Role of histidine 42 in ascorbate peroxidase
Kinetic analysis of the H42A and H42E variants
Latesh Lad
1
, Martin Mewies
1
, Jaswir Basran
2
, Nigel S. Scrutton
2
and Emma L. Raven
1
1
Department of Chemistry and
2
Department of Biochemistry, University of Leicester, UK
To examine the role of the distal His42 residue in the cata-
lytic mechanism of pea cytosolic ascorbate peroxidase, two
site-directed variants were prepared in which His42 was
replaced with alanine (H42A) or glutamic acid (H42E).
Electronic spectra of the ferric derivatives of H42A and
H42E (pH 7.0, l ¼ 0.10
M
,25.0°C) revealed wavelength
maxima [k
max
(nm):397,509,% 540
sh
, 644 (H42A); 404, 516,
% 538
sh

1
and K
d
were, respectively,
4.3 ± 0.2 s
)1
and 30 ± 2.0 m
M
(H42A) and 28 ± 1.0 s
)1
and 0.09 ± 0.01 m
M
(H42E). Photodiode array experi-
ments for H42A revealed wavelength maxima for this
intermediate at 401 nm, 522 nm and 643 nm, consistent
with the formation of a transient [H42A–H
2
O
2
] species. Rate
constants for Compound I formation for H42A were
independent of pH, but for rAPX and H42E were pH-
dependent [pK
a
¼ 4.9 ± 0.1 (rAPX) and pK
a
¼ 6.7 ± 0.2
(H42E)]. The results provide: (a) evidence that His42 is
critical for Compound I formation in APX; (b) confirmation
that titration of His42 controls Compound I formation and

O
2
in plants and algae using ascorbate as a
source of reducing equivalents [11,12]. APX was known
from sequence comparisons [13] to contain the same active-
site Trp residue (Trp179) as is used by CcP (Trp191) during
catalysis. With high-resolution structural information avail-
able for the recombinant pea cytosolic enzyme (rAPX) [14]
(Fig. 1), APX has provided a new opportunity to reassess
the functional properties of CcP and to determine whether it
is indeed representative of class I peroxidases. As detailed
functional information has emerged, however, it seems that
APX has several rather curious features of its own, and, in
some ways, more questions have been raised than answered.
(In fact, even the current classification of APX as a class I
enzyme has been recently questioned [15].) For example,
Trp179 in APX is not a necessary requirement for oxidation
of ascorbate [16] and there is general agreement from kinetic
[17–19] and EPR data [20] that the initial product
(Compound I) of the reaction of APX with H
2
O
2
is a
porphyrin p-cation intermediate and not a protein-based
trytophan radical. Equally intriguing is the existence of a
Correspondence to E. L. Raven, Department of Chemistry,
University of Leicester, University Road, Leicester LE1 7RH, UK.
Fax: + 44 (0)116 2523789, Tel.: + 44 (0)116 2522099,
E-mail: [email protected]

2
O
2
À!
k
1
Compound I þ H
2
O ð1Þ
Compound I þ HS À!
k
2
Compound II þ S

ð2Þ
Compound II þ HS À!
k
3
APX þ S

þ H
2
O ð3Þ
Although Eqn (1) is commonly written as a single step,
experimental [23–26] and theoretical [27–30] evidence sug-
gests that this is an over-simplification. A more complex
mechanism, as first suggested by Poulos and Kraut [31] –
involving binding of neutral peroxide to the enzyme,
concomitant proton transfer from the bound peroxide to
the distal histidine residue, followed by O–O bond cleavage

