Vol. 168, No. 3, 1990 May 16, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1311-1317
ONE-ELECTRON REDUCTION OF CHLOROPEROXIDASE GENERATED ELECTRONS Diana
Metodiewa*
and Ii.
BY RADIOLYTICALLY
Brian
Dunford
*Institute of Applied Radiation Chemistry, Technical University of m2 93-950 md2, Poland Department of Chemistry, Edmonton, Alberta, Received
March
University of Alberta, Canada T6G 2G2
7, 1990
SUMMARY: Upon irradiation of aqueous ethylene glycol/water solutions of native chloroperoxidase (CPO) with 6oCo-Y rays at 11X one observes the one-electron reduction of the enzyme active site by radiolytically generated thermolyzed electrons. In the present study the first absorption spectrum of a low-spin ferrous form of CPO is reported which has peaks at 438, 532 and 563 nm, similar to those observed previously for cytochrome P-450. All previously described ferrous forms of CPO are high spin. In order to observe the final results of the CPO reaction with electrons, the spectral changes of native enzyme after room temperature-y-irradiation have also been investigated. Evidence of changes is also presented probably connected with disruption of with decrease of enzyme activity. the tertiary structure of enzyme, correlated 01990
Academic
Press,
Inc.
Chloroperoxidase EC 1.11.1.10)
(CPO)
horseradish
as well
Current
cytochrome
in
the
(3-5)
y-irradiation
P-450
classical
in
prompted
its brominate
we present
of frozen
ethylene
efficient
electron
addition
to the
electron
reduction
of
enzyme
at
ferrous
species.
The
ratio
of
the
IIK
and of
are
low/high
its
unstable
that
low
is
temperature
of CPO result
The products of
addition
(6).
solutions
a mixture spin
reactions
one-electron
center.
and organic
catalase
CPO and
evidence
glycol/water heme iron
properties
y-radiolysis
first
catalase,
and iodinate
investigate
of low-temperature
to
physical
reactions
us to
oxidoreductase,
a similarity
peroxidase
one-electron
of CPO by application communication
with
can chlorinate,
as catalyze
compounds
In this (77K)
and
interest
short-lived products
a glycohemoprotein
The enzyme
activity.
substances
(2).
is
peroxidase
catalytic
peroxide
(chloride:hydrogen
low-
dose
and
of the
one-
high
spin
dependent. 0006-291X/90
1311
in
Some $1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
168, No. 3, 1990
experiments electrons
(in
have
also
been
the
presence
AND BIOPHYSICAL
performed
to
of OH' scavenger)
observe
RESEARCH COMMUNICATIONS the
CPO reaction
with
at room temperature.
EXPERIMENTAL Chloroperoxidase (CPO) from Caldariomyces fumago, a purified suspension 0.1 M NaIQP04 was purchased from the Sigma Chemical Company and used as by using a molar received. The enzyme concentration was calculated absorptivity of 9.12x104 M-l cm-l at 400 run (7). The chlorination of P-chlorodimedone was used as an assay of enzyme activity (8). All other chemicals were of analytical grade. Optical absorption spectra were determined at 77K or at room temperature with a Beckman Acta M IV spectrophotometer equipped with a thermo-regulated The reaction mixtures were made anaerobic by purging with nitrogen assembly. gas (30 min). All low-temperature spectra were determined with buffered enzyme solutions (50 mM potassium phosphate buffer, pH 6.4) in 50% ethylene glycol frozen to form a clear glass. In the optical experiments disk-shaped ice samples abour 1 mm thick and 5 mm in diameter were prepared at 77X. The samples were irradiated in the dark by 6oCo y-rays at the temperature of The dose rate was 1.6 Gy/sec. The liquid nitorgen or at room temperature. solvent-trapped electrons were removed by bleaching of samples (10 min) at 77K with visible light (1 > 450 MI). The number of electrons formed by irradiation at room temperature was calculated from ferrous dosimetry calibrations using G(Fe3+) = 15 . 5 . in
RESULTS Exposure
of
y-rays
at
maxima
at 423,
la)
disappearing:
is
aqueous
77K results 438,
ethylene
in the 532,
glycol
formation
of new spectral
563 and 584 nm (Fig.
a significant
300
decrease
400
The
reduction
solutions
irradiation concentration
St
of
Fe(III)CPO
2).
