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Cytochrome c interaction with membranes
.kRCHIVk:S
OF
BIOCHl?YISTRY
.IND
BIOPHYSICS
Cytochrome I. Use of a Fluorescent
c Interaction Chromophore
with Artificial JANE
VAn’DERI
Johnson
164, 219-229 (1973)
with
in the Study of Cytochrome
and Mitochondrial
RL4RIA
Membranes
ERECI%IiA,
c Interaction
Membranes’ .4ND
BRITTON
CHANCE
Research Foundation, Department of Biophysics and Physical Biochemistry, I’nirersity of Pennsylvania, Philadelphia, Pennsykjania I.9104 Received
August
21, 1972
Quenching of 12.(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS3 fluorescence is biphasic (tm below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids. Addition of cytochrome c to @ochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytoehrome c-depleted mitochondria is 1.8 X 106 M, while the affinity constant for reduced cytochrome c is 0.5 X lo6 M. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance wit,h midpoint potential differences between the bound and free forms.
Cytochrome c is unique among the mitochondrial respiratory eleckon carriers in its extractability and ability to restore both electron transport and ATP synthesis in the deficient mit’ochondria (1). i(‘urthermore, cytochrome c forms lipid complexes which provide an excellent model system for the st’udy of interactions between lipid and protein components of membranes (2-6). Two methods have been generally used to measure cytochromct c binding to phospholipids. In one, binding of cytochrome c is measured by extraction of the proteinlipid complex into an organic phase (3, 4); in the other, cyt’ochromc c is incorporated 1 This work was supported by USPHS GM12202. Computer time was supported by NIH Grant No. RR-15, using the Digital Equipment Corporation PDP-6 Computer of the University of Pennsylvania Medical School Computer Facility. * To whom correspondence should be addressed. 3 Abbreviations used: AS, 12.(9-anthrogl) stearic acid; MOPS, rnorpholilioprop:~rle sulfonate. 219 Copyright .\I1 rights
0 1973 by Academic Press, of reproduction in an>- funn
Inc. rcscrved
into multilayered phospholipid vesicles, and the complex is separated from the excess of cyt80chrome c by centrifugation (5, 6). n‘either of these methods yields information on the dynamics of cytochrome c interaction with membranes, and trapping of cytochrome c between the multilayered vesicles rather than binding to the lipid may be the actual parameter measured. This report describes a method to measure cyt!ochrome c binding to sonicated phospholipid vesicles and to mitochondrial membranes using resonance energy transfer from the excited state of a fluorescent chromophore to cytochrome c with subsequent quenching of fluorescence. The parameters of cytochrome c binding to artificial phospholipid membranes and to mitochondria are compared and discussed in terms of the mode of action of cytochrome c in the mitochondrial respiratory chain. A similar approach has been used by Fromberg to measure the distances between cytochrome
220
VANDERKOOI,
ERECIr;JSKA,
c and fluorescent chromophores in oriented lipid multilayers (7). MATERIALS Horse heart cytochrome c type VI was obtained from Sigma Chemical Company (St. Louis, MO). Bovine cardiolipin was obtained from Nutritional Biochemical Company (Cleveland, OH), and egg lecithin was a gift of Dr. T. Gulik-Krzywicki (Lab. de Biochimie, C.N.R.S., Gif-sur-Yvette, France). The purity of the lipids was checked by thinlayer chromatography on Silica Gel G plate (A. H. Thomas Company, Philadelphia, PA) using chloroform: methanol: ammonia (65: 25:4) as solvent followed by staining with iodine vapor. The cardiolipin appeared as one spot. Lecithin contained about 10% contamination, tentatively identified as diglyceride and lysolecithin. Lipid concentration was determined by weighing or by analyzing for phosphate (8). 12-(9-anthroyl) stearic acid (AS), obtained as a gift from Drs. A. S. Waggoner and L. Stryer (9), appeared as one spot on thin-layer chromatography under uv illumination. EXPERIMENTAL