2
O
2
.
MATERIALS AND METHODS
Materials
L
-Ascorbic acid (Aldrich Chemical Co.), guaiacol, imida-
zole, 1,2-dimethylimidazole (Sigma Chemical Co.) and the
chemicals used for buffers (Fisher) were of the highest
analytical grade (more than 99% purity) and used without
further purification. H
2
O
2
solutions were freshly prepared
by dilution of a 30% (v/v) solution (BDH): exact concen-
trations were determined using the published absorption
coefficient (e
240
¼ 39.4
M
)1
Æcm
)1
[38]). Aqueous solutions
were prepared using water purified through an Elgastat
Option 2 water purifier, which itself was fed with deionized
water. All pH measurements were made using a Russell pH-
electrode attached to a digital pH-meter (Radiometer

(dotted lines) are indicated.
Ó FEBS 2002 Catalytic mechanism of ascorbate peroxidase (Eur. J. Biochem. 269) 3183
sequence data were analysed using the program SeqED
(Applied Biosystems). Individual mutations were confirmed
by sequencing across the whole rAPX-coding gene.
Bacterial fermentation of cells and purification of rAPX
were carried out according to published procedures [7].
Enzyme purity was assessed by examination of the A
Soret
/
A
280
value; in all cases an A
Soret
/A
280
value > 1.9 for rAPX,
H42A and H42E was considered pure. Enzyme purity was
additionally assessed using SDS/PAGE, and the prepara-
tions were judged to be homogeneous by the observation of
a single band on a Coomassie Blue-stained reducing SDS/
polyacrylamide gel. Enzyme concentrations (pH 7.0,
l ¼ 0.10
M
, 25.0 °C) were determined using the pyridine
haemochromagen method [39]: absorption coefficients were
e
403
¼ 88 m
M

for rAPX below, and used to calibrate the spectrometer in
the range 600–1400 m/z. Protein samples were introduced
into the instrument at a flow rate of 5 lLÆmin
)1
. Trace salt
was removed using a Centricon-10 concentrator (Amicon)
and successive centrifugation and dilution with highly
purified water (Elgastat). Samples (% 2mgÆmL
)1
,20lL)
were then diluted 10-fold with a solution of 50 : 50 (v/v)
acetonitrile/water containing 0.1% acetic acid.
Steady-state measurements
Stock solutions of
L
-ascorbic acid, guaiacol, H
2
O
2
and
enzyme were prepared in sodium phosphate (l ¼ 0.10
M
,
pH 7.0, 25.0 °C). Enzyme assays were performed in a 1-mL
quartz cuvette: various concentrations of substrate and
25 n
M
enzyme were preincubated for 3 min in buffer and
the reaction was initiated by the addition of H
2

rate of activity (l
M
)1
Æs
)1
) by the micromolar concentration
of enzyme; values for K
m
were determined by a fit of the
data to the Michaelis–Menten equation using a nonlinear
regression analysis program (Grafit32 version 3.09b;
Erithacus Software Ltd). All reported values are the
mean of three independent assays. Errors on k
cat
and K
m
are estimated to ± 5% and ± 10%, respectively. For
pH-dependent assays, a mixed sulfonic acid buffer system
(l ¼ 95–110 m
M
depending on the exact pH) that buffered
over the entire pH range was used; reactions were initiated
by the addition of H
2
O
2
(to 0.10 m
M
). In these cases,
[

water bath (± 0.1 °C). Reported values of k
obs
are an
average of at least three measurements. All curve fitting was
performed using the Grafit software package. All data were
analysed using nonlinear least-squares regression analysis
on an Archimedes 410–1 microcomputer using Spectra-
kinetics software (Applied Photophysics). Pseudo-first-
order rate constants for the formation of Compound I
(k
1,obs
) were monitored at 403 nm (rAPX), 404 nm (H42E)
and 397 nm (H42A), in single mixing mode by mixing
enzyme (0.5–1.0 l
M
) with various concentrations of H
2
O
2
.
Absorbance changes were independent of [H
2
O
2
]; observed
changes in absorbance were 97–99% of the calculated
values. The pH-jump method was used to examine the
pH-dependence of Compound I formation, to avoid enzyme
instability problems below pH 5 and above pH 8.5. Enzyme
samples were prepared in water, adjusted to pH 7 with trace