Thus
of absorption
500 Wavelength
Fig.1:
solutions
containing species the
CPO to with
native intensity
absorption enzyme
by
600
electrons in ethylene glycol/water were obtained before (a) and after with 20 tbt and 50 (c) kGy of 6oCo rays at 77K. Enzyme 0.5 mM; 0.05 M potassium phosphate buffer (pli 6.4).
1312
(Fig.
at 400 nm
(nm)
77K. The spectra
60~0
700
Vol.
168, No. 3, 1990
BIOCHEMICAL
I 300
I
I 400
AND BIOPHYSICAL
I
I 500
600
WaveLength
Fig.2:
mM.
registered
(Fig.
reduction
of
electron
capture
mentioned
that
in
the
of
irradiated
dose
ferric
dark
markedly
lb,c).
This
provided
enzyme,
taking
into
at the
(Fig.
one
as the
a unique
lb,c)
of
a small
The optical
shoulder
presence
of OH' radical
optical
spectrum
irradiation
latter
(Fig.
the
intensity
transfer accompanied
aqueous
band
solution
of
2b)
shows
of the
(400
normally high-spin
by a decrease
the
univalent
probability small.
of two-
It
on irradiation
should
be
of CPO at
observed
after
spectrum
(Fig.
50 kGy (Fig.
obtained,
changes
CPO taken
is
is
spectral
scavenger)
band
occur
20 to
bands
the
negligibly
those
from
of
Soret
which
for
77~
photo-bleaching lb)
lc).
centered
is
changed
At the at
430,
latter 532
and
at 423 nm.
absorption
deaerated
is
that
radiation-modified
increased
evidence
account
resemble
absorption
buffered
at
closely
is
clear
center
changes
The
dose set
active
spectral
samples.
563 nm with
nm) ,
7
b-m 1
Optical absorption spectral changes associated with y-irradiation of deaerated aqueous solutions of CPO at room temperature. The spectra were obtained before (a) and after irradiation with 3.0 kGy of %o rays (bf. Enzyme concentration 6.9 p&l; t-butanol 0.1 M; 0.05 potassium phosphate buffer (pH 6.4); electrons produced 4.7
was
RESEARCH COMMUNICATIONS
have
associated
enzyme also
been
immediately
a decrease run)
and
associated heme complex
of enzyme
activity 1313
room
the
visible
region
(about
(Fig.
(Fig. in
absorbance
of native
CPO). 29%).
of
temperature
absorbance
with
y-irradiation
investigated
before
in in
at
with
2a)
the
W
at from These
(in
the
2).
The
and region
650
after (280
nm (this
the changes
charge were
Vol. 168, No. 3, 1990 1.
T8blo
BIOCHEMICAL
Comparison
of
Maxima
Absorption
Chloroperoxidase
(CPO)
CPO Native Chemically
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and
400,515; 409, shoulders
Reduced
Cvtochrome
423,
438,
P-450
Reduced
Comparisons radiolytically
563,
shoulder
535,
Reduced
Radiolytically
550
maxima
416,
Chemically
450;
Absorption
of
absorption
reduced
products
423,
554
530,
563
maxima are
References
at
4,g this
584
work
References
649
15
native
of
4,s
422
(run)
570,
Reduced
P-450
544, 585, 652 at approximately
532,
Native
and
maxima (run)
and Radiolytically
Native
Cytochrome
Absorption
Reduced
of
summarized
and
CPO
in Table
its
chemically
or
1.
DISCUSSION The for
low-temperature
studying
one-electron
intermediates.
being
after
electron
molecular
trapped
in
the
at
that
motion
attachment
to
are
that
results
in
products
of the
to
efficient
Data
one-electron
reaction
an open
formation with
and protein this of
transient nitrogen
and
the
macromolecular
minor
relaxation
can occur
all
reactions
involving
thermolyzed
site (11).
glassy to
the
states
and
(12-14).
