 |
INTRODUCTION |
Cytochrome c (cyt
c)1 is a
mitochondrial peripheral membrane protein functioning in the
respiratory chain in the inner mitochondrial membrane, shuttling
electrons from cyt c reductase to cyt c oxidase. An acidic phospholipid, cardiolipin, either alone or complexed with cyt c oxidase, provides the membrane-binding site (1, 2). ATP can modulate the electron transfer rate between cyt c and its redox partners (3, 4). However, whether these effects are due to nucleotide binding to cyt c or to cyt
c oxidase, or both remains unclear as both cyt c
and cyt c oxidase have been shown to contain at least one
nucleotide-binding site (5, 6). In cyt c part of the high
affinity ATP-binding site is constituted by the invariant
Arg91 (5, 7-10), and binding of ATP to this decreases the
rate of electron flow through the mitochondrial electron transport
chain (9). Accordingly, when the ATP-binding site of cyt c
is occupied by a covalently bound ATP, the electron-transfer activity
of cyt c with reductase and oxidase are inhibited to 41 and
11-15%, respectively, of the values measured for the native protein
(3). However, the redox potential of the above modified cyt
c remains close to the value of the native form (11).
Several studies have indicated ATP-induced changes in the structure of
cyt c. For example, auto-oxidation of reduced cyt
c takes place upon its gel filtration in the presence of ATP
(7), whereas this is not observed in the absence of the nucleotide
(12). ATP has also been shown to reduce the thermal stability of cyt
c (13).
Interestingly, release of cyt c from mitochondria to
cytoplasm has been found to be of critical importance in processes
connected to programmed cell death (apoptosis), raising a novel and
central point of interest in its properties (14, 15, 16). Furthermore, this release represents in most cases the commitment step for the full
activation of the cell death program (17). Outlines of the apoptotic
function of cyt c have been elucidated lately. In cytoplasm
cyt c forms a complex with a protein called Apaf-1 and
caspase-9, and formation of this "apoptosome" complex leads to the
activation of the cascade of proteases executing apoptosis in cells.
The presence and hydrolysis of ATP or dATP are required for the
formation of the apoptosome and thus also make these nucleotides mediators of the programmed cell death (18). The concentration range of
ATP present in cytoplasms of living cells is millimolar (19), which is
sufficient for its binding to cyt c (7). Based on the
recognition of different conformations of cyt c by
monoclonal antibodies, it was recently suggested that the apoptotically
active cyt c is membrane-bound (20). The combined effects of
ATP on cyt c and its membrane binding properties could thus
provide a possible mechanism to regulate the activity of cyt
c in triggering apoptosis.
The membrane association of cyt c has been extensively
studied (21). We have provided evidence for two distinct acidic
phospholipid-binding sites in cyt c and have nominated these
as the A- and C-sites (22). Accordingly, negative surface charge
density of the liposomes, pH, and ionic strength together determine
whether cyt c is bound electrostatically via its A-site or
by hydrogen bonding via its C-site (22, 23). Interaction of cyt
c with lipids has been suggested to involve additionally a
hydrophobic interaction between the protein and an acidic phospholipid
due to the so-called extended lipid anchorage (24, 25). In this
mechanism one of the acyl chains of acidic phospholipid is accommodated
within a hydrophobic channel in cyt c (26), whereas the
other chain(s) of the glycerophospholipid remain(s) in the lipid
bilayer. ATP is able to dissociate the A-site- but not the
C-site-mediated interaction of cyt c with acidic
phospholipids (22, 23, 27).
The effects of ATP on cyt c associated with liposomes
containing acidic phospholipids have indicated that the nucleotide may induce conformational changes in this protein (22). We report here
evidence for a change in the conformation of cyt c to be induced by ATP, revealed by CD measurements in the Soret region, and
time-resolved fluorescence spectroscopy of a cyt c analog with a Zn2+-substituted heme moiety. Resonance energy
transfer measurements demonstrate this effect of ATP to be strongly
attenuated for the lipid bound [Nle91]cyt c in
which the high affinity ATP-binding site is abrogated.
 |
EXPERIMENTAL PROCEDURES |
Materials--
1-Palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3- phosphoglycerol
(PPDPG) was purchased from K&V Bioware (Espoo, Finland). Horse
heart cyt c (type VI, oxidized form), egg PG, and egg PC were from Sigma. The Na2 salt of ATP was from Roche
Molecular Biochemicals. No impurities were detected in the above lipids upon thin layer chromatography on silicic acid using
chloroform/methanol/water/ammonia (65:20:2:2, v/v) as the solvent
system and examination of the plates for pyrene fluorescence or after
iodine staining. All other reagents were of reagent grade from Sigma.
