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Cytochrome c . I. Effect of Cl− on the conformational transitions of ferricytochrome c at acid pH
BIOCHEMICAL
Vol. 31, No. 4, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
'XCOCHROME 2. I. EFFECTOF Cl- ON THE COIWOWIONAL TF&NSI'lTONSOF FERRICYTOCHROME 2 AT ACID pH
Daniel Fung and Serge Vinogradov Biochemistry
Department, School of Medicine
Wayne State University,
Detroit,
Michigan,
48207
Received April 15, 1968
The visible
absorption spectrum of ferricytochrome
over the pH range 0 - 14 five occurring
at pH 0.42,
The transition
2.5,
different
9.5
types, with transitions
and 12.76
from the type III,
(Theorell
of the central
c.
this transition
(Bull and Breese, 1966).
coordination
should occur at pH 2.12.
below neutrality
have recently
Their relationships
is
complex of
change accompanies
Boeri et al.
(1953) have
shown that in the absence of any anion, the transition to type II
It
absorption spectrum
An appreciable conformational
ferricy-tochrome
1941).
2 present
occurs at pH 2.5.
accompanied by profound changes in the visible from a disruption
and Bkesson,
low spin ferricytochrome
at neutral pH, to the high spin type II,
resulting
2 exhibits
from type III
Other conformational
transitions
been reported by Flatmark (1964,
to the transitions
observed earlier
1966).
remains unclear.
The weak absorption-band present in the spectrum of ferricytochrome 2 at 695 mu is known
molecule (Schejter
to be sensitive
et al.,
1963).
to conformational
It
in the
disappears in the presence of
denaturing agents, of ligands capable of subsituting two ligands in positions
changes
5 and 6 of the central
for one of the
coordination
complex
BIOCHEMICAL
Vol. 31, No. 4, 1968
of ferricytochrome
transfer
2, ana on increase in temperature (Schejter
Recently,
George, 1964) .
transition
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
this band has been assigned to a charge
(Eaton and Hochstrasser, 1967).
We report below somepreliminary ana extrinsic
and
results on the effect
KC1 on the intensities
bands of horse ferricytochrome
of low pH
of the 695 mu and 620 mu absorption
c.
Horse heart cytochrome 2 was Sigma type VI used without further purification, and
or type III,
Walasek, 1967).
distilled
chromatographed on Amberlite (X-50 (Margoliash
The cytochrome 2 was thoroughly
water prior to each experiment.
dialyzed against
The concentrations
from 0.5 to 2 x 10D4M. The spectrophotometric
titrations
ranged
were performed
on a Cary model 15 spectrophotometer and a glass apparatus consisting of a 2 cm quartz cell and a thermostatted
reservoir
fitted
with a
Radiometer glass-calomel electrode GK 220, and with microburettes the addition
of acid or base.
The assembly was maintained at 25M.l'C.
The pH was measured by meansof a Radiometer PHM4 pH meter. electrode was standardized before and after of standard solutions,
for
The glass
each experiment with a series
pH 1.68, 2.00, 3.00, 4.01 and 7.00 (Harleco Co.).
The solution of the ferricytochrome
2 was circulated
continuously
through
the cell placed in the sample compartment of the spectrophotometer, allowing simltaneous measurementof pH and absorption spectra. solutions were titrated The titrations
The
with concentrated HCl or H PO and 10 N KOH. 3
were performed starting
4
at neutral pH and proceeding
towards low PH. Figure 1 shows a typical ferricytochrome variation Figure 2.
spectrophotometric
titration
of horse
2 dissolved in water, with cont. HCl or $Ff$.
The
of absorbance at 695 mu and 620 mu with pH is shown in The reversibility
whereas reverse titrations forward titration.
of the titrations at 695 mu failed
at 620 mu was acceptable to reproduce exactly
Figures 3 and 4 show the spectra and titration
597
the
BIOCHEMICAL
Vol. 3 1, No. 4, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
1.5
1.3 1
I.1 -
s 0.9 t!