The
found
to be
at 77% were reduced
shown
of hemoprotein
centers
provide
of high-
1314
has been
original
iron
and
native
(11,13,15).
communication
electrons
primary,
solutions
heme
of
of It
of hemoproteins
moieties
a mixture
the
question
addition reduction
in
method
liquid
to
and
from
nonequilibrium
of ligands
the
center
migration
electron
presented
only
a useful
involving
in
leads
matrix:
redox
enzymes
enzyme
temperature
of low-temperature
stabilized,
conformations
of
is
(10).
still
y-irradiation
solutions of
solution
the
electron
heme is
glassy
reaction
rigid
suppressed of
the
accepted
kinetically
a fairly
addition
The mechanism
CPO upon
of
r-irradiation
system
of
reduction
Quenching
subsequent
indicating
radiolysis
spectral
and low-spin
at 77K
(Fig. 1).
ferrous
evidence forms
of
Vol.
168, No. 3, 1990 The
comparison
radiolytically species,
with
spin
ferrous
(Fig.
lc).
absorption
maxima
Similar
documented
similarities
the
center
(15)
with
nm (Fig.
ligands
in
the
cytochrome iron(I1)
is
additional d-orbital
obtained field
is
indicate
thiolate the
P-450
frozen
of
of
the for
active Hz0
six
ligands,
spheres
at
530-532
of
and axial
CPO as
As a rule
a sufficiently
P450
same two
site
(2).
well
same low-spin
of the
and
are
cytochrome
maxima
iron
unusually
coordination
presence
the
causes
of
display
previously
the
presence
enzyme
(21. There
formation
ligand
strength
important
center
of
other
nature
of
CPO sixth
of
proximal low
described
spin in
With CPO becomes ferric
observation
ligand
Whatever
of the
ferrous
the
this
will
of
the
ferric
ion form
similar
low
in
spin
not
five,
as
the
large
split
in
the
high not
of
spin observe
until
(2):
peroxidase
in
in
the
ferrous measurable
1315
the
unusual
mixture,
fifth unusual
CPO (Table formation
1). of
spin
form
with
traces
In
support
the
sixth
spectral
In compar-
same technique
properties
along
the
of an equally
by the
low
of
and
(2,15,16).
optical
active
an interesting
a reflection p-450
the proof
be
CPO, the
formed
different the
to
ligand native
6 in further
continue
of CPO are
electrons
species
of
position
to cytochrome
has distinctly
dominant
one does
vacant
nature
horseradish
excess
CPO and
is
low-spin
paper
a large
coordination
arises,
environment
this
that
the
of
ferrous
the
note
peroxidases
position
properties unusual
to
ferric
study.
coordination
spin
in
P-450
absorption
of
ferric
y-radiolysis
forms
observed
sphere
to be low-
energies. It
ison
The results
a proximal
ligand
cytochrome
high-spin
been
of new
(15).
indicative
coordination
P450:
P-450
in
and
appearance
high-spin
previously
we observe
CPO as has
chemically
the
native
CPO and cytochrome
Here
(2,16).
1).
indicates
from
their
characteristically
560-562
area
in
native,
532 and 563 run, considered
CPO and
between
from
438,
obtained
heme proteins
of
clearly
of cytochrome
distortions
species
at
were
solutions
iron
1)
radiolytically
results
most
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
characteristics
CPO (Table
rhombic
ferrous
spectral
CPO, produced
Unlike
of
of
reduced
water/glycerol
strong
BIOCHEMICAL
(17). of
so-called
ferrous of of
highthis high-
Vol.
BIOCHEMICAL
168, No. 3, 1990
spin
marker
band
transition native
in
at
the
high-spin
ferric
the
with
native
the
CPO (Table The data
the
active
process
in
the
may
structure
our
here
to
of
an electronic
650 nm band
the
at room
is
spectral
typical
It
unstable
only
changes
temperature.
short-lived
indicate
in
formation
of
the
of
of
is
CPO
known
species
(3)
at
disappearance
into
free
the
iron
orbital
absorbance
of
Further
studies
low-spin from
active
must
room
some of
reduction ferrous
center, of
and localization re-oxidation
be a complicated
process
binding,
as suggested
a decrease
The
undefined
fast
disruption
of
CPO.
a primary,
partial
one observes
of
the
which
tertiary
previously
of enzyme
of
(19).
activity
and a
2).
on the
heme enzymes
one-electron
The process
(11).
of heme-protein
(Fig.
of
at the
changes,
suggestion
that
electrons
CPO at room temperature
of this
of
given
clearly
migration
protein
in
reduction
nm. The
2 show
Fig.
