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Heme-heme interactions in cytochrome c oxidase; Effects of photodissociation of the CO compound
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HEME-HEME INTERACTIONSIN CYTOCHROME c OXIDASE; EFFECTSOF PHOTODISSOCIATION OF THE Cz COMPOUND* John S. Leigh, Jr. and David F. Wilson Johnson Research Foundation Department of Biophysics and Physical Biochemistry University of Pennsylvania Philadelphia, Pennsylvania 19104 Received
July
24,
1972 I
SUMMARY
The binding of CO to reduced cytochrome a results in the heme of oxidized cytochrome 2 changing from high spin to low sp -3 n. Photodissociation of the COat lOoK not only shifts the epr signal of the low spin heme to a higher magnetic field but also changes someof the low spin heme to high spin heme. Thus"heme-hemeinteraction"can occur even at lOoK.
c
INTRODUCTION Cytochrome c oxidase functions as the terminal electron donor of the mitochonaria1 electron
transport
chain.
Acting both as the catalyst
(l-3 - for review, see 4) and as a "free-energy (5,6),
its
conserving site"
which are conventionally dation-reduction
two
distinguishable
The 0x14
of cytochromes a and z3 at pH 7.2 are approxi-
mately +220 mV and +380 mV respectively cytochrome g3 is identified
ob-.
hememoieties (1,4)
designated as cytochrome 5 and cytochrome s3 (1).
midpoint potentials
+
for ATP synthesis
reaction mechanismhas been studied for many years. Spectrophotometric
servations have established the existence of
ally,
for oxygen reduction
(6,7) in uncoupled mitochondria.
Operation-
. +
as the cytochrome which reacts with molecular oxy-
gen and which is the CObinding pigment (l-4).
In addition,
the hemoprotein contains a
two moles of copper per mole of cytochromes 2 and z3 (4).
I
Recent experimental evidence that the two hemeunits are not independent of one another has come from spectrophotometric heme interaction coefficient
has been demonstrated:
(7) and epr (8-10) studies.
1) by the effect
Heme-
on the a-band extinction
of reduced cytochrome 5 (when CObinds to cytochrome s3);
2) by a shift
* Supported by National Science Foundation Grant GB 28125. D.F. Wilson is the recipient of U.S. Public Health Service Career Development Award l-KO4-G&18154.
Copyright AN rights
0 19 72 by Academic Press. Inc. of reproduction in any form reserved.
1266
~
BIOCHEMICAL
Vol. 48, No. 5, 1972
in the oxidation-reduction tochrome 23);
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
midpoint potential
3) by a shift
of cytochrome 5 (when CObinds to cy-
in the oxidation-reduction
chrome-3 a in the presence of azide;
midpoint potential
4) by epr spectral
of cyto-
changes of the high spin
hemea components on reduction of cytochrome z3 (8,9) and 5) by epr spectral changes of the low spin heme5 signals in the presence of COand azide under controlled redox conditions
(8).
In this paper we report the effects on the epr spectral
characteristics
from cytochrome z3 was originally
of COphotodissociation
of cytochrome 5.
The photodissociation
is essentially
At low temperatures the
remains quite rapid with high quantum efficiency
irreversible
of CO
discovered by Warburg and Negelein (2) and has been
studied at low temperatures by Chance and coworkers (11). photodissociation
from cytochrome a3
but the reaction
below s160°K (12).
MATERIALSANDMETHODS epr measurementswere performed with a Varian E-3 spectrometer equipped for low temperature operation.
The sample tube (quartz 4mmOD, 3mmID) was positioned in a
simple, unsilvered "flow-through" and liquid
helium flow (l-3 litersfhr.)
an efficient, Pa.).
quartz dewar (J.F.
flexible,
Illumination
liquid
was delivered
helium transfer
Calif.)
from the storage dewar through
line (Air Products, Inc.,
was provided by an unfiltered
spectra were obtained by subtraction
Scanlon, Co., Whittier,
Allentown,
Unitron Lamp (40 watts).
Difference
of the desired signals with a Varian C-1024 com-
puter. Oxidation-reduction
potentials
were measured by the method of Dutton (13,14).
