BIOCHEMICAL
Vol. 79, No. 3, 1977
PROBING AXIAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LIGANDS IN FERRIC HAEMOPROTEINS:
AN ESR STUDY OF MYOGLOBIN AND HORSERADISH PEROXIDASE IN Hzl’O S. Vuk-PavloviE*, Departments
of Chemical
The Weizmann
Received
September
Y. Siderer Immunology
Institute
and Biochemistry
of Science,
Rehovot,
Israel
22,1977 SUMMARY
Substitution of Hz160 by Hzl’ 0 induces a substantial broadening of the high-field line in the electron-spin resonance spectrum of ferric myoglobin due to the presence of Hzl’O at the axial ligand-site. Computer simulations of the experimental spectra yielded the values of the reciprocal relaxation time Tz-l= 7.8 G and the “0-hyperfine coupling constant A = 18 f 1 G. Under identical experimental conditions no effect of Hzl’O was observed in horseradish peroxidase. The latter finding excludes the possibility that a water molecule is liganded to the peroxidase haem-iron and supports either the idea that both axial ligands are amino acid residues or that the haem in ferric horseradish peroxidase is pentacoordinate. INTRODUCTION In order haemoproteins of axial
to explain
the specificities
take
it
ligands
sixth (i)
of various
exists ligand
for is
Peroxidases
axial
ligand
amino
acids
molecule
(2).
coordinated these
less
compounds
as the
certain, of the
but
(ii)
ferric
Distal
fifth
ion
ferric
by hydrogen
hypotheses type
in which for
the identity
ligand
(1).
The nature
proved
of the
forward:
i.e.
both
by atoms from neighbouring (4).
ferric (iii)
enzyme is Recently
enzyme has been proposed
has been decisively
in
much
have been put
of the acid
catalysts
peroxide,
haemoproteins,
are occupied
to an amino
haem in high-spin
hypotheses
evidence
haem-ligand
several
(sixth)
hydrogen-bounded
reactions
In the case of peroxidases,
aromatic
histidine
to obtain
are of the “close-crevice” sites
(3)
is necessary
to the haem-iron.
the oxidations evidence
part,
of various
by direct
(5).
observation
a water
also
penta-
Neither
of
of the sixth
ligand. The water molecule coordinated proteins may be observed by X-ray presence
by spectroscopic
been proposed technique * Permanent Yugoslavia.
Copyright All rights
recently
capable
at the sixth crystallography
methods, that
data
are generally
solvent-proton
to resolve
whether
magnetic
a water
address: Institute of Immunology, Correspondence should be forwarded
0 1977 by Academic of reproducrion in any
Press, Inc. form reserved.
ligand (6),
molecule
site of ferric but when probing less
decisive.
relaxation is
haemoits
might
coordinated
It
has
be the at the
Rockefellerova 2, 41000 to this address.
Zagreb,
885 1SSN
0006-291X
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
I 3350
I 3400
3450
3500
3400
MAGNETK
3450
35m
FIELD / G
parts (g=!) of ESR spectra of aquo complex of ferric Fig. 1. High-f:$d myoglobin in Hz 0 and 50%HP 0. a) Experimental spectra taken at X-band, microwave power 5 mW, modulation amplitude 2.5 G, temperature 10 K, protein concentration 0.5 mM. The amplitude of the spectrum in H2l'O has been adjusted to the Hz160-spectrum by increasing receiver gain. b) Corresponding simulated ESRspectra. Best-fit parameters are T2-l = 7.8 G and A = 18 f 1 G.
axial ligand-site
(7), but this view has been subsequently criticized
(8).