-
+z 5:
_
2
0.7 -
0.5 -
0.3 -
0.1 -
! Wavelength,
FIGURE
Forward
1.
titration
concentrated
mp
of horse H3FQ4 in the
heart
ferricytochrome
absence
of extrinsic
2 with KCl.
Temperature 25" C. pH: A-4.01, B-3.27, C-3.04, ~-2.87, ~-2.76, F-2.69, G-2.63, ~-2.54, I-2.49, J-2.43, K-2.36, L-2.28,
respectively,
curves,
presence
spin
for
N-2.02,
horse
o-1.80.
ferricytochrome
ctitrated
in the
of 1 M KCl.
In the high
M-2.20,
absence type
of extrinsic
ferricytochrome
salt,
the
c as indicated
transition by the
from
low
increase
spin in the
to
BIOCHEMICAL
Vol. 3 1, No. 4, 1968
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
2.0
3.0
4.0
PH
Plot
FIGUFZ 2.
of absorbance
and reverse
intensity
of the 620 mu band,
The midpoint 2.4
- 2.6.
the
entire
of this
point
only
from type
point
the
forward.
(data
spanning
III
the transition
the transition two types
completed
of Figure
by pH 1.8
to type
II
at 755 - 760 mu remains
at 645 mu remains
Assuming
involving
pH for
is effectively
transition
The isosbestic pH range
isosbestic range.
versus
titrations.
(Figure
constant
over
to be a simple
of ferricytochrome
l),
occurs
- 2.0. at pH
constant while
over
the
a more restricted protonation
pH
equilibrium
c molecules
c1 +nH+-c -2 the
experimental
titration
curves
variation with
presence of the
decreasing
of KC1 the intensities
pH, gives
rectified
(A2 - A)/(A
log In the
were
single
of the
- Al)
transition
using = pK'
the
relationship
- npH
exhibited
by the
695 mu and 620 mu absorption
way to two transitions,
599
one at a higher
peaks and
1)
Vol. 31, No. 4, 1968
BIOCHEMICAL
I
I I 650 700 750 Wavelength, mp
600
FIGURE 3.
Forward
titration
concentrated
3
I I 650 700 750 Wavelength, mp
600
of horse PO4 in the
c; pH: ~-4.48,
25'
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
heart
ferricytochrome of 1 M KCl.
presence
~-4.n,
c-3.85,
D-3.73,
2 with Temperature
E-3.64,
F-3.54,
G-3.40, H-3.30, I-3.10, J-2.86, K-2.53, L-1.86, M-1.47, N-1.27, 0-1.02, p-0.84, ~-0.68, R-0.56, S-0.41, T-0.22.
the
other
at a lower
pH than
the
first
transition
occurs
occurs
The maximum of the
begins
to shift
acidity
below
below
pH 2.5
below
pH 1.9.
titration
gradually pH 2.
to higher
curves
high-spin
The isosbestic wavelengths
are given
while
on 695 mu data
towards
The results
3 and 4).
(Figures
at pH 3.4 - 3.6,
0.8 based
at a pH of about
620 mu data.
previously
of the in Table
higher
wavelengths
and then rectification
600
second
and at ca.
absorption
point
1.
the
band with
at 645 mu begins remains
constant
of the
In 1 M KC1 transition 1.0 based at 620 mu increasing to shift at 675 mu
spectrophotometric
on
Vol. 31, No. 4, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
695 rnp
0.30,1
0.90 -
0.70 -
620 rnp
a50 I
I 1.o
I
I
2.0
3.0
I 4.0
PH
FIGURE4.
Plot of absorbance versus pH for the forward titration (data of Figure 4).
TABLE Effect transition
of extrinsic
1
Cl- on the conformational
of horse ferricytochrome
2 at acid pH
Decrease in absorbance at 695 mu pH midpoint"
n
Increase in absorbance at 620 mu pH midpoint"
n
In water
2.58
2.7
2.50
2.6
In 1 M KC1
3.58 0.78
1.5 1.5
3.51 0.95
1.5 1.6
a Obtained graphically.
