Spectroscopic and kinetic properties of the horseradish
peroxidase mutant T171S
Evidence for selective effects on the reduced state of the enzyme
Barry D. Howes
1
, Nigel C Brissett
2
, Wendy A. Doyle
2
, Andrew T. Smith
2
and Giulietta Smulevich
1
1 Dipartimento di Chimica, Universita
`
di Firenze, Italy
2 Department of Biochemistry, School of Life Sciences, University of Sussex, Brighton, UK
Horseradish peroxidase (HRPC) is a member of class
III of the plant peroxidase superfamily and is cap-
able of utilizing hydrogen peroxide to oxidize a wide
range of phenols, anilines and other synthetic sub-
strates [1]. Historically, it is has been the subject of
extensive spectroscopic and functional studies [2–4]
and is the archetypal enzyme on which many of our
ideas of biological oxidation reactions have been
based [1]. More recently this has involved the
detailed characterization of mutants [2–4] designed to
probe various aspects of its catalytic mechanism and
spectroscopic properties. Detailed structural informa-
tion for the enzyme and the catalytic intermediates
in all five oxidation states is now available [5]. In

onstrated that proximal structural features can also exert an important
influence in determining the electronic structure of the haem pocket. To
extend our understanding of the significance of proximal characteristics in
regulating haem properties the proximal Thr171Ser mutant has been con-
structed. Thr171 is an important linking residue between the structural
proximal Ca
2+
ion and the proximal haem ligand, in particular the methyl
group of Thr171 interdigitates with other proximal residues in the core of
the enzyme. Although the mutation induces no significant changes to the
functional properties of the enzyme, electronic absorption and resonance
Raman spectroscopy reveal that it has a highly selective affect on the
reduced state of the enzyme, effectively stabilizing it, whilst the electronic
properties of the Fe(III) state unchanged and essentially identical to those
of the native protein. This results in a significant change in the Fe
2+
⁄ Fe
3+
redox potential of the mutant. It is concluded that the unusual properties
of the Thr171Ser mutant reflect the loss of a structural restraint in the
proximal haem pocket that allows ‘slippage’ of the proximal haem ligand,
but only in the reduced state. This is a remarkably subtle and specific effect
that appears to increase the flexibility of the reduced state of the mutant
compared to that of the wild-type protein.
Abbreviations
ABTS, 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonate); BHA, benzhydroxamic acid; APX, ascorbate peroxidase; CCP, cytochrome c
peroxidase; CIP, Coprinus cinereus peroxidase; HRPC, horseradish peroxidase C; LS, low spin; MOPS, 3-morpholinopropanesulfonic acid;
TcAPXII, cationic ascorbate peroxidase isoenzyme II from tea; PG, pyrolytic graphite; RR, resonance Raman; 5-c, 5-coordinate; HS, high spin;
QS, quantum mechanically mixed spin; SCE, standard calomel electrode; TBMPC, tributylmethyl phosphonium chloride; F221M, Phe221Met
mutant HRPC; T171S, Thr171Ser mutant HRPC.

been replaced by a serine residue. Ser differs from Thr
only by the absence of a methyl group and so repre-
sents a very subtle change, a change that is naturally
present in other fungal peroxidases belonging to class
II, such as lignin peroxidase [3]. This region of the
structure has particular relevance in both enzymes
because of its potential to provide structural coupling
between the proximal Ca
2+
ion and the residues of the
active site, most notably the distal His (H170 in
HRPC, Fig. 1). The effects of the Thr171Ser mutant
have proven to be particularly intriguing and specific
to the reduced state of the enzyme. The Fe(II) state of
the enzyme has features in common with both the
Phe221Met mutant and Ca-depleted proteins whilst
the Fe(III) state is essentially identical to that of the
wild-type protein. We conclude that the properties of
the T171S mutant reflect the loss of a structural
restraint in the proximal haem pocket that results in
unusually subtle and selective effects that are mediated
exclusively on the reduced state of the enzyme. We
hypothesize that this residue imposes a degree of rigid-
ity to the structure of the reduced state of class III
peroxidases.
Results and Discussion
Table 1 shows some of the functional parameters asso-
ciated with the T171S mutant. Its ability to react with
hydrogen peroxide to form Compound I, as measured
by the second order rate constant for Compound I for-

e
a
(mM
)1
Æcm
)1
) k
1
(M
)1
Æs
)1
)
Turnover
no. (s
)1
)
K
d
(BHA, lM)
Recombinant
wild-type
a
98 ± 3 (1.7 ± 0.1) · 10
7
560
b
2.7 ± 0.3
c
F221M

