Effect of Heme Iron Valence State on the Conformation of
Cytochrome c and Its Association with Membrane
Interfaces
A CD AND EPR INVESTIGATION*
Iseli L.
Nantes
,
Maria R.
Zucchi§,
Otaciro R.
Nascimento§, and
Adelaide
Faljoni-Alario¶
From the
Centro Interdisciplinar de
Investigação Bioquímica (CIIB), Prédio I,
Sala 1S-15, Diretoria de Pesquisa e Pós-Graduação,
Universidade de Mogi das Cruzes (UMC), CP 411, Mogi das Cruzes,
São Paulo, CEP 08780-911, Brazil, § Grupo de
Biofísica, Instituto de Física de São Carlos,
Universidade de São Paulo-São Carlos, CP 369, São
Paulo, CEP 13560-970 Brazil, and ¶ Departamento de
Bioquímica, Instituto de Química, Universidade de
São Paulo,
CP 26077 São Paulo, CEP 05513-970, Brazil
Received for publication, July 17, 2000, and in revised form, October 10, 2000
 |
ABSTRACT |
Recently cytochrome c has been
mentioned as an important mediator in the events of cellular oxidative
stress and apoptosis. To investigate the influence of charged
interfaces on the conformation of cytochrome c, the CD and
magnetic circular dichroic behavior of ferric and ferrous cytochrome
c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and
dicetylphosphate liposomes was studied in the 300-600 and 200-320 nm
wavelength region. EPR spectra demonstrate that the association of
cytochrome c with membranes promotes alterations of the
crystal field symmetry and spin state of the heme Fe3+. The
studies also include the effect of Pi, NaCl, and
CaCl2. Magnetic circular dichroism and CD results show that
the interaction of both ferrous and ferric cytochrome c
with charged interfaces promotes conformational changes in the
-helix content, tertiary structure, and heme iron spin state.
Moreover, the association of cytochrome c with different
liposomes is sensitive to the heme iron valence state. The more
effective association with membranes occurs with ferrous cytochrome
c. Dicetylphosphate liposomes, as a negatively
charged membrane model, promoted a more pronounced conformational
modification in the cytochrome c structure. A decrease in
the lipid/protein association is detected in the presence of increasing
amounts of CaCl2, NaCl, and Pi, in response to
the increase of the ionic strength.
 |
INTRODUCTION |
Cytochrome c is a peripheral membrane protein, and its
interaction with negatively charged interfaces is a well known
phenomenon (1, 2). The binding of cytochrome c with
phospholipid bilayers probably encompasses electrostatic and
hydrophobic interactions (3) and induces conformational alterations in
the protein (4, 5). In this regard, several works have been concerned
with structure-function relationships of cytochrome c (1, 3, 6). Besides its well established role as an electron carrier in the
mitochondrial respiratory chain, cytochrome c also exhibits oxidase/peroxidase activity on several substrates, including
t-butylhydroperoxide, aldehydes, and
-diketones (7-11).
Cytochrome c associated with the inner mitochondrial
membrane or with dicetylphosphate
(DCP)1 liposomes can oxidize
diphenylacetaldehyde and methylacetoacetone (9, 10). This
reaction produces excited species that, in mitochondria, lead to
oxidative injury of the organelle. The requirement of Pi
for the occurrence of this reaction was attributed exclusively to the
increase in the rate of aldehyde enolization catalyzed by this anion
(12). Kowaltowski et al. (13) recently have proposed
a model for phosphate-stimulated lipid peroxidation. In this model,
high phosphate concentration and Ca2+ cooperate in reactive
oxygen species-mediated mitochondrial damage. The importance of
electronically excited triplet states in biological events has been
extensively discussed by Cilento and Adam (14).
The recent demonstration that the apoptosis cascade involves cytochrome
c-promoted caspase activation (15, 16) shows that cytochrome
c plays a broader role in the cells than electron transport in the respiratory chain. In this regard, evidence has been provided for a relationship between apoptosis, the Ca2+-induced
permeability transition pore (17), and hydrogen peroxide production
(18).
In the present work we examine the different types of cytochrome
c/membrane association influenced by the heme iron redox state, membrane charge, and ionic strength. For this purpose, CD and
magnetic circular dichroism (MCD) have been used to detect the
conformational alterations in the protein induced by the interfacial microenvironment. These techniques are sensitive to the secondary and
tertiary structures of the protein and also reflect the relative configuration of the prosthetic group within the protein. EPR was
employed to refine the results, providing accurate information about
the iron valence, spin state, and change in local symmetry of the iron
crystal field around iron.
 |
MATERIALS AND METHODS |
Chemicals--
Cytochrome c (horse heart, type III),
HEPES, phosphatidylcholine (egg yolk), phosphatidylethanolamine (ovine
brain, type II-S), cardiolipin (bovine heart), and dicetylphosphate
were purchased from Sigma.
