(Received for publication, June 1, 1995; and in revised form, August 10, 1995)
From the
Mössbauer and electron paramagnetic
resonance (EPR) spectroscopies were used to characterize the diheme
cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two
distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for
the two heme groups. The Paracoccus peroxidase can be
non-physiologically reduced by ascorbate. Mössbauer
investigation of the ascorbate-reduced peroxidase shows that only one
heme (the high potential heme) is reduced and that the reduced heme is
diamagnetic (S = 0). The other heme (the low potential
heme) remains oxidized, indicating that the enzyme is in a mixed
valence, half-reduced state. The EPR spectrum of the half-reduced
peroxidase, however, shows two low spin ferric species with g = 2.89 (species I) and g
= 2.78 (species II). This EPR
observation, together with the Mössbauer result,
suggests that both species are arising from the low potential heme.
More interestingly, the spectroscopic properties of these two species
are distinct from that of the low potential heme in the oxidized
enzyme, providing evidence for heme-heme interaction induced by the
reduction of the high potential heme. Addition of calcium ions to the
half-reduced enzyme converts species II to species I. Since calcium has
been found to promote peroxidase activity, species I may represent the
active form of the peroxidatic heme.
A cytochrome c peroxidase has recently been isolated
from Paracoccus denitrificans(1) , and initial
characterization has been performed using UV-visible, nuclear magnetic
resonance (NMR) and electron paramagnetic resonance (EPR)
spectroscopies(2, 3, 4) . The Paracoccus peroxidase is a periplasmic protein with a molecular mass of 40
kDa(2) . The physiological electron donor is most probably the
soluble cytochrome c from the same
organism(5) . In many respects, this enzyme appears to be
closely related to the peroxidase isolated from Pseudomonas
aeruginosa, which has been extensively studied both
spectroscopically and
kinetically(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
Similar to the Pseudomonas enzyme(11) , the Paracoccus peroxidase contains two c-type hemes with
well separated midpoint redox potentials(2) . For the high
potential heme of the P. denitrificans enzyme, the redox
potential was determined to be +176 mV. For the low potential
heme, the redox potential was estimated to be within the range of
-200 to -100 mV(2) . Based on the spectroscopic and
biochemical results(2) , together with analogies drawn from the
studies of P. aeruginosa peroxidase, it is concluded that the
low potential heme is the peroxidatic center and the high potential
heme functions as an electron transfer site.
Spectroscopic
investigations of the Paracoccus peroxidase have revealed a
complex activation mechanism that involves changes in the redox and
spin state of the heme groups(2, 3, 4) . As
purified, the peroxidase is in the oxidized form where both hemes are
in the ferric state. At room temperature, the high potential heme is at
least partly high spin (S = 5/2) and the low potential
heme is low spin (S = 1/2). At low temperatures, both
hemes are low spin exhibiting typical low spin ferric EPR
signals(2) . A NMR peak at 90 ppm, assignable to the
-CH
group of an axial methionine ligand, was observed
and suggested to be associated with the high potential heme, indicating
that the high potential heme has a methionine-histidine axial
coordination. The coordination of the low potential heme is not yet
confirmed but has been proposed to be either bis-histidine or
histidine-lysine by analogy to the Pseudomonas peroxidase. The
oxidized form of the enzyme is catalytically inactive (17) and
does not bind exogenous ligands such as cyanide. Addition of ascorbate
reduces the high potential heme to the ferrous state, and a
half-reduced mixed valence form of the enzyme is attained. The reduced
high potential heme is low spin (S = 0)(4) .
This half-reduced form of the enzyme is catalytically active for the
reduction of hydrogen peroxide (17) and readily binds exogenous
ligands such as cyanide(2) . Addition of sodium dithionite
generates a fully reduced all ferrous form of the enzyme, which is a
catalytically irrelevant species. Similar characteristics have also
been found for other diheme bacterial peroxidases such as the
peroxidases from P.
aeruginosa(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) and Pseudomonas stutzeri(18) . In addition the Paracoccus enzyme was found to bind calcium
ions(2, 3, 17) . Other peroxidases, such as
lignin(19) , manganese(20) , and horseradish
peroxidases(21, 22, 23) , have also been
found to contain bound calcium ions. Binding of calcium to the Paracoccus peroxidase promotes its activation (17) and
appears to induce conformational changes surrounding the immediate
environment of the low potential heme(2, 3) . In the
presence of calcium, the low potential heme of the half-reduced enzyme
becomes high spin at room temperature, suggesting a more open
configuration for the heme site. The low potential heme is therefore
ready for substrate binding. The binding of calcium also increases the
midpoint redox potential of the high potential heme by approximately 50
mV, which should facilitate its reduction(2) .