alkaline pH. Addition of saturating amounts of BHA
did not reveal any spectral differences between the wild
type and the mutant (data not shown). Furthermore,
the electronic absorption spectra of the Thr171Ser
mutant and wild-type HRPC at pH 10.1 were also
identical (data not shown), indicating that the mutant
binds a hydroxyl group at alkaline pH, forming a
6-coordinate low spin (LS) haem species in an identical
way to the wild type [16,17]. The pK
a
for the alkaline
transition being similar to that of the wild type, % 11.1
[18]. Finally, comparison of the X-ray structures of the
oxidized forms of the native [5,19] and the T171S
mutant (protein databank code: 1GW2.pdb) did not
reveal any significant differences between the two pro-
teins. These observations are consistent with the very
subtle nature of the mutation, i.e. the loss of a single
methyl group, depicted in green in Fig. 1.
In marked contrast, for the reduced state compar-
ison of the RR spectra of the Thr171Ser mutant and
wild type reveal very significant differences. Figures 2
and 3 show the electronic absorption and RR spectra,
respectively, of the T171S mutant at pH 6.8 and 8.9
and wild type (pH 6.8) in the Fe(II) state. The previ-
ously characterized proximal pocket mutant F221M
(pH 6.8) [14] and the Ca-depleted protein (pH 6.8) [13]
are also shown for comparison. The electronic absorp-
tion spectrum of the T171S mutant is characteristic of
a 5-c HS haem, as previously established for the wild-

the wild-type protein.
Wavenumber /cm
-1
Fig. 3. Resonance Raman spectra of ferrous HRPC. Buffers as
reported in Fig. 2. Experimental conditions: 5 cm
)1
resolution;
441.6 nm excitation wavelength; concentration of 50 l
M,10s⁄
0.5 cm
)1
collection interval, 20 mW laser power at the sample
(wild type, pH 6.8); concentration of 45 l
M,12s⁄ 0.5 cm
)1
collec-
tion interval, 20 mW laser power at the sample (T171S, pH 6.8);
concentration of 40 l
M,26s⁄ 0.5 cm
)1
collection interval, 20 mW
laser power at the sample (T171S, pH 8.9); concentration of 70 l
M,
5s⁄ 0.5 cm
)1
collection interval, 20 mW laser power at the sample
(F221M, pH 6.8); concentration of 40 l
M,12s⁄ 0.5 cm
)1
collection

[22,23] to out-of-plane modes of the porphyrin ring
itself together with the bending modes of the propionyl
and vinyl substituents of the haem.
Class III peroxidases normally exhibit only one
Fe-Im band, in contrast to the class I and II peroxidases
that have two Fe-Im bands resulting from the tauto-
merism of the imidazole N
d
proton with respect to the
donor and acceptor atoms of the proximal His and
Asp H-bond [11]. The only exception to this is the cat-
ionic ascorbate peroxidase isoenzyme II from tea
(TcAPXII); this shows two Fe-Im stretches at 233 and
249 cm
)1
. This is a rather anomalous hybrid peroxi-
dase, that exhibits the spectroscopic characteristics and
substrate preferences of both class I and class III per-
oxidases [24]. As in ascorbate peroxidase (APX) [25],
the absence of a decrease of the I
220
⁄ I
247
intensity ratio
between the two bands observed for the Thr171Ser
mutant, upon raising the pH, suggests that the two
species are independent and not in equilibrium, as is
thought to be the case for CCP [23] and Coprinus cine-
reus peroxidase (CIP) [26]. The frequencies of the two
m(Fe-Im) stretching modes at 220 cm

(217 cm
)1
) [13]. In the second case the opposite effect
is seen, which is much less pronounced (band at
247 cm
)1
).
The redox potential for the Thr171Ser mutant
(E ¼ )32 ± 7 mV vs. SCE) was determined to be
significantly less negative than that of the wild-type
(E ¼ )133 ± 7 mV vs. SCE). The increase in the
redox potential compared to the wild type is in
accord with the observation of a m(Fe-Im) mode in
Thr171Ser at a markedly lower frequency than in
the wild-type protein (220 cm
)1
) (Fig. 3). In fact, a
greater imidazolate character, stabilizing the higher
oxidation state, leads to a decrease of the redox
potential of the heme iron. However, it is not pos-
sible to make a direct correlation between the magni-
tude of the changes in Fe-His band frequencies and
the redox potential values. This is exemplified by the
case of CCP and its mutants D235E, D235N and
D235A [27]. The H-bond between the proximal His
and Asp235 is completely lost when Asp235 is
replaced by the nonbonding residues Asn and Ala,
but the D235E mutation results only in a very small
displacement of the carboxylate group. Nevertheless,
in all three cases the RR frequency of the Fe-His

