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A study of the horseradish peroxidase catalytic
site by FTIR spectroscopy
W.J. Ingledew*1 and P.R. Rich†
*School of Biology, University of St. Andrews, St. Andrews KY16 9ST, U.K., and †Glynn Laboratory of Bioenergetics, Department of Biology,
University College London, Gower Street, London WC1E 6BT, U.K.
Abstract
Vibrational changes in the catalytic site of horseradish peroxidase were investigated by FTIR (Fouriertransform infrared) spectroscopy in the 1000–2500 cm−1 range. Difference spectra were generated by
photolysis of the haemII-CO compound at different pH/pD values. The spectra report on the fine structure
around the catalytic site and show vibrational changes of protein backbone, amino acid residues and
cofactors. Assignments of the FTIR vibrations can be made based upon known crystal structures, comparisons
with absorption frequencies and extinction coefficients of model amino acids and cofactors, effects of H2 O/
2
H2 O exchange and changes of pH/pD. Concomitant with the photolysis of the CO ligand are changes
due to haem and protein vibrations, predominant among which are arginine and histidine residue
vibrations.
HRPC (horseradish peroxidase C) is an enzyme that catalyses
the oxidation of a range of organic compounds by hydrogen
peroxide and is a model system for studies on redox catalysis.
The crystal structure of the enzyme, with various ligands and
in different stages of its catalytic cycle, has been reported
[1–4]. The active site contains haem (Fe-protoporphyrin IX)
proximally ligated to a histidine (His170 ) residue, with the
substrate-binding site close to the distal face of the haem
and comprising histidine (His42 ), arginine (Arg38 ) and phenylalanine (Phe41 ) residues. The histidine residue has a pK a of 7.5,
which is shifted to 8.8 when CO is bound to the reduced enzyme [5]. A comparison of the reduced and the reduced plus
CO structures at high and low pH is informative for assignment of vibrations in the CO photolysis difference spectra.
The crystal structure of the reduced, CO ligated enzyme has
been published recently [4], and we have also modelled this
structure. In the low-pH form in the distal pocket, the CO
oxygen has possible H-bonding partners of the His42 and
the εNH of the Arg38 guanidinium in a dynamic network in
which a putative water molecule offers alternate H-bonding
for His42 (see [3] and model 5c in [6]). At high pH, the
His42 does not hydrogen bond with the CO and the principal
partner is the εNH of the Arg38 guanidinium.
Materials and methods
Preparation of HRPC for spectroscopy
HRPC (Armoracia rusticana), type VI-A (P-6782) (molecular mass 33 722 Da) was purchased from Sigma. The buffers
Key words: FTIR spectroscopy, haemII-CO compound, horseradish peroxidase, photolysis.
Abbreviations used: FTIR, Fourier-transform infrared; HRPC, horseradish peroxidase C.
1
To whom correspondence should be addressed (email [email protected]).
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used were either 100 mM CHES, 100 mM KCl and 0.05 mM
CaCl2 (pH/pD 9.5) or 100 mM Mes, 100 mM KCl and
0.05 mM CaCl2 (pH/pD 6.0). Approximately 6 mg of crystalline HRPC was dissolved in 500 µl of appropriate buffer
and concentrated to approx. 50 µl using centrifugal concentrators (Vivascience, Lincoln, U.K.). The sample was rediluted to 500 µl and the process repeated three times. The
final concentration of the enzyme in IR samples was approx. 3.5 mM HRPC. For the 2 H2 O samples, the 2 H2 O buffer
was prepared by freeze-drying the equivalent H2 O buffer, dissolving the resulting salts in 2 H2 O, refreeze-drying and redissolving the salts in 2 H2 O for a second time.
The pD was measured using a pH electrode and applying the formula pD = pH reading + 0.4 [7]. Samples were
placed on a CaF2 window, reduced with appropriately
buffered sodium dithionite and saturated with CO, before
placing a second window over the sample and sealing with
vacuum grease. FTIR (Fourier-transform infrared) photolysis difference spectra were obtained as described previously [8,9].
Molecular modelling
The PDB (Protein Data Bank) file of the crystal structure
of the CO compound of HRPC is not yet available although
the structure has been recently described [4]. Hence, for the
purpose of comparison and discussion, it was modelled using
the package ‘Whatif’ developed by Vriend and co-workers
[10]. The starting templates were PDB files H58 and H57.
A comprehensive discussion of the modelling process for
H-bonding within possible CO compound conformers has
been published by Dalasto et al. [6]. The active site of the
modelled structure closely resembles the recently published
structure [4].