Preparation of Liposomes--
Lipids were dissolved in
chloroform and mixed in this solvent to obtain the desired
compositions. PPDPG (X = 0.01) was used as the
fluorescent lipid probe. The solvent was removed under a stream of
nitrogen, and the lipid residue was subsequently maintained under
reduced pressure for at least 2 h. The dry lipids were then hydrated in 20 mM Hepes, 0.1 mM EDTA, pH 7.0 or
4.0, at room temperature to yield a lipid concentration of 1 mM. To obtain unilamellar vesicles, the hydrated lipid
dispersions were extruded with a LiposoFast small-volume homogenizer
(Avestin, Ottawa, Canada). Samples were subjected to 19 passes through
two polycarbonate filters (100 nm pore size, Nucleopore,
Pleasanton, CA) installed in tandem (28). Minimal exposure of the
lipids to light was ensured throughout the above procedure.
Subsequently, the liposome solution was divided into proper aliquots
and diluted with the buffer to a final lipid concentration of 25 µM.
Steady-state Fluorescence Measurements--
The lipid binding of
cyt c was assessed as described previously (22-24, 29, 30)
by monitoring resonance energy transfer (31, 32) between the pyrene
containing lipid PPDPG and the heme of cyt c. The
measurements where conducted with a PerkinElmer Life Sciences LS50B
spectrofluorometer using 2.5 and 4.0 nm band passes for excitation and
emission beams, respectively. The excitation wavelength was 344 nm, and
the quenching of PPDPG fluorescence by the heme of cyt c was
measured at 398 nm. Two ml of liposome solution were placed into a
magnetically stirred 4-window quartz cuvette in a holder thermostated
with a circulating water bath at 25 °C. Subsequently, ATP was
included to yield up to 5 mM final concentration, and then
5- or 10-µl aliquots of a 20-40 µM solution of native
or Arg91-modified cyt c were added, and the
quenching of pyrene fluorescence by the heme of cyt c was
observed. In experiments at pH 4 ATP was added after the addition of
cyt c. Changes in fluorescence were allowed to stabilize for
~40 s, and then the intensity of pyrene monomer fluorescence was
recorded. Because of the low concentrations of both lipids and cyt
c generally utilized, minimal interference by the inner
filter effect was expected. The merits as well as limitations of the
use of pyrene-labeled lipids in energy transfer measurements have been
discussed elsewhere (22, 33-35).
Formation of a hexagonal HII phase has been demonstrated as
a consequence of the cyt c-cardiolipin interaction (36).
Accordingly, although cardiolipin is likely to constitute the
membrane-binding site for cyt c in the inner mitochondrial
membrane (1, 2), the present experiments were conducted using PG as the
acidic phospholipid so as to avoid ambiguities in the interpretation of
the results arising from the formation of a HII phase.
Except for interference presumably arising from the formation of the inverted hexagonal phase as well as peculiarities in the extent of
protonation of the two vicinal phosphates of cardiolipin, we have found
no evidence for significant differences in the binding of cyt
c to these two lipids, cardiolipin and PG.
Circular Dichroism Spectroscopy--
CD spectra were collected
with an Olis RSF 1000F CD spectrophotometer (On-line Instrument Systems
Inc., Bogart, GA). Soret region CD spectra in the 380-460 nm region
were recorded with 10-mm path length cells containing 20 mM
Hepes, 0.1 mM EDTA, pH 7.0, 10 µM cyt
c, LUVs, and different concentrations of ATP where indicated. Measurement cell was thermostated to 25 °C with a
circulating water bath. Final spectra, representing the average of at
least three tracings, were corrected for the background. CD is
expressed as difference of the extinction coefficients for left and
right circularly polarized light calculated per heme.
Time-resolved Fluorescence Spectroscopy of
[Zn2+-Heme]cyt c--
The intrinsic Trp fluorescence in
cyt c is nearly imperceptible due to quenching by the
vicinal strongly absorbing heme, preventing the use of this fluorescent
amino acid as an intrinsic probe for the conformation of cyt
c. Substitution of Zn2+ for Fe2+ in
the porphyrin of cyt c yields an intensely fluorescent
derivative of cyt c (37). This analog has been characterized
in considerable detail and has been shown to resemble closely the
parent protein in most qualities thus representing a good model to
study the conformation of cyt c (38). This cyt c
derivative was prepared from horse cyt c according to
Vanderkooi and Erecinska (39). Iron-free cyt c was
first made, which was subsequently treated with ZnCl2 to
yield [Zn2+-heme]cyt c (37). Also in the
[Zn2+-heme]cyt c the energy transfer was very
efficient, and practically no Trp emission could be measured (data not shown).