CPO results
and loosening
decrease
been
CPO are
conformational
In support
in
in
energy
ferrous
involve
spectra
involve
to the
transient
of
disappearance
(2,lE).
generated
forms
of
lowest
650
1)
has
the
RESEARCH COMMUNICATIONS
1).
site
attachment
CPO (Fig.
presented
must
after
about
electrons
ferrous
temperature:
at
attention
reaction
that
nm (18)
vicinity
Considerable after
638
AND BIOPHYSICAL
mechanism
and
by electrons
intermediate
and superoxide
product anion
formation are
in
in
progress
laboratories. ACKNOWLEDGMENTS This
work
and NSERC (grant
was carried A1248)
out
under
the
projects
CPBP 01.19.14.12
of Poland
of Canada. REFERENCES
1. 2.
3. 4. 5.
Dawson, J.H., Trudell, J-R., Barth, G., Linder, R.E., Bunneberg, R.E., Djerassi, C., Chiang, R., Hager, L.P. (1976) J. Am. Chem. Sot. 98, 3709-3712. Dunford, H.B., Araiso, T., Job, D., Ricard, J., Rutter, R., Hager, L.P., Wever, R., Kast, W.M., Boelens, R., Ellfolk, N., Ronnberg, M. (1982) in The Biological Chemistry of Iron (Dunford, H.B., Dophin, D., Raymond, K.N. and Sieker, L., eds.) D. Reidel Publishing Company, pp.337-355. Nakajima, R., Yamazaki, J., Griffin, B.W. (1985) Biochem. Biophys. Res. comm. 128, 1-6. Lamb&r, A.M., Dunford, H.B. (1985) Eur. J. Biochem. 147, 93-96. Netodiewa, D., Pickard, M., Dunford, H.B. (1989) Biochem. Biophys. Res. Conun. 159, 1086-1092. 1316
Vol. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Symcns, M.C.R., West, D.X., Wilkinson (1973) J.C.S. Comm. 917. Hollenberg, P.F., Hager, L.P., Blumberg, W.E., Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807. Hager, L.P., Moris, D.R., Brown, F-S., Eberwein, H. (1966) J. Biol. Chem. 241, 1769-1777. Moris, D.R., Hager, L.P. (1966) J. Biol. Chem. 241, 1763-1769. in Radiation Chemistry of Frozen Aqueous Solutions, Kevan, L. (1968) ~~-21-71 (Stein, G. ed.) The Weixmann Science Press of Israel. Buxton, G.V. (1984) Adv. Inorg. Bioeng. Mech. 3, 131-173. Blumenfeld, L.A., Davidov, R.M., Magonov, S.N., Nilu, R.O. (1974) FEBS Lett. 45, 256-259. Gasyna, 2. (1979) FEBS Lett. 106, 213-218. Davidov, R.M. (1980) Biofizyka 25, 203-208. Davidov, R.M., Greshner, Z., Magonov, S-N., Rukpaul, K., Blumenfeld, L.A. (1978) Dokl. AN SSSR 241, 707-709. Hollenberg, P.F., Hager, L.P. (1973) J. Biol. Chem. 248, 2630-2633. Magonov, S.N., Arutiuxian, A.M., Blumenfeld, L.A., Davidov, R.M., Sharonov, U.A. (1977) Dokl. AN SSSR 238, 695-698. Sono, M., Dawson, Y.H., Hall, K. and Hager, L.P. (1986) Biochemistry 25, 347-356. Metodiewa, D., Gebicka, L. and Bachman, S. (1987) J. Radioanal. Nucl. Chem. Lett. 119, 87-95.