601
BIOCHEMICAL
Vol. 3 1, No. 4, 1968
The transition type II-type
type
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-type
III
II
has n = 1.14, and the transition
I which occurs at pH 0.42, has n = 1.53 (Theorell
and
The values of n obtained spectrophotometrically
iikesson, 1941). the conformational
transitions
of ferricytochrome
for
2 at acid pH apparently The
vary with the wavelength at which the absorbance data is obtained. simplest interpretation relate
at present of the effect
the two observed transitions,
the transitions If this
type III-+type
interpretation
KC1 is to
at pH 3.5 and at ca. pH 0.8,
II
and type II-type
I,
to
respectively.
is accepted, the conclusion then is that the
presence of an excess of extrinsic II
of extrinsic
forms of ferricytochrome
Cl- ions stabilizes
5 relative
the type I and
to type III.
This work was supported in part by Research Grant HE 11620-01 the National Institutes
of Health, U. S. Public Health Service,
from and by a
grant from the Michigan Heart Association.
REFERENCES hesson, A. and Theorell, Boeri,
E., Ehrenberg, A., Paul, K. G. and Theorell,
Biophys. Acta, l2, Bull,
H., (1941), J. Am. Chem. Sot.,
63, 1812.
H., (1953), Biochim.
275.
H. and Breese, K., (1966)) Biochem. Biophys. Res. Comm.,&,
Eaton, W. M. and Hochstrasser, R. M., (1967), J. Chem. Phys., g, Flatmark,
T.,
(1964), Acta Chem. Scand., l.8, 2269.
Flatmark,
T.,
(1966), ibid.,
Margoliash,
'74. 2533.
2C, 1470.
E. and Walasek, 0. S., (1967), Methods in Enzymology, Estabrook,
R. W. and Pullman, M. E., eds., Academic Press, vol. Schejter,
A. and George, P., (1964), Biochemistry,
Schejter,
A., Glauser, S.C., George, P. and Margoliash,
Biochim. Biophys. Acta, 2,
641.
602
10, p. 339,
2, 1045. E., (1965),
Vol. 31, No. 4, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
'XCOCHROME 2. I. EFFECTOF Cl- ON THE COIWOWIONAL TF&NSI'lTONSOF FERRICYTOCHROME 2 AT ACID pH
Daniel Fung and Serge Vinogradov Biochemistry
Department, School of Medicine
Wayne State University,
Detroit,
Michigan,
48207
Received April 15, 1968
The visible
absorption spectrum of ferricytochrome
over the pH range 0 - 14 five occurring
at pH 0.42,
The transition
2.5,
different
9.5
types, with transitions
and 12.76
from the type III,
(Theorell
of the central
c.
this transition
(Bull and Breese, 1966).
coordination
should occur at pH 2.12.
below neutrality
have recently
Their relationships
is
complex of
change accompanies
Boeri et al.
(1953) have
shown that in the absence of any anion, the transition to type II
It
absorption spectrum
An appreciable conformational
ferricy-tochrome
1941).
2 present
occurs at pH 2.5.
accompanied by profound changes in the visible from a disruption
and Bkesson,
low spin ferricytochrome
at neutral pH, to the high spin type II,
resulting
2 exhibits
from type III
Other conformational
transitions
been reported by Flatmark (1964,
to the transitions
observed earlier
1966).
remains unclear.
The weak absorption-band present in the spectrum of ferricytochrome 2 at 695 mu is known
molecule (Schejter
to be sensitive
et al.,
1963).
to conformational
It
in the
disappears in the presence of
denaturing agents, of ligands capable of subsituting two ligands in positions
changes
5 and 6 of the central
for one of the
coordination
complex
BIOCHEMICAL
Vol. 31, No. 4, 1968
of ferricytochrome
transfer
2, ana on increase in temperature (Schejter
Recently,
George, 1964) .
transition
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
this band has been assigned to a charge
(Eaton and Hochstrasser, 1967).