Mechanisms of Bioenergetic Membrane Proteins
Figure 1 Light-induced FTIR difference spectra of HRPC due to CO
Figure 2 The pH (pD) induced differences in the light-induced CO
photolysis
Light minus dark photolysis difference spectra at pH 6.0 (A), pD 6.0 (B),
photolysis FTIR difference spectra of HRPC
Isotopic difference spectrum at pH/pD 6.0 (pH 6.0 photolysis difference
pH 9.5 (C) and pD 9.5 (D). Samples of the CO adduct of fully reduced
HRPC were prepared as described in the Materials and methods section.
After sufficient time for equilibration and settling at 288 K, repetitive
spectrum minus pD 6.0 photolysis difference spectrum) (A) and pH/pD
9.5 (pH 9.5 photolysis difference spectrum minus pD 9.5 photolysis difference spectrum) (B). pH difference spectrum (pH 6.0 photolysis difference
light/dark cycles were recorded and averaged. Photolysis was achieved
with light from a 250 W lamp, filtered through BG39, heat and water
filters and delivered to the sample by fibre optics. The CO photolysis
spectrum minus pH 9.5 photolysis difference spectrum) (C) and pD
difference spectrum (pD 6.0 photolysis difference spectrum minus
pD 9.5 photolysis difference spectrum) (D). (E) Spectrum D–spectrum
spectra shown are averages of 2000 individual photolysis spectra (each
consisting of 100 averaged interferograms at 4 cm−1 ). Each spectrum is
a light minus dark difference spectrum from which 50% of a dark minus
C (this is also the same as spectrum B–spectrum A).
dark control has been subtracted to allow for any non-photochemical
drift (cf. [8]).
Results and discussion
CO photolysis difference spectra, the haemII-CO
stretch and the protein and cofactor vibrations
at pH/pD 6.0 and 9.5
Figure 1 shows the CO photolysis difference spectrum of the
haemII-CO stretch of HRPC at pH (pD) 6.0 and 9.5.
The most prominent features are the troughs of the ν CO
stretch of the haemII-CO at approx. 1934 and 1905 cm−1 .
These minima are listed in Table 1 with approximate percent-
age area of the total under each component. Both, polarization
of the CO molecule at the haem and bending of the haem-CO
by steric interactions influence the position of the absorption.
H-bonding of the CO to His42 and Arg38 would result in a
lowering of the stretching frequency compared with a single
H-bond. Thus the simplest explanation of the HPRC ν CO
spectra is that at pH 6.5, the 1905 cm−1 species is H-bonded
to both the εNH of Arg38 and the NH of His42 , and the
1934 cm−1 species is H-bonded only to the εNH of Arg38 .
Deprotonation of His42 at more alkaline pH will preclude its
H-bonding with CO, so in the pH 9.5 spectra the CO can
only H-bond with the εNH of Arg38 , thus the predominance
of the 1933 cm−1 form at this pH (the ferrous enzyme
exhibits a pK a of ∼ 7.5 that is shifted to 8.8 on CO binding
[5,11,12]).
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Table 1 Frequencies of significant features of the four difference spectra in Figure 1
pH 6.0
Peak
pD 6.0
Trough
2137
Peak
Trough
2137
1934 (37%)
Peak
pD 9.5
Trough
2137
1932 (40%)
1905 (62%)
1693
1684
pH 9.5
1694
1679
1903 (59%)
1688
1659
1651
1641
1675
Peak
Trough
Tentative assignments
1931 (85%)
CO photolysis state
Haem FeII-CO
2137
1933 (73%)
1905 (27%)
1708
1708
1679
1692
1676
1651
1619
1636
1558
1538
1903 (14%)
1702
1664
Haem FeII-CO
Haem propionate
ν as guanidinium
1663
1651
1617
1620
1548
1641
1558
1539
1524
1404
1331
1220
1147
1117
1098
1061
1041
1597
1548
1608
1645
1558
1538
1524
1440
1524
1460
1339
1245
1404
1331
1220
1339
1245
1404
1331
1220
1090
1147
1117
1098
1090
1147
1117
1098
1011
1011
ν as guanidinium
ν s guanidinium
1573
1558
1538
1524
Haem
Haem
1459
1334
1245
1404
1331
1220
1090
1147
1117
1098
1340
1245
Haem
Haem
Haem/histidine
1090
Haem
Histidine
Histidine
1048
1011
Also apparent in Figure 1 are vibrational changes due to
perturbation of protein (backbone and residues) and haem
due to photolysis of the haemII-CO compound. The major
peaks and troughs are marked on the spectra and tabulated
in Table 1. Possible assignments of some of these features are
discussed below.
