Allosteric modulation of Euphorbia peroxidase by nickel ions Francesca Pintus 1 , Anna Mura 1 , Andrea Bellelli 2 , Alessandro Arcovito 3 , Delia Spano ` 1 , Anna Pintus 1 , Giovanni Floris 1 and Rosaria Medda 1 1 Department of Applied Sciences in Biosystems, University of Cagliari, Cagliari, Italy 2 Department of Biochemical Sciences ‘A. Rossi Fanelli’, University of Rome ‘La Sapienza’ and CNR Center of Molecular Biology, Rome, Italy 3 Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Rome, Italy Peroxidases are considered to act as antioxidant enzymes, protecting cells, tissues and organs against the toxic effects of peroxides, but some of them are also directly involved in the synthesis of important metabolites, particularly in plants [1,2]. The super- family of heme peroxidases can be grouped into three classes. Class I contains bacterial and plant intracellular enzymes from mitochondria and chlo- roplasts, such as ascorbate peroxidase and cyto- chrome c peroxidase. Class II consists of secreted fungal peroxidases, e.g. manganese peroxidase and lignin-degrading peroxidase. Class III contains secreted plant peroxidases, exemplified by horseradish peroxidase (EC 1.11.1.7, donor hydrogen peroxide oxidoreductase), of which the best known isoform is isozyme C (HRP-C) [3–5]. The native enzyme is generally considered to contain high-spin Fe 3+ in a protoporphyrin IX pentacoordinated to a ‘proximal’ Keywords calcium; heme proteins; hydrogen peroxide; nickel; peroxidase Correspondence R. Medda, Dipartimento di Scienze Applicate ai Biosistemi, Citta ` Universitaria, I–09042 Monserrato, CA, Italy Fax: +39 070 6754523 Tel: +39 070 6754517 E-mail: rmedda@unica.it (Received 13 November 2007, revised 7 January 2008, accepted 9 January 2008) doi:10.1111/j.1742-4658.2008.06280.x A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron–protoporphyrin IX pentacoordinated with a histidine ‘proximal’ ligand as heme prosthetic group. In addition, the purified peroxidase con- tained 1 mole of endogenous Ca 2+ per mole of enzyme, and in the pres- ence of excess Ca 2+ , the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni 2+ causes reversible inhibition, whereas, in the presence of excess Ca 2+ ,Ni 2+ leads to an increase of the catalytic activity of Euphorbia peroxidase. UV ⁄ visible absorption spectra show that the heme iron remains in a quantum mechan- ically mixed-spin state as in the native enzyme after addition of Ni 2+ , and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni 2+ . In the presence of H 2 O 2 and in the absence of a reducing agent, Ni 2+ decreases the catalase-like activity of Euphorbia peroxidase and accel- erates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni 2+ affects the H 2 O 2 -binding site and inhibits the formation of com- pound I. In the presence of excess Ca 2+ ,Ni 2+ accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni 2+ -binding sites with opposite func- tional effects. Abbreviations ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid); ELP, Euphorbia latex peroxidase; HRP-C, horseradish peroxidase isozyme C; SVD, singular value decomposition. FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS 1201 histidine ligand. This histidine functions to stabilize the higher oxidation states of the iron atom [6], while another histidine, known as the ‘distal’ ligand, functions as an acid–base catalyst to accept one pro- ton from the peroxide. Native HRP-C also contains 2 mol Ca 2+ ⁄ mol enzyme, with the binding sites being known as proximal and distal, according to their location relative to the porphyrin plane [7,8]. The role of these ions has been intensively investi- gated, and, in general, Ca 2+ has been proposed to maintain the heme pocket structure associated with high catalytic activities [9–11]. We have previously isolated a class III peroxidase from the latex of the Mediterranean shrub Euphorbia characias, and some of its physicochemical characteristics have been reported [12]. The purified enzyme shows a very low specific activity for classic peroxidase substrates, but the catalytic efficiency is enhanced by three orders of magnitude in the presence of Ca 2+ . In this study, we investigated the effect of Ni 2+ on the catalytic pathways of native Euphorbia latex peroxidase (ELP) and in the presence of Ca 2+ . The enzyme is strongly inhibited by Ni 2+ in the absence of Ca 2+ , whereas, in the presence of both Ca 2+ and Ni 2+ , the enzy- matic activity is enhanced, and we present a possible explanation for this singular mechanism. Results The catalytic cycles of ELP are now established [13] and are similar to the well-known mechanisms of HRP-C [14]. Pathway I The initial reaction of hydrogen peroxide with HRP-C (PrIXFe III ) forms a ferric hydroperoxide, called com- pound 0 (PrIXFe III –OOH) [15–17], and involves the distal histidine as a general