In order to probe spectroscopically the presence of the axially liganded water molecule we utilized the nuclear spin of oxygen-17 in Hzl'O. Similarly to fluoride in fluoro complexes (9), "0 has an effect especially in the high-field part of the electron-spin resonance spectrum of aquo complexes of ferric haemoproteins. This part of the spectrum is broadened by the superhyperfine interaction present at the s.xth
of "0 nuclear spin and ds iron-electrons when Hzl'O is coordination site. In this communication we present
results of our study of Hzl'O interaction in ferric peroxidase. EXPERIMENTAL
myoglobin and horseradish
Horse heart myoglobin (Sigma,Type III) and horseradish peroxidase (Sigma, Type VI, Lot No. 65C-9530-l) were used without further purification. Lyophilized proteins were dissolved in 100 ml4sodium phosphate, pH 6.0, to a final concentration of 0.5 mM. These solutions were subsequently freeze-dried and redissolved in an equal amount of either Hz160 or Hz0 50%enriched in I'0 (Heavy Oxygen Plant, Weizmann Institute of Science). The ESRspectra were taken by a Varian E-12 spectrometer at X-band (9.35 GHz) operating under nonsaturating Measurementswere performed below 10 K conditions of microwave irradiation. using a home-madeliquid helium flow system. RESULTS Since the axially liganded water in ferric myoglobin has been observed by X-ray diffraction (6), this protein was chosen to investigate the effect of the presence of Hzl'O at the axial ligand site. The powder spectrum of high-
886
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I I200
2200 MAGNETIC
3200 FIELD / G
Fig. 2. ESR spectrum of horseradish peroxidase same conditions as in Fig. 1. Insert: enlarged spectra in H2160 (lower curve) and 50% Hzl’O.
in H2160 recorded high-field parts
spin
anisotropic.
aquo complex
of ferric
myoglobin
is
arises from the difference in absorption the haem-plane (g=6) and perpendicular large
zero-field
non-zero
splitting
nuclear
as exemplified “F
(1=1/2)
ive
also
spins by the
(9).
Therefore,
aquo complex by water
part
spectrum
experimental choosing
in Hal60
swept
myoglobin
were
g-values
spectrum
recorded
possessing
absorption
around
the nuclear
of the
spectrum
derivative in Fig.
spin
g=2, of
to be sensit-
la.
through the
(g=2)
of the
Substitution
broadening
of amplitude
interpreted
and varying
Thus,
in Ha 160.
part
of ligands
with
a substantial
by a decrease
spectra
spectra,
this
is shown
170 produces
correspondence to experimental corrected transition probability, field
in the
interaction
of the ESR absorption
accompanied
appropriate
The effect
remarkable
we expect
50% in
powder
anisotropy
of Ha170.
of myoglobin
enriched
This
when the magnetic field is applied in it (g=2), and is due, in turn, to the (10).
especially
superhyperfine
to the presence
The high-field
of the
parameters is
highly
under the of the
(Fig.
of H2160
of this
region
These
la).
computer
line-width
ferric
simulation
parameter
by
until
full
spectra was achieved. The program includes according to Aasa and Wnng8rd (11)) for
a value
The simulated
in the presence
of Ta-1 high-field
= 7.8 G was obtained line
is
for
shown in Fig.
of Hz 170 was simulated
the
ferric lb.
The
by superposition
of
two different properly weighted spectra: (i) of the spectrum in H2160 described above, and (ii) of the spectrum in Hz170 with the same parameters as the previous (I = S/2).
one,
In the
but
split
case of
into
a sextet
170-broadened
due to the nuclear
spectrum
887
the best
spin
of
correspondence
170
BIOCHEMICAL
Vol. 79, No. 3, 1977
between
the
value
experimental
very
coupling
to mention
sensitive
observed
spectra
expanded-scale 50% in
“0
also
changes
of this
of the
2.
broadening
both
It
It
the is
high-field
line
is
deviations
from the
parameter.
in Hal60
spectra
in Fig.
using
lb).
simulated
in significant
peroxidase
part
any pronounced
of the
of A resulting
depicted
has been obtained
A = 18 2 1 G (Fig.
the width
of herseradish
high-field
spectra
constant
that
even upon minor
are
induce
here
to the value
The spectrum
not
and simulated
of the hyperfine
important
AND BIOPHYSICAL RESEARCH COMMJNlCATlONS
is
is
shown in Fig.
An
in H2160 and H20 enriched
readily
as in the
2.
apparent
that
case of myoglobin
“0
(Fig.
does la).