ively, and k is the rate constant (either second-order, k
1
,for
rAPX or limiting first-order, k¢
1
, for H42A/H42E) for
Compound I formation. Formation of Compound I in the
presence of exogenous imidazole was carried out using
single-wavelength mode (397 nm), where one syringe con-
tained H42A (1 l
M
)andtheotherH
2
O
2
(0.5–35 m
M
)inthe
presence of either imidazole or 1,2-dimethylimidazole
(20 m
M
) (relatively low concentrations of exogenous imi-
dazole and a high buffer concentration were used to
minimize the effect of fluctuating imidazole levels on the
ionic strength and pH and to prevent binding of
the imidazole to the haem). Time-dependent spectra of the
various reactions were obtained by multiple-wavelength
stopped-flow spectroscopy using a photodiode array detec-
tor and
X

(nm) (e (m
M
)1
Æcm
)1
)) ¼ 420 (102), 540, 574
sh
; H42E–CN
k
max
(nm) (e (m
M
)1
Æcm
)1
)) ¼ 420 (109), 540, 573
sh
] similar
to those for the corresponding derivative of rAPX [rAPX–
CN k
max
(nm) (e (m
M
)1
Æcm
)1
)) ¼ 419 (104), 539, 572
sh
]. The
spectra of both ferric H42A and H42E were, on the other

, K
m
and the arithmetically calcu-
lated selectivity coefficient, k
cat
/K
m
) for oxidation of
L
-ascorbic acid and guaiacol by rAPX and H42E are shown
in Table 2. (The oxidation of
L
-ascorbic acid by rAPX does
not obey standard Michaelis kinetics [8,22,43] and, in this
case, data were fitted to the Hill equation. Oxidation of
guaiacol by rAPX obeys Michaelis kinetics and data were
fitted to the Michaelis–Menten equation. The origin of the
different concentration-dependencies for these two sub-
strates is not known. Oxidation of both
L
-ascorbic acid and
guaiacol by H42E was observed to obey Michaelis–Menten
kinetics.) For both substrates, k
cat
values for H42E are % 50-
fold lower than for rAPX, with K
m
values largely unaffected
(about threefold lower for H42E). The H42A variant was
inactive with both

)1
Æcm
)1
) for the ferric and Compound I derivatives of rAPX, H42A
and H42E.
Derivative rAPX H42A H42E
Fe
III
403(88), 506, 540
sh
, 636 397(83), 509, %540, 644 404(95), 516, 538
sh
, 639
Compound I 404(59), 529, 583
sh
, 650 403(62), 534, 575
sh
, 645 404(66), 530, 573
sh
, 654
Ó FEBS 2002 Catalytic mechanism of ascorbate peroxidase (Eur. J. Biochem. 269) 3185
Pre-steady-state kinetics
Spectra of the transient Compound I intermediates of
H42A and H42E, formed by reaction of the ferric deriva-
tives with 10 equivalents of H
2
O
2
, were obtained by
photodiode array experiments. These preliminary experi-

and [21,22]) and pAPX [17], a linear dependence on [H
2
O
2
]
is observed; in this work, a second-order rate constant of
(6.1 ± 0.1) · 10
7
M
)1
Æs
)1
was derived for reaction of rAPX
with H
2
O
2
. Saturation behaviour of the kind exhibited by
H42A and H42E is consistent with a mechanism involving a
pre-equilibrium step which precedes Compound I forma-
tion (Eqns 5 and 6):
E þ H
2
O
2
ÀÀ*
)ÀÀ
K
a
X ð5Þ

in Eqn (5) (K
d
¼ 1/K
a
)andk¢
1
is the limiting first-order rate
constant at high peroxide concentrations. A fit of these data
for H42A and H42E to Eqn (7) (Fig. 3) yields values for k¢
1
and K
d
of 4.3 ± 0.2 s
)1
and 30 ± 2.0 m
M
, respectively
(H42A) and 28 ± 1.0 s
)1
and 0.09 ± 0.01 m
M
, respect-
ively (H42E). These data pass through the origin, indicating
that the second step of the reaction is irreversible. At low
concentrations of peroxide, where a linear dependence is
observed, it is possible to extract an approximate value for
the second-order rate constant for reaction with H
2
O
2