PROCEDURES
Aqueous suspensions of phospholipids were prepared by sonicating the lipids in the presence of AS with a Branson sonifier for 5 min at 50 W followed by centrifugation at 100,OOOgfor 15 min to remove undispersed lipid. Fluorescence was measured at 90” to the exciting beam using a Hitachi MPF-2A fluorescence spectrometer. The sample compartment was maintained at 22-24°C with the use of circulating water. Pigeon heart mitochondria were prepared according to the method of Chance and Hagihara (10). Cytochrome c was extracted from mitochondria essentially by the method of Jacobs and Sanadi (1) with the modification described by Boveris et al. (11). Cytochrome c concentration was determined by reduced minus oxidized difference spectra using 19.7 as extinction coefficient (12). Oxygen uptake was (ESSO-540mM-l cm-l) measured at 24°C using a Clark oxygen electrode in 0.2 M mannitol-0.05 M sucrose-O.040 M morphoM phosphate medium, linopropane sulfonate-0.010 pH 7.0, with succinate plus glutamate as substrate. Cytochrome c binding was determined as the amount of cytochrome c found in the mitochondrial pellet after centrifuging down the mitochondria (10 min at SOOOgat 4°C) from the suspending medium in which the fluorescence assays had been done. The pellet was suspended in 0.05 M phosphate buffer containing 1% Triton X-100. The concentration of cytochrome c was estimated from
AND
CHANCE
the difference between total oxidized (+5 mM ferricyanide) - total reduced (+ dithionite). The measurements were done using a Johnson Foundation dual-wavelength spectrophotometer. The concentration of free cytochrome c was determined by measuring the amount found in the supernatant fluid from the 8OOOgcentrifugation. The recoveries were 95-100yc under all conditions. Dat,a concerning the quenching of AS fluorescence by cytochrome c were analyzed according to Fiister’s theory of dipole-dipole energy transfer (13). RO , the distance in Angstroms at which transfer efliciency is 5Ooj, was calculated by
Ro = (JKZQor~-4)1’6(9.79x 103), factor for dipole-dipole where K2 is the orientation transfer, Qo is the unquenched quantum yield of AS, n is the refractive index of the medium, and J is the spectral overlap integral (14). In our calculations KZ is taken to be 35, 1~ is assumed to be 1.4, and J was calculated by
J=
s
F(x)&)V dX . F(X) clX J
where F(A) is the AS fluorescence intensity in absence of quencher and e(x) is the cytochrome c extinction coefficient at wavelength (X) (15). For quenching of AS fluorescence by cytochrome c we calculate a J value of 5.55 X lo-l4 cm3 M-I for oxidized cytochrome c and 5.76 X lo-l4 cm3 M-I for reduced cytochrome c. The quantum yield &a of AS in cardiolipin was calculated to be 0.4, using 1-anilino-S-naphthalene sulfonate as a standard (16). Ro , the distance at which AS fluorescence is half-maximally quenched is calculated to be 49 .i for oxidized and 49.2 A for reduced cytochrome c. These values depend upon assumptions made in the calculations with regard to both K, the orientation factor and n, the refractive index, and must be regarded as tentative in the absence of precise data. The AS fluorescence lifetime was measured on an Ortec photon counting fluorescent lifetime instrument equipped with a Corning 760 filter for excitation, and a Corning 373 filter for emission. The oscilloscope tracings were photographed and read into the computer via an automatic graphic record digitizer, and the data analyzed by nonlinear regression to a variable number of exponentials. Normally a single exponential was tried first, and if the deviations showed good evidence of two species, two exponentials were tried and always found sufficient to fit the data.