DISCUSSION Judging acquo
from
complex
sensitive
the profound
of ferric
method
containing
for
ligands
horseradish
is would
has been
considered
sequences
basis
of magnetic
However, ities. about ligand
can be ruled
peroxidases
there
(3).
experiments
out in
similar
(12,13).
in the present
properties
substrates
ways. the
is
effect
sixth-ligand A tyrosine
in the
phenolate
recently (1).
be consistent
also
iron,
proposed In the with
a
a possibility raised recently
on the
the pentacoordinated
discriminate
in
The first
between
the
haem.
two possibil-
favor
Using
of either
higher
study,
of the
of described
experiments
using one,
enrichments
the method
“0
have been of
"0
alternatives.
as a probe introduced
and even
may be sensitive
in the
We thank
Dr.
R. Poupko
temperatures
to monitor
at “O-nucleus. Such changes may result from effects on the haem due, for instance, to the medium,
the presence
of allosteric
for
his
generous
and friendly
effecters help
especially in the computer simulations of the reading and Mrs. E. Majerowich for typing the
gratefully organization.
of
only
lower
enough
study
and/or
and inhibitors.
of the work, Wherland for S.V.-P. Biology
(S),
of oxygen-
is no tyrosine
with the possibility,
data
cannot
but
would
contact The second
to the present
changes of the spin density direct or protein-mediated altered
our data
dichroism
resonance
metalloproteins, recently
(3,11),
et al
reactions
residue.
in
Obviously, more data are needed in order to reach a definite conclusion actual sixthligand of the iron, but a water molecule as the axial
Electron-spin
than
candidate
distal
acid
spectrum
to be a
of any similar
(2) where
as a likely
in the
and/or
The lack
structure
structure
of the
appears
in two different
by an amino
circular
the present
the
haemoproteins.
plant
region
of identity
be occupied
of five
by Ricard
parallel substitution
can be interpreted
crevice”
involved
discussed
investigation
in ferric
also
case of “closed carboxylate
in the isotopic
the “closed-crevice”
position distal
the
peroxidase
possibility
effect
myoglobin,
acknowledges
a long-term
888
fellowship
in all
spectra, Dr. manuscript.
by European
stages S.
Molecular
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2.
Welinder, K.G. and Mazza, R. (1977) Eur. J. Biochem., 73, 353-358.
3.
Ricard, J., 566-578.
4. 5.
Yamada, H., and Yamazaki, I. (1974) Arch. Biochem. Biophys., 165, 728-738. Kobayashi, N., Nozawa, T., and Hatano, M. (1977) Biochim. Biophys. Acta, 493, 340-351. Kendrew, J.C., Watson, H.C., Strandberg, B.E., Dickerson, R.E., Phillips, D.C., and Shore, V.C. (1961) Nature, 190, 666-670. Lanir, A., and Schejter, A. (1975) Biochem. Biophys. Res. Commun.,62, 199-203. Vuk-Pavlovi:, S., and Benko, B. (1975) Biochem. Biophys. Res. Commun.,66, 1154-1159. Kotani, M., and Morimoto, H. (1967) in "Magnetic Resonancein Biological Systems", (Ehrenberg, A., MalmostrBm, B.G., and V!lnng&d, T., eds.) pp. 135-140, PergamonPress, Oxford.
6. 7. 8. 9.
George, P., and Lyster, 1013-1029.
R.L.J.
(1958) Proc. Nat. Acad. Sci. U.S.A.,
Mazza, G., and Williams, R.J.P.
44,
(1972) Eur. J. Biochem., 28,
10.
Bennett, J.E., Gibson, F.J., and Ingram, D.J.E. (1957) Proc. Roy. Sot. (London) A240, 67-82. 11. Aasa, R., and VRnnggrd, T. (1975) J. Magn. Reson., 19, 308-315. 12. Phelps, C., Forlani, L., and Antonini, E. (1971) Biochem. J., 124, 605-614. 13. Gupta, R.K., Mildvan, A.S., Yonetani, T., and Srivastava, T.S. (1975) Biochem. Biophys. Res. Commun.,67, 1005-1012. 14.
Deinum, J.S.E.,
and V%ng&d, T. (1975) FEBSLetters,
889
58, 62-65.