The samples were prepared by allowing the stirred roughly 50-50 mixture of (02-free) adjusting
the redox potential
suspensions to equilibrate
argon gas and COfor at least 15 minutes and then
of the anaerobic mitochondrial
or submitochondrial pre-
parations to approximately +300 mV, such that cytochrome z3 was essentially duced and cytochrome 2 was 90%oxidized
(see ref.
diators were 40 uM phenazine methosulfate, cyanide.
with a
The oxidation-reduction
reme-
40 uM diaminodurene and 100 PM ferri-
The entire procedure was done essentially
1267
8).
all
in the dark and samples were
BIOCHEMICAL
Vol. 48, No. 5, 1972
quickly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
frozen in an isopentane-methylcyclohexane
The samples were maintained in the dark at liquid
bath maintained at about -192'C. nitrogen temperature until
mea-
surement. Pigeon heart mitochondria were prepared by the method of Chance and Hagihara (15: Submitochondrial particles of Lindsay --et al.
were prepared from pigeon heart mitochondria by the method
(16).
RESULTS A typical
low temperature epr spectrum of submitochondrial particles
the dark at +300 mV in the presence of CO is shown in Fig. IA.
taken in
The cytochrome 3
is present as the ferrous low spin CO compoundand has no measurable epr absorbance. The g6 signals are due to ferric ferric
high spin cytochrome 2 and the g3 signals are due to With the temperature maintained at lOoK,
low spin cytochrome 5 and 5 (8,9).
the sample was illuminated illumination,
the spectrum shown in Fig. 1B was obtained.
both the high-spin at 83.
for a few seconds with white light.
After
cessation of
Changesmay be noted in
(g6) components of cytochrome 5 and in the low-spin heme region
These changes are more clearly
shown in the difference
spectra of Figs. 2
and 3. The difference before illumination
spectrum in the g6 region obtained by subtracting from the signal after
illumination
(Fig. 2) shows the "creation"
of new high-spin heme components. At least two different signals may be characterized
by different
the signal
signals result.
degrees of "rhombicity".
The
The most asym-
metric signal which comprised the outermost peaks shows g, = 6.58 and g = 5.40. Y The other, more syouaetric, signal has principle g value peaks at g, = 6.20 and = 5.83. The spectrum represents approximately a doubling in the amount of epr% observable, high-spin heme. Difference in line position ward higher field Fig. 3.
spectra in the low spin hemeregion around g3 show an apparent shift of the large peak at g, = 3.05. after
illumination
resulting
The peak shifts
in the difference
The most prominant features of the difference
1268
about 10 gauss tospectrum shown in
spectrum are a trough at
BIOCHEMICAL
Vol. 48, No. 5, 1972
1000
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Iso0
2ooo
2500
H (gauss)
Figure 1. Typical epr spectra obtained from a single sample of submitochondrial particles before (A) and after (B) low temperature photodissociation. The particles, 30 mg protein/ml, were poised at +300 mVprior to freezing. The mediumcontained 0.2 M mannitol, 0.5 M sucrose, and 40 mMNa-morpholinopropane sulfonate at pH 7.0. The spectra were obtained from a single scan at microwave power 50 raw, frequency 9.15 GHz, time constant 1 sec., and a modulation amplitude of 40 gauss. The sample temperature was lOoK.
g = 3.07 and peak at g = 2.99.
The ratio
the peak is approximately
The area of the trough and the peak represent about
9% and 7%of the total
1.3.
of area contained in the trough to that in
g3 signal respectively.
net decrease in the observable low-spin signal. 2% of the overall
g3 peak.
Thus, illumination
This net decrease represents about
It should be noted that the g3 peak is only partially
to cytochrome 2 and contains major contributions
results in all respects except that the lower available preparations results
due
from cytochrome 2 and sl also (8,9).
Experiments with mitochondria and submitochondrial particles
tion in mitochondrial
causes an apparent
yield
identical
cytochrome oxidase concentra-
in lower signal-to-noise
ratios
in the ob-
served spectra. DISCXJSSION When the epr signals of cytochrome oxidase are measured at various oxidationreduction potentials,
the reduction of cytochrome s3 (Em = 385 mV> is accompaniedby
1269
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
9 100
9.0
600
80
70
800
65
60
55
1000
5p
1200
4.5
1400
1600
H (gauss)
Figure 2. Typical epr difference spectrum (after illumination minus before illumination) of the high-spin heme region. The sample conditions were the sameas in Fig. 1.