BINDING
OF CYTOCIIROMIS
RESULTS
Quenching of AS Fluorescence in the Preserxe of Phospholipid Dispersions by Cytochrome c Addition of cytochrome c to a dispersion of cardiolipin-AS results in decreased izS fluorcscencc intensity. The fluorescence excitation and emission spectra of AScardiolipin dispersion in the presence and absence of cytochrome c are presented in Fig. 1. For comparison, t)he spectra mcasurcd in the presence of cyt’ochrome c have been recorded using an instrumental gain t,hree t’imes higher than that of the unquenched AS. Although the fluorescence yield of the AS-cardiolipin dispersion is sizably quenched by t’he presence of 1 PM cytochromc c, the fluorescence spectrum remains unchanged. This result eliminates a trivial reabsorption mechanism and suggests t’hat a nontrivial transfer of electronic excitation energy between the molecular electronic system of AS to cytochrome c is the mechanism of quenching (17). Consistent with this interpret’at’ion is a decrease in the lifetime of the excited state under conditions of fluorescence quenching. In Fig. 2, the decay curves of AS fluorescence in the presence and absence of cyt80chrome c are compared. The
c
221
fluorescence decay of 10 ~L\I AS in 1 mg cardiolipin/ml can be represented by a single exponential with a lifetime of 5.9 nsec, indicative of one population of AS molecules. The lifetime of the quenched sample is best fitted by two decay curves, with lifetimes of 5.5 nsec and 1.7 nsec contributing 30 and 70 %, respect,ively, to the signal. The existence of two lifet,imes indicates that there are two populations of AS molecults which are located at different distances from the quencher. AS located in the out’er layer of t’he bimolecular leaflet composing the cardiolipin vesicle is quenched upon cytochrome c binding and result’s in a shortened lifetime (less than 2 nsec). AS located on the inner surface of the bimolecular leaflet is not quenched osince it lies at a distance greater than 49 A which is t)he calculated distance for half maximum quenching (cf. Experiment’al Procedures). (The AS chromophore located on the inner surface is calculated to be about 75 ,& from the heme assuming a diameter of 30 8 for cytochromc c (lS), a t,hickncss of 60 d for the bimolecular leaflet, and the position of t’he A! chromophore in the membrane to be 15 A from the aqueous interface (9).) In the absence of cytochrome c the fluorescence intensity of AS-cardiolipin dispersions is linearly proportional to both t,he
FIG. 1. Uncorrected excitation and emission spectra of AS-cardiolipin suspensions in t,he presence and absence of cytochrome c. The medium contained 1 mM PO, buffer, pH 7.0, 1 mg/ml cardiolipin and 30 PM AS. Excitation wavelength for emission curve: 360 nm; emission wavelength for excitation curve: 460 nm. -, no additions; -----, 1 PM ferricytochrome c added. Instrumental gain was three times that of the unquenched sample.
222
VANDERKOOI,
ERECIfiSKS,
AND CHANCE
016
012
020
FIG. 3. Fluorescence of AS-cardiolipin mixed vesicles in the presence of cytochrome C. Medium contained 10 rnM PO, , pH 4.5. Cardiolipin was added from a stock solution containing 1 mM cardiolipin and 20 ~RI ilS. n , no cytochrome C; 0, 0.2 ~ZVX oxidized cytochrome c; A, 1.0 PM oxidized cytochrome c.
Factors Injluencing the Fluorescence Quenching of BS-phospholipid Dispersions by Cytoch rome c “XC
“WC
FIG. 2. Fluorescent lifetime of AS in cardiolipin micelles. A and B. Medium contained 10 mM PO 1, pH 6.5,l mg cardiolipin/ml, and 10b~ AS. Squares refer to experimental values. The line is a theoretical curve for exponential decay assuming alifetime of 5.9 nsec. C and D. Medium contained 10 mM PO1, pH 6.5, and 1 mg cardiolipin/ml, 10 pM AS, and 2 PM ferricytochrome c. The line is a theoretical curve for exponential decay assumingalifetime of 3.0 nsec. E and F. Same as C and D, assuming two lifetimes of 1.7 and 5.5 nsec. Inset A. Photograph of the oscilloscope tracing of A (upper) and C (lower).