Vol. 79, No. 3, 1977
PROBING AXIAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LIGANDS IN FERRIC HAEMOPROTEINS:
AN ESR STUDY OF MYOGLOBIN AND HORSERADISH PEROXIDASE IN Hzl’O S. Vuk-PavloviE*, Departments
of Chemical
The Weizmann
Received
September
Y. Siderer Immunology
Institute
and Biochemistry
of Science,
Rehovot,
Israel
22,1977 SUMMARY
Substitution of Hz160 by Hzl’ 0 induces a substantial broadening of the high-field line in the electron-spin resonance spectrum of ferric myoglobin due to the presence of Hzl’O at the axial ligand-site. Computer simulations of the experimental spectra yielded the values of the reciprocal relaxation time Tz-l= 7.8 G and the “0-hyperfine coupling constant A = 18 f 1 G. Under identical experimental conditions no effect of Hzl’O was observed in horseradish peroxidase. The latter finding excludes the possibility that a water molecule is liganded to the peroxidase haem-iron and supports either the idea that both axial ligands are amino acid residues or that the haem in ferric horseradish peroxidase is pentacoordinate. INTRODUCTION In order haemoproteins of axial
to explain
the specificities
take
it
ligands
sixth (i)
of various
exists ligand
for is
Peroxidases
axial
ligand
amino
acids
molecule
(2).
coordinated these
less
compounds
as the
certain, of the
but
(ii)
ferric
Distal
fifth
ion
ferric
by hydrogen
hypotheses type
in which for
the identity
ligand
(1).
The nature
proved
of the
forward:
i.e.
both
by atoms from neighbouring (4).
ferric (iii)
enzyme is Recently
enzyme has been proposed
has been decisively
in
much
have been put
of the acid
catalysts
peroxide,
haemoproteins,
are occupied
to an amino
haem in high-spin
hypotheses
evidence
haem-ligand
several
(sixth)
hydrogen-bounded
reactions
In the case of peroxidases,
aromatic
histidine
to obtain
are of the “close-crevice” sites
(3)
is necessary
to the haem-iron.
the oxidations evidence
part,
of various
by direct
(5).
observation
a water
also
penta-
Neither
of
of the sixth
ligand. The water molecule coordinated proteins may be observed by X-ray presence
by spectroscopic
been proposed technique * Permanent Yugoslavia.
Copyright All rights
recently
capable
at the sixth crystallography
methods, that
data
are generally
solvent-proton
to resolve
whether
magnetic
a water
address: Institute of Immunology, Correspondence should be forwarded
0 1977 by Academic of reproducrion in any
Press, Inc. form reserved.
ligand (6),
molecule
site of ferric but when probing less
decisive.
relaxation is
haemoits
might
coordinated
It
has
be the at the
Rockefellerova 2, 41000 to this address.
Zagreb,
885 1SSN
0006-291X
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
I 3350
I 3400
3450
3500
3400
MAGNETK
3450
35m
FIELD / G
parts (g=!) of ESR spectra of aquo complex of ferric Fig. 1. High-f:$d myoglobin in Hz 0 and 50%HP 0. a) Experimental spectra taken at X-band, microwave power 5 mW, modulation amplitude 2.5 G, temperature 10 K, protein concentration 0.5 mM. The amplitude of the spectrum in H2l'O has been adjusted to the Hz160-spectrum by increasing receiver gain. b) Corresponding simulated ESRspectra. Best-fit parameters are T2-l = 7.8 G and A = 18 f 1 G.
axial ligand-site
(7), but this view has been subsequently criticized
(8).