Liposome Preparation--
DCP liposomes were prepared in HEPES
buffer according to Mortara et al. (19). DCP was suspended
in HEPES buffer and sonicated for 20 min with a microtip-equipped
Cole-Parmer 4710 series ultrasonic processor at an output of 60 watts. In the preparation of
phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PC/PE/CL;
15/12/9 molar ratio) liposomes, the lipids were first dissolved in
chloroform and the chloroform evaporated with N2, and the
resulting test tube film was stored at
20 °C until use. Prior to
the experiments, cold HEPES buffer was added to the tube, mixed with a
Vortex mixer, and sonicated at 60 watts on ice in 30-s bursts with
1-min cooling intervals. Liposome preparations were centrifuged
at 8000 × g and 4 °C for 1 h to precipitate
the Ti released by the sonicator tip. Similar procedures were used in
the preparation of CL and PE liposomes.
CD and MCD Spectrometry--
CD and MCD measurements of
cytochrome c (10 µM) were conducted in a Jasco
J-720 spectropolarimeter. For the MCD the magnetic field was 870 mT and
the optical path 5 mm. The spectra were obtained at room temperature,
pH 7.4.
EPR Spectrometry--
EPR measurements of cytochrome
c (300 µM) were obtained by using an X-band
Varian E-109 spectrometer and/or an X-band Bruker ELEXSYS E580
spectrometer, equipped with a helium cryostat and temperature
controller from Oxford Company under the following conditions: gain,
5 × 103; modulation amplitude, 1.0 mT; microwave
power, 4 milliwatt; temperature, 11 K; and time constant, 0.064 s.
After the addition of cytochrome c in different
medium, 120 µl of the mixture was quickly introduced
into an EPR quartz tube, cooled in liquid nitrogen, and then
transferred to the helium cryostat into the EPR cavity to obtain the spectra.
 |
RESULTS |
Liposome-induced Structural Modifications in the Cytochrome c Heme
Group--
In Fig. 1, the MCD spectra
(top) and UV-visible absorbance (bottom) of
ferric cytochrome c in HEPES buffer (solid line),
PC/PE/CL (dashed line), and DCP liposomes
(dotted line) are shown. A Cotton effect in the Soret band,
with a positive band at 400 nm and a negative band at 418 nm, is
exhibited in the MCD spectra. The DCP-bound cytochrome c
spectrum shows a decrease and a hypsochromic effect in the
negative band with a minimum at 414 nm. The UV-visible spectra
also show a hypsochromic effect in the presence of DCP liposomes.

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Fig. 1.
MCD (top) and UV-visible
(bottom) spectra of ferric cytochrome
c. The MCD spectra (top) and
UV-visible absorbance (bottom) of ferric cytochrome
c in HEPES buffer, pH 7.4, at room temperature (solid
line), PC/PE/CL (dashed line), and DCP liposomes
(dotted line) are shown. The magnetic field is 8.6 mT and the optical path is 0.5 cm.
|
|
The same experiments were carried out with ferrous cytochrome
c. Fig. 2 shows that the
spectrum of ferrous cytochrome c in 10 mM HEPES,
pH 7.4, is quite different from that obtained with ferric cytochrome
c. The ferrous cytochrome c MCD spectrum only exhibits a positive band at 418 nm (Fig. 2, top), which
accompanies the bathochromic effect observed in the Soret band (Fig. 2,
bottom). Furthermore, a remarkable Cotton effect appears in
the 549-nm band that characterizes the ferrous cytochrome c
visible spectrum. In the presence of PC/PE/CL liposomes, the ferrous
cytochrome c MCD and UV-visible spectra are very similar to
those obtained with the oxidized hemeprotein (Fig. 1). The spectrum of
ferrous cytochrome c obtained in the presence of DCP
liposomes suggests a partitioning of the protein between the vesicles
and the aqueous medium.

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Fig. 2.
MCD (top) and UV-visible
(bottom) spectra of ferrous cytochrome
c. The MCD spectra (top) and
UV-visible absorbance (bottom) of ferrous cytochrome
c in HEPES buffer, pH 7.4, at room temperature (solid
line), PC/PE/CL (dashed line), and DCP liposomes
(dotted line) are shown. The magnetic field is 8.6 mT, and
the optical path is 0.5 cm.