In this paper, a Mössbauer study of the P. denitrificans cytochrome c peroxidase is described. Both the oxidized and ascorbate-reduced forms of the enzyme were examined. Effects of the binding of calcium to the heme sites were investigated by both Mössbauer and EPR techniques. Intricate conformational changes induced by calcium binding to promote enzymatic activation are also suggested by this study.
Cells of P. denitrificans (L.M.D. 52.44) were grown
under low oxygen tension, and the periplasmic cytochrome c peroxidase was purified as described previously(1) . The
enzyme is isolated in the oxidized state. The growth medium was either
enriched with the Mössbauer isotope Fe
(95% plus enrichment) or contained natural abundance
Fe
(2.2%). The protein concentration of the purified enzyme solutions was
determined by using
= 250 mM
cm
at 409 nm (oxidized enzyme)(1) .
The half-reduced form of the enzyme was achieved by anaerobic addition
of a concentrated solution of sodium ascorbate. The enzyme was
incubated under argon atmosphere for 60 min prior to calcium addition.
Mössbauer spectra of samples with a protein
concentration of 1 mM in 10 mM Hepes (pH 6.4) and a
volume of 350-400 µl were recorded, either on a weak field
Mössbauer spectrometer equipped with a Janis 8DT
variable temperature cryostat or on a strong field
Mössbauer spectrometer equipped with a Janis 12
CNDT/SC SuperVaritemp cryostat containing an 8-tesla superconducting
magnet. Standard transmission Mössbauer
measurements were made with a 50-mCi Co(Rd) source driven
by a Doppler velocity transducer operating in the constant acceleration
mode. The velocity scale was calibrated using room temperature spectra
of a metallic iron foil, and the zero velocity of the
Mössbauer spectra refers to the centroid of these
spectra. EPR measurements were performed on a Bruker ER 200-SRC
spectrometer with an Oxford Instrument ESR-9 continuous flow cryostat.
For the EPR samples, the protein concentration was about 200 µM in 10 mM Hepes buffer (pH 6.4), and the volume was
200-250 µl.
Fig. 1A shows the EPR spectrum of the
oxidized cytochrome c peroxidase from P. denitrificans recorded at 8 K. This spectrum is presented for the reason of
clarity and for the purpose of comparison with those of the
half-reduced state but is similar to the earlier published spectrum (2) in that two sets of resonances corresponding to the two c-type hemes were detected. The resonances at g = 3.00, g
= 2.27, and g
= 1.44 (not
shown) were assigned to the low potential heme and the signal at g
= 3.41 to the high potential
heme(2) . As with other low spin ferric hemes with a large g
, the other two resonances for the high
potential heme are not detected. It was assumed that g
was
2.0, and the corresponding g
was
estimated to be
0.6 according to the equation, g
2 + g
2 + g
2 = 16(24) . The weak signal
observed at g = 6 represents a minor high spin ferric
heme component, possibly associated with the high potential heme, which
is in a high spin-low spin equilibrium at room temperature(7) .
Figure 1:
EPR spectra of
cytochrome c peroxidase from P. denitrificans. A, oxidized enzyme; B, half-reduced enzyme (0.4
mM ascorbate); C, half-reduced enzyme in the presence
of 20 mM Ca. Experimental conditions:
temperature, 8 K; microwave frequency, 9.43 GHz; microwave power, 2
milliwatts; modulation amplitude, 1 mT; receiver gain, 8
10
.
Incubation of the oxidized sample with ascorbate for 60 min under an
argon atmosphere yielded a half-reduced peroxidase, which exhibits the
EPR spectrum shown in Fig. 1B. ()In this
half-reduced enzyme, the high potential heme is reduced, as expected
from its redox behavior and as supported by the absence of the g
= 3.41 signal. Most interestingly, the
low potential heme in this half-reduced enzyme displays two sets of EPR
signals corresponding to two different species, both of which are
distinct from that of the low potential heme in the oxidized enzyme.