In order to probe spectroscopically the presence of the axially liganded water molecule we utilized the nuclear spin of oxygen-17 in Hzl'O. Similarly to fluoride in fluoro complexes (9), "0 has an effect especially in the high-field part of the electron-spin resonance spectrum of aquo complexes of ferric haemoproteins. This part of the spectrum is broadened by the superhyperfine interaction present at the s.xth
of "0 nuclear spin and ds iron-electrons when Hzl'O is coordination site. In this communication we present
results of our study of Hzl'O interaction in ferric peroxidase. EXPERIMENTAL
myoglobin and horseradish
Horse heart myoglobin (Sigma,Type III) and horseradish peroxidase (Sigma, Type VI, Lot No. 65C-9530-l) were used without further purification. Lyophilized proteins were dissolved in 100 ml4sodium phosphate, pH 6.0, to a final concentration of 0.5 mM. These solutions were subsequently freeze-dried and redissolved in an equal amount of either Hz160 or Hz0 50%enriched in I'0 (Heavy Oxygen Plant, Weizmann Institute of Science). The ESRspectra were taken by a Varian E-12 spectrometer at X-band (9.35 GHz) operating under nonsaturating Measurementswere performed below 10 K conditions of microwave irradiation. using a home-madeliquid helium flow system. RESULTS Since the axially liganded water in ferric myoglobin has been observed by X-ray diffraction (6), this protein was chosen to investigate the effect of the presence of Hzl'O at the axial ligand site. The powder spectrum of high-
886
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I I200
2200 MAGNETIC
3200 FIELD / G
Fig. 2. ESR spectrum of horseradish peroxidase same conditions as in Fig. 1. Insert: enlarged spectra in H2160 (lower curve) and 50% Hzl’O.
in H2160 recorded high-field parts
spin
anisotropic.
aquo complex
of ferric
myoglobin
is
arises from the difference in absorption the haem-plane (g=6) and perpendicular large
zero-field
non-zero
splitting
nuclear
as exemplified “F
(1=1/2)
ive
also
spins by the
(9).
Therefore,
aquo complex by water
part
spectrum
experimental choosing
in Hal60
swept
myoglobin
were
g-values
spectrum
recorded
possessing
absorption
around
the nuclear
of the
spectrum
derivative in Fig.
spin
g=2, of
to be sensit-
la.
through the
(g=2)
of the
Substitution
broadening
of amplitude
interpreted
and varying
Thus,
in Ha 160.
part
of ligands
with
a substantial
by a decrease
spectra
spectra,
this
is shown
170 produces
correspondence to experimental corrected transition probability, field
in the
interaction
of the ESR absorption
accompanied
appropriate
The effect
remarkable
we expect
50% in
powder
anisotropy
of Ha170.
of myoglobin
enriched
This
when the magnetic field is applied in it (g=2), and is due, in turn, to the (10).
especially
superhyperfine
to the presence
The high-field
of the
parameters is
highly
under the of the
(Fig.
of H2160
of this
region
These
la).
computer
line-width
ferric
simulation
parameter
by
until
full
spectra was achieved. The program includes according to Aasa and Wnng8rd (11)) for
a value
The simulated
in the presence
of Ta-1 high-field
= 7.8 G was obtained line
is
for
shown in Fig.
of Hz 170 was simulated
the
ferric lb.
The
by superposition
of
two different properly weighted spectra: (i) of the spectrum in H2160 described above, and (ii) of the spectrum in Hz170 with the same parameters as the previous (I = S/2).
one,
In the
but
split
case of
into
a sextet
170-broadened
due to the nuclear
spectrum
887
the best
spin
of
correspondence
170
BIOCHEMICAL
Vol. 79, No. 3, 1977
between
the
value
experimental
very
coupling
to mention
sensitive
observed
spectra
expanded-scale 50% in
“0
also
changes
of this
of the
2.
broadening
both
It
It
the is
high-field
line
is
deviations
from the
parameter.
in Hal60
spectra
in Fig.
using
lb).
simulated
in significant
peroxidase
part
any pronounced
of the
of A resulting
depicted
has been obtained
A = 18 2 1 G (Fig.
the width
of herseradish
high-field
spectra
constant
that
even upon minor
are
induce
here
to the value
The spectrum
not
and simulated
of the hyperfine
important
AND BIOPHYSICAL RESEARCH COMMJNlCATlONS
is
is
shown in Fig.
An
in H2160 and H20 enriched
readily
as in the
2.
apparent
that
case of myoglobin
“0
(Fig.
does la).