Themechanisticschemeimplicatedbytheabovedata
suggested the accumulation of a reaction intermediate, the
conversion of which to product was rate-limiting at high
peroxide concentrations. To examine the nature of this
intermediate, photodiode array experiments were carried
out for H42A (Fig. 4). Intermediate spectra were obtained
from a spectrally deconvoluted model: A fi B fi C,
where A corresponds to ferric H42A, B corresponds to the
intermediate (tentatively assigned as the [H42A–H
2
O
2
]
complex, vide infra) and C corresponds to Compound I.
Wavelength maxima for the proposed intermediate were at
401 nm, 522 nm and 643 nm. The model yielded rate
constants for each step: k
A
(A fi B) and k
B
(B fi C) of
Fig. 3. Dependence of k
1,obs
on [H
2
O
2
] concentration for the reaction of
H42A (A) and H42E (B) with H
2

)1
) K
m
(m
M
)
k
cat
/K
m
(m
M
)1
Æs
)1
) k
cat
(s
)1
) K
m
(m
M
)
k
cat
/K
m
(m
M

diate, the absorbance–time trace shows a lag phase before
Compound I formation, providing further evidence to
support the proposed intermediate; the filled circles in this
inset are simulated data points derived from the model and
show good agreement between the simulated and experi-
mental data. For H42E, no intermediate complex could be
detected under these conditions.
The pH-dependences of the observed rate constants for
Compound I formation for rAPX, H42A and H42E
enzymes are shown in Fig. 5. Nonlinear dependencies with
zero intercepts on [H
2
O
2
] were observed at all pH values for
H42A and H42E. The use of acetate and nitrate buffers has
been avoided to prevent anionic effects previously observed
in pH-dependent studies on HRP and CcP [44–46].
Compound I spectra for each enzyme were identical at all
pHs (data not shown). Determination of second-order (k
1
)
and limiting first-order (k¢
1
) rate constants for rAPX and
H42E, respectively, revealed pH-dependent behaviour for
Compound I formation in both cases (Fig. 5A,C). These
datawerefittedtoasingle-protonprocess(Eqn4),andpK
a
values for rAPX and H42E of 4.9 ± 0.1 and 6.7 ± 0.2,

spectrum of ferric H42A; the dotted and dashed lines show the inter-
mediate spectra derived from the model, and represent the spectra of
the proposed [H42A–H
2
O
2
] complex and Compound I, respectively.
(B) Inset: time course at 342 nm. (d) Simulated data points derived
from the model calculated using the
PROKIN
software.
Fig. 5. pH-dependence of Compound I formation for (A) rAPX, (B)
H42A and (C) H42E. The solid line for rAPX and H42E represents a fit
of the data to the Henderson–Hasselbach equation for a single proton
process (Eqn 4); data for H42A were fitted to y ¼ c. Conditions:
l ¼ 0.10
M
,5.0°C.
Ó FEBS 2002 Catalytic mechanism of ascorbate peroxidase (Eur. J. Biochem. 269) 3187
l ¼ 0.20
M
,5.0°C, pH 7.0, [H42A] ¼ 1 l
M
;Fig.6).
[Higher concentrations of imidazole were not experiment-
ally accessible for two main reasons: (a) high concentra-
tions of either imidazole led to large changes in both ionic
strength and solution pH; (b) binding of imidazole to the
iron is observed at higher concentrations, leading to
enzyme inhibition.] The spectrum of Compound I formed