The observed g values are 2.89, 2.32, and 1.51 for species I
and 2.78, 2.40, and 1.58 for species II. Also, these signals are sharp
in comparison with that of the low potential heme in the oxidized
enzyme. These observations indicate a subtle heme-heme interaction in
which the reduction of the high potential heme induces a structural
modification at and around the low potential heme. The relative
proportion of species I and II was found to be dependent on the enzyme
preparation and is probably due to the different amounts of residual
calcium bound to the purified enzyme (see below). For the sample shown
in Fig. 1B, the relative concentration of species I to
species II was estimated to be approximately 3 to 2 from the relative
intensity of the peaks at g
= 2.89 and
2.78, using the method of Aasa and
Vänngård(25) . Addition of calcium
ions to this half-reduced state converts species II (g
= 2.78) into species I (g
=
2.89), resulting in an EPR spectrum showing a majority of species I (Fig. 1C). Consequently, both the reduction of the high
potential heme and the binding of calcium ions to the protein have
effects on the low potential heme. These effects could include protein
conformation changes or changes in the coordination structure such as
an increase in histidinate character of the proximal ligand, which is
observed in some eukaryotic peroxidases(19, 23) . It
is interesting to note that, for peroxidases isolated from P.
aeruginosa and P. stutzeri, the half-reduced enzymes
display EPR spectra showing only one low spin ferric heme species.
Either these proteins do not require Ca
or they bind
Ca
so strongly that it is not lost during
purification.
To further characterize the heme groups in the Paracoccus peroxidase, Mössbauer
measurements were performed. We will first present the data recorded at
200 K, where the electronic relaxation time is fast in comparison with
the nuclear precession, resulting in the observation of quadrupole
doublets for the heme irons regardless of their oxidation states. Under
such conditions, data analysis is simpler. Fig. 2shows the 200
K spectra of the Paracoccus peroxidase in its oxidized
(spectrum A), ascorbate-reduced (spectrum B), and
ascorbate-reduced-plus-calcium (spectrum C) states. The
oxidized peroxidase shows two partially resolved quadrupole doublets (Fig. 2A) corresponding to the two inequivalent c-type hemes. The data were least-squares fitted assuming two
doublets with equal intensity and line width. For the half-reduced
samples, the relative proportion of the two species of the low
potential heme determined from the EPR data was used for the analysis
of the Mössbauer data (Fig. 2, B and C). The results of the analysis are plotted as solidlines in Fig. 2, and the parameters
obtained are listed in Table 1. The good agreement between the
data and the fits indicates that the Mössbauer data
are consistent with the EPR findings. Also, this analysis allows
unambiguous assignments of the parameters to the two different hemes.
For the high potential heme, the parameters (E
= 2.01 mm/s and
= 0.24 mm/s for the oxidized S = 1/2 state and
E
=
1.27 mm/s and
= 0.42 mm/s for the reduced S = 0 state) are common for low spin heme compounds and are
consistent with a methionine-histidine
coordination(26, 27, 28, 29, 30, 31, 32) (see also Table 2). The quadrupole splittings for
the low potential heme (2.45 mm/s in the oxidized enzyme and 2.46 mm/s
in the calcium-bound enzyme), however, are atypically large (see Table 2). The origin and significance of these unusually large
values are currently unclear.
Figure 2:
High temperature
Mössbauer spectra of cytochrome c peroxidase from P. denitrificans. A, oxidized
enzyme; B, half-reduced enzyme (6 mM ascorbate); C, half-reduced enzyme in the presence of 17 mM Ca. The data were recorded at 200 K in the
absence of an applied field. The solidlines are
least-squares fits of the experimental spectra (see text). The
quadrupole doublets originating from the high potential (HP)
and low potential (LP) hemes are indicated by brackets.
At 4.2 K, the electronic relaxation is
slow and the oxidized peroxidase exhibits Mössbauer
spectrum with magnetic hyperfine structures as expected for low spin
ferric heme compounds. Fig. 3shows the
Mössbauer spectra of the oxidized P.