FTIR haemII-CO photolysis spectra of HRPC at
pH 6.0 (pD 6.0) and pH 9.5 (pD 9.5)
Figure 2 shows the isotopic (pH/pD) and pH and pD
(6.0–9.5) double difference spectra (differences between the
photolysis difference spectra shown in Figure 1). In the pH/
pD difference spectra, only those vibrations of the photolysis
difference spectrum that are pH/pD-sensitive will appear
and in the pH double difference spectra only those that are
pH-sensitive will appear. Figure 2(A) shows the difference
between photolysis difference spectra at pH 6.0 and pD 6.0;
it has fewer principle features than the parent spectra (Figures 1A and 1B) and is dominated by arginine guanidinium
and histidine (His170 , His around 1229 cm−1 ; and His42 ,
His-H+ possibly at 1060 cm−1 although this vibration is
normally expected at 1090 cm−1 . Figure 2(B) shows the difference between the pH 9.5 and pD 9.5 photolysis difference
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Histidine
spectra. It is dominated by arginine guanidinium vibrations,
whereas putative His-H+ vibrations at 1060 cm−1 are much
reduced. Figure 2(C) shows the difference between the
pH 6.0 and pH 9.5 photolysis difference spectra, it is
again dominated by arginine guanidinium (showing that
deprotonation of His42 profoundly affects the vibrational
modes of Arg38 ) and histidine vibrations. The sharp feature
at 1548/1539 cm−1 (1549/1533 cm−1 in Figure 2D) is due to
protonation/deprotonation but the species cannot be readily
identified. Figure 2(D) shows the difference between the pD
6.0 and pD 9.5 photolysis difference spectra, it is dominated
by arginine guanidinium vibrations. The difference between
Figures 2(C) and 2(D) is shown in Figure 2(E) (which is also
the same as Figure 2B minus Figure 2A). This spectrum is
interesting in terms of what is lost in the subtraction, such as
the vibrations at 1651/1642 (1652/1643) cm−1 and 1549/1539
(1549/1533) cm−1 , which show elements that are pH/pDsensitive but not (or only weakly) pH/pD exchange-sensitive.
The catalytic pocket
The haemIX
Changes in vibrations associated with the haem group upon
photolysis of its FeII-CO adduct are expected. The most
Mechanisms of Bioenergetic Membrane Proteins
easily resolved of these are expected to be the propionic acid
vibrations that occur around 1700 cm−1 . These are noted
in Table 1. Based on other haem data, it is very likely
that pH/pD-insensitive bands at 1548/1524/1404/1331,1245–
1220/1147 are due to haem ring modes [13,14].
appear to contribute significantly to the photolysis difference
spectra. The perturbation of these vibrations is predictable
from a comparison of the structures of the reduced and
reduced plus CO forms of HRP.
Phe41
We are grateful to M. Iwaki (Department of Biology, University
College London, London, U.K.) for access to unpublished data on
the FTIR of model compounds.
Phenylalanine residues have a moderately intense (pH/pDinsensitive) ring band at 1494 cm−1 (molar absorption
coefficient (ε) = 80 M−1 · cm−1 ) [15,16]. There is no indication
of a vibration in the difference spectra, so it appears that Phe41
is relatively unperturbed by photolysis of the CO compound.
Arg
38
Arginine residues have strong vibrations in the guanidinium group, which is shown in the difference spectra.
The guanidinium ν as (CN3 H5 + ) absorbs around 1672–
1673 cm−1 (1608 cm−1 in 2 H2 O), and the ε value is strong
(∼460 M−1 · cm−1 in both media). The guanidinium ν s
(CN3 H5 + ) is expected around 1635 cm−1 (1586 cm−1 in
2
H2 O) [15,17]. Vibrations in the difference spectra can be
assigned to this group and are indicated in Table 1.
His42 and His170
Histidine residues have different ionizations (denoted as
His− , His and His-H+ ) and several vibrational modes, most
of these are only moderately strong, but several occur in
the relatively uncluttered region of the spectrum below
1300 cm−1 [15,16], where they can be diagnostic.
At pH 6.5, the fully protonated (His-H+ ) form of His42
will predominate but at pH 9.5 it will be the neutral (His)
form that predominates, with vibrations around 1117, 1098
and 1011 cm−1 . His170 , which is the proximal haem ligand, is
expected to be in the neutral form at both pH values.
In conclusion, the vibrational difference spectra of HRPC
are dominated by vibrational changes attributable to arginine
(Arg38 ), histidine (His42 and His170 ) and the haem (ν CO ,
propionate and the haem macrocycle). Phe41 does not
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Received 29 April 2005
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