1317
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1311-1317
ONE-ELECTRON REDUCTION OF CHLOROPEROXIDASE GENERATED ELECTRONS Diana
Metodiewa*
and Ii.
BY RADIOLYTICALLY
Brian
Dunford
*Institute of Applied Radiation Chemistry, Technical University of m2 93-950 md2, Poland Department of Chemistry, Edmonton, Alberta, Received
March
University of Alberta, Canada T6G 2G2
7, 1990
SUMMARY: Upon irradiation of aqueous ethylene glycol/water solutions of native chloroperoxidase (CPO) with 6oCo-Y rays at 11X one observes the one-electron reduction of the enzyme active site by radiolytically generated thermolyzed electrons. In the present study the first absorption spectrum of a low-spin ferrous form of CPO is reported which has peaks at 438, 532 and 563 nm, similar to those observed previously for cytochrome P-450. All previously described ferrous forms of CPO are high spin. In order to observe the final results of the CPO reaction with electrons, the spectral changes of native enzyme after room temperature-y-irradiation have also been investigated. Evidence of changes is also presented probably connected with disruption of with decrease of enzyme activity. the tertiary structure of enzyme, correlated 01990
Academic
Press,
Inc.
Chloroperoxidase EC 1.11.1.10)
(CPO)
horseradish
as well
Current
cytochrome
in
the
(3-5)
y-irradiation
P-450
classical
in
prompted
its brominate
we present
of frozen
ethylene
efficient
electron
addition
to the
electron
reduction
of
enzyme
at
ferrous
species.
The
ratio
of
the
IIK
and of
are
low/high
its
unstable
that
low
is
temperature
of CPO result
The products of
addition
(6).
solutions
a mixture spin
reactions
one-electron
center.
and organic
catalase
CPO and
evidence
glycol/water heme iron
properties
y-radiolysis
first
catalase,
and iodinate
investigate
of low-temperature
to
physical
reactions
us to
oxidoreductase,
a similarity
peroxidase
one-electron
of CPO by application communication
with
can chlorinate,
as catalyze
compounds
In this (77K)
and
interest
short-lived products
a glycohemoprotein
The enzyme
activity.
substances
(2).
is
peroxidase
catalytic
peroxide
(chloride:hydrogen
low-
dose
and
of the
one-
high
spin
dependent. 0006-291X/90
1311
in
Some $1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
168, No. 3, 1990
experiments electrons
(in
have
also
been
the
presence
AND BIOPHYSICAL
performed
to
of OH' scavenger)
observe
RESEARCH COMMUNICATIONS the
CPO reaction
with
at room temperature.
EXPERIMENTAL Chloroperoxidase (CPO) from Caldariomyces fumago, a purified suspension 0.1 M NaIQP04 was purchased from the Sigma Chemical Company and used as by using a molar received. The enzyme concentration was calculated absorptivity of 9.12x104 M-l cm-l at 400 run (7). The chlorination of P-chlorodimedone was used as an assay of enzyme activity (8). All other chemicals were of analytical grade. Optical absorption spectra were determined at 77K or at room temperature with a Beckman Acta M IV spectrophotometer equipped with a thermo-regulated The reaction mixtures were made anaerobic by purging with nitrogen assembly. gas (30 min). All low-temperature spectra were determined with buffered enzyme solutions (50 mM potassium phosphate buffer, pH 6.4) in 50% ethylene glycol frozen to form a clear glass. In the optical experiments disk-shaped ice samples abour 1 mm thick and 5 mm in diameter were prepared at 77X. The samples were irradiated in the dark by 6oCo y-rays at the temperature of The dose rate was 1.6 Gy/sec. The liquid nitorgen or at room temperature. solvent-trapped electrons were removed by bleaching of samples (10 min) at 77K with visible light (1 > 450 MI). The number of electrons formed by irradiation at room temperature was calculated from ferrous dosimetry calibrations using G(Fe3+) = 15 . 5 . in
RESULTS Exposure
of
y-rays
at
maxima
at 423,
la)
disappearing:
is
aqueous
77K results 438,
ethylene
in the 532,
glycol
formation
of new spectral
563 and 584 nm (Fig.
a significant
300
decrease
400
The
reduction
solutions
irradiation concentration
St
of
Fe(III)CPO
2).