wild-type protein.
In contrast to the present study, in previous cases
where the proximal site of HRPC has been modified
by mutation [14] or Ca-depletion [13] significant chan-
ges in the properties of the ferric form of the protein
has been detected. Even so, the changes detected in the
haem cavity of the reduced state appear more promin-
ent. In both cases significant structural alterations to
the protein conformation were indicated, not only by
marked changes in the geometric disposition of the
proximal His and Asp residues, affecting the imidazo-
late character of the His, but also by the formation of
a LS species. The latter indicating the probable bind-
ing of His42 to the haem iron, i.e. a major collapse or
rearrangement of the distal cavity has taken place.
Hence, the overall conclusion that may be drawn is
that modification of the proximal cavity of HRPC by
mutation or Ca
2+
ion removal has a significant impact
on the properties of His170. The strength of the hydro-
gen bond between the proximal His and Asp residues,
and thus the imidazolate character of the His is expec-
ted to modulate not only the strength of the Fe-Im
bond but also the stability of the different oxidation
states. In fact, the potential sensitivity and dependence
of enzyme properties on the structural characteristics
of the proximal domain is demonstrated by the mark-
edly less negative (by approximately 100 mV) redox
potential of the T171S mutant compared to the wild-

involved the use of the pSD18 template. Oligonucleotide
primer WDHRP9 (5¢-GAGTGTCCGGAGGCCACAGCT
TTGG-3¢; where mutated bases are shown in bold) was
designed for the point mutation at position 171 and to over-
lap the BspEI site. WDHRP10 (5¢-CATAGGGATCCTT
ATTAAGAGTTGC-3¢) was designed to overlap the BamHI
site at the 3¢ end of the gene. A mutant DNA insert (430 bp)
was generated by PCR. The purified fragment was inserted
into the cloning vector pBGS19 via ‘blunt-ended’ ligation
and checked by automated DNA sequencing (Applied Bio-
systems, Foster City, CA, USA). Only the expected muta-
tion was detected. The plasmid insert was digested using
BspEI and BamHI and ligated in frame into pSD18 [32] cut
with the same restriction enzymes. The whole HRPC insert
was then excised from pSD18 with NdeI and BamHI and
ligated into the expression vector pFLAG1 at the unique
NdeI and BglII sites.
Expression of HRPC in E. coli W31110, isolation of
inclusion bodies, refolding and purification of the wild-type
protein and the Phe221Met and Thr171Ser mutants were
carried out as previously described [14,32,33]. Purified
recombinant enzyme Thr171Ser was stored at )80 °Casa
frozen solution in 10 mm Mops buffer at pH 7.0.
Steady-state turnover with 2,2¢-azinobis-(3-ethyl-
benzothiazoline-6-sulphonate) (ABTS)
Peroxidase activity was determined in 50 mm phos-
phate ⁄ citrate buffer pH 5.0 at 25 °C, by measuring the
increase in absorbance at 405 nm given by the formation
of the 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonate)
(ABTS) cation radical product with 1.5 mm H

Determination of dissociation constants for
benzhydroxamic acid
The dissociation constants (K
d
) of complexes formed
between resting state enzymes and benzhydroxamic acid
were determined by titration of the Soret region of the vis-
ible spectrum as described previously [33]. K
d
values were
calculated by fitting the data to Eqn. (1) using a weighted
least squares error minimization procedure.
A ¼ 2A
1
L=fðL þ K
d
þ PÞþ½ðL þ K
d
þ PÞ
2
À 4PL
1=2
gð1Þ
The absorbance change at 408 nm resulting from benzhydr-
oxamic acid of concentration L, binding to a total protein
concentration P, was determined, while allowing the
remaining K
d
and maximum absorbance change at satura-
tion (A

nics. To minimize local heating of the protein by the laser
beam, the sample was cooled by a gentle flow of N
2
gas
passed through liquid N
2
. RR spectra were calibrated to an
accuracy of 1 cm
)1
for intense isolated bands, with indene
as the standard for the high-frequency region and with
indene and CCl
4
for the low-frequency region.
Redox potential measurements
The redox potential measurements were made by firstly
embedding the protein in a tributylmethyl phosphonium
chloride (TBMPC) membrane followed by immobilization
on a pyrolytic graphite (PG) electrode surface as previously
described [34]. DC cyclic voltammograms were run in previ-
ously degassed 0.1 m sodium phosphate, pH 7.0. Measure-
ments were carried out at 25 °C in a glass microcell
(sample volume, 1 mL). During the measurements the
anaerobic environment was maintained by a gentle flow of
high-purity grade nitrogen just above the surface of the
solution. A PG electrode (AMEL, Milan, Italy) was the
working electrode, a saturated calomel electrode (AMEL)
was the reference and a Pt ring the counter-electrode. An
Amel 433 ⁄ W multipolarograph (Milan, Italy) interfaced
with a PC as data processor was employed for voltammet-