, K
d
¼ 30 ± 2.0 m
M
(sodium
phosphate, pH 7.0 l ¼ 0.20
M
], indicates that imidazole
and 1,2-dimethylimidazole lead to % 10-fold and % 40-fold
increases in k¢
1
, respectively, with the values for K
d
largely
unaffected. [These values are slightly different from
those determined above (k¢
1
¼ 4.3 ± 0.2 s
)1
and
K
d
¼ 30 ± 2.0 m
M
), because the two determinations were
carried out at a slightly different ionic strengths.]
In parallel steady-state experiments ([H42A] ¼ 100 n
M
,
[guaiacol] ¼ 30 m

for rAPX) to generate a low-spin
H42A–imidazole derivative (k
max
¼ 412, 533 and 565
sh
nm)
that inhibited activity. Guaiacol activities in the presence of
1,2-dimethylimidazole were % 10-fold higher than for
imidazole itself and were observed to increase linearly with
increasing 1,2-dimethylimidazole concentration (1–40 m
M
);
no evidence for saturation was observed in this case and
addition of 1,2-dimethylimidazole to H42A did not generate
an observable low-spin derivative.
DISCUSSION
To investigate the catalytic role of the conserved His42
residue in APX catalysis, two site-directed variants were
prepared in which the distal histidine was replaced by
alanine (H42A) and glutamic acid (H42E) residues. The
results provide: (a) unambiguous evidence that His42 is
critical for efficient Compound I formation in APX; (b)
confirmation that titration of this residue controls the rate
constant for Compound I formation and an assignment of
the pK
a
for this group; (c) mechanistic evidence for an
intermediate before Compound I formation; (d) spectro-
scopic evidence on the nature of this intermediate. The
detailed implications of these data are discussed below.

Michaelis–Menten kinetics. Although it was initially pro-
posed that the origin of the sigmoidal dependence may arise
from the dimeric structure of the enzyme, site-directed
mutagenesis work [43] has indicated that this is unlikely to
be the case. It is possible that nonspecific effects, perhaps
involving radical chemistry associated with oxidation of
ascorbate, or the existence of more than one substrate
binding site, may be influential. A sigmoidal dependence on
substrate concentration is not observed for H42E-catalysed
oxidation of
L
-ascorbate, an observation that we are not, at
present, able to fully rationalize. Replacement of the distal
His42 by alanine renders the H42A enzyme essentially
inactive, which is presumably directly linked to the very
poor reactivity of this variant with H
2
O
2
.
Fig. 6. Dependence of k
1,obs
, the pseudo-first-order rate constant for
Compound I formation in H42A, on H
2
O
2
concentration in the absence
and presence of exogenous imidazoles (20 m
M

protein radical chemistry involving a Trp amino acid occurs
under these conditions, leading to a permanent alteration of
the haem structure that is reflected in the absorption
maxima of the final (ferric-like) decay product. For H42A
and H42E in the absence of substrate, Compound I does
not spontaneously convert into a Compound II species
as observed for rAPX, and the Compound I intermediates
for both enzymes slowly generate species with a slightly
red-shifted Soret band [k
max
(H42A) ¼ 405 nm; k
max
(H42E) ¼ 406 nm] compared with the ferric state. These
maxima are analogous to those observed for the decay of
rAPX (k
max
¼ 406 nm), suggesting that similar radical
chemistry may occur in the variants, although this has not
been examined in detail in this work. The failure to observe
Compound II for the two variants under these conditions is
intriguing (particularly as H42E is clearly active against
both
L
-ascorbate and guaiacol) and has been noted
previously for the H42E [49,50] and H42A and H42V [51]
variants of HRP. These data suggest that the distal histidine
residue stabilizes Compound II through a hydrogen-bond-
ing structure involving the N
e
of His42 and the ferryl