Thus
of absorption
500 Wavelength
Fig.1:
solutions
containing species the
CPO to with
native intensity
absorption enzyme
by
600
electrons in ethylene glycol/water were obtained before (a) and after with 20 tbt and 50 (c) kGy of 6oCo rays at 77K. Enzyme 0.5 mM; 0.05 M potassium phosphate buffer (pli 6.4).
1312
(Fig.
at 400 nm
(nm)
77K. The spectra
60~0
700
Vol.
168, No. 3, 1990
BIOCHEMICAL
I 300
I
I 400
AND BIOPHYSICAL
I
I 500
600
WaveLength
Fig.2:
mM.
registered
(Fig.
reduction
of
electron
capture
mentioned
that
in
the
of
irradiated
dose
ferric
dark
markedly
lb,c).
This
provided
enzyme,
taking
into
at the
(Fig.
one
as the
a unique
lb,c)
of
a small
The optical
shoulder
presence
of OH' radical
optical
spectrum
irradiation
latter
(Fig.
the
intensity
transfer accompanied
aqueous
band
solution
of
2b)
shows
of the
(400
normally high-spin
by a decrease
the
univalent
probability small.
of two-
It
on irradiation
should
be
of CPO at
observed
after
spectrum
(Fig.
50 kGy (Fig.
obtained,
changes
CPO taken
is
is
spectral
scavenger)
band
occur
20 to
bands
the
negligibly
those
from
of
Soret
which
for
77~
photo-bleaching lb)
lc).
centered
is
changed
At the at
430,
latter 532
and
at 423 nm.
absorption
deaerated
is
that
radiation-modified
increased
evidence
account
resemble
absorption
buffered
at
closely
is
clear
center
changes
The
dose set
active
spectral
samples.
563 nm with
nm) ,
7
b-m 1
Optical absorption spectral changes associated with y-irradiation of deaerated aqueous solutions of CPO at room temperature. The spectra were obtained before (a) and after irradiation with 3.0 kGy of %o rays (bf. Enzyme concentration 6.9 p&l; t-butanol 0.1 M; 0.05 potassium phosphate buffer (pH 6.4); electrons produced 4.7
was
RESEARCH COMMUNICATIONS
have
associated
enzyme also
been
immediately
a decrease run)
and
associated heme complex
of enzyme
activity 1313
room
the
visible
region
(about
(Fig.
(Fig. in
absorbance
of native
CPO). 29%).
of
temperature
absorbance
with
y-irradiation
investigated
before
in in
at
with
2a)
the
W
at from These
(in
the
2).
The
and region
650
after (280
nm (this
the changes
charge were
Vol. 168, No. 3, 1990 1.
T8blo
BIOCHEMICAL
Comparison
of
Maxima
Absorption
Chloroperoxidase
(CPO)
CPO Native Chemically
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and
400,515; 409, shoulders
Reduced
Cvtochrome
423,
438,
P-450
Reduced
Comparisons radiolytically
563,
shoulder
535,
Reduced
Radiolytically
550
maxima
416,
Chemically
450;
Absorption
of
absorption
reduced
products
423,
554
530,
563
maxima are
References
at
4,g this
584
work
References
649
15
native
of
4,s
422
(run)
570,
Reduced
P-450
544, 585, 652 at approximately
532,
Native
and
maxima (run)
and Radiolytically
Native
Cytochrome
Absorption
Reduced
of
summarized
and
CPO
in Table
its
chemically
or
1.
DISCUSSION The for
low-temperature
studying
one-electron
intermediates.
being
after
electron
molecular
trapped
in
the
at
that
motion
attachment
to
are
that
results
in
products
of the
to
efficient
Data
one-electron
reaction
an open
formation with
and protein this of
transient nitrogen
and
the
macromolecular
minor
relaxation
can occur
all
reactions
involving
thermolyzed
site (11).
glassy to
the
states
and
(12-14).