A commercial laser spectrometer (Photon Technology International,
Ontario, Canada) was used to measure fluorescence lifetimes. A train of
500-ps pulses at a repetition rate of 10 Hz was produced by a nitrogen
laser, pumping a dye (rhodamine 6G, Merck, 5 mM solution in
methanol) laser and followed by frequency doubling to yield the
excitation pulses at 298 nm. Excitation maximum for the
[Zn2+-heme]cyt c is at approximately 415 nm.
In order to maximize the spectral separation between the excitation and
emission bands, we used energy transfer from Trp for the excitation of
the Zn-porphyrin, i.e. exciting the single Trp residue at
295 nm. Fluorescence from the Zn-porphyrin at 640 nm is thus due to
Förster type resonance energy transfer (31, 32) from the Trp
residue (donor) to the Zn-porphyrin (acceptor) of cyt c. Two
ml of 10 µM [Zn2+-heme]cyt c in
20 mM Hepes, 0.1 mM EDTA, pH 7.0, was placed in a magnetically stirred 4-window quartz cuvette in a holder thermostated with a circulating water bath at 25 °C. For the measurement of fluorescence lifetimes, the average of five emission decay curves at
640 nm was analyzed by the non-linear least squares method and fit to a
sum of exponentials using the software provided by the instrument
manufacturer. Instrument response functions were measured separately
using aqueous glycogen solution. The validity of the fit of a
particular model was judged by the value of the reduced
2 (40, 41).
Fractional intensities I(t) were calculated according to
Equation 1,
|
(Eq. 1)
|
where
i is amplitude and
i is lifetime.
Fluorescence emission decay for [Zn2+-heme]cyt
c in solution had an average lifetime
of ~4.2
ns. The emission decays for [Zn2+-heme]cyt c
could be best fitted to a two-exponential process, yielding short
(
1) and long (
2)
lifetime components (Table I).
At this point it is relevant to note that differences were evident in
the lifetimes of different [Zn2+-heme]cyt c
preparations. Yet, the qualitative changes measured under the different
conditions used were highly reproducible.
Semisynthesis and Characterization of [Nle91]cyt
c--
Solid-phase peptide synthesis was used to first obtain a
synthetic fragment representing residues 66-104 of the horse cyt c sequence, with the naturally occurring Arg91
replaced by norleucine, i.e. with a straight aliphatic chain instead of the alkylguanidine. Methods for monitoring reaction progress, resin cleavage, deprotection, and purification of peptides were as described previously (42). Glu (the C-terminal residue) was
coupled via a 4-carboxyamidomethyl-benzyl ester to a cross-linked polystyrene resin (0.4 mmol), and the peptide chain was extended with
symmetrical anhydrides of side chain-protected N-t-Boc amino acids (or 1-hydroxybenzotriazole esters of Boc-Asn or Boc-Glu) in an
automated system. Purity was checked by analytical reversed-phase HPLC,
amino acid analysis, and mass spectrometry. The 39-residue peptide (163 mg) had the expected amino acid composition and a molecular mass of
4529.9 ± 0.8 (calculated 4530.4). The complementary fragment
1-65 with the covalently linked heme at Cys14 and
Cys17 was prepared by CNBr cleavage from natural horse cyt
c. Its purification and coupling at equimolar ratio to
synthetic 66-104 to create the semisynthetic holocytochrome were as
reported previously (42). After ligation the product was separated from
unreacted peptides by Sephadex G50 gel exclusion chromatography in 7%
HCOOH, with a crude yield of 50%. The protein was renatured by buffer
exchange from 8 M urea into 50 mM potassium
phosphate, pH 7, and finally purified by low pressure cation-exchange
chromatography. The semisynthetic cyt c analog revealed a
single component by HPLC and was 97% reducible by ascorbate. The
UV-visible absorption spectra for both oxidation states of the analog
were fully superimposable with those of the native protein (data not
shown). The above indicate no significant disruption of the
coordination sphere or the environment of the heme in the interior of
the protein.
Upon elution of the [Nle91]cyt c in both
oxidation states on a Waters 600E HPLC system using an SP5PW
cation-exchange column and a gradient of phosphate buffer (40-400
mM, pH 7), the elution times were exactly as expected on
the basis of deletion of a single positive charge (Table II), mirroring
the absence of the basic guanidino group (42). Titrations of the
disappearance of the 695 nm charge transfer band that signals heme
iron-Met80 ligation with pH change were performed in 50 mM phosphate buffer (43). A minimal change was noted in the
resistance of the protein to the conformational transition
precipitating the ligand exchange reaction at alkaline pH (44), but a
somewhat larger effect on the more complex denaturation process at acid
pH was observed (12). It is possible that in the native structure the
proximity of the guanidino group to the carboxylate of
Glu69 could be stabilizing at low pH, forming an ion pair
in the absence of ATP.
