Báo cáo khoa học: Spectroscopic and kinetic properties of the horseradish peroxidase mutant T171S Evidence for selective effects on the reduced state of the enzyme potx

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

contrast to globins, the preferred resting state of per-
oxidases is the oxidized ferric state. The structural
factors, particularly those in the proximal environ-
ment of the haem that underlie the relative stability
Keywords
Fe-His stretch; haem peroxidase;
horseradish peroxidase; resonance Raman;
redox potential
Correspondence
A. T. Smith, Department of Biochemistry,
School of Life Sciences, John Maynard
Smith Building, University of Sussex,
Falmer, Brighton BN1 9QG, UK
Fax: +44 1273 678433
Tel: +44 1273 678863
E-mail:
G. Smulevich, Dipartimento di Chimica,
Universita
`
di Firenze, Via della Lastruccia 3,
50019 Sesto Fiorentino (FI), Italy
Fax: +39 0554573077
Tel: +39 0554573083
E-mail: giulietta.smulevich@unifi.it
(Received 17 June 2005, revised 11 August
2005, accepted 30 August 2005)
doi:10.1111/j.1742-4658.2005.04943.x
Studies on horseradish peroxidase C and other haem peroxidases have been
carried out on selected mutants in the distal haem cavity providing insight
into the functional importance of the distal residues. Recent work has dem-

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.

5514 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS
of the ferrous ⁄ ferric state of HRPC, are not under-
stood.
Spectroscopic and functional studies have concentra-
ted predominantly on the role of residues in the distal
haem cavity that provide an insight into the role of
key catalytic residues [6–9]. A significant outcome of
many of these studies [6,10–12] has been the discovery
of coupled effects that can be understood on the basis
of a hydrogen bonding network that links the distal
and proximal halves of the protein.
It is perhaps surprising that the proximal domain
has received relatively little attention compared to the
extensive studies referred to above on the distal cavity.
Recent studies, including the removal of the proximal
structural Ca
2+
ion [13] and the construction of a
mutant in which the proximal Phe221 residue was
replaced by Met [14], have demonstrated the ‘sensitivity’
of the proximal region in determining the electronic
and structural properties of the haem pocket.
In the present study we attempt to obtain further
insights into the extent to which the proximal environ-
ment influences the electronic and functional properties
of the haem. The proximal residue Thr171 that pro-
vides two bonds to the proximal Ca
2+
ion and is adja-
cent in sequence to the active site residue His170, has

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-

mation, was identical to the wild-type enzyme. Other
functional parameters such as steady state turnover
with 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulpho-
nate) (ABTS) as substrate and the ability to bind the
aromatic donor molecule benzhydroxamic acid (BHA),
itself a sensitive indicator of the integrity of the distal
haem pocket [3,4] were also essentially unaffected.
Hence, the mutation causes no gross change in the
functional properties of the enzyme and any catalytic
consequences of the mutation are at best slight.
This finding is strengthened by the electronic absorp-
tion and resonance Raman (RR) spectra of the oxid-
ized wild-type HRPC and the Thr171Ser mutant (data
not shown), which clearly show that mutation does
Fig. 1. The structural features of the proximal haem pocket of
HRPC showing the haem, the proximal Ca
2+
(grey sphere) H170,
and T171. The methyl group of T171S is shown as a green sphere,
it can be seen to interdigitate with F172, F229, G168, D230, I228
and is directly constrained by the carbonyl oxygen of G168. Inclu-
sion of F221 obscures the local structure in the proximity of T171
and for clarity is omitted.
Table 1. Comparison of functional parameters for HRPC proximal
pocket mutants. Kinetic parameters were determined as described
in the Experimental procedures section. Earlier values for the
F221M mutant which stacks against the proximal His (Fig. 1) are
shown for comparison.
Enzyme
variant

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

a
110 ± 3 (1.5 ± 0.1) · 10
7
510 4.3 ± 0.1
T171S 106 ± 2 (1.6 ± 0.1) · 10
7
607 ± 35 2.5 ± 0.5
c
a
Values from [14].
b
Values from [32].
c
Determined in 25 mM
Mops pH 7.0 supplemented with 100 lM CaCl
2
.
B. D. Howes et al. Selective effects on the reduced state of HRPC
FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS 5515
not affect either the coordination or the spin states of
the haem system. The Thr171Ser mutant contains a
substantially 5-coordinate (5-c) quantum mechanically
mixed spin (QS) haem essentially identical to the wild-
type protein [4,8,11,15].
Additional evidence that in the oxidized state the
structural properties of the HRPC haem cavity are
essentially unaffected by the Thr171Ser mutation was
obtained from comparison of the electronic absorption
and RR spectra of the wild type and Thr171Ser either
in the presence of the aromatic donor BHA or at