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To improve the comprehension of the association of cytochrome
c with the charged interfaces, spectra of cytochrome
c were recorded with increasing amounts of membrane. Fig. 4
shows the alterations in the MCD band in the Soret region that
accompany the progressive association of the hemeprotein with DCP
liposomes. In fact, the association of ferric cytochrome c
with DCP liposomes promotes a decrease of the molar ellipticity of the
negative band at 415 nm (Fig. 3,
inset) associated with a
hypsochromic effect (Fig. 3). In contrast, increasing the amount of
PC/PE/CL liposomes does not promote any significant alteration in the
ferric cytochrome c MCD spectra (not shown).

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Fig. 3.
MCD spectra of cytochrome c
in the presence of different DCP concentrations.
Inset, the normalized molar ellipticity ( ) 415 nm
in each lipid concentration. The experiments were carried out in HEPES
buffer, pH 7.4, at room temperature. The magnetic field is 8.6 mT, and
the optical path is 0.5 cm.
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Unlike ferric cytochrome c, PC/PE/CL liposomes promote
striking modifications of the MCD spectra of ferrous cytochrome
c in both the Soret and Q bands (Fig.
4). The inset of Fig. 4 shows that the association of ferrous cytochrome c with PC/PE/CL
liposomes exhibits a sigmoidal behavior. The same experiment realized
with DCP liposomes also leads to remarkable modifications in the
ferrous cytochrome c spectra (Fig.
5); however, this titration does not show
a sigmoidal behavior (Fig. 5, inset).

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Fig. 4.
MCD spectra of cytochrome c
at different PC/PE/CL concentrations. Inset, the
normalized molar ellipticity ( ) to 400 and 550 nm at each
lipid concentration. The lipid/protein ratios used were 0, 7.5, 15, 30, 60, and 120. The experiments were carried out in PC/PE/CL liposomes in
the presence of HEPES buffer, pH 7.4, at room temperature. The magnetic
field is 8.6 mT, and the optical path is 0.5 cm.
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Fig. 5.
MCD spectra of cytochrome c
at different DCP concentrations. Inset, the
normalized molar ellipticity ( ) at 400 and 550 nm at each
lipid concentration. The lipid/protein ratios used were 0, 7.5, 15, 30, 60, and 150. The experiments were carried out in PC/PE/CL liposomes in
the presence of HEPES buffer, pH 7.4, at room temperature. The magnetic
field is 8.6 mT, and the optical path is 0.5 cm.
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EPR spectra of ferric cytochrome c obtained with increasing
amounts of PC/PE/CL liposomes, shown as the lipid/protein ratio (Fig.
6A), corroborate the
insignificant alteration in the cytochrome c heme iron as
indicated by the MCD results. Above the lipid/protein ratio of 24, it
is possible to see the appearance of a small quantity of high spin form
(g = 6.0, low field region). Above the lipid/protein ratio of 12, the two narrow signals (g = 4.3, around 160 mT and g = 2.0, around 330 mT) appear, provided by the reaction of cytochrome c heme iron with cardiolipin peroxide derivatives and a
radical product, respectively. EPR spectra obtained with increasing
amounts of DCP liposomes are in agreement with the MCD spectra obtained under similar conditions (Fig. 6B). Above the lipid/protein
ratio of 12, a high spin form (g
= 6.0, low field
region, and g
= 2.0, high field region) is observed that
becomes predominant at higher lipid/protein ratios. A different signal
of low spin state superimposed on the signal of the protein in
homogeneous medium can be easily identified in the 220 mT
region. At the lipid/protein ratio of 24, the concomitant existence of
the three species can be clearly identified.

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Fig. 6.
A, EPR spectra of ferric
cytochrome c in PC/PE/CL liposomes at different
lipid/protein ratios showing the low spin form of free cytochrome
c (0) and bound cytochrome c (lipid/protein
ratios, 12, 24, 36, 48, and 60). Two paramagnetic species of bound
cytochrome c are detected: a high spin state with rhombic
symmetry (~150 mT) and a high spin state with axial symmetry (~110
mT). The narrow line at ~330 mT corresponds to a
lipid-derived radical, a.u., arbitrary units. B, EPR
spectra of ferric cytochrome c in DCP liposomes at different
lipid/protein ratios, showing the low spin form of free cytochrome
c (0) and bound cytochrome c (12, 24, 36, 48, and
60). Two paramagnetic species of bound cytochrome c are
detected: high spin state (lowest field line and highest field line)
and low spin state clearly visualized at ratio 36. EPR spectra were
obtained at a temperature of 11 K, with 1.0 mT of field modulation and
4.0 milliwatts of microwave power in X-band.