The association of ATP with [Nle91]cyt c was
assessed by two methods. First, elution times on an ATP-agarose column
equilibrated in 35 mM phosphate were measured. The observed
retention times in this column vary widely for different cyt
c analogs and provide a measure of the affinity of the
protein for ATP (7). This method revealed [Nle91]cyt
c to bind ATP substantially less effectively than the native protein, and the elution time from a column with immobilized ATP is
half that of cyt c (12 versus 24 min). Second,
equilibrium gel filtration of both native protein and the
Nle91 analog was undertaken in 10 mM Tris
cacodylate buffer, pH 6.95, containing 0.1 mM ATP, as
described by Corthésy and Wallace (5). This method gave the
nucleotide/cyt c stoichiometry of 0.37 for the analog and
1.14 for the native protein. [Nle91]cyt c thus
represents a model for the parent protein in which the only function
compromised is ATP binding.
 |
RESULTS |
Effects of ATP on the Binding of cyt c to Liposomes--
At
neutral pH ATP dissociates cyt c from membranes with a low
content (X = 0.20) of acidic phospholipids (22, 23).
This A-site interaction of the protein with lipids is mainly
electrostatic in nature, and the dissociation has been concluded to
result from a competition between ATP and the deprotonated phosphate
group of acidic lipids for the binding to cyt c (22). In the
presence of ATP the ionic strength required to dissociate cyt
c from the inner mitochondrial membrane decreases (3) in
keeping with our results on the binding of cyt c to
liposomes in vitro (23). If the content of acidic
phospholipids in membranes is increased or, alternatively, the pH of
the medium is lowered at a low acidic phospholipid content in the
liposomes, the membrane association of cyt c changes from A-
to C-site interaction (22, 23). The C-site interaction of cyt
c is not dissociated by nucleotides, and elevated ionic
strength is able to dissociate the protein only if it causes the
deprotonation of acidic phospholipids, thus changing the
interaction from C- to A-site.
We have previously reported the C-site interaction of cyt c
with liposomes at acidic pH to be modulated by ATP, and we have suggested a conformational change in the membrane-bound protein to be
induced by the nucleotide (22). This is seen in the data measured at
neutral pH and at XPG = 1.0 (Fig.
1, panel A). Accordingly, efficiency of quenching of pyrene fluorescence due to cyt c
bound to membranes was augmented in the presence of increasing
nucleotide concentrations; this effect was saturated at 2 mM ATP. We have suggested the enhanced fluorescence
quenching to be due to an ATP-induced conformational change in cyt
c, which then results in a more efficient resonance energy
transfer (i.e. dipole-dipole coupling) between pyrene and
the heme of cyt c (23). The latter could be caused by an
altered orientation of the heme of the membrane-bound cyt c
with respect to the relaxation dipole of pyrene. Instead of increasing
the negative surface charge density of liposomes at neutral bulk pH by
increasing XPG, the C-site interaction of cyt c
can also be achieved at XPG = 0.2 by decreasing pH to 4.0, thus promoting the protonation of the acidic phospholipids (22). A
conformational change in cyt c due to ATP was also indicated under these conditions (pH 4.0 and at XPG = 0.2) by the
increase in fluorescence quenching due to the addition of ATP after the membrane association of cyt c had commenced (Fig. 1,
panel B).

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Fig. 1.
Effect of ATP on the C-site membrane
association of native cyt c liposomes.
Panel A, binding of cyt c in the presence of ATP
to liposomes containing XPG = 1.0, pH 7. The concentration
of ATP was 0 ( ), 1 ( ), or 2 ( ) mM. Panel
B, effect of subsequently added ATP on C-site bound cyt
c. The protein was added prior to the addition of ATP to a
final concentration of 0.15 µM. The mole fraction of PG
in PC liposomes was 0.20 at pH 4. Total lipid concentration in 20 mM Hepes, 0.1 mM EDTA was 25 µM.
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CD Spectroscopy of Membrane-bound cyt c--
The circular
dichroism of heme group in cyt c was measured in order to
monitor structural changes in cyt c. The exact underlying mechanism resulting in optical activity in the Soret region near 400 nm
is uncertain, and the peaks in the cyt c spectrum cannot be
assigned with absolute certainty to specific chemical moieties. Nevertheless, CD spectra of this region are strongly dependent on the
immediate conformational environment of the heme group and provide a
sensitive indicator of small scale conformational changes in the
protein (45). Denaturation studies have indicated that the trough at
420 nm relates to the heme iron-Met (80) bond in cyt c
(46). The spectrum of cyt c in solution (Fig.