9 34
33
1900
3.2
2000
3.1
3.0
2100
2.9
2200
2,8
2300
, 2400
H (gauss)
Figure 3. Typical epr difference spectrum (after illumination minus before illuminaSample conditions were the sameas in Fig. 1 tion) of the low-spin heme region. except that the microwave power was 4.0 mwand the time constant was 0.3 sec. In the computer 8 scans of the spectrum before illumination were accumulated and subtracted from 8 scans after illumination in order to increase the signal to noise ratio.
the appearance of the high spin ferric in the medium, the ferric
heme signal of cytochrome 5.
If CO is present
cytochrome a observed when 5 is reduced is changed from
a high spin form to a mixture of high and low spin forms (8).
Thus the irreversible
photodissociation the ferric
of CO from reduced cytochrome a might result in a conversion of -3 cytochrome 2 from low spin to high spin if this is kinetically possible.
The results
in Figs. 1 through 3 show that at lOoK, the low to high spin transition
is largely
prevented by the low temperature and that the high spin componentwhich
1270
BIOCHEMICAL
Vol. 48, No. 5, 1972
a2,+-co hv lOOK 3+ +a;
aL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a? a:‘+$+
*
+a;
tco- Warmer
a2+ 3 3+ 0”
Figure 4. A schematic representation of the reactions accompanying the photodissociation of CO from cytochrome oxidase in the experiments described. The CO is initially bound to reduced cytochrome 3 and oxidized cytochrome a is present p$marily as a low spin (-$+) species in equilibrium with a small amount of high spin (3 ) species. Photodissociation at lOoK yields free cytochrome a~, a slig&decrease in low spin cytochrome a and a new high spin species of cytochrome a (a ). Warming the sample allows it to attain the state in the absence of COfor whit-It the cytochrome a is essen tially all high spin (8).
does appear is different
from that observed for comparable samples in the absence of
CO. A reasonable interpretation
of these results is that the heme groups of cyto-
chromes a and 53 are in very close proximity,
possibly aligned in such a way that
the principal
is located "between" the two hemes.
The failure
reactive
site
(for CO, 02 etc.)
to observe epr spectra of ferric
hemein the fully
oxidase (with the exception of a small g3 signal (8,lO) dipolar and/or antiferromagnetic
exchange interactions.
chrome s3 from high or low spin ferric ance of a signal from high-spin, Interactions
) could arise from magnetic Thus the conversion of cyto-
to low spin ferrous would result
ferric
of interactions
transmitted by the protein.
could have very low or zero activation
changes observed at lOoK (Fig. 4).
in the appear-
cytochrome a.
between the hemes (7-9,16) may consist partially
actions and partially hemeinteractions
oxidized cytochrome
These include shifts
of direct The direct
interheme-
energy and represent the in the epr spectrum of the
low spin hemeand someconversion of low spin to high spin heme. The heme-hemeinteractions
requiring
protein rearrangements would be expected to have much higher
activation
energies and would be "frozen
out" at lOoK (Fig.
4).
The latter
reactions
would be required to complete the conversion of the hemeof cytochrome a from the low spin form to high spin form and to allow the high spin form to change into the conformation present in the absence of CO (8). Carbon monoxide is a competitive
inhibitor
with oxygen (2,3) in respiration
and it would be reasonable to propose that the transformations
observed using COare
also present in the reactions of cytochrome oxidase with oxygen.