ke H,
Henriksen A & Hajdu J (2002) The catalytic pathway of
horseradish peroxidase at high resolution. Nature 417,
463–468.
6 Smulevich G, Paoli M, Burke JF, Sanders SA,
Thorneley RNF & Smith AT (1994) Characterization
of recombinant horseradish peroxidase C and three
site-directed mutants, F41V, F41W and R38K, by
resonance Raman spectroscopy. Biochemistry 33,
7398–7407.
B. D. Howes et al. Selective effects on the reduced state of HRPC
FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS 5519
7 Howes BD, Rodriguez-Lopez JN, Smith AT & Smule-
vich G (1997) Mutation of distal residues of horseradish
peroxidase: influence on substrate binding and cavity
properties. Biochemistry 36, 1532–1543.
8 Howes BD, Heering HA, Roberts TO, Schneider-
Belhadadd F, Smith AT & Smulevich G (2001) Muta-
tion of residues critical for benzohydroxamic acid bind-
ing to horseradish peroxidase isoenzyme C. Biopolymers
62, 261–267.
9 Heering AH, Indiani C, Regelsberger G, Jakopitsch C,
Obinger C & Smulevich G (2002) New insights into the
heme cavity structure of catalase-peroxidase: a spectro-
scopic approach to the recombinant Synechocystis
enzyme and selected distal cavity mutants. Biochemistry
41, 9237–9247.
10 Smulevich G, Mauro JM, Fishel LA, English A, Kraut
J & Spiro TG (1988) Heme pocket interactions in cyto-
chrome c peroxidase studied by site-directed mutagen-

complexes of metmyoglobin, methemoglobin, and horse-
radish peroxidase at room and low temperatures.
Biochemistry 33, 4577–4583.
18 Marklund S, Ohlsson P-I, Opara A & Paul K-G (1974)
The substrate profiles of the acidic and slightly basic
horseradish peroxidases. Biochim Biophys Acta 350,
304–313.
19 Gajhede M, Schuller DJ, Henriksen A, Smith AT &
Poulos TL (1997) Crystal structure of horseradish per-
oxidase C at 2.15 A
˚
resolution. Nat Struct Biol 4, 1032–
1038.
20 Teraoka J & Kitagawa T (1981) Structural implications
of the heme-linked ionization of horseradish peroxidase
probed by the Fe-histidine stretching Raman line. J Biol
Chem 256, 3969–3977.
21 Kitagawa T (1988) The heme protein structure and the
iron histidine stretching mode. In Biological Applications
of Raman Spectroscopy III (Spiro TG, ed.), pp. 97–131.
Wiley and Sons, New York, USA.
22 Hu S, Smith KM & Spiro TG (1996) Assignment of
protoheme resonance Raman spectrum by heme labeling
in myoglobin. J Am Chem Soc 118, 12638–12646.
23 Smulevich G, Hu SZ, Rodgers KR, Goodin DB, Smith
KM & Spiro TG (1996) Heme–protein interactions in
cytochrome c peroxidase revealed by site-directed muta-
genesis and resonance Raman spectra of isotopically
labeled hemes. Biospectroscopy 2, 365–376.
24 Heering HA, Jansen MAK, Thorneley RNF & Smule-

ley RNF & Rich PR (1998) Redox- and anion-linked
protonation sites in horseradish peroxidase: analysis
of distal haem pocket mutants. Biochem J 330, 303–
309.
31 Tanaka M, Ishimori K, Mukai M, Kitagawa T &
Morishima I (1997) Catalytic activities and structural
properties of horseradish peroxidase distal His42 Glu or
Gln mutant. Biochemistry 36, 9889–9898.
Selective effects on the reduced state of HRPC B. D. Howes et al.
5520 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS
32 Smith AT, Santama N, Dacey S, Edwards M, Bray RC,
Thorneley RNF & Burke JF (1990) Expression of a
synthetic gene for horseradish peroxidase in Escherichia
coli and folding and activation of the recombinant
enzyme with Ca
2+
and heme. J Biol Chem 265, 13335–
13343.
33 Smith AT, Sanders SA, Thorneley RNF, Burke JF &
Bray RC (1992) Characterisation of a haem active site
mutant of horseradish peroxidase, Phe41 fi Val, with
altered reactivity towards hydrogen peroxide and redu-
cing substrates. Eur J Biochem 207, 507–519.
34 Ferri T, Poscia A & Santucci R (1998) Direct electro-
chemistry of membrane-entrapped horseradish peroxi-
dase. Part I: a voltammetric and spectroscopic study.
Bioelectrochem Bioenerg 44, 177–181.
35 Murray RW (1984) Chemically modified electrodes. In
Electroanalytical Chemistry, Vol. 13 (Bard, AJ, ed.),
pp. 205–207. Marcel Dekker, New York, USA.