and % 10
2
-fold decrease has occurred for H42A and H42E,
respectively. These data provide convincing evidence to
support a key catalytic role for His42 and indicate that the
glutamic acid residue is able to replace the distal histidine
residue such that reasonably efficient Compound I forma-
tion is effected. The corresponding H42A and H42E
variants in HRP have been shown to lower the rate
constant for Compound I formation by six and four
orders of magnitude, respectively [49–51,57], although no
evidence for an intermediate was found for these variants.
Large effects on the rate constant of Compound I
formation have also been reported for other His42 variants
of HRP [58–60] and for CcP [61,62].
The pre-steady-state data (Fig. 3) are consistent with a
mechanism involving formation of an enzyme–substrate
intermediate, the conversion of which into product is rate-
limiting at high concentrations of peroxide. The simplest
mechanism that is consistent with these data is described by
Eqns (5) and (6). Although mechanisms involving conform-
ational gating of the reaction at high peroxide concentra-
tions [55] or before Compound I formation [63] are
possible, the observation of a spectroscopically distinct
intermediate for H42A provides good evidence for the
proposed mechanism (but leaves this interpretation slightly
more open for H42E). This enzyme intermediate has
wavelength maxima at 401 nm, 522 nm and 643 nm and
we assign the intermediate as arising from a transient
[H42A–H

As such, only the linear part of the nonlinear k
1,obs
vs.
[H
2
O
2
] dependence is experimentally accessible for rAPX,
and O–O bond cleavage is not rate-limiting under any
experimental conditions. [In fact, for all APXs [17–19] and
variants of rAPX [16,21,22] examined so far, a linear
dependence on [H
2
O
2
] is observed and second-order rate
constants are derived (k
1
% 10
7
M
)1
Æs
)1
).]Indeed,this
intermediate has eluded detection for this very reason in
other peroxidase systems and its exact structure is still not
clear. For example, Baek & Van Wart [23,24] identified an
intermediate, designated Compound 0 and proposed to be a
hyperporphyrin (Fe

(pK
a
¼ 11.6), which dictates
that the peroxide molecule is predominantly in the proto-
nated form under the conditions used in this work
Ó FEBS 2002 Catalytic mechanism of ascorbate peroxidase (Eur. J. Biochem. 269) 3189
(deprotonation of the bound hydroperoxide species is
assumed to occur before O–O bond cleavage).
The pH-dependent data for Compound I formation,
Fig. 5, are particularly informative. The absence of a pH-
dependence for H42A unambiguously assigns this residue as
being responsible for the pH-dependent reaction between
rAPX and H
2
O
2
.ThepK
a
of His42 can also be derived
directly (pK
a
¼ 4.9) from the rAPX data and indicates that
this group must be deprotonated for efficient reaction with
H
2
O
2
(Scheme 1). By analogy with the rAPX data, the new
pK
a

¼ 2.5–5.3
[66,67]), myeloperoxidase (pK
a
¼ 4.0 [68,69]) and Coprinus
cinereus peroxidase (pK
a
¼ 5.0 [70]) and have been assigned
as arising from titration of the distal residue. For peroxid-
ases that exhibit pH-independent kinetics, for example,
lignin [71] and manganese peroxidases [72], titration of the
distal histidine presumably still influences Compound I
formation, but the pK
a
is not experimentally accessible. For
CcP, examination of the role of the distal histidine residue in
Compound I formation has been complicated by specific
buffer effects that alter the kinetic profile [46,62]. Where pH-
dependent rate constants have been reported, the pK
a
values
(pK
a
¼ 5.4 [62], 4.0 [62]) are in a similar range to those
reported here for rAPX.
The catalytic deficiency of H42A can be partially com-
pensated for by the use of exogenous imidazole [57]; we
observed 1,2-dimethylimidazole to be more effective than
imidazole in recovering activity against guaiacol. This is
probably related to the relative affinities of the two imidazole
derivatives for binding to the haem iron: binding of imidazole

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