1271
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
Keilin, D. and Hartree, E.F. (1939) Proc. Roy. Sot. London, Ser. B. 125, 171. Warburg, 0. and Negelein, E. (1929) Biochem. 2. 214, 64. Keilin, D. (1927) Nature, London 119, 670. Lemberg, R. (169) Physiol. Rev. 49, 48. Nielsen, S.O. and Lehninger, A.L. (1955) J. Biol. Chem.215, 555. Wilson, D.F. and Dutton, P.L. (1970) Arch. Biochem. Biophys. 136, 583. Wilson, D.F., Lindsay, J.G. and Brocklehurst, E.S. (1972) Biochim. Biophys. Acta 256, 277. Wilson, D.F. and Leigh, J.S., Jr. (1972) Arch. Biochem. Biophys. 150, 154. Wilson, D.F., Leigh, J.S., Jr., Lindsay, J.G. and Dutton, P.L. In: Oxidases and Related Redox Systems II (T.E. King, Mason, H.S. and Morrison, M., eds.) Williams and Wilkins, 1972. van Gelder, B.F. and Beinert, 8. (1969) Biochim. Biophys. Acta 189, 1. Chance, B., Schoener, B. and Yonetani, T. (1965)In: Oxidases and Related Redox Systems (T.E. King, H.S. Mason and M. Morrison, eds), John Wiley, New York p. 609. Yonetani, T., ibid. p. 614. Dutton, P.L. (1971) Biochim. Biophys. Acta 226, 63. Wilson, D.F., Ereci&ska, M., Dutton, P.L. and Tsudzuki, T. (1970) Biochem. Biophys. Res. Commun.61, 1273. Chance, B. and Hagihara, B. (1961) Proceedings Fifth International Congress on Biochemistry (A.N. Sissakian, ed.) Vol. 5, p, 3, Pergamon Press, N.Y. Lindsay, J.G., Dutton, P.L. and Wilson, D.F. (1972) Biochemistry, 11, 1937.
1272
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HEME-HEME INTERACTIONSIN CYTOCHROME c OXIDASE; EFFECTSOF PHOTODISSOCIATION OF THE Cz COMPOUND* John S. Leigh, Jr. and David F. Wilson Johnson Research Foundation Department of Biophysics and Physical Biochemistry University of Pennsylvania Philadelphia, Pennsylvania 19104 Received
July
24,
1972 I
SUMMARY
The binding of CO to reduced cytochrome a results in the heme of oxidized cytochrome 2 changing from high spin to low sp -3 n. Photodissociation of the COat lOoK not only shifts the epr signal of the low spin heme to a higher magnetic field but also changes someof the low spin heme to high spin heme. Thus"heme-hemeinteraction"can occur even at lOoK.
c
INTRODUCTION Cytochrome c oxidase functions as the terminal electron donor of the mitochonaria1 electron
transport
chain.
Acting both as the catalyst
(l-3 - for review, see 4) and as a "free-energy (5,6),
its
conserving site"
which are conventionally dation-reduction
two
distinguishable
The 0x14
of cytochromes a and z3 at pH 7.2 are approxi-
mately +220 mV and +380 mV respectively cytochrome g3 is identified
ob-.
hememoieties (1,4)
designated as cytochrome 5 and cytochrome s3 (1).
midpoint potentials
+
for ATP synthesis
reaction mechanismhas been studied for many years. Spectrophotometric
servations have established the existence of
ally,
for oxygen reduction
(6,7) in uncoupled mitochondria.
Operation-
. +
as the cytochrome which reacts with molecular oxy-
gen and which is the CObinding pigment (l-4).
In addition,
the hemoprotein contains a
two moles of copper per mole of cytochromes 2 and z3 (4).
I
Recent experimental evidence that the two hemeunits are not independent of one another has come from spectrophotometric heme interaction coefficient
has been demonstrated:
(7) and epr (8-10) studies.
1) by the effect
Heme-
on the a-band extinction
of reduced cytochrome 5 (when CObinds to cytochrome s3);
2) by a shift
* Supported by National Science Foundation Grant GB 28125. D.F. Wilson is the recipient of U.S. Public Health Service Career Development Award l-KO4-G&18154.
Copyright AN rights
0 19 72 by Academic Press. Inc. of reproduction in any form reserved.
1266
~
BIOCHEMICAL
Vol. 48, No. 5, 1972
in the oxidation-reduction tochrome 23);
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
midpoint potential
3) by a shift
of cytochrome 5 (when CObinds to cy-
in the oxidation-reduction
chrome-3 a in the presence of azide;
midpoint potential
4) by epr spectral
of cyto-
changes of the high spin
hemea components on reduction of cytochrome z3 (8,9) and 5) by epr spectral changes of the low spin heme5 signals in the presence of COand azide under controlled redox conditions
(8).
In this paper we report the effects on the epr spectral
characteristics
from cytochrome z3 was originally
of COphotodissociation
of cytochrome 5.
The photodissociation
is essentially
At low temperatures the
remains quite rapid with high quantum efficiency
irreversible
of CO
discovered by Warburg and Negelein (2) and has been
studied at low temperatures by Chance and coworkers (11). photodissociation
from cytochrome a3
but the reaction
below s160°K (12).