2, panel A) was similar to
data published previously (46, 47) and was composed of a distinct
shoulder at 390 nm and a trough at ~420 nm. The spectrum measured in
the presence of 3 mM ATP was identical to the spectrum of
free cyt c, indicating no changes in the heme environment
upon interaction of ATP with cyt c in solution.

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Fig. 2.
Effect of ATP on the Soretvregion
(380-440 nm) CD spectra of cytochrome c. cyt
c concentration was 10 µM, and where
indicated, LUVs (250 µM total lipid) and/or ATP (1 or 3 mM) were added. The mole fraction of POPG in the LUVs was
either 0.2 (panel B) or 1.0 (panel C) so as to
bind cyt c to membrane via A- and C-site, respectively.
Medium consisted of 20 mM Hepes, 0.1 mM EDTA,
pH 7.0. Panel A, cyt c in buffer in the presence
of either 0 ( ) or 3 mM ( ) ATP. Panel B,
A-site membrane-associated cyt c with liposomes containing
XPG = 0.2 in the absence of ATP ( ) or with 1 mM nucleotide ( ). Panel C, c-site
membrane-associated cyt c with liposomes containing
XPG = 1.0 without ( ) and with 3 mM ( )
ATP.
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|
The spectrum for A-site membrane-bound cyt c
(XPG = 0.2, Fig. 2, panel B) displayed similar
features as free cyt c but with decreased peak intensities.
In the presence of 1 mM ATP cyt c remains
membrane-bound (Fig. 3, panel
B), yet should also bind ATP based on the equilibrium gel
filtration measurements (5). However, 1 mM ATP caused no
changes in the spectrum. In contrast, the CD spectrum of cyt
c bound to liposomes via the C-site at XPG = 1.0 (Fig. 2, panel C) was very different from the free cyt c in solution as well as from the A-site bound protein. More
specifically, a wide positive band was centered at 390 nm, where a
shoulder was seen in the spectrum of free cyt c, and a sharp
negative deflection was measured at 410 nm, with an opposite peak
centered at 420 nm. C-site lipid binding thus seems to change the heme
environment dramatically, suggesting the conformation of C-site
membrane-bound cyt c to be different from both free or
A-site-bound cyt c. Importantly, CD spectra revealed 3 mM ATP to cause a further conformational change in the
C-site membrane-interacting protein (Fig. 2, panel C),
evident as diminished intensities of the peaks at 390 and 410 nm and an
emerging shoulder at ~405 nm.

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Fig. 3.
Effect of ATP on the A-site
membrane association of native and [Nle91]cyt
c. Panel A, quenching of pyrene fluorescence as a
function of native ( ) and [Nle91]cyt c
( ). Panel B, dissociation of membrane-bound cyt
c by ATP. The mole fraction of PG in PC liposomes was 0.20. Total lipid concentration in 20 mM Hepes, 0.1 mM EDTA, pH 7, was 25 µM.
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|
ATP-induced Changes in the Fluorescence of
[Zn2+-Heme]cyt c--
The above CD data reveal ATP to
induce conformational changes in lipid-bound cyt c (22). A
fluorescent cyt c analog with the heme iron substituted by
Zn2+ described has been previously (37). We utilized
time-resolved fluorescence spectroscopy to monitor structural changes
in this cyt c derivative induced by ATP and upon lipid
binding, reflected in the lifetimes of Zn-porphyrin fluorescence at 640 nm. One mM ATP induces a small yet significant increase in
the average lifetime
, from 4.2 to
4.7 ns. Also the
component lifetimes
1 and
2 were sensitive to the presence of ATP and
were prolonged from 1.8 to 2.0 and from 9.1 to 11.2 ns, respectively,
with only minor changes in the fractional intensities of the decay
components. Compared with the above modest effects of ATP on
[Zn2+-heme]cyt c in solution, much more
pronounced effects were evident upon binding to liposomes. These
changes also depend on the type of lipid binding involved, determined
by the content of the acidic phospholipid POPG in the liposomes (Table
I). Accordingly, the values for the
average lifetime
of the Zn-porphyrin fluorescence were
reduced in the presence of liposomes from 4.2 ns for free cyt
c to
2.0 and
3.5 ns at XPG = 0.20 (A-site)
and XPG = 1.0 (C-site), respectively. Significant changes
were observed also in the component lifetimes of the Zn-porphyrin
emission in the presence of liposomes (Table I). In brief, at
XPG = 0.20 the values for
1 and
2 were 0.6 and 2.6 ns, respectively, whereas the corresponding values for [Zn2+-heme]cyt c
bound to liposomes via the C-site at XPG = 1.0 were considerably longer, 1.5 and 8.0 ns. Pronounced differences were seen
also in the fractional intensities of the component lifetimes, and at
XPG = 0.20 ~70% of the excited fluorophore relaxes back to the ground state via the process with the longer lifetime. At
XPG = 1.0 the opposite was evident, a conformation with the shorter lifetime representing the dominant species (Table I).
