Effect of Calcium, Other Ions, and pH on the Reactions of Barley Peroxidase with Hydrogen Peroxide and Fluoride
CONTROL OF ACTIVITY THROUGH CONFORMATIONAL CHANGE*

Christine B. Rasmussen, Alexander N. P. Hiner, Andrew T. SmithDagger , and Karen G. Welinder§

From the Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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).
<UP>Peroxidase</UP>+<UP>ROOH </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB></UL></LIM> <UP>cpd I</UP>+<UP>ROH</UP>
<UP>cpd I</UP>+<UP>AH </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM> <UP>cpd II</UP>+<UP>A<SUP>·</SUP></UP>
<UP>cpd II</UP>+<UP>AH </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> <UP>Peroxidase</UP>+<UP>A<SUP>·</SUP></UP>+<UP>H<SUB>2</SUB>O</UP>
<UP><SC>Reaction</SC> 1</UP>
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 pi  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).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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,
A=2A<SUB>∝</SUB>L/((L+K<SUB>d</SUB>+P)+((L+K<SUB>d</SUB>+P)<SUP>2</SUP>−(4PL))<SUP>1/2</SUP>) (Eq. 1)
where A is the change observed, Aproportional to 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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).

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).
E<SUB><UP>A</UP></SUB>+<UP>H<SUB>2</SUB>O<SUB>2</SUB> → cpd I</UP>+<UP>H<SUB>2</SUB>O</UP> (Eq. 2)
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,
E<SUB><UP>S</UP></SUB>+<UP>H<SUB>2</SUB>O<SUB>2</SUB> ↔ </UP>X → <UP>cpd I</UP>+<UP>H<SUB>2</SUB>O</UP> (Eq. 3)
following Michaelis-Menten type kinetics,
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>cat</UP></SUB>[<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>]/(K<SUB>m</SUB>+[<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>]) (Eq. 4)
or an initial slow conformational change of an inactive state EI preceding cpd I formation,
E<SUB><UP>I</UP></SUB> ↔ E<SUB><UP>S</UP></SUB>+<UP>H<SUB>2</SUB>O<SUB>2</SUB> → cpd I</UP> (Eq. 5)
with the rates ki, k-i, and k1s [H2O2] and
k<SUB><UP>obs</UP></SUB>=k<SUB>i</SUB>[<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>]/((k<SUB>i</SUB>+k<SUB><UP>−</UP>i</SUB>)/k<SUB>1<UP>s</UP></SUB>+[<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>]) (Eq. 6)
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.).

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.

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).


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Scheme 1.   Kinetically defined forms of BP 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.

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 beta  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.

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.

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.

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.

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.

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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 Cepsilon -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 Nepsilon 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.

    ACKNOWLEDGEMENTS

Extensive discussions with Drs. A. Henriksen (University of Copenhagen), G. Smulevich (University of Florence), and H. B. Dunford (University of Alberta) are gratefully acknowledged.

    FOOTNOTES

* This research was supported by European Community Human Capital and Mobility Program Grant ERBCHRX-CT92-0012 (to K. G. W. and A. T. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex BN1 9QG, United Kingdom.

§ To whom correspondence should be addressed. Tel.: 45 35322077; Fax: 45 35322075; E-mail: welinder{at}biobase.dk.

1 The abbreviations used are: CCP, yeast cytochrome c peroxidase; cpd I and cpd II, compound I and II, respectively; BP 1, barley grain peroxidase isoenzyme 1; CIP, C. cinereus peroxidase; HRP C, horseradish peroxidase isoenzyme C.

2 The same study gave two Ca2+ for cationic peanut peroxidase as observed by x-ray crystallography of this isoenzyme (17).

3 The present data show that 20% of BP 1 would be in the EA state under the conditions used in Ref. 9 and increase the rate k1 at low H2O2 concentration (Table I) from 0.1 µM-1 s-1 to 16 × 0.2 congruent  µM-1 s-1 as was observed for ferulic acid and caffeic acid (9). However, approximately 7 µM-1 s-1 found for coniferyl alcohol suggests a small effect of this substrate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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