MATERIALSANDMETHODS epr measurementswere performed with a Varian E-3 spectrometer equipped for low temperature operation.
The sample tube (quartz 4mmOD, 3mmID) was positioned in a
simple, unsilvered "flow-through" and liquid
helium flow (l-3 litersfhr.)
an efficient, Pa.).
quartz dewar (J.F.
flexible,
Illumination
liquid
was delivered
helium transfer
Calif.)
from the storage dewar through
line (Air Products, Inc.,
was provided by an unfiltered
spectra were obtained by subtraction
Scanlon, Co., Whittier,
Allentown,
Unitron Lamp (40 watts).
Difference
of the desired signals with a Varian C-1024 com-
puter. Oxidation-reduction
potentials
were measured by the method of Dutton (13,14).
The samples were prepared by allowing the stirred roughly 50-50 mixture of (02-free) adjusting
the redox potential
suspensions to equilibrate
argon gas and COfor at least 15 minutes and then
of the anaerobic mitochondrial
or submitochondrial pre-
parations to approximately +300 mV, such that cytochrome z3 was essentially duced and cytochrome 2 was 90%oxidized
(see ref.
diators were 40 uM phenazine methosulfate, cyanide.
with a
The oxidation-reduction
reme-
40 uM diaminodurene and 100 PM ferri-
The entire procedure was done essentially
1267
8).
all
in the dark and samples were
BIOCHEMICAL
Vol. 48, No. 5, 1972
quickly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
frozen in an isopentane-methylcyclohexane
The samples were maintained in the dark at liquid
bath maintained at about -192'C. nitrogen temperature until
mea-
surement. Pigeon heart mitochondria were prepared by the method of Chance and Hagihara (15: Submitochondrial particles of Lindsay --et al.
were prepared from pigeon heart mitochondria by the method
(16).
RESULTS A typical
low temperature epr spectrum of submitochondrial particles
the dark at +300 mV in the presence of CO is shown in Fig. IA.
taken in
The cytochrome 3
is present as the ferrous low spin CO compoundand has no measurable epr absorbance. The g6 signals are due to ferric ferric
high spin cytochrome 2 and the g3 signals are due to With the temperature maintained at lOoK,
low spin cytochrome 5 and 5 (8,9).
the sample was illuminated illumination,
the spectrum shown in Fig. 1B was obtained.
both the high-spin at 83.
for a few seconds with white light.
After
cessation of
Changesmay be noted in
(g6) components of cytochrome 5 and in the low-spin heme region
These changes are more clearly
shown in the difference
spectra of Figs. 2
and 3. The difference before illumination
spectrum in the g6 region obtained by subtracting from the signal after
illumination
(Fig. 2) shows the "creation"
of new high-spin heme components. At least two different signals may be characterized
by different
the signal
signals result.
degrees of "rhombicity".
The
The most asym-
metric signal which comprised the outermost peaks shows g, = 6.58 and g = 5.40. Y The other, more syouaetric, signal has principle g value peaks at g, = 6.20 and = 5.83. The spectrum represents approximately a doubling in the amount of epr% observable, high-spin heme. Difference in line position ward higher field Fig. 3.
spectra in the low spin hemeregion around g3 show an apparent shift of the large peak at g, = 3.05. after
illumination
resulting
The peak shifts
in the difference
The most prominant features of the difference
1268
about 10 gauss tospectrum shown in
spectrum are a trough at
BIOCHEMICAL
Vol. 48, No. 5, 1972
1000
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Iso0
2ooo
2500
H (gauss)
Figure 1. Typical epr spectra obtained from a single sample of submitochondrial particles before (A) and after (B) low temperature photodissociation. The particles, 30 mg protein/ml, were poised at +300 mVprior to freezing. The mediumcontained 0.2 M mannitol, 0.5 M sucrose, and 40 mMNa-morpholinopropane sulfonate at pH 7.0. The spectra were obtained from a single scan at microwave power 50 raw, frequency 9.15 GHz, time constant 1 sec., and a modulation amplitude of 40 gauss. The sample temperature was lOoK.
g = 3.07 and peak at g = 2.99.
The ratio
the peak is approximately
The area of the trough and the peak represent about
9% and 7%of the total
1.3.
of area contained in the trough to that in
g3 signal respectively.
net decrease in the observable low-spin signal. 2% of the overall
g3 peak.