The
found
to be
at 77% were reduced
shown
of hemoprotein
centers
provide
of high-
1314
has been
original
iron
and
native
(11,13,15).
communication
electrons
primary,
solutions
heme
of
of It
of hemoproteins
moieties
a mixture
the
question
addition reduction
in
method
liquid
to
and
from
nonequilibrium
of ligands
the
center
migration
electron
presented
only
a useful
involving
in
leads
matrix:
redox
enzymes
enzyme
temperature
of low-temperature
stabilized,
conformations
of
is
(10).
still
y-irradiation
solutions of
solution
the
electron
heme is
glassy
reaction
rigid
suppressed of
the
accepted
kinetically
a fairly
addition
The mechanism
CPO upon
of
r-irradiation
system
of
reduction
Quenching
subsequent
indicating
radiolysis
spectral
and low-spin
at 77K
(Fig. 1).
ferrous
evidence forms
of
Vol.
168, No. 3, 1990 The
comparison
radiolytically species,
with
spin
ferrous
(Fig.
lc).
absorption
maxima
Similar
documented
similarities
the
center
(15)
with
nm (Fig.
ligands
in
the
cytochrome iron(I1)
is
additional d-orbital
obtained field
is
indicate
thiolate the
P-450
frozen
of
of
the for
active Hz0
six
ligands,
spheres
at
530-532
of
and axial
CPO as
As a rule
a sufficiently
P450
same two
site
(2).
well
same low-spin
of the
and
are
cytochrome
maxima
iron
unusually
coordination
presence
the
causes
of
display
previously
the
presence
enzyme
(21. There
formation
ligand
strength
important
center
of
other
nature
of
CPO sixth
of
proximal low
described
spin in
With CPO becomes ferric
observation
ligand
Whatever
of the
ferrous
the
this
will
of
the
ferric
ion form
similar
low
in
spin
not
five,
as
the
large
split
in
the
high not
of
spin observe
until
(2):
peroxidase
in
in
the
ferrous measurable
1315
the
unusual
mixture,
fifth unusual
CPO (Table formation
1). of
spin
form
with
traces
In
support
the
sixth
spectral
In compar-
same technique
properties
along
the
of an equally
by the
low
of
and
(2,15,16).
optical
active
an interesting
a reflection p-450
the proof
be
CPO, the
formed
different the
to
ligand native
6 in further
continue
of CPO are
electrons
species
of
position
to cytochrome
has distinctly
dominant
one does
vacant
nature
horseradish
excess
CPO and
is
low-spin
paper
a large
coordination
arises,
environment
this
that
the
of
ferrous
the
note
peroxidases
position
properties unusual
to
ferric
study.
coordination
spin
in
P-450
absorption
of
ferric
y-radiolysis
forms
observed
sphere
to be low-
energies. It
ison
The results
a proximal
ligand
cytochrome
high-spin
been
of new
(15).
indicative
coordination
P450:
P-450
in
and
appearance
high-spin
previously
we observe
CPO as has
chemically
the
native
CPO and cytochrome
Here
(2,16).
1).
indicates
from
their
characteristically
560-562
area
in
native,
532 and 563 run, considered
CPO and
between
from
438,
obtained
heme proteins
of
clearly
of cytochrome
distortions
species
at
were
solutions
iron
1)
radiolytically
results
most
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
characteristics
CPO (Table
rhombic
ferrous
spectral
CPO, produced
Unlike
of
of
reduced
water/glycerol
strong
BIOCHEMICAL
(17). of
so-called
ferrous of of
highthis high-
Vol.
BIOCHEMICAL
168, No. 3, 1990
spin
marker
band
transition native
in
at
the
high-spin
ferric
the
with
native
the
CPO (Table The data
the
active
process
in
the
may
structure
our
here
to
of
an electronic
650 nm band
the
at room
is
spectral
typical
It
unstable
only
changes
temperature.
short-lived
indicate
in
formation
of
the
of
of
is
CPO
known
species
(3)
at
disappearance
into
free
the
iron
orbital
absorbance
of
Further
studies
low-spin from
active
must
room
some of
reduction ferrous
center, of
and localization re-oxidation
be a complicated
process
binding,
as suggested
a decrease
The
undefined
fast
disruption
of
CPO.
a primary,
partial
one observes
of
the
which
tertiary
previously
of enzyme
of
(19).
activity
and a
2).
on the
heme enzymes
one-electron
The process
(11).
of heme-protein
(Fig.
of
at the
changes,
suggestion
that
electrons
CPO at room temperature
of this
of
given
clearly
migration
protein
in
reduction
nm. The
2 show
Fig.