base. The transiently pro- tonated histidine acts as a general acid to protonate the leaving hydroxide and generates the green enzyme intermediate compound I (PrIX •+ Fe IV =O 2) ), with both of the oxidizing equivalents of H 2 O 2 transferred to the enzyme. In the presence of reducing substrate molecules (DH 2 ), which can be a wide variety of organic and inorganic compounds, the red com- pound II (PrIXFe IV =O) is produced by the first elec- tron transfer from DH 2 to compound I. Compound II then reverts to the resting state by a successive one- electron reaction transfer from DH 2 . The reduction of compound II to resting enzyme is the rate-limiting step, so that the rate of this catalytic cycle usually depends on the nature of the reducing substrate. Pathway II In the absence of reducing substrate and in the pres- ence of an excess of H 2 O 2 , HRP-C reacts differently: decomposition of H 2 O 2 to oxygen in a catalase-like two-electron process, or via two single-electron trans- fers in which compound II (PrIXFe IV =O), com- pound III (PrIXFe II –O 2 or PrIXFe III –O 2 ) ) and superoxide radical anion are formed [18]. Pathway III From pathway II, a competing enzyme inactivation takes place with the formation of verdohemo- chrome P670. The complex between HRP–C com- pound I and peroxide has been identified as a pivotal point connecting the three simultaneous pathways. Thus, the complex compound I–H 2 O 2 appears to be the central motif from which the partition between turnover (catalase activity and compound III forma- tion) and inactivaction take place. Pathway I – reaction of ELP with H 2 O 2 and reducing substrates Kinetic parameters In 100 mm Tris ⁄ HCl buffer (pH 7.0), the value of K m for 2,2¢ -azinobis(3-ethylbenzo-6-thiazolinesulfonic acid (ABTS) at saturating concentrations of H 2 O 2 (25 mm) was shown to be 0.55 mm (Table 1), whereas the K m for H 2 O 2 , at saturating concentrations of ABTS (10 mm), was calculated to be 0.12 mm. At saturating concentrations of H 2 O 2 and ABTS, native ELP showed a k cat value of approximately 170 s )1 . Effect of Ca 2+ on the enzyme activity The effect of added Ca 2+ on ELP activity was investi- gated using ABTS as substrate. When native ELP was incubated for 10 min in the presence of Ca 2+ , an acti- vation was observed (Fig. 1) that showed a maximum at 10 mm Ca 2+ . Moreover, the value of K m for ABTS at saturating concentrations of H 2 O 2 was shown to be 0.4 mm (Table 1), whereas the K m for H 2 O 2 , at satu- rating concentrations of ABTS, was calculated to be 0.074 mm. At saturating concentrations of both the substrates, a k cat value of 270 s )1 was measured. After dialysis against 100 mm Tris ⁄ HCl buffer or filtration through a Sephacryl S-200 column, the activating effect was lost. The effect of Ni 2+ in the absence of added CaCl 2 was determined by inverse plots of the steady-state kinetics at saturating concentrations of ABTS (10 mm) Nickel ions and Euphorbia peroxidase F. Pintus et al. 1202 FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS with H 2 O 2 as the varied substrate. The inverse plots, linear with a common ordinate intercept (supplemen- tary Fig. S1A), indicate that this metal ion is a com- petitive inhibitor of H 2 O 2 .AK i of 0.82 mm was calculated from the replot of the slope (K m,app ⁄ V max ) versus [Ni 2+ ] (supplementary Fig. S1B). This method does not yield the true equilibrium dissociation con- stant of the enzyme–inhibitor complex in the absence of substrates, but estimates the apparent K i in the absence of H 2 O 2 and in the presence of 10 mm ABTS. The effect of Ni 2+ on the oxidation of ABTS as the varied substrate, at two concentrations of H 2 O 2 (5 and 25 mm), was also investigated. The low K m for H 2 O 2 guarantees that both concentrations are saturating in the absence of Ni 2+ . The inverse plots at various Ni 2+ concentrations in the presence of 5 and 25 mm H 2 O 2 (supplementary Fig. S2A,C) were linear and parallel, indicating that Ni 2+ is an uncompetitive inhibitor of ABTS. Apparent K i values of 1.36 and 6.36 mm were calculated from the replot of intercepts (1 ⁄ V max ) versus [Ni 2+ ] using data for 5 and 25 mm H 2 O 2 respectively (supplementary Fig. S2B,D). These results are consis- tent with Ni 2+ competing with a fixed concentration of H 2 O 2 present in this assay. Thus, the apparent K i includes a function of the ratio [H 2 O 2 ] ⁄ K m,H 2 O 2 (whereas it does not contain contribution from the concentration of ABTS). Under the experimental con- dition explored, it may be assumed that the affinity for each substrate is independent of the other one, and the apparent K i is linearly dependent on the concentration of H 2 O 2 (supplementary Fig. S3) and scarcely or not at all dependent on the concentration of ABTS (sup- plementary Fig. S3, inset). Finally, when the effect of Ni 2+ was studied at