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As described above the association of ferrous cytochrome c
with charged interfaces leads to MCD and UV-visible spectral changes very similar to those that occur upon conversion of ferrous cytochrome c to ferric cytochrome c (compare Figs. 1 and 2).
EPR measurements with liposome-bound ferrous cytochrome c
exclude the possibility of conversion of cytochrome c from
ferrous to ferric after association with membranes because no EPR
signal was observed. Moreover, the MCD spectral changes are reversible
when liposome-bound cytochrome c is dissociated from the
PC/PE/CL membrane by increasing the ionic strength as shown in Fig.
7A.

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Fig. 7.
A, MCD spectra of ferrous cytochrome
c at different ionic strengths. Arrow a, the
spectra obtained under the following conditions: 10 mM
HEPES + 100 mM NaCl, 10 mM HEPES, 2 mM PC/PE/CL + 100 mM NaCl, 2 mM
PC/PE/CL + 35 mM Pi. Arrow b, the
spectrum obtained in 0.8 mM PC/PE/CL. Arrow c,
the spectrum obtained in 2 mM PC/PE/CL. The experiments
were carried out at pH 7.4 at room temperature. The magnetic field is
8.6 mT, and the optical path is 0.5 cm. B, MCD spectra of
ferrous cytochrome c at different ionic strengths.
Arrow a, the spectrum obtained in 10 mM HEPES.
Arrow b, the spectra obtained in 2 mM PC/PE/CL
(lipid/protein ratio, 150:1) + 1 mM CaCl2 or
0.8 mM PC/PE/CL (lipid/protein ratio, 60:1). Arrow
c, the spectrum obtained in 2 mM PC/PE/CL. The
experiments were carried out at pH 7.4 at room temperature.
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As previously mentioned (13), Ca2+ promotes modifications
in the organization of lipid bilayers. However, the results obtained from CaCl2 titration indicate that, in this model, the
effect of Ca2+ is restricted to an increase of the ionic
strength, promoting the dissociation of the hemeprotein from the
PC/PE/CL membrane (Fig. 7B). In this regard, the addition of
1 mM CaCl2 to 13.5 µM ferrous
cytochrome c bound to 2 mM PC/PE/CL liposomes
(lipid/protein ratio, 150:1), a condition in which all
cytochrome c molecules are initially membrane-bound, leads
to a partial dissociation of the protein similar to that of a
lipid/protein ratio of 60:1.
The Lipid-induced Structural Modifications of Cytochrome c Extend
to the Secondary Structure Content--
The MCD spectra in Figs. 1 and
2 reveal that the association of cytochrome c with lipid
bilayers promotes alterations in the structure of the heme group of
this protein. To assess whether this effect of cytochrome
c/lipid association extends to the protein fraction,
the UV CD spectra of cytochrome c were obtained in these three different media. Analysis of the spectra (not shown) indicates that the association of cytochrome c with charged interfaces
promotes modifications in the secondary structure. The spectra have
been analyzed using the Selcon method (20, 21) to obtain fractions of
and
and the remainder secondary structure, and these
parameters are compared with those obtained from crystallographic data
(Tables I and II). In accordance with the
data obtained in the Soret and visible region, the UV CD spectra reveal
that DCP promotes the most significant modification in both ferrous
(Table I) and ferric (Table II)
cytochrome c secondary structure.
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Table I
Secondary structure fraction obtained from far UV () CD spectra
of ferrous cytochrome c in different media
The CD analysis of cytochrome c proceeded at pH 7.4. The
Sreerama and Woody analyses (20, 21) of the cytochrome c
spectra (not shown) are given below.
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Table II
Secondary structure fraction obtained from far UV () CD spectra
of ferric cytochrome c in different media
The CD analysis of cytochrome c proceeded at pH 7.4. The
Sreerama and Woody analyses (20, 21) of the cytochrome c
spectra (not shown) are given below.
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 |
DISCUSSION |
The Association of Cytochrome c with Liposomes Promotes
Conformational Changes in the Protein--
The MCD spectra of ferric
and ferrous cytochrome c reveal that the association of this
hemeprotein with vesicles promotes conformational changes in the heme
group and protein fraction (Figs. 1 and 2; Tables I and II). However,
the comparison of Figs. 1 and 2 reveals that only the association with
DCP liposomes promotes significant conformational alterations in both
ferric and ferrous cytochrome c.