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Table I
Fluorescence lifetimes of [Zn2+-heme]cyt c, at excitation and
emission wavelengths of 295 and 640 nm, respectively
is the average fluorescence lifetime. 1 and
2 represent the short and long lifetime components derived
from fitting the curves to two-exponential decays, and C1 and
C2 are the fractional intensities (in percentage) of
1 and 2, respectively. [Zn2+-heme]cyt
c concentration was 10 µM, and where
indicated, 1 mM ATP and/or LUVs (250 µM total
lipid) were added. Mole fraction XPG of the acidic phospholipid
POPG in the LUVs was either 0.20 or 1.0 as indicated. Medium consisted
of 20 mM Hepes, 0.1 mM EDTA, pH 7.0.
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In keeping with the ATP-induced conformational changes observed by CD
also the fluorescence decays of lipid-associated
[Zn2+-heme]cyt c were also strongly influenced
by ATP. At XPG = 0.20 the lipid-induced changes in the
fluorescence lifetimes appear to be somewhat reduced by ATP, and in the
presence of 1 mM ATP the value for
of A-site
lipid-bound cyt c was prolonged from 2.0 to 2.7 ns (Table
I). For the C-site interaction of [Zn2+-heme]cyt
c with liposomes (XPG = 1.0), the nature of the
interaction seems to change drastically in the presence of ATP.
Accordingly, the value for
of [Zn2+-heme]cyt
c associated to liposomes via its C-site is 3.6 ns in the
absence of ATP and 8.3 ns with 1 mM nucleotide, in keeping with ATP-induced conformational changes. Also the component lifetimes as well as the fractional intensities were affected by the nucleotide (Table I). More specifically, in the presence of 1 mM ATP,
the values for
1 and
2 were 1.2 and 5.1 ns at XPG = 0.20 (A-site interaction) and 1.8 and 10.5 ns for XPG = 1.0 (C-site interaction). ATP thus appears to partially counteract
the effect of A-site-mediated lipid binding on the conformation
of cyt c.
Association of Nle91-cyt c to Liposomes--
The high
affinity ATP-binding site in cyt c has been demonstrated
previously to involve the invariant Arg91 (5, 7-10). In
order to study the possibility that the observed changes induced by ATP
in lipid association of cyt c would be due to this site, we
studied the liposome association of the mutant [Nle91]cyt
c in which the high affinity binding site for ATP is
abrogated. Accordingly, we compared the effects of ATP on the membrane
association of native cyt c and the
[Nle91] analog. The A-site interaction of cyt
c with liposomes was not affected by the lack of the
Arg91 guanidino group, and at XPG = 0.20 and pH 7.0 membrane association of native cyt c and its
Nle91 derivative were indistinguishable (Fig. 3). However,
compared with the native protein somewhat less ATP was required to
dissociate the modified protein. This difference is likely to reflect a
small fraction of C-site-associated cyt c present at
XPG = 0.20 at neutral pH for which a slight
apparent increase in membrane association due to ATP occurred (Fig. 3,
panel B). These data also show the high affinity
ATP-binding site at Arg91 to be distinct from the
lipid-binding A-site, in keeping with our previous results (24,
27).
In the absence of ATP the C-site-mediated membrane association of the
modified protein was similar to the native protein indicating that the
Arg91
Nle modification caused no detectable change in
this interaction of cyt c with lipids (Fig.
4, panel A). However,
comparison of these data with those of Fig. 1, panel A,
shows that 2 mM ATP had a significantly reduced effect on
the apparent affinity of [Nle91]cyt c for neat
PG liposomes. Likewise, the effect of ATP on the fluorescence quenching
by the modified protein was significantly smaller. Yet, the fact that
even for the Arg91
Nle mutant a slightly enhanced
quenching was seen suggests that the deletion of the guanidino group
leaves intact the contribution of the other components of the binding
site (48) and that the loss of ATP binding to the mutated site is not
complete (Fig. 4, panel A), confirming the affinity column
data (Table II). Accordingly, compared
with the wild type cyt c., subsequent addition of ATP to
C-site-bound Nle91 at XPG = 0.2, pH 4.0, showed
diminished yet still noticeable increase in the quenching by ATP (Fig.
4, panel B). These data provide evidence for the high
affinity nucleotide-binding site involving Arg91 to be
required for the ATP-induced conformational changes in cyt c
and the enhancement of energy transfer from the membrane-contained pyrene to the cyt c heme in the presence of ATP.