satu- rating concentrations of ABTS (10 mm) and H 2 O 2 (25 mm), a plot of k cat as a function of [Ni 2+ ] looks like a rectangular hyperbola (Fig. 2A), and the data can be fitted to Eqn (1): k cat ¼ k cat;0 À k cat;0 À k cat;1 1 þðK i;app =½Ni 2þ Þ ð1Þ where k cat,0 and k cat,¥ are the values of k cat in the absence and at saturating concentration of Ni 2+ respectively. The apparent K i,app is numerically equal to the Ni 2+ concentration that produces 50% of the maximum inhibition, but the estimated value contains the contribution of both substrates and their affinity constants (or their K m ). This method yields K i,app = 8.3 mm, which is quite consistent with the data in Fig. 2A, given the expected effect of the com- peting H 2 O 2 . It is important to note that the apparent inhibition constants (supplementary Figs S1 and S2) have been calculated under the assumption that satura- tion with Ni 2+ is not coupled to the complete inactiva- tion of the enzyme, whereas the same assumption was not applied to estimate K i,app (the dependence of K i,app on [H 2 O 2 ] is also reported in supplementary Fig. S3). The fact that Ni 2+ does not completely abolish the peroxidase activity of ELP is a demonstration that the mechanism of inhibition is not classic competition for the active site, but results from a strong unfavorable allosteric coupling, as to be expected on the basis of chemical considerations. When native ELP was incubated for 10 min in the presence of Ca 2+ (10 mm) and Ni 2+ (30 mm), the value of K m for ABTS at saturating concentrations of H 2 O 2 was shown to be 2.8 mm (Table 1), whereas the K m for H 2 O 2 at saturating concentrations of ABTS was calculated to be 0.06 mm. At variance with its inhibitory effect observed in the absence of Ca 2+ , under these experimental conditions Ni 2+ increases the Table 1. Kinetic parameters of ELP native enzyme, in the presence of 10 m M Ca 2+ and in the concomitant presence of 10 mM Ca 2+ and 30 mM Ni 2+ . Buffer used: 100 mM Tris ⁄ HCl buffer (pH 7.0). ELP ELP ⁄ Ca 2+ ELP ⁄ Ca 2+ ⁄ Ni 2+ K m ABTS (mM) a 0.55 ± 0.02 0.4 ± 0.015 2.8 ± 0.17 k cat (s )1 ) 170 ± 11 270 ± 21 980 ± 53 k cat ⁄ K m ABTS (m M )1 Æs )1 ) 311 675 350 K m H 2 O 2 (mM) b 0.12 ± 0.018 0.074 ± 0.005 0.06 ± 0.004 k cat ⁄ K m H 2 O 2 (mM )1 Æs )1 ) 1425 3650 16 330 a Using a saturating concentration of H 2 O 2 (25 mM). b Using a satu- rating concentration of ABTS (10 m M). Fig. 1. Effect of Ca 2+ concentration on ELP activity. The buffer used was 100 m M Tris ⁄ HCl (pH 7.0). The continuous curve repre- sents the theoretical binding isotherm fit to the data. F. Pintus et al. Nickel ions and Euphorbia peroxidase FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS 1203 k cat value from 270 to 980 s )1 . After dialysis against 100 mm Tris ⁄ HCl buffer (pH 7.0), or filtration through a Sephacryl S-200 column, the activating effect was lost. Figure 2B shows the effect of different concentra- tions of Ni 2+ on peroxidase activity in the presence of Ca 2+ (10 mm). As shown in Table 2, although 50 lm Ca 2+ did not have an activating effect on ELP (Fig. 1), it can pro- tect the enzyme by Ni 2+ inactivation. This can be explained by assuming that both metals compete for the same site, with Ca 2+ showing more affinity than Ni 2+ . This hypothesis is confirmed by kinetics experi- ment (see below). Other divalent cations such as Zn 2+ ,Cd 2+ and Mn 2+ gave rise to less inactivation of the enzyme, with a half-maximal inactivation obtained with 50 mm metal ions, whereas Mg 2+ had no effect on ELP activ- ity (not shown). Moreover, Zn 2+ ,Cd 2+ and Mn 2+ showed very little, if any, activation of the enzyme in the presence of 10 mm Ca 2+ . The lack of efficient inactivation by other metal ions might simply be related to their different ionic radii. Spectrophotometric features The electronic absorption spectrum of ELP showed maxima at 278, 401, 498 and 637 nm in 100 mm Tris ⁄ HCl buffer (pH 7.0) (Fig. 3A). Compound I with characteristic absorption maxima at 278, 398, 544, 577 and 651 nm was produced by addition of an equimolar amount of H 2 O 2 . Compound II with characteristic absorption maxima at 278, 413, 541 and 576 nm was generated from compound I by the addition of one equivalent of ascorbic acid (Fig. 3A). The absorbance spectra in the presence of 10 mm Ca 2+ were very simi- lar to that of the native enzyme (not shown). In the presence of 30 mm Ni 2+ , whereas the addition of H 2 O 2 generated compound I as observed in the native enzyme, the addition of ascorbic acid to compound I did not lead to the formation of compound II, but a progressive compound I absorption decrease, leading to the native spectrum, was observed (Fig. 3B). In this process, two clear isosbestic points were observed at 540 and 450 nm. Stopped-flow determinations Two types of experiment were carried out to investi- gate the