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-

type protein (Fig. 2) [20], while the blue-shift of the
Soret and the changes in the visible region of the
Ca-depleted and F221M spectra indicate a more or less
marked presence of LS haem in these proteins. In
agreement with the absorption spectra, the high fre-
quency RR spectra of Fe(II) Thr171Ser at pH 6.8 and
8.9 for 441.6 nm excitation (data not shown) are very
similar to those of the wild-type enzyme at pH 6.8 [13]
and indicative of a 5-c HS haem species. However, the
low frequency region of the RR spectrum of the
T171S variant differs markedly from that of the parent
enzyme. In particular, the wild-type protein is charac-
terized by an intense band at 244 cm
)1
(Fig. 3), that
has been assigned to the m(Fe-Im) stretching mode
between the haem iron atom and the imidazole ligand
(Im) of the proximal histidine residue [20]. This mode,
which is only active in the 5-c HS Fe(II) state in
peroxidases is at higher frequencies than found in
other haem proteins as a consequence of the strong
Fig. 2. Electronic absorption spectra of 40 lM ferrous HRPC. Wild-
type protein at pH 6.8 in 25 m
M MOPS, T171S at pH 6.8 in
100 m
M citrate and pH 8.9 in 100 mM glycine, F221M at pH 6.8 in
100 m
M citrate, Ca-depleted at pH 6.8 in 5 mM EDTA, 50 mM
Tris ⁄ HCl. The visible region is expanded 8-fold. The path length of
the cuvette was 1 mm for all spectra. The ordinate scale refers to

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

interval, 30 mW laser power at the sample (Ca-depleted, pH 6.8).
Selective effects on the reduced state of HRPC B. D. Howes et al.
5516 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS
hydrogen bond between the proximal His170 and
Asp247 residues (Fig. 1). This strong hydrogen bond
imparts a pronounced imidazolate character to the
proximal His [21]. It is evident from Fig. 3 that the RR
spectrum of the Thr171Ser mutant at pH 6.8 is very
similar to that of the wild-type except for the three
bands at 220, 247 and 276 cm
)1
, all of which shift to
lower frequencies upon raising the pH to 8.9. The pH
sensitivity of these frequencies is a common character-
istic of all peroxidases and is a consequence of the
strong H-bond between the proximal His and Asp resi-
dues that is weakened at alkaline pH. Therefore, on
the basis of their sensitivity to pH, the bands at 220
and 247 cm
)1
are assigned to two m(Fe-Im) modes.
The band at 276 cm
)1
is assigned to an internal vibra-
tional mode of the imidazole ligand, which is enhanced
by coupling with the Fe-Im mode, as previously
observed for the F221M HRPC mutant [14]. The fre-
quencies of the remaining bands in the RR spectrum,
which are unaffected by the pH, are assigned by anal-
ogy to myoglobin and cytochrome c peroxidase (CCP)

[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

)1
(downshifted
24 cm
)1
compared to wild-type) and 247 cm
)1
(up shif-
ted 3 cm
)1
compared to wild-type) are very close to
those found for the F221M mutant. These are distin-
guished by the strength of the hydrogen bond between
the proximal His and the Asp carboxylate side chain.
In structural terms, these observations could be related
to changes in the steric constraints operating at the
proximal His and ⁄ or Asp residues induced by the
T171S mutation. It appears as though the C
a
back-
bone in the His170 region may be more mobile due to
the absence of the methyl group at position 171, T171
presumably normally constrains any ‘slippage’ of the
adjacent His170 (Fig. 1). Interestingly, the introduction
of this flexibility into the proximal cavity structure
leads to two populations of molecules. In the first case
the Fe-Im bond strength is decreased (band at
220 cm
)1
), this is similar to the situation that arises
upon removal of the proximal structural calcium ion

(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

band is at 205 cm
-1
(CCP wild type, 246 ⁄ 233 cm
-1
),
suggesting that in the three mutants the proton is no
longer shared between residue 235 and His175. How-
ever, the redox potentials of the mutants increase
compared to wild type by 70 (D235E), 104 (D235N)
and 105 (D235A) mV. The redox potential depends
on a number of other factors such as the electro-
static, Van der Waals and hydration status of the
haem environment that also vary with the peroxidase
under consideration, while the Fe-His frequency is
primarily dependent on the strength of the Fe-His
bond and hence on the status of the proximal His
H-bond. It appears that one can readily rationalize
the general trends but not the magnitude of the chan-
ges seen.
Entropy factors can in principle play an important
part in determining the redox potential of HRPC. In
fact, contrary to the situation found for electron
transfer proteins, reduction of HRPC leads to a
marked increase in entropy [28]. Thus, the greater
flexibility of the proximal cavity structure evident in
B. D. Howes et al. Selective effects on the reduced state of HRPC
FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS 5517
the ferrous state of the mutant may contribute to an
increase in the disorder and hence entropy of the
reduced state of the mutant compared to that of the