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Fig. 4.
Effect of ATP on the C-site membrane
association of [Nle91]cyt c
liposomes. Panel A, binding of
[Nle91]cyt c in the presence of ATP to
liposomes containing XPG = 1.0. The concentration of ATP
was 0 ( ), 1 ( ), or 2 ( ) mM. Comparison of
Nle91 and wt cyt cs (displayed in Fig. 1,
panel A) is aided by taking into account that the graphs for
cyt c, and the mutants recorded in the absence of ATP are
practically indistinguishable. Panel B, effect of
subsequently added ATP on the C-site-bound [Nle91]cyt
c ( ) compared with native cyt c ( ), data
taken from Fig. 1, panel B. The proteins were added prior to
the addition of ATP to a final concentration of 0.15 µM.
The mole fraction of PG in PC liposomes was 0.20, pH 4. Total lipid
concentration was 25 µM in 20 mM Hepes, 0.1 mM EDTA.
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|
 |
DISCUSSION |
Interactions of cyt c with lipids have been thoroughly
investigated, and cyt c is generally taken as a paradigm for
an electrostatically membrane-bound peripheral protein bearing a net
positive charge and associating with acidic phospholipids (21). Changes
in the conformation of cyt c upon binding to phospholipid
membranes have been described using a variety of techniques including
tryptophan fluorescence, CD (49), NMR (50-53), Fourier
transform infrared spectroscopy and differential scanning calorimetry
(54), resonance Raman spectroscopy (55), and surface plasmon resonance
spectroscopy on supported lipid bilayers (56). In brief, these studies
have revealed that binding of cyt c to acidic phospholipids
induces a conformation with less organized tertiary structure (49-53)
and reduced thermal stability (54) but with native-like helical secondary structure (49). The lipid-induced conformational changes were
demonstrated to depend on the negative surface charge density in the
membrane (54). Likewise, cardiolipin was more effective than
phospholipids with a single negative charge (51). The above is in
keeping with the C-site of cyt c to be involved in mediating phospholipid-induced conformational alterations (23). Three different
membrane-bound conformations for cyt c have been described (57), and these authors demonstrated the presence of an
electrostatically bound cyt c and two conformationally
different membrane-bound types of cyt c that were not
dissociable from the membrane by increasing ionic strength.
Interestingly, the efficiency in electron transport was also different
for the latter two conformations (57).
The present data confirm changes in the conformation of cyt
c upon its binding to liposomes. Most important, we also
demonstrate that further conformational alterations are induced by ATP
in cyt c bound to phospholipids. These ATP-induced changes
in the conformation of liposome-associated cyt c were
further dependent on the lipid composition. Accordingly, they were
observed in CD spectra only when using liposomes composed of the acidic
PG. Under these conditions cyt c binds to membranes via the
so-called C-site and attaches to the protonated head groups of PG. The
C-site has been suggested to involve residue Asn52 and a
fatty acid-binding hydrophobic cavity in cyt c (24). C-site
lipid bound cyt c is not detached from liposomes by ATP, in
contrast to the A-site-bound protein (23).
Although the heme moiety as such is not optically active in solution,
its interactions with the neighboring amino acid residues and
distortions in its planarity render heme-containing proteins optically
active, with the characteristic CD spectra in the Soret region near 400 nm. These CD spectra thus provide information on the variation of
environment of the heme group and thus reflect the conformation of cyt
c. These spectra demonstrate pronounced differences in the
conformations of cyt c bound to liposomes via the C- and
A-sites as well as the conformation of cyt c in solution. Moreover, the structure of cyt c bound to liposomes via the
C-site can be further altered by ATP, whereas no such effect was seen on the protein in solution or when interacting with lipids by its
A-site. Although these CD spectra cannot be assigned to specific conformational features, qualitative properties may be discussed. The
distinct shape of the spectrum for the C-site-bound cyt c with sharp peaks and rotational strength equal to the unbound protein
suggests an organized conformation instead of a molten globule-like
state. This is in contrast to lipid-induced unfolding of cyt
c in the presence of phospholipid vesicles (49) revealing a
Soret region CD spectrum similar to guanidine hydrochloride denatured
species (46). However, in the latter study, conditions different from
those used here were employed. More specifically, these authors used
phosphatidylserine as the acidic phospholipid and at high concentration
(several mM) which could explain the denaturing effect.