fundamental steps of the catalytic cycle of ELP. First, the resting ferric enzyme was mixed with H 2 O 2 in the absence of the reducing substrate. Second, the enzyme was mixed with H 2 O 2 and ascorbate; by repeating this experiment several times over some min- utes, we checked that H 2 O 2 was not consumed by ascorbate while waiting in the instrument. Both experi- ments were carried out at several concentrations of Fig. 2. Effect of Ni 2+ concentration on ELP activity. Euphorbia per- oxidase (2.5 n M) was incubated with NiCl 2 concentrations from 1 to 40 m M, in 100 mM Tris ⁄ HCl buffer (pH 7.0) in the absence (A) or in the presence (B) of 10 m M Ca 2+ . Table 2. Kinetic parameters of ELP native enzyme in the presence of different concentrations of Ca 2+ and in the concomitant pres- ence of 30 m M Ni 2+ . Buffer used: 100 mM Tris ⁄ HCl buffer (pH 7.0) at saturating concentrations of H 2 O 2 (25 mM) and ABTS (10 mM). k cat (s )1 ) without Ni 2+ k cat (s )1 )+Ni 2+ (30 mM) ELP (native enzyme) 170 ± 11 40 ± 8 ELP + 50 l M Ca 2+ 170 ± 11 170 ± 11 ELP + 1 m M Ca 2+ 208 ± 38 630 ± 48 ELP + 4 m M Ca 2+ 250 ± 42 750 ± 68 ELP + 10 m M Ca 2+ 270 ± 21 980 ± 53 Nickel ions and Euphorbia peroxidase F. Pintus et al. 1204 FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS each substrate, both in the presence and the absence of Ni 2+ and Ca 2+ (Fig. 4). The determination of the rate constants of the single elementary steps of the catalytic cycle is difficult and model-dependent; nevertheless, the experimental data reported in Fig. 4 suggest the following. (a) In the absence of both Ca 2+ and Ni 2+ , the formation of compound I is strongly biphasic, and two second-order processes are evident and described by a sum of two exponentials (k 1 =80s )1 , k 2 = 0.5 s )1 ) with similar amplitudes (Fig. 4A). This result is in agreement with the hypothesis of two slowly interconverting forms of the enzyme as already established [12]. (b) Addition of Ni 2+ in the absence of Ca 2+ shifts the conformational equilibrium in favor of the slowly reacting form of the enzyme, so that the amplitude of the faster process is reduced to almost zero (Fig. 4B). The time course can be described by two second-order processes with the same rate constants as those reported in Fig. 4A, but the quickly reacting form is almost negligible. More- over, the final spectrum collected is not that of a pure compound I species, perhaps as a consequence of par- tial formation of compound II. (c) In the presence of Ca 2+ and in the absence of Ni 2+ , the opposite effect is observed and the enzyme is almost entirely stabilized in the quickly reacting form (Fig. 4C). A single sec- ond-order process is sufficient to describe the time course, with the same rate constant as the fast one from Fig. 4A, and the final species, as implied by the last spectrum collected, is pure compound I. (d) Finally, as shown in Fig. 4D, when both Ni 2+ and Ca 2+ are present, the Ca 2+ effect dominates, and the enzyme is almost entirely converted to the quickly reacting form. A second kinetic process, different from the one assigned to the slowly reacting species forming compound I, is observed with a rate constant of 4 s )1 . This process is probably due to partial formation of compound II, which seems to be favored by the pres- ence of Ni 2+ , in agreement with the same effect observed in the experiment reported in Fig. 4B, where only Ni 2+ was present. Intrinsic fluorescence and CD spectroscopy Euphorbia latex peroxidase showed a fluorescence emis- sion spectrum with a maximum at 336 nm when excited at 295 nm. The spectrum was independent of the presence of Ca 2+ or Ni 2+ . The CD spectra of native ELP show no significant modification upon addition of 10 mm Ca 2+ or 30 mm Ni 2+ or in the presence of both ions, either at 195 and 210 nm or in the Soret region, indicating that gross changes in the secondary or tertiary structure did not occur (results not shown). Laser photolysis experiments Euphorbia peroxidase, reduced with sodium dithionite, was equilibrated with CO and submitted to flash pho- tolysis. Difference spectra of the photoproduct with respect to the CO-bound form were collected using a fast-pulsed CCD camera. Singular value decomposition (SVD) analysis was applied to a matrix containing as a column a difference spectrum and as a row the time course at a single wavelength. The rate of CO recombi- nation at pH 7.0 was derived by globally fitting the v1 Fig. 3. Absorption spectra of Euphorbia peroxidase (4.2 lM). (A) Compound I is formed after addition of an equimolar amount of H 2 O 2 to the native enzyme (4.2 lM). Compound II is formed after addition of one equivalent of ascorbic acid to compound I. (B) Euphorbia peroxidase in the presence of 30 m M Ni 2+ before and after addition of an equimolar amount of H 2 O 2 . Arrows indicate the disappearance of