denitrificans peroxidase recorded with the presence of a 50-mT ()field applied parallel (spectrum A) and
perpendicular (spectrum B) to the
-beam and with an 8-T
parallel field (spectrum C). Similar to the high temperature
data, these low temperature spectra can also be analyzed as a
superposition of two components of equal intensity corresponding to the
two low spin ferric hemes. The electronic g values have been
determined from the EPR measurements, and the parameters,
E
and
, at 4.2 K can be extrapolated
from the high temperature spectra. The only unknown parameters for the
analysis of the low temperature data are the magnetic hyperfine
coupling tensor A, which can be estimated from a ligand field theory
commonly applied to low spin ferric heme complexes(33) . In
this theory the t
orbitals are assumed to be well
isolated from the e
orbitals and are split by the
tetragonal and rhombic crystal fields,
and V,
respectively. The spin-orbit interaction mixes the t
orbitals, resulting in three Krammers doublets. The observed g values and the low temperature Mössbauer
spectra are originating from the ground doublet. If the g values of a low spin ferric compound are known, this theory can be
used to estimate the corresponding A tensor (34) and
the crystal field parameters
and V(33, 35, 36) in the units of the
spin-orbit coupling constant
. Since the g values of the
two hemes in the oxidized peroxidase are known from the EPR
measurements, this theory was used to obtain initial guesses for the A values in the analysis of the low temperature
Mössbauer spectra. These A values were
then allowed to vary until a reasonable match between the theoretical
simulations and experimental data was obtained. Results of such an
analysis are shown as solidlines in Fig. 3,
and the parameters obtained are listed in Table 3along with the
theoretical values. The good agreement between the simulated and the
experimental spectra indicates that the data support the presence of
two distinct low spin ferric hemes in the oxidized peroxidase. The less
than desirable fit at the outer absorption region is due to a
distribution of the crystal field strength, which is not included in
the analysis. Distributions of
and V have generally been
used to explain the EPR and Mössbauer line shapes
of low spin ferric complexes(37) .
Figure 3:
Low temperature
Mössbauer spectra of oxidized cytochrome c peroxidase from P. denitrificans. The data were recorded
at 4.2 K with a 50-mT magnetic field applied parallel (A) and
perpendicular (B) to the -beam and an 8-T magnetic field
applied parallel to the
-beam (C). The solidlines plotted over the experimental spectra are
theoretical simulations using the parameters listed in Table 3.
The simulations of each spectral component in the presence of a 50-mT
parallel field are also shown on top of spectrum A (solidline, high potential heme; dashedline,
low potential heme).
Since the crystal field
parameters and V can be readily determined from the EPR g values and since these parameters are expected to be
affected by the ligands, EPR spectroscopy has been used for axial
ligand assignment for proteins containing low spin ferric protoheme IX.
It was first demonstrated by Blumberg and Peisach (35) that
proteins containing protoheme IX with the same axial ligands tend to
have
and V values that are clustered in a plot of the
rhombic (V/
) versus the tetragonal (
/
)
fields. This method of axial ligand assignment, however, was later
found to contain ambiguities, particularly in the case of bis-histidine
heme proteins(38, 39) . Another method that has been
proposed for heme axial ligand identification uses magnetic circular
dichroism spectroscopy to measure the near infrared
to d charge transfer bands, the frequencies of which are sensitive to
the axial ligation(38) . In a study of 34 low spin ferric heme
proteins, Gadsby and Thomson (38) established a linear
correlation between the charge transfer transition energy and the
electronic hole energy of the t
orbital (E
=
/3 + V/2).
Consequently, E
can also be used for axial ligand
assignment. Of the 34 heme proteins investigated, the Met/His ligation
shows lower E
(1.5
- 1.9
) than
that of the His/His ligation (2.0
- 2.3
). In Table 3, the experimentally determined and the theoretically
estimated A values agree very well, suggesting that the theory
used in our analysis describes the electronic properties of the hemes
in the oxidized P. denitrificans peroxidase quite well. Using
the theoretical values of
and V, the energy E
for the high potential and the low potential
heme was found to be 1.15
and 1.91
, respectively. Earlier NMR
measurements (2, 3) have established methionine
ligation to the high potential heme, and the value 1.15
is
therefore the smallest E
ever reported for a
Met/His ligation. Using the formula derived by Gadsby and Thomson (38) , 1.15
for E
corresponds to a
charge transfer energy in the order of 5300 cm
. It
would be interesting to perform magnetic circular dichroism
measurements to investigate whether the correlation between the E
and the charge transfer energy holds for the
high potential heme. The value 1.91
obtained for the low potential
heme falls into the range of Met/His axial ligation and is slightly
outside of the range reported for bis-histidine heme proteins. It is,
however, important to point out that axial ligand assignments using EPR
and Mössbauer spectroscopy contain large
uncertainties and that methionine ligation to the low potential heme is
not supported by NMR measurements(3, 4) .
On the
basis of their amino acid sequence work on the P.
aeruginosa enzyme, Ellfolk et al. (40) proposed that a C-terminal domain contained a heme
coordinated by a histidine and Met-254 (289 in P. denitrificans cytochrome c peroxidase numbering) and an N-terminal
domain contained a heme coordinated by a histidine and either His-240
(His-275 in P. denitrificans cytochrome c peroxidase
numbering) or a lysine. Extension of sequence analysis to the enzyme
from P. denitrificans, the open reading frame f465 on the Escherichia coli chromosome and the Mau G proteins ()revealed that Met-289 and His-275 were indeed conserved
but also Met-129 was a conserved feature of the N-terminal domain.