Thus, illumination
This net decrease represents about
It should be noted that the g3 peak is only partially
to cytochrome 2 and contains major contributions
results in all respects except that the lower available preparations results
due
from cytochrome 2 and sl also (8,9).
Experiments with mitochondria and submitochondrial particles
tion in mitochondrial
causes an apparent
yield
identical
cytochrome oxidase concentra-
in lower signal-to-noise
ratios
in the ob-
served spectra. DISCXJSSION When the epr signals of cytochrome oxidase are measured at various oxidationreduction potentials,
the reduction of cytochrome s3 (Em = 385 mV> is accompaniedby
1269
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
9 100
9.0
600
80
70
800
65
60
55
1000
5p
1200
4.5
1400
1600
H (gauss)
Figure 2. Typical epr difference spectrum (after illumination minus before illumination) of the high-spin heme region. The sample conditions were the sameas in Fig. 1.
9 34
33
1900
3.2
2000
3.1
3.0
2100
2.9
2200
2,8
2300
, 2400
H (gauss)
Figure 3. Typical epr difference spectrum (after illumination minus before illuminaSample conditions were the sameas in Fig. 1 tion) of the low-spin heme region. except that the microwave power was 4.0 mwand the time constant was 0.3 sec. In the computer 8 scans of the spectrum before illumination were accumulated and subtracted from 8 scans after illumination in order to increase the signal to noise ratio.
the appearance of the high spin ferric in the medium, the ferric
heme signal of cytochrome 5.
If CO is present
cytochrome a observed when 5 is reduced is changed from
a high spin form to a mixture of high and low spin forms (8).
Thus the irreversible
photodissociation the ferric
of CO from reduced cytochrome a might result in a conversion of -3 cytochrome 2 from low spin to high spin if this is kinetically possible.
The results
in Figs. 1 through 3 show that at lOoK, the low to high spin transition
is largely
prevented by the low temperature and that the high spin componentwhich
1270
BIOCHEMICAL
Vol. 48, No. 5, 1972
a2,+-co hv lOOK 3+ +a;
aL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a? a:‘+$+
*
+a;
tco- Warmer
a2+ 3 3+ 0”
Figure 4. A schematic representation of the reactions accompanying the photodissociation of CO from cytochrome oxidase in the experiments described. The CO is initially bound to reduced cytochrome 3 and oxidized cytochrome a is present p$marily as a low spin (-$+) species in equilibrium with a small amount of high spin (3 ) species. Photodissociation at lOoK yields free cytochrome a~, a slig&decrease in low spin cytochrome a and a new high spin species of cytochrome a (a ). Warming the sample allows it to attain the state in the absence of COfor whit-It the cytochrome a is essen tially all high spin (8).
does appear is different
from that observed for comparable samples in the absence of
CO. A reasonable interpretation
of these results is that the heme groups of cyto-
chromes a and 53 are in very close proximity,
possibly aligned in such a way that
the principal
is located "between" the two hemes.
The failure
reactive
site
(for CO, 02 etc.)
to observe epr spectra of ferric
hemein the fully
oxidase (with the exception of a small g3 signal (8,lO) dipolar and/or antiferromagnetic
exchange interactions.
chrome s3 from high or low spin ferric ance of a signal from high-spin, Interactions
) could arise from magnetic Thus the conversion of cyto-
to low spin ferrous would result
ferric
of interactions
transmitted by the protein.
could have very low or zero activation
changes observed at lOoK (Fig. 4).
in the appear-
cytochrome a.
between the hemes (7-9,16) may consist partially
actions and partially hemeinteractions
oxidized cytochrome
These include shifts
of direct The direct
interheme-
energy and represent the in the epr spectrum of the
low spin hemeand someconversion of low spin to high spin heme. The heme-hemeinteractions
requiring
protein rearrangements would be expected to have much higher
activation
energies and would be "frozen
out" at lOoK (Fig.
4).
The latter
reactions
would be required to complete the conversion of the hemeof cytochrome a from the low spin form to high spin form and to allow the high spin form to change into the conformation present in the absence of CO (8). Carbon monoxide is a competitive
inhibitor
with oxygen (2,3) in respiration
and it would be reasonable to propose that the transformations
observed using COare
also present in the reactions of cytochrome oxidase with oxygen.
1271
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
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