Most important, we could demonstrate the presence of ATP to have
further effects on the CD spectra (Fig. 2), thus revealing an
ATP-induced conformational change in lipid-bound cyt c. The
ATP-induced effect observed in the resonance energy transfer between
the heme moiety of cyt c and the membrane-embedded lipid
(Fig. 4.) could thus be explained by these conformational species
having different properties as acceptors in the resonance energy
transfer process. Except for decreased amplitudes of the peaks, the CD
spectra for the A-site membrane-bound cyt c is identical to
that of unbound cyt c in solution. The difference in
amplitudes could be explained by more intense fluctuations in the heme
environment of the A-site membrane-interacting protein, where the
time-averaged structure would be identical to that of the free cyt
c. Unfortunately, the amounts of protein required for
measuring CD spectra for Nle91 cyt c were
prohibitively high.
Measurement of fluorescence lifetimes provides an additional and
extremely sensitive tool to study changes in the immediate microenvironment of a fluorophore (58). For this purpose we used the
intensely fluorescent [Zn2+-heme]cyt c (38).
In addition to confirming that lipid binding induces structural changes
in [Zn2+-heme]cyt c and demonstrating that ATP
causes conformational changes in this protein, the present data reveal
that ATP has pronounced additional effects on the structure of
lipid-bound cyt c. Accordingly, our results reveal four
different conformations for membrane-bound cyt c as follows,
depending on the content of the acidic phospholipid in the membrane and
the presence of ATP. At low content of acidic phospholipid and at
neutral pH the cyt c is bound to lipids electrostatically via the A-site (23). Whereas higher concentrations of ATP (>2 mM) reverse this interaction, low [ATP] <2
mM appears to bind to the lipid-associated cyt c
and to induce a conformational change, as demonstrated by the
fluorescence lifetimes and fractional intensities for
[Zn2+-heme]cyt c (Table I). At either
XPG = 1.0 or at XPG = 0.20 and at acidic pH the
interaction of cyt c involves protonated acidic phospholipids and has been nominated as C-site binding (23). Judged
from time-resolved fluorescence, the conformations of cyt c
bound to lipids via the C-site and A-site are different. Also, in cyt
c bound to liposomes via the C-site interaction, further structural changes are caused by ATP, evident both in the fluorescence lifetime data for [Zn2+-heme]cyt c (Table I)
as well as in the resonance energy transfer between the fluorescent
membrane probe and wt-cyt c (Fig. 1). More specifically, for
the C-site-bound cyt c the changes induced by ATP result in
an augmented resonance energy transfer between the fluorescent
pyrene-containing lipid analog PPDPG and the heme moiety of wt-cyt
c. This enhanced resonance energy transfer could result from
an altered orientation of the dipoles involved due to changing
orientation of the heme with respect to the membrane plane or reduced
distance between the dipoles (22).
A high affinity binding site for ATP has been described and shown to
involve the invariant Arg91 (5, 7-10). The involvement of
Arg91 in binding of ATP and consequent modulation of
electron transfer by the protein has been investigated previously by
semisynthetic analogs of cyt c in which this single arginine
residue of the 66-104 peptide was chemically modified by
cyclohexane-1,2-dione prior to ligation with the 1-65 peptide (10).
The [Nle91] analog used here binds ATP better than
the previously described N7,N8-(1,2-dihydroxycyclohex-1,2-ene)diyl-L-arginine
91-cyt c (7). This difference probably reflects the fact
that the latter modification introduces a bulky group, which prevents
the approach of the nucleotide to the other moieties constituting this
site. Because of this, the choice of norleucine as the substitution for
arginine was made specifically with steric considerations in mind, as
the side chain of residue 91 (Fig. 5)
resides in the hydrophobic face of an amphipathic helix and is fully
buried, with the exception of the guanido head group. A straight
aliphatic chain of norleucine should thus fulfill the space-filling
role of this residue. This analog had strongly reduced affinity for
ATP, whereas its other characteristics remained unaffected. In order to
study whether the above site was responsible also for the ATP-induced
changes in cyt c bound to lipids, we examined the effect of
ATP on the liposome association of the [Nle91]cyt
c. We have previously demonstrated that the interactions of
[Nle91]cyt c with lipids differ from those of
the wt-cyt c with lack of aggregation by
[Nle91] of LUV composed of acidic phospholipid
(27). Most important, compared with the wild type the strongly reduced
effect of ATP on the resonance energy transfer between the heme moiety
of [Nle91]cyt c and the membrane-embedded
fluorescent lipid (Fig. 4, panel B) strongly suggests that
the invariant Arg91 is indeed important in mediating the
conformational changes induced by ATP.

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Fig. 5.
Molecular model for the binding of the
ATP (green) to a high-affinity binding site in yeast
ferrocytochrome c (48). Panel A,
interacting side chains are colored in orange, and the heme
(brown) is shown for spacial reference. Oxygens in ATP
molecule are colored dark green and phosphorus atoms are
medium green. Panel B, cytochrome c
presented in conventional orientation.
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