absence of added Ca2+. Furthermore, it must be
considered that the Ca2+ content of manganese peroxidase
(33) and anionic peanut peroxidase (34)2 was 3.9 and 5 mol/mol,
respectively, indicating that additional Ca2+ binding sites
may exist in these peroxidases.
Against this background, we will consider two alternative sites
responsible for Ca2+-induced activation in BP 1, either
binding at the canonical distal Ca2+ site or at a novel
unidentified site. In favor of the canonical distal site is the fact
that this provides the simplest explanation. However, a
Kd value of 4 mM rather than 0.1 µM and the substitution of Ca2+ with the
larger Sr2+ or Ba2+ ions would give a distal
site very different from that of cationic peanut and horseradish
peroxidases (17, 18). A novel alternative site, in addition to a distal
Ca2+ with properties similar to those of cationic peanut
peroxidase and HRP C, must be compatible with the insignificant change
of the absorption spectrum of BP 1 observed in the presence of
Ca2+, which demonstrates that there is no structural change
near the heme iron to perturb the iron coordination or spin state. The exact location of the activating Ca2+ site must await
crystallographic studies in the presence of Sr2+ or
Ba2+.
Other BP 1 Complexes--
Crystallographic (16) and kinetic
evidence demonstrate several well defined states of BP 1 and a uniquely
adaptable distal domain in the presence of cations. In addition to
those already discussed, BP 1 shows a significant spectral change in
the presence of ammonium sulfate or ammonia (data not shown) similar to
that observed for Coprinus cinereus (synonymous with
Arthromyces ramosus) peroxidase (35). It was also noted
previously (9) that the absorption spectrum of BP 1 changes slightly in
the presence of ethanol (Kd = 2 mM) and
ferulic acid (Kd = 7 µM). The binding
of phenolic substrate was thought to account for a 10-40-fold increase
in the rate of cpd I formation observed in steady-state analyses at pH
3.96 with ferulic acid, caffeic acid, and coniferyl alcohol; however,
these experiments were performed in 1 mM CaCl2
(9). Reinterpretation of these data in the light of the present data
suggests that the presence of calcium can explain the ferulic acid and
caffeic acid results but not the coniferyl alcohol
data.3
Biological Function of BP 1--
BP 1 is highly regulated at the
gene level, since mRNA for BP 1 is expressed only for 2 days 15 days after flowering and only in the starchy endosperm of the barley
seed (36). The protein product is very stable, however, and BP 1 is the
most abundant peroxidase of mature barley grain. Protein signals target
BP 1 for the secretory pathway (N-terminal signal peptide) and onward to the vacuole (C-terminal propeptide) (37). In the present work, we
document that plant peroxidase activity can also be strongly regulated
at the enzyme level through protein conformational changes induced by
pH and Ca2+. Still, however, there is no evidence to
suggest the major biological role of BP 1. BP 1 seems to be well suited
to functioning within the vacuole at the low pH and high
Ca2+ and phenolics concentrations often present in this
organelle (38) and requires only hydrogen peroxide for the initiation of catalysis.
Extensive discussions with Drs. A. Henriksen (University of Copenhagen), G. Smulevich
(University of Florence), and H. B. Dunford (University of
Alberta) are gratefully acknowledged.