We report below somepreliminary ana extrinsic
and
results on the effect
KC1 on the intensities
bands of horse ferricytochrome
of low pH
of the 695 mu and 620 mu absorption
c.
Horse heart cytochrome 2 was Sigma type VI used without further purification, and
or type III,
Walasek, 1967).
distilled
chromatographed on Amberlite (X-50 (Margoliash
The cytochrome 2 was thoroughly
water prior to each experiment.
dialyzed against
The concentrations
from 0.5 to 2 x 10D4M. The spectrophotometric
titrations
ranged
were performed
on a Cary model 15 spectrophotometer and a glass apparatus consisting of a 2 cm quartz cell and a thermostatted
reservoir
fitted
with a
Radiometer glass-calomel electrode GK 220, and with microburettes the addition
of acid or base.
The assembly was maintained at 25M.l'C.
The pH was measured by meansof a Radiometer PHM4 pH meter. electrode was standardized before and after of standard solutions,
for
The glass
each experiment with a series
pH 1.68, 2.00, 3.00, 4.01 and 7.00 (Harleco Co.).
The solution of the ferricytochrome
2 was circulated
continuously
through
the cell placed in the sample compartment of the spectrophotometer, allowing simltaneous measurementof pH and absorption spectra. solutions were titrated The titrations
The
with concentrated HCl or H PO and 10 N KOH. 3
were performed starting
4
at neutral pH and proceeding
towards low PH. Figure 1 shows a typical ferricytochrome variation Figure 2.
spectrophotometric
titration
of horse
2 dissolved in water, with cont. HCl or $Ff$.
The
of absorbance at 695 mu and 620 mu with pH is shown in The reversibility
whereas reverse titrations forward titration.
of the titrations at 695 mu failed
at 620 mu was acceptable to reproduce exactly
Figures 3 and 4 show the spectra and titration
597
the
BIOCHEMICAL
Vol. 3 1, No. 4, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
1.5
1.3 1
I.1 -
s 0.9 t!
-
+z 5:
_
2
0.7 -
0.5 -
0.3 -
0.1 -
! Wavelength,
FIGURE
Forward
1.
titration
concentrated
mp
of horse H3FQ4 in the
heart
ferricytochrome
absence
of extrinsic
2 with KCl.
Temperature 25" C. pH: A-4.01, B-3.27, C-3.04, ~-2.87, ~-2.76, F-2.69, G-2.63, ~-2.54, I-2.49, J-2.43, K-2.36, L-2.28,
respectively,
curves,
presence
spin
for
N-2.02,
horse
o-1.80.
ferricytochrome
ctitrated
in the
of 1 M KCl.
In the high
M-2.20,
absence type
of extrinsic
ferricytochrome
salt,
the
c as indicated
transition by the
from
low
increase
spin in the
to
BIOCHEMICAL
Vol. 3 1, No. 4, 1968
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
2.0
3.0
4.0
PH
Plot
FIGUFZ 2.
of absorbance
and reverse
intensity
of the 620 mu band,
The midpoint 2.4
- 2.6.
the
entire
of this
point
only
from type
point
the
forward.
(data
spanning
III
the transition
the transition two types
completed
of Figure
by pH 1.8
to type
II
at 755 - 760 mu remains
at 645 mu remains
Assuming
involving
pH for
is effectively
transition
The isosbestic pH range
isosbestic range.
versus
titrations.
(Figure
constant
over
to be a simple
of ferricytochrome
l),
occurs
- 2.0. at pH
constant while
over
the
a more restricted protonation
pH
equilibrium
c molecules
c1 +nH+-c -2 the
experimental
titration
curves
variation with
presence of the
decreasing
of KC1 the intensities
pH, gives
rectified
(A2 - A)/(A
log In the
were
single
of the
- Al)
transition
using = pK'
the
relationship
- npH
exhibited
by the
695 mu and 620 mu absorption
way to two transitions,
599
one at a higher
peaks and
1)
Vol. 31, No. 4, 1968
BIOCHEMICAL
I
I I 650 700 750 Wavelength, mp
600
FIGURE 3.