DISCUSSION Judging acquo
from
complex
sensitive
the profound
of ferric
method
containing
for
ligands
horseradish
is would
has been
considered
sequences
basis
of magnetic
However, ities. about ligand
can be ruled
peroxidases
there
(3).
experiments
out in
similar
(12,13).
in the present
properties
substrates
ways. the
is
effect
sixth-ligand A tyrosine
in the
phenolate
recently (1).
be consistent
also
iron,
proposed In the with
a
a possibility raised recently
on the
the pentacoordinated
discriminate
in
The first
between
the
haem.
two possibil-
favor
Using
of either
higher
study,
of the
of described
experiments
using one,
enrichments
the method
“0
have been of
"0
alternatives.
as a probe introduced
and even
may be sensitive
in the
We thank
Dr.
R. Poupko
temperatures
to monitor
at “O-nucleus. Such changes may result from effects on the haem due, for instance, to the medium,
the presence
of allosteric
for
his
generous
and friendly
effecters help
especially in the computer simulations of the reading and Mrs. E. Majerowich for typing the
gratefully organization.
of
only
lower
enough
study
and/or
and inhibitors.
of the work, Wherland for S.V.-P. Biology
(S),
of oxygen-
is no tyrosine
with the possibility,
data
cannot
but
would
contact The second
to the present
changes of the spin density direct or protein-mediated altered
our data
dichroism
resonance
metalloproteins, recently
(3,11),
et al
reactions
residue.
in
Obviously, more data are needed in order to reach a definite conclusion actual sixthligand of the iron, but a water molecule as the axial
Electron-spin
than
candidate
distal
acid
spectrum
to be a
of any similar
(2) where
as a likely
in the
and/or
The lack
structure
structure
of the
appears
in two different
by an amino
circular
the present
the
haemoproteins.
plant
region
of identity
be occupied
of five
by Ricard
parallel substitution
can be interpreted
crevice”
involved
discussed
investigation
in ferric
also
case of “closed carboxylate
in the isotopic
the “closed-crevice”
position distal
the
peroxidase
possibility
effect
myoglobin,
acknowledges
a long-term
888
fellowship
in all
spectra, Dr. manuscript.
by European
stages S.
Molecular
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2.
Welinder, K.G. and Mazza, R. (1977) Eur. J. Biochem., 73, 353-358.
3.
Ricard, J., 566-578.
4. 5.
Yamada, H., and Yamazaki, I. (1974) Arch. Biochem. Biophys., 165, 728-738. Kobayashi, N., Nozawa, T., and Hatano, M. (1977) Biochim. Biophys. Acta, 493, 340-351. Kendrew, J.C., Watson, H.C., Strandberg, B.E., Dickerson, R.E., Phillips, D.C., and Shore, V.C. (1961) Nature, 190, 666-670. Lanir, A., and Schejter, A. (1975) Biochem. Biophys. Res. Commun.,62, 199-203. Vuk-Pavlovi:, S., and Benko, B. (1975) Biochem. Biophys. Res. Commun.,66, 1154-1159. Kotani, M., and Morimoto, H. (1967) in "Magnetic Resonancein Biological Systems", (Ehrenberg, A., MalmostrBm, B.G., and V!lnng&d, T., eds.) pp. 135-140, PergamonPress, Oxford.
6. 7. 8. 9.
George, P., and Lyster, 1013-1029.
R.L.J.
(1958) Proc. Nat. Acad. Sci. U.S.A.,
Mazza, G., and Williams, R.J.P.
44,
(1972) Eur. J. Biochem., 28,
10.
Bennett, J.E., Gibson, F.J., and Ingram, D.J.E. (1957) Proc. Roy. Sot. (London) A240, 67-82. 11. Aasa, R., and VRnnggrd, T. (1975) J. Magn. Reson., 19, 308-315. 12. Phelps, C., Forlani, L., and Antonini, E. (1971) Biochem. J., 124, 605-614. 13. Gupta, R.K., Mildvan, A.S., Yonetani, T., and Srivastava, T.S. (1975) Biochem. Biophys. Res. Commun.,67, 1005-1012. 14.
Deinum, J.S.E.,
and V%ng&d, T. (1975) FEBSLetters,
889
58, 62-65.