The Association of Cytochrome c with an Inner Mitochondrial
Membrane Model Is a Cooperative Process--
The plot of molar
ellipticity at 415 nm for ferric cytochrome c (Fig. 3,
inset), or at 400 and 550 nm for ferrous cytochrome c, versus DCP concentration reveals a hyperbolic
curve. The same curves obtained for PC/PE/CL-bound ferrous cytochrome
c are sigmoidal, suggesting that binding of this hemeprotein
to an inner mitochondrial mimetic membrane is a cooperative process. As
a result, the association of cytochrome c to PC/PE/CL might
alter the membrane structure, favoring the attachment of other
cytochrome c molecules.
The Association of Cytochrome c with Charged Interfaces Is
Modulated by the Heme Iron Oxidation State--
Whereas the MCD and
EPR spectra of ferric cytochrome c in PC/PE/CL liposomes
(Figs. 1 and 6A) suggest a lipid/protein interaction with
little protein modification, Fig. 2 indicates an interaction with strong modification in the case of PC/PE/CL liposomes, when the
heme iron of cytochrome c is in oxidation state II. In the case of DCP liposomes, the MCD and EPR spectra suggest a strong modification in both ferric and ferrous cytochrome c (Figs.
3, 5, and 6B). The titration of ferrous cytochrome
c with DCP liposomes exhibits protein partitioning between
the aqueous and liposomal media (Fig. 5). Increasing the
DCP/protein ratio up to 320:1 does not modify the spectra (not shown)
from those obtained with a 120:1 lipid/protein ratio of 120:1 (Fig. 5).
This partitioning suggests a weak lipid-protein interaction.
The Association of Ferric and Ferrous Cytochrome c with Charged
Interfaces Promotes Spin State Changes in the Heme Iron--
The
comparison of the MCD and absorbance spectra of ferric cytochrome
c in homogeneous medium (Fig. 1, top and
bottom, respectively, solid lines) with
those of ferrous cytochrome c in PC/PE/CL liposomes (Fig. 2,
top and bottom, respectively, dashed
lines) might suggest, at first glance, that the association of
cytochrome c with biological mimetic membranes leads to the
oxidation of the heme iron. Iwase et al. (22) propose
a reaction of ferrous cytochrome c and cardiolipin with
production of the monoepoxide of linoleic acid and oxidation of the
heme iron. The authors suggest a change of the heme iron redox state
based only on the absorbance spectral changes (loss of the 549-nm band)
observed after the addition of cytochrome c to a
cardiolipin-containing medium. However, our results show that the
detachment of the protein from DCP or PC/PE/CL liposomes induced by
increasing the ionic strength with NaCl or Pi (Fig. 7A) recovers the ferrous cytochrome c spectra.
This clearly indicates the absence of oxidation of heme iron by
phospholipids. This result was corroborated by titration with
CaCl2 (Fig. 7B), which concomitantly reverts the
ferrous cytochrome c MCD spectra in the presence of lipid
membranes. This spectral modification must therefore be explained by
environmental modifications in the heme iron crevice. One possibility
is a change in the heme iron spin state modulated by conformational
changes controlling the heme iron crystal field. In homogeneous medium
the absorbance spectrum of ferric cytochrome c exhibits a
695-nm charge transfer band, indicative of the heme iron sixth
coordination position with methionine 80 (7) that disappears in
the presence of charged interfaces (10). This charged
interface-promoted loss of the cytochrome c sixth
coordination position significantly enhances the peroxidase/oxidase
activity of this protein (8-11). The comparison of the results
obtained with ferric cytochrome c bound to DCP and PC/PE/CL
liposomes provides the following information. (i) The secondary
structure modifications are more significant in DCP vesicles than
PC/PE/CL vesicles. (ii) Contrary to that observed in PC/PE/CL vesicles,
the Soret band of the MCD spectrum in DCP vesicles shows a clear
difference when compared with homogeneous medium. (iii) Unlike in
PC/PE/CL vesicles, the EPR spectra in DCP vesicles clearly show the
presence of both spin states (low and high). The existence of this high
spin state species has been described in the literature as a denatured
cytochrome c (23). However, the addition of NaCl promotes
the detachment of the hemeprotein from the membrane as observed by the
decrease of the signal of the high spin state form and the recuperation
of the signal of the native low spin state form (not shown). This is in
agreement with the MCD results obtained with ferrous MCD (Fig. 7,
A and B). Moreover, the analysis of the UV CD
spectra of ferrous and ferric cytochrome c is
incompatible with protein denaturation, as shown in Tables I and II.