CPO results
and loosening
decrease
been
CPO are
conformational
In support
in
in
energy
ferrous
involve
spectra
involve
to the
transient
of
disappearance
(2,lE).
generated
forms
of
lowest
650
1)
has
the
RESEARCH COMMUNICATIONS
1).
site
attachment
CPO (Fig.
presented
must
after
about
electrons
ferrous
temperature:
at
attention
reaction
that
nm (18)
vicinity
Considerable after
638
AND BIOPHYSICAL
mechanism
and
by electrons
intermediate
and superoxide
product anion
formation are
in
in
progress
laboratories. ACKNOWLEDGMENTS This
work
and NSERC (grant
was carried A1248)
out
under
the
projects
CPBP 01.19.14.12
of Poland
of Canada. REFERENCES
1. 2.
3. 4. 5.
Dawson, J.H., Trudell, J-R., Barth, G., Linder, R.E., Bunneberg, R.E., Djerassi, C., Chiang, R., Hager, L.P. (1976) J. Am. Chem. Sot. 98, 3709-3712. Dunford, H.B., Araiso, T., Job, D., Ricard, J., Rutter, R., Hager, L.P., Wever, R., Kast, W.M., Boelens, R., Ellfolk, N., Ronnberg, M. (1982) in The Biological Chemistry of Iron (Dunford, H.B., Dophin, D., Raymond, K.N. and Sieker, L., eds.) D. Reidel Publishing Company, pp.337-355. Nakajima, R., Yamazaki, J., Griffin, B.W. (1985) Biochem. Biophys. Res. comm. 128, 1-6. Lamb&r, A.M., Dunford, H.B. (1985) Eur. J. Biochem. 147, 93-96. Netodiewa, D., Pickard, M., Dunford, H.B. (1989) Biochem. Biophys. Res. Conun. 159, 1086-1092. 1316
Vol. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Symcns, M.C.R., West, D.X., Wilkinson (1973) J.C.S. Comm. 917. Hollenberg, P.F., Hager, L.P., Blumberg, W.E., Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807. Hager, L.P., Moris, D.R., Brown, F-S., Eberwein, H. (1966) J. Biol. Chem. 241, 1769-1777. Moris, D.R., Hager, L.P. (1966) J. Biol. Chem. 241, 1763-1769. in Radiation Chemistry of Frozen Aqueous Solutions, Kevan, L. (1968) ~~-21-71 (Stein, G. ed.) The Weixmann Science Press of Israel. Buxton, G.V. (1984) Adv. Inorg. Bioeng. Mech. 3, 131-173. Blumenfeld, L.A., Davidov, R.M., Magonov, S.N., Nilu, R.O. (1974) FEBS Lett. 45, 256-259. Gasyna, 2. (1979) FEBS Lett. 106, 213-218. Davidov, R.M. (1980) Biofizyka 25, 203-208. Davidov, R.M., Greshner, Z., Magonov, S-N., Rukpaul, K., Blumenfeld, L.A. (1978) Dokl. AN SSSR 241, 707-709. Hollenberg, P.F., Hager, L.P. (1973) J. Biol. Chem. 248, 2630-2633. Magonov, S.N., Arutiuxian, A.M., Blumenfeld, L.A., Davidov, R.M., Sharonov, U.A. (1977) Dokl. AN SSSR 238, 695-698. Sono, M., Dawson, Y.H., Hall, K. and Hager, L.P. (1986) Biochemistry 25, 347-356. Metodiewa, D., Gebicka, L. and Bachman, S. (1987) J. Radioanal. Nucl. Chem. Lett. 119, 87-95.
1317