Although NMR evidence seems to preclude both the C- and N-terminal
domains having methionine coordination, there is clearly potential for
this in the sequence, and the presence of His-275 as a possible ligand
leads to a number of permutations of coordination possibilities in the
two redox states. Some of these possibilities are indicated in Fig. 4.
Figure 4: Proposed models of the two domains of P. denitrificans cytochrome c peroxidase. The cytochrome c peroxidase is represented as a two-domain structure. In model A, the heme domain with His (H)-Met (M) coordination of the heme accepts the incoming electron and can therefore be considered high potential. Entry of the electron results in dissociation of His-275 (H*) from the heme of the second domain and allows access by hydrogen peroxide. HP, high potential; LP, low potential. In model B, both hemes are coordinated by methionine in the oxidized state. After reduction of one heme, the methionine of the second heme dissociates from the iron and allows access for hydrogen peroxide. In this model, His-275 (H*) is not a heme ligand but still plays a role as a distal catalytic residue. In model C, the entry of an electron to center 1 causes a change in the properties of both hemes. Center 1 switches from high to low potential and reduces center 2, which becomes coordinated by a methionine. Center 1 itself becomes the peroxidatic center.
Fig. 5shows the Mössbauer
spectra of the half-reduced peroxidase in the absence (spectrum A) and presence (spectra B and C) of
calcium. The data were recorded at 4.2 K with a 50-mT (spectra A and B) and a 8-T magnetic field (spectrum C)
applied parallel to the -beam. These spectra clearly demonstrate
that in the ascorbate-reduced peroxidase, one heme is reduced to a low
spin ferrous (S = 0) state and the other remains
oxidized. The reduced heme (presumably, the high potential heme)
displays a quadrupole doublet with parameters (
E
= 1.23 ± 0.03 mm/s and
= 0.46 ±
0.02 mm/s) typical of low spin ferrous hemes. Addition of calcium has
no effect on this doublet. The oxidized heme (presumably, the low
potential heme) exhibits a hyperfine split spectrum, which is
distinguishable from that of the reduced heme. To analyze the spectral
component of the oxidized heme, the contribution of the reduced heme
was first removed, and the remaining spectral component was analyzed
using the method described above. We began by analyzing the spectrum of
the sample with calcium present, since it contains mostly species I (g
= 2.89). The parameters obtained for
species I were then used in the analysis of the spectra of the sample
without added calcium where both species I and II are present. The
spectral data for species I and II are not sufficiently resolved to
allow reliable determination of the hyperfine parameters for species
II. The theoretical A values for species II and the
concentration ratio of species I to species II obtained from the EPR
measurements were used in the analysis. The results are listed in Table 4. The solidlines plotted in Fig. 5are simulations using a 50/50 oxidized/reduced heme ratio.
Good agreement is observed between the experiments and the simulations,
indicating that only one heme is reduced in these half-reduced protein
samples with or without added calcium.
Figure 5:
Mössbauer spectra of
half-reduced Paracoccus cytochrome c peroxidase in
the absence (A) and presence (B and C) of
added calcium. The data were recorded at 4.2 K with a 50-mT (A and B) and an 8-T (C) magnetic field applied
parallel to the -beam. The solidlines are
theoretical simulations using the parameters listed in Table 3.
In these simulations, the contributions from the oxidized and the
reduced hemes are assumed to be equal. The relative proportions of
species I and II for the oxidized low potential heme were taken from
the EPR results. Diamagnetism was assumed for the reduced high
potential heme.
From these
Mössbauer and EPR observations, the following
conclusions can be drawn. The addition of ascorbate to the peroxidase
reduces the high potential heme only. Reduction of the high potential
heme affects the conformation surrounding the low potential heme,
resulting in two low spin ferric states (species I and II) that are
distinguishable from that in the oxidized form. The addition of
Ca converts species II into species I. Since calcium
has been found to promote the peroxidase activity, the low spin species
I probably represents the peroxidatic heme in its active form.
In summary, Mössbauer and EPR spectroscopy have been used to characterize the two heme groups of P. denitrificans cytochrome c peroxidase in its oxidized and half-reduced forms. It was found that the spectroscopic properties of the peroxidatic heme can be affected by the reduction of the high potential heme and by the binding of calcium to the peroxidase, providing further evidence for the long range heme-heme interactions observed in the bacterial cytochrome c peroxidases.