These results could be explained if we assume the interaction between
the charged vesicles and protein as a mechanism of modulation of the
crystal field of the iron moiety removing the sulfur atom from the heme
iron coordination sphere and changing the spin state from low to high
form. If this assumption is correct, similar behavior should occur with
ferrous cytochrome c. In this case, the UV CD and MCD
results show that both vesicle types produce significant changes in the
cytochrome c secondary and tertiary structure (Tables I and
II; Figs. 2, 4, and 5). In the case of the MCD results for ferrous
cytochrome c, drastic modifications in the 550-nm range are
observed when the protein is associated with the membrane. To decide
whether this modification reflects the change of heme iron spin state, we compared these results with MCD of human met-, oxy-, and
deoxyHb (not shown), the heme iron valence and spin states of
which are well known (24). This comparison shows that ferrous
cytochrome c heme iron in homogeneous medium is in
the low spin state (S = 0) and exhibits a strong signal
at 550 nm in MCD spectra. When associated with the membranes,
ferrous cytochrome c heme iron is in the high spin state
form (S = 2). In this case, the strong signal at 550 nm
in the MCD spectra disappears. The similarity of the ferrous cytochrome
c high spin state MCD and absorbance spectra (membrane
bound) with the ferric cytochrome c MCD and absorbance
spectra (compare Figs. 1 and 2) could induce the erroneous interpretation that cytochrome c oxidation is promoted by
the membrane phospholipids (22).
Possible Implications of the Different Types of Ferrous and Ferric
Cytochrome c Association with Membranes--
One important aspect of
the above results is the fact that only ferrous cytochrome c
exhibits an association with biological membranes accompanied by
significant structural alterations in the protein. Moreover, only the
association of ferrous cytochrome c with mimetic biological
membranes exhibits cooperative behavior, suggesting conformational
changes in the lipid bilayer. This high specificity for heme iron
valence in determining the type of interaction and the affinity of
cytochrome c for biological membranes suggests a
relationship to the role played by cytochrome c in cell
apoptosis. In this regard, this could be the point where the
participation of cytochrome c in the respiratory chain and
in apoptosis shows overlap of their regulatory mechanisms. Recently,
several reports have correlated the cytochrome c redox state
with its upstream (cytochrome c release from inner
mitochondrial membrane) (25-28) and downstream (cytochrome
c-mediated caspases in the cytosol) (29, 30) mechanism of
participation in apoptosis. Although there is not agreement in the
literature as to whether reactive oxygen species are the cause or the
consequence of cytochrome c release, cytochrome c
oxidants such as singlet oxygen (27) or ONOO- (26) have been
consistently pointed out to be upstream apoptosis inducers. In this
context, ceramide, an apoptosis inducer, has a high affinity only for
ferric cytochrome c, promoting its detachment from the inner
mitochondrial membrane (25). On the other hand, there is evidence
pointing to ferric cytochrome c as the caspase activator in
the cytosol (25, 30). In any event, a sustained steady state of
cytochrome c in the ferric form seems to favor its
detachment from the inner mitochondrial membrane and the caspase
activation in the cytosol. Thus, the ability of cytochrome c
to participate in the caspase activation might be dependent on the
association of this protein with membranes and/or protein complexes, as
suggested by recent literature (29). In this context, it is important
to verify whether the association of cytochrome c with
protein complexes, such as apoptosomes (29), is dependent on the heme
iron valence state and association with membranes.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Frank Herbert Quina
for the critical reading of this manuscript and to Derminda Isabel
Moraes, Andressa Patrícia Alves Pinto, and Roberto
Fukuhara for technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from Conselho Nacional de
Desenvolvimento Cientifico e Tecnológico, Fundação de
Amparo ao Ensino e Pesquisa da Universidade de Mogi das Cruzes,
and Fundação de Amparo à Pesquisa do Estado de
São Paulo.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
55-11-3818-3810; Fax: 55-11-3815-5579; E-mail:
afalario@iq.usp.br.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M006338200
 |
ABBREVIATIONS |
The abbreviations used are:
DCP, dicetylphosphate;
MCD, magnetic circular dichroism;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine;
CL, cardiolipin;
mT, millitesla.
 |
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