compound I after addition of one equivalent of ascorbic acid. Buffer used: 100 m M Tris ⁄ HCl (pH 7.0). F. Pintus et al. Nickel ions and Euphorbia peroxidase FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS 1205 and v2 columns of the experiments carried out both in the presence and in the absence of NiCl 2 (30 mm) (Fig. 5). The recombination of reduced ELP with CO is described by a sum of two exponentials. This behav- ior, surprising for a monomeric hemoprotein, had already been observed by us [12] and attributed to the presence of two slowly interconverting conformations of the enzyme. We can now confirm and extend our previous interpretation, as follows: (a) two bimolecular rebinding processes are observed, with rate constants of 4.7 · 10 5 m )1 Æs )1 and 7 · 10 4 m )1 Æs )1 that are in agreement with those derived in our previous paper [12] – as shown in Fig. 5B for the experiment in the presence of both Ca 2+ and Ni 2+ , these two spectra are slightly different and testify to the presence of the two interconverting forms; (b) the experimental data can be satisfactorily described under the assumption that the rate constants of the two conformations are insensitive to the ionic composition of the medium; and (c) Ca 2+ and Ni 2+ bind differentially to the two conformations of ELP and bias their equilibrium, thus changing the relative amplitude of the two processes (Fig. 5C,D). The rebinding of photolyzed CO was faster in the absence of Ca 2+ than in its presence, whereas Ni 2+ increased the fraction of the quickly reacting confor- mation in the absence of Ca 2+ and had the opposite effect in its presence (data not shown). Fig. 4. Time course of the absorbance changes recorded in a stopped-flow apparatus. Selected absorbance spectra at t = 2 ms (a), 63 ms (b), 300 ms (c) and 10 s (d), collected during the reaction of ELP with H 2 O 2 . Experimental conditions: [H 2 O 2 ]=16lM and [ELP] = 4 lM after mixing, T =21°C, in 100 m M Tris ⁄ HCl buffer (pH 7.0). Insets: time course recorded at k = 401 nm. (A) In the absence of added Ca 2+ and Ni 2+ . (B) In the presence of Ni 2+ 30 mM. (C) In the presence of Ca 2+ 10 mM; the spectrum at t = 10 s containing contributions from com- pound II is omitted. (D) In the presence of Ca 2+ 10 mM and Ni 2+ 30 mM; the spectrum at t = 10 s containing contributions from compound II is omitted. Nickel ions and Euphorbia peroxidase F. Pintus et al. 1206 FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS Pathway II – catalase-like activity – kinetics Euphorbia latex peroxidase was shown to be able to produce molecular oxygen when incubated with H 2 O 2 (0.5–40 mm) in the absence of reducing substrates [13]. The effect of added Ca 2+ and Ni 2+ on ELP catalase- like activity was investigated. In the concomitant pres- ence of 40 mm H 2 O 2 and 10 mm Ca 2+ or 30 mm Ni 2+ , oxygen production by the catalase-like activity of ELP seemed to be reduced to 55%. Similar results were obtained in the presence of both Ca 2+ and Ni 2+ (data not shown); the decrease in ELP activity depended on the concentration of Ca 2+ and ⁄ or Ni 2+ . Pathway III – enzyme inactivation – spectro- photometric features The addition of excess H 2 O 2 (enzyme ⁄ H 2 O 2 ratio 1 : 1000) to the native ELP generated compound III, with absorption maxima at 277, 413, 542 and 576 nm. This compound was not stable, and its spectrum bleached within a few minutes, this being accompanied Fig. 5. Time course of CO recombination as observed after photolysis. Time course and difference spectra collected after laser photolysis of CO-reduced ELP complex in 100 m M Tris ⁄ HCl buffer (pH 7.0) containing 10 mM Ca 2+ in the presence or in the absence of 30 mM Ni 2+ . Sam- ples were equilibrated with 1 atm CO. (A) Difference spectra were arranged into a matrix and submitted to an SVD analysis (see Experimen- tal procedures); V 1 , the first column of the resulting V matrix, was globally fitted with a sum of two exponentials; o, in the absence of Ni 2+ ; h, in the presence of Ni 2+ 30 mM; solid lines correspond to the global fit with observed rate constants of k 1 = 4.7 · 10 5 M )1 Æs )1 and k 2 =7· 10 4 M )1 Æs )1 . Under the present experimental conditions, the half-times of the two processes correspond to 1.5 and 9.5 ms respec- tively. (B) Reconstructed difference spectra of the fast and slow CO-rebinding processes, in the presence of Ni 2+ . (C) As (B), but the spec- trum of the fast component has been multiplied by a factor of 2.8 in order to demonstrate the incomplete superimposition with that of the slow component; the small difference suggests that the quickly and slowly reacting forms of ELP differ because of a minor perturbation of the heme environment. (D) Reconstructed difference spectra of the fast and slow CO-rebinding processes, in the absence of Ni 2+ ; compari- son with (B) demonstrates that Ni 2+ strongly