The composit8ion of t)he phospholipid dispersion profoundly affects cytochrome c quenching of AS fluorescence. Figure 4 shows t’hat quenching of AS fluorescence by oxidized cytochrome c is more extensive in t,he presence of negatively charged cardiolipin dispersions t’han in zwitterionic egg lecithin dispersions. In mixed micelles of egg lecithin and cardiolipin the quenching of AS fluorescence is int,ermediate to that observed with either
cardiolipin
cytochrome
or egg lecithin.
c is somewhat
Reduced
less effective
than the oxidized form; this is due to a lower affinity of t,he reduced cytochrome for the
lipid dispersions, as demonstrated by double chromophore and lipid concentration (Fig. 3). In the presence of 0.2 PM and 1.0 PCIRI reciprocal plot’s (Fig. 4B). Consistent with the coulombic nature of oxidized cytochrome c, the fluorescence cytochromc c binding, the degree of quenchintensity of AS is quenched at low lipid ing of AS fluorescence is dependent upon concentration; however, at higher lipid of the medium concentrations, it is linear to the concen- the pH and salt concentration as well as the ionic character of artificial tration of AS and cardiolipin. Extrapolatmembranes. The magnitude ing the linear portion of the curve to zero phospholipid of quenching, and by extrapolation, the fluorescence intensity yields a value which degree of binding, is greatest at acidic is an ‘expression of the binding of cytopH values while at high pH values cytochrome c to cardiolipin. Using the conditions chrome c has litt’lc effect’ on the AS fluoresdescribed in the legend of Fig. 3, it was found cence (Fig. a). The isoelectric point of cytothat 1 molecule of cytochrome c was bound chrome c is reported to be 10.7 (19) and the per 80 molecules of cardiolipin.
BINDING
pM
02;
B
Cyt
OF CYTOCHROME
c
r)
’ .
FIG. 4. Effect of cytochrome c on AS fluorescence in cardiolipin egg lecithin mixed micelles. A. Oxidized cytochrome c was added to a medium containing 10 mM PO, , pH 6.9, and phospholipid vesicles prepared in the presence of AS to give 5 PM final concentration and the following concentrations of lipid: 0-0, 0.5 mg cardiolipin/ml; H, 0.25 mg cardiolipin/ml and 0.25 mg egg lecithin/ml; A----A, 0.5 mg egg lecithin/ml. Excitation: 360 nm; emission 460 nm. B. Doublereciprocal plot of the percentage quenching of AS fluorescence in the presence of oxidized (0) and reduced (0) cytochrome c. Conditions the same as above. Medium contained 0.25 mg cardiolipin and 0.25 mg egg lecithin/ml.
pK of cardiolipin is expected to be below 3 (20) ; thus, the positively charged protein is able to bind to the negatively charged lipid. Interestingly, there is little quenching of AS fluorescence by cytochrome c in the presence of egg lecithin, which is zwitterionic between pH values of 3.5-11.5 (al), suggesting that cytochrome c cannot bind to the highly charged surface of egg lecithin. These results are consistent with those reported by other workers (5, 22, 23). The fluorescence quenching of the AS-cardiolipin complex by cytochrome c is characterized by a fast’ phase, followed by a slower phase. An attempt was made t’o resolve the kinetics of the fast phase with use of stopped-flow mixing apparatus (inset
c
223
of Fig. 5). The fast phase of quenching is completed within the 20-msec mixing time of the instrument., while the slower phase is undetecbable in the time scale of this recording. The quenching of the fluorescence of AScardiolipin mixtures by cytochrome c is reversed by altering the pH. In Fig. 6 we see the results of an experiment in which the pH was rapidly changed from 2.0 to 11.3 by addition of concentrated NaOH, resulting in increased fluorescence. This could be reversed by the addition of HCI. The marked dependence of AS fluorescence quenching by cytochrome c on the pH of the medium cannot be explained by the shifts in absorpbion spectrum of cytochrome c it’self (24) since t’hey are not large enough