Forward
titration
concentrated
3
I I 650 700 750 Wavelength, mp
600
of horse PO4 in the
c; pH: ~-4.48,
25'
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
heart
ferricytochrome of 1 M KCl.
presence
~-4.n,
c-3.85,
D-3.73,
2 with Temperature
E-3.64,
F-3.54,
G-3.40, H-3.30, I-3.10, J-2.86, K-2.53, L-1.86, M-1.47, N-1.27, 0-1.02, p-0.84, ~-0.68, R-0.56, S-0.41, T-0.22.
the
other
at a lower
pH than
the
first
transition
occurs
occurs
The maximum of the
begins
to shift
acidity
below
below
pH 2.5
below
pH 1.9.
titration
gradually pH 2.
to higher
curves
high-spin
The isosbestic wavelengths
are given
while
on 695 mu data
towards
The results
3 and 4).
(Figures
at pH 3.4 - 3.6,
0.8 based
at a pH of about
620 mu data.
previously
of the in Table
higher
wavelengths
and then rectification
600
second
and at ca.
absorption
point
1.
the
band with
at 645 mu begins remains
constant
of the
In 1 M KC1 transition 1.0 based at 620 mu increasing to shift at 675 mu
spectrophotometric
on
Vol. 31, No. 4, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
695 rnp
0.30,1
0.90 -
0.70 -
620 rnp
a50 I
I 1.o
I
I
2.0
3.0
I 4.0
PH
FIGURE4.
Plot of absorbance versus pH for the forward titration (data of Figure 4).
TABLE Effect transition
of extrinsic
1
Cl- on the conformational
of horse ferricytochrome
2 at acid pH
Decrease in absorbance at 695 mu pH midpoint"
n
Increase in absorbance at 620 mu pH midpoint"
n
In water
2.58
2.7
2.50
2.6
In 1 M KC1
3.58 0.78
1.5 1.5
3.51 0.95
1.5 1.6
a Obtained graphically.
601
BIOCHEMICAL
Vol. 3 1, No. 4, 1968
The transition type II-type
type
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-type
III
II
has n = 1.14, and the transition
I which occurs at pH 0.42, has n = 1.53 (Theorell
and
The values of n obtained spectrophotometrically
iikesson, 1941). the conformational
transitions
of ferricytochrome
for
2 at acid pH apparently The
vary with the wavelength at which the absorbance data is obtained. simplest interpretation relate
at present of the effect
the two observed transitions,
the transitions If this
type III-+type
interpretation
KC1 is to
at pH 3.5 and at ca. pH 0.8,
II
and type II-type
I,
to
respectively.
is accepted, the conclusion then is that the
presence of an excess of extrinsic II
of extrinsic
forms of ferricytochrome
Cl- ions stabilizes
5 relative
the type I and
to type III.
This work was supported in part by Research Grant HE 11620-01 the National Institutes
of Health, U. S. Public Health Service,
from and by a
grant from the Michigan Heart Association.
REFERENCES hesson, A. and Theorell, Boeri,
E., Ehrenberg, A., Paul, K. G. and Theorell,
Biophys. Acta, l2, Bull,
H., (1941), J. Am. Chem. Sot.,
63, 1812.
H., (1953), Biochim.
275.
H. and Breese, K., (1966)) Biochem. Biophys. Res. Comm.,&,
Eaton, W. M. and Hochstrasser, R. M., (1967), J. Chem. Phys., g, Flatmark,
T.,
(1964), Acta Chem. Scand., l.8, 2269.
Flatmark,
T.,
(1966), ibid.,
Margoliash,
'74. 2533.
2C, 1470.
E. and Walasek, 0. S., (1967), Methods in Enzymology, Estabrook,
R. W. and Pullman, M. E., eds., Academic Press, vol. Schejter,
A. and George, P., (1964), Biochemistry,
Schejter,
A., Glauser, S.C., George, P. and Margoliash,
Biochim. Biophys. Acta, 2,
641.
602
10, p. 339,
2, 1045. E., (1965),