favors the slowly CO-binding conformation. F. Pintus et al. Nickel ions and Euphorbia peroxidase FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS 1207 by the formation of inactive verdohemochrome P670 (Fig. 6A) and by a loss of enzyme activity (not shown). When the same experiment was performed in the presence of 10 mm Ca 2+ the disappearance of compound III was slower than that observed in the absence of Ca 2+ , and the formation of verdohemo- chrome P670 reached a maximum after 20 min and slowly evolved to produce a bleaching inactive species with t ½ = 48 h at 4 °C (Fig. 6B). In the presence of 30 mm Ni 2+ , the formation of verdohemochrome P670 was elusive (Fig. 6C), and the formation of a new shoulder with a maximum at 619 nm was observed (Fig. 6C, inset). Discussion A cationic peroxidase extracted from the latex of the perennial Mediterranean shrub E. characias has one strongly bound endogenous Ca 2+ , as determined by atomic absorption measurements. Removal of that Ca 2+ after incubation with 6 m guanidine hydrochlo- ride and 10 mm EDTA results in changes in the elec- tronic structure of the heme iron, and the activity of Ca 2+ -free enzyme was approximately 2% that of the native enzyme (data not shown). However, in Tris ⁄ HCl buffer (pH 7.0), upon addition of a second Ca 2+ to native ELP, a small increase of the k cat value and a decrease of K m for H 2 O 2 was observed. Ni 2+ is shown to be an effective reversible inhibitor of Euphorbia peroxidase. When H 2 O 2 was used as the varied substrate, an apparent competitive inhibition was seen and a K i $ 8.2 · 10 )4 m was calculated from the replot of the slope (K m,app ⁄ V max ) versus [Ni 2+ ]. The binding of Ni 2+ does not result in a perturbation of the heme Soret band, and this suggests that Ni 2+ is not bound at a site close enough to the heme for the electronic absorption band of the prosthetic group to be affected. Thus, the inhibitory effect has to be attrib- uted to a strong allosteric coupling, and implies that the enzyme is stable in two conformations whose equi- librium is biased by Ni 2+ and Ca 2+ , as confirmed by the analysis of stopped-flow experiments. The allosteric conformation stabilized by Ni 2+ forms compound I more slowly than the other conformation, as shown by kinetic experiments. Competition between Ni 2+ and ABTS is not observed, suggesting that the two alloste- ric conformations do not discriminate for this ligand. The determination of the rate constants from our experiments shows the following. The second-order rate constant for the formation of compound I (k 1 ) depends on Ca 2+ and Ni 2+ , being much faster in the presence of Ca 2+ and lower in the presence of Ni 2+ . Both ions certainly bind to one site that affects the Fig. 6. Absorption spectra of 4 lM native ELP and after addition of 5m M H 2 O 2 both in the absence and in the presence of Ca 2+ ,in 100 m M Tris ⁄ HCl buffer (pH 7.0). (A) Different spectra were recorded immediately after addition of H 2 O 2 and at intervals of 5 min. (B) Spectra as in (A) in the presence of 10 m M Ca 2+ . Inset: amplified spectra in the range from 450 to 800 nm to show P670 species. (C) Spectra as in (A) in the presence of 30 m M Ni 2+ . Inset: amplified spectra in the range from 450 to 800 nm to show 619 nm absorption species. Nickel ions and Euphorbia peroxidase F. Pintus et al. 1208 FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS H 2 O 2 -binding site, which is compensated for by the presence of an excess of that substrate. ELP remains active after incubation with high concentrations of Ni 2+ , confirming that the inhibition is allosteric rather than competitive. The results reported above indicate that the combinations of enzyme with H 2 O 2 and with Ca 2+ or Ni 2+ are not independent, so that the free enzyme, the enzyme–Ca 2+ complex and the enzyme– Ni 2+ complex have different affinities for the sub- strate. In the presence of Ca 2+ , the Euphorbia peroxi- dase is completely converted into the more active form, whereas in the presence of Ni 2+ , the protein is converted into the less active form (Fig. 7). It is known that the formation rate of compound I depends on the catalytic reactivity of the distal histi- dine of the heme pocket, acting as a general acid–base catalyst; thus, allosteric conformational changes could involve the position of this residue and easily explain the observed effects. Euphorbia peroxidase reacts with an excess of H 2 O 2 , forming compound I and then the resting enzyme with production of molecular oxygen. Moreover, during the reaction, the enzyme becomes inactive, suggesting that H 2 O 2 acts as a mechanism-based inactivator. In the presence of H 2 O 2 and Ca 2+ or Ni 2+ , the catalase-like activity of ELP is reduced to 55%, and this confirms the interaction of both ions in compound I formation. Finally, the formation of the inactive verdohemo- chrome P670 species is achieved after incubation of ELP with an excess of H 2 O 2 . In the absence of Ca 2+ and in the presence of Ni 2+ , verdohemochrome P670 is present to a much smaller extent, probably due to the smaller population of its precursor compound I, and a new species accumulates with a shoulder at 619 nm. At present, we cannot assign this species any structure. When Ni 2+ is added in the presence of Ca 2+ ,itis clearly seen that Ni 2+ enhances the enzymatic activity, suggesting a possible additional interaction. This result is explained by the presence of a second binding site for Ni 2+ (but not for Ca 2+ ) with an opposite effect to the other one. UV ⁄ visible absorption spectra show that the heme iron of ELP is pentacoordinated and the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ca 2+ or Ni 2+ . Only minor changes in the secondary or tertiary struc- ture of the protein could be detected by fluorescence or CD measurements in the presence of Ca 2+ or Ni 2+ . Thus, structural changes associated with activation or inhibition by these effectors must be quite subtle. As observed by laser photolysis experiments, the re- binding of photolyzed CO was faster in the absence of Ca 2+ than in its presence, whereas Ni 2+ increased the fraction of the quickly reacting conformation in the absence of Ca 2+ and had the opposite effect in its presence. These observations are again compatible with the hypothesis that over the concentration range of the two cations explored, the equilibria take place as shown in Fig. 7. Figure 7 includes: the high-affinity Ca 2+ -binding site (so-called constitutive Ca 2+ ) (site 1, red), the low-affin- ity binding site for Ca 2+ , at which Ni 2+ can also bind and exert its inhibitory effect (site 2, green), and a sec- ond Ni 2+ -binding site with activating effect, where no competition with Ca 2+ is observed (site 3, blue). Bind- ing of Ca 2+ to the site (site 2) biases the equilibrium in favor of the quickly reacting form, whereas binding of Ni 2+ to the same site has the opposite effect. Bind- ing of Ni 2+ to the site (site 3) biases the equilibrium in favor of the slowly reacting form. Under the assump- tion that the allosteric equilibrium is maintained even in the ferrous CO-bound state, we suggest that the conformation that reacts slowly with CO is also the most catalytically active, and our experiments on fer- rous ELP confirm that Ni 2+ exerts opposite effects in the absence and in the presence of Ca 2+ . Moreover, the slowly and quickly CO-rebinding forms of ELP show subtle but consistent spectroscopic differences, as shown in Fig. 5. Fig. 7. The scheme shows the native enzyme, in which the proxi- mal Ca 2+ is always present. The red rectangle represents the con- stitutive Ca 2+ -binding site; the green rectangle represents the binding site for both Ca 2+ and Ni 2+ with opposite effects, and the blue rectangle is the solely Ni 2+ -activating binding site. QRF is the quickly reacting form of the enzyme and SRF the slowly reacting one. F. Pintus et al. Nickel ions and Euphorbia peroxidase FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS 1209 In conclusion, the above results confirm and extend our previous finding that ELP, although monomeric, is an allosteric enzyme that is finely tuned by divalent cations. The concentration of Ca 2+ in the latex is surely sufficient to saturate the high-affinity site and probably also the low-affinity one. Although nickel is an essential trace element for animals and plants, it is one of the heavy metals with toxic effects, and it is well known that antioxi- dant enzymes play a key role in protection against heavy metal toxicity. In Euphorbia latex, Ni 2+ is present in very low amounts, below the detection limit as observed by atomic absorption. Nevertheless, Ni 2+ , if present in the environment and uploaded by the plant, could further increase the activity of the peroxidase. Thus, ELP is probably a positively mod- ulated (rather than inhibited) enzyme. Experimental procedures Materials ABTS, nickel chloride and ascorbic acid were pur- chased from Sigma (St Louis, MO, USA). H 2 O 2 was obtained from Merck (Darmstadt, Germany), and an e 240 = 43.6 m )1 Æcm )1 was used to determine its concentra- tion. All chemicals were obtained as pure commercial prod- ucts and used without further purification. Enzyme Peroxidase from E. characias latex (RZ v alue A 401 ⁄ A 278 =2.7) was purified as previously described [12]. The enzyme concentration was determined spectrophotometrically using an e 401 = 130.7 mm )1 Æcm )1 . Peroxidase activity Activity measurements were made in 100 mm Tris ⁄ HCl buffer (pH 7.0) at 25 °C, using 25 mm H 2 O 2 and 10 mm ABTS, by following the increase in absorbance at 415 nm resulting from the formation of the ABTS cation radical product (e 415 =36mm )1 Æcm )1 ). Catalytic center activity (k cat ) was defined as (mol substrate consumed) ⁄ (mol active sites) · s )1 . The value of K m for ELP using varying reducing sub- strate concentrations at saturating concentrations of H 2 O 2 (25 mm), or varying concentrations of H 2 O 2 at saturating concentrations of reducing substrate (10 mm ABTS), was calculated from initial velocity data fitted to the Michaelis– Menten equation by nonlinear regression and by double reciprocal plots by Michaelis–Menten analysis in 100 mm Tris ⁄ HCl buffer (pH 7.0) at 25 °C. The k cat ⁄ K m value was also used as a more useful measure of substrate specificity. The effects of Ni 2+ on ELP activity were examined in buffers with or without NiCl 2 or NiSO 4 . Inactivation of ELP was monitored as follows. Stock protein solutions (3 lm) were incubated in 100 mm Tris ⁄ HCl buffer (pH 7.0) in a water bath at 25 °C. At given time intervals, aliquots of enzyme mixture were taken and assayed for peroxidase activity as reported above. The activity was expressed as a percentage of remaining activity. The inhibition constant (K i )ofNi 2+ was determined from a Dixon plot [19]. The effects of Ni 2+ on ELP was monitored in both the presence and absence of Ca 2+ . Kinetic parameters were cal- culated as the mean of at least five different measurements. Catalase-like activity Catalase-like activity of ELP was determined as oxygen production from H 2 O 2 using a Clark-type electrode coupled to an OXYG1 Hansatech oxygraph (Hansatech Instruments Ltd, King’s Lynn, UK). Nitrogen was bubbled through the reaction medium to remove the dissolved oxygen. A base- line increase in oxygen concentration of less than 0.5 lmÆ min )1 in the absence of enzyme was obtained. The tempera- ture of the reaction chamber was controlled at 25 °C using a Haake circulating water bath. The reaction medium (total volume of 1 mL) contained different H 2 O 2 concentrations in 100 mm Tris ⁄ HCl buffer (pH 7.0) at 25 °C. The reac- tions were started by the addition of ELP. The initial rate of oxygen production was calculated from the slope of the trace observed on ELP addition after discounting the rapid rise in oxygen due to the introduction of a small amount of gas into the enzyme solution. The K m value for H 2 O 2 was obtained from a double reci- procal plot in 100 mm Tris ⁄ HCl buffer (pH 7.0) at 25 °C. Kinetic parameters were calculated as the mean of at least five different measurements. Spectrophotometry Absorption spectra and data from all activity assays were obtained with an Ultrospec 2100 spectrophotometer (Bio- chrom Ltd, Cambridge, UK) using cells with a 1 cm path length. Fluorescence spectra Fluorescence spectra were obtained using a Perkin- Elmer LS-3 spectrofluorimeter (Perkin-Elmer Ltd, Beacons- field, UK). CD spectroscopy CD spectra were measured with a Jasco J-715 spectropola- rimeter (Jasco Ltd, Hachioji City, Tokyo, Japan). A 9.2 lm solution of ELP in 100 mm Tris ⁄ HCl buffer (pH 7.0) was Nickel ions and Euphorbia peroxidase F. Pintus et al. 1210 FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... 7.0), 5.8 nm Euphorbia peroxidase, and 5 mm H2O2 NiCl2 concentrations of 1 mm (d), 5 mm ( ) and 10 mm ( ) were used (B) Replot of the 1 ⁄ Vmax values of the inverse plot versus [Ni2+] (C) as in (A), but 25 mm H2O2 and NiCl2 concentrations of 0 (D), 1 mm (d), 5 mm ( ) and 10 mm ( ) were used (D) Replot of the 1 ⁄ Vmax values of the inverse plot versus [Ni2+] Fig S3 Plot of the apparent Ki of Ni2+ versus... S1 Inverse plot of 1 ⁄ rate versus 1 ⁄ [H2O2] (A) Reaction mixtures contained, in 1 mL of 100 mm Tris ⁄ HCl buffer (pH 7.0) and 4 nm Euphorbia peroxidase NiCl2 concentrations of 0 mm ( ), 1 mm (s), 2 mm (d), 3 mm (h) and 4 mm ( ) were used (B) Replot of the slope of the inverse plot versus [Ni2+] Fig S2 Inverse plot of 1 ⁄ rate versus 1 ⁄ [ABTS] (A) Reaction mixtures contained, in 1 mL of 100 mm Tris... second harmonic with a wavelength of Nickel ions and Euphorbia peroxidase 532 nm and E = 10–80 mJÆpulse)1) was focused onto one face of a 1 cm fluorescence cuvette sealed to a Thunberg tube containing the desired solution and gas phase The transmittance of the sample was monitored using either of two detection systems Single-wavelength time courses were ˚ recorded by an optical line arranged at 90 A... 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The effect of Ni 2+ in the absence of added CaCl 2 was determined by inverse plots of the steady-state kinetics at saturating concentrations of ABTS (10 mm) Nickel ions and Euphorbia peroxidase. Allosteric modulation of Euphorbia peroxidase by nickel ions Francesca Pintus 1 , Anna Mura 1 , Andrea Bellelli 2 , Alessandro. catalytic pathways of native Euphorbia latex peroxidase (ELP) and in the presence of Ca 2+ . The enzyme is strongly inhibited by Ni 2+ in the absence of Ca 2+ , whereas, in the presence of both Ca 2+ and