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-

type protein.
Mutations of distal residues can give rise to a sub-
stantial reduction of the catalytic activity if they have
a direct impact on the disposition of the catalytically
important His42 and Arg38 residues [29,30]. However,
although the Fe-Im bands of many distal mutants
undergo a small but significant shift to lower frequen-
cies compared to the wild-type protein, their redox
potentials are virtually unaffected [30,31]. It is appar-
ent that the effects resulting from proximal changes
in HRPC, particularly those affecting the Ca
2+
ion
site are far reaching. This underlines the importance
of long range interactions originating form the prox-
imal cavity in fine tuning the properties of the haem,
most notably the haem iron redox potential. The
most significant finding in this study is the effect of a
single mutation on the structural constraints of the
protein, whereby a relatively minor alteration to
the proximal cavity is capable of selectively stabilizing
the reduced state of the enzyme but is having essen-
tially no detectable affect on the oxidized form of the
enzyme.
Experimental procedures
Site-directed mutagenesis and expression of
recombinant proteins
A PCR-based method was used for site-directed mutagenesis
that utilized the synthetic HRP C gene [32] exactly as des-
cribed in [14]. The construction of the Thr171Ser mutant

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

2
O
2
and
0.3 mm ABTS as described in [32].
Measurement of the second-order rate constants
for Compound I formation
The rate of Compound I formation (k
1
) was determined in
10 mm sodium phosphate buffer pH 7.0 (l ¼ 100 mm) and
25 °C under pseudo-first-order conditions (Applied Photo-
physics SX18MV stopped-flow system; Leatherhead, UK)
by following the decrease in absorbance at 395 nm. Time
courses were fitted to single exponentials and the rate
constants (k
obs
) determined from the fits. Values for k
obs
were plotted against H
2
O
2
concentration and linear pseudo-
first order plots were obtained over the substrate range
studied. The k
1
value was obtained from the gradient.
Selective effects on the reduced state of HRPC B. D. Howes et al.
5518 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS

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

1
) to float.
Resonance Raman and electronic absorption
spectroscopy
For resonance Raman and electronic absorption spectro-
scopy the experimental conditions were as reported in the
captions to figures. Samples of ferrous enzymes for electronic
absorption and resonance Raman spectroscopy were pre-
pared by addition of 2 lL of dithionite (20 mgÆmL
)1
)to
50 lL of deoxygenated peroxidase solution. Benzhydroxamic
acid complexes were prepared by adding aliquots of 0.2 m
benzhydroxamic acid (Sigma, St Louis, MO, USA) in 10 mm
MOPS pH 7.0 to the enzyme samples, to a final (saturating)
concentration of 5 mm.
Electronic absorption spectra, measured with a Cary 5
spectrophotometer, were recorded both prior to and after
RR measurements. No degradation was observed under the
experimental conditions used. RR spectra were obtained at
room temperature with excitation from the 406.7 nm line of
aKr
+
laser (Coherent, Innova 90 ⁄ K, Santa Clara, CA,
USA), and from the 441.6 nm line of a HeCd laser
(Liconix, xxxx, xxxx). The back-scattered light from a
slowly rotating NMR tube was collected and focused into a
computer-controlled double monochromator (Jobin-Yvon
HG2S, xxxx, xxxx) equipped with a cooled photomultiplier
(RCA C31034A, xxxx, xxxx) and photon counting electro-

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-

ric measurements. The potentials reported in the text are
referenced to the standard calomel electrode (SCE). The
redox potential of the wild-type protein determined by this
method is approximately 100 mV less negative than that
determined using potentiometry [30,34]. However, differ-
ences of this order between the values of the redox poten-
tial of proteins measured using cyclic voltammetry and
potentiometry have been noted previously [35].
Acknowledgements
This work was supported by the EU Biotechnology
Programme, ‘Towards Designer Peroxidases’ BIO4-
CT97-2031 (to G.S. and A.T.S.), Italian CNR and ex
60% (to G.S) and the BBSRC under B17590 to A.T.S.
The authors acknowledge the COST action D21 ‘Met-
allo Enzymes and Chemical Biomimetics’ for support-
ing the exchange among the different laboratories. The
authors are grateful to Prof. R. Santucci for carrying
out the redox potential measurements.
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