Báo cáo khoa học: Allosteric modulation of Euphorbia peroxidase by nickel ions pdf

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:
(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
placed in strain-free 0.1 mm (for far-UV spectra) and
1.0 cm (for Soret spectra) quartz cuvettes, and spectra were
measured between 260 and 190 nm or 460 and 350 nm with
the following setup: bandwidth 2 nm, time constant 2 s,
scan rate 20 nmÆmin
)1
and N
2
purging rate 25 LÆmin
)1
.
Eight spectra corrected for background (buffer and salt)
were averaged and successively smoothed with an eight-
point Savisky Golay smoothing procedure. The CD spec-
trum was normalized as De units using the molar extinction
coefficient e
280
=3· 10
4
m

)1
Æcm
)1
.
Stopped-flow experiments
Stopped-flow experiments were carried out using an
Applied Photophysics MV 17 apparatus (Leatherhead,
UK), equipped with an observation chamber with a 1 cm
path length and either a monochromator and a photomulti-
plier tube (for single-wavelength measurements) or a spec-
trometer and a photodiode array detector (for rapid
acquisition of absorbance spectra over the range 250–
800 nm). Single-wavelength measurements were analyzed by
a least squares minimization routine (developed using the
Borland Pascal 7.0 compiler) capable of fitting any desired
theoretical model to the experimental data, using either
analytical or numerical integration. Time-resolved absorp-
tion spectra were deconvoluted by SVD performed with the
program matlab (The Math Works, Natick, MA, USA).
This analysis requires arrangement of time-resolved spectra
into a matrix A, with each column a spectrum and each
row a time course or a titration profile at a single wave-
length. The application of SVD yielded three matrices, U, S
and V, such that A = U · S · V
T
. U and V are ortho-
gonal matrices, whereas S is diagonal, with its nonzero ele-
ments arranged in decreasing order. Each column of matrix
U · S represented a pseudospectrum (i.e. the absorption of
one of the spectroscopic components required to approxi-

mate the original data; only the first few columns were sig-
nificant, with the last ones corresponding to negligibly
small S values). Each column of matrix V represented the
time course or titration profile of the corresponding column
of U · S and was analyzed by ordinary nonlinear minimi-
zation routines (using either matlab or dedicated programs
developed with the Borland Pascal Compiler) as previously
described [20], to obtain the pertinent equilibrium or kinetic
constants and the amplitudes of the V elements. The fitted
V amplitudes (V
fit
) were used to calculate the spectra of
each chemical species involved in the reaction in accordance
with the formula: A
fit
=U· S · V
fit
T
.
Laser photolysis experiments
Laser photolysis experiments were carried out using an
instrument developed in our laboratory [21]. Briefly, the
5 ns pulse of a Nd:YAG laser (Quanta Systems, Milan,
Italy; we used the second harmonic with a wavelength of
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
˚
with respect
to the laser pulse, consisting of a 100 W lamp (Oriel, Strat-
ford, CT) a blue glass filter (Corning Glassware, Corning,
NY, USA), the sample holder, a second filter, a monochro-
mator (Spex, Edison, NJ, USA) and a photomultiplier
module (model H6870; Hamamatsu, Shimokanzo, Shi-
zuoka-ken, Japan). The output current of the photomulti-
plier was converted to voltage by a Tektronix ADA400
amplifier and read in a Tektronix TDS360 digital oscillo-
scope (Tektronix, Beaverton, OR, USA). The use of a fas-
ter current-to-voltage amplifier [model AM3100 (Analogue
Modules USA), capable of time resolution up to 350 MHz]
allowed us to explore the nanosecond and microsecond
time regime. Full spectra at time delays after the laser pulse
ranging from 5 ns to 100 ms or more were collected using
an optical line oriented at approximately 165 A
˚
from the
laser beam, consisting of a 300 W lamp (ILC Technology,
Sunnyvale, CA, USA), an Acton Princeton 320 PI Spec-
trometer (Princeton Instruments, Trenton, NJ, USA), and a
pulsed CCD camera controlled by an FG100 pulse genera-
tor (Princeton Instruments). Both setups are capable of
collecting data over the sub-microsecond time regime;
however, no spectroscopic changes could be detected in this
time window, so no such data are presented in this work.
The spectra obtained by the CCD camera were analyzed

using the svd algorithm as detailed above.
Metal content
Ca
2+
and Ni
2+
contents were determined by atomic
absorption using a Perkin-Elmer 3030 apparatus.
Acknowledgements
This study was partially supported by PRIN 2006
(Progetti di ricerca di interesse nazionale) funds and by
a grant from ‘Fondazione Banco di Sardegna’, Sassari.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Inverse plot of 1 ⁄ rate versus 1 ⁄ [H
2
O
2
]. (A)
Reaction mixtures contained, in 1 mL of 100 mm
Tris ⁄ HCl buffer (pH 7.0) and 4 nm Euphorbia peroxi-
dase. NiCl
2
concentrations of 0 mm ( ), 1 mm (s),
2mm (d), 3 mm (h) and 4 mm (
) were used. (B) Re-
plot of the slope of the inverse plot versus [Ni
2+
].
Fig. S2. Inverse plot of 1 ⁄ rate versus 1 ⁄ [ABTS]. (A)
Reaction mixtures contained, in 1 mL of 100 mm
Tris ⁄ HCl buffer (pH 7.0), 5.8 nm Euphorbia peroxi-

dase, and 5 mm H
2
O
2
. NiCl
2
concentrations of 1 mm
(d), 5 mm (
) and 10 mm ( ) were used. (B) Replot of
the 1 ⁄ V
max
values of the inverse plot versus [Ni
2+
]. (C)
as in (A), but 25 mm H
2
O
2
and NiCl
2
concentrations
of 0 (D), 1 mm (d), 5 mm (
) and 10 mm ( ) were
used. (D) Replot of the 1 ⁄ V
max
values of the inverse
plot versus [Ni
2+
].
Fig. S3. Plot of the apparent K

i
of Ni
2+
versus [H
2
O
2
].
Corresponding ABTS concentrations were: 10 mm (d);
no ABTS (s) (extrapolated values). Inset: the same
data plotted as a function of the ABTS concentrations.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Nickel ions and Euphorbia peroxidase F. Pintus et al.
1212 FEBS Journal 275 (2008) 1201–1212 ª 2008 The Authors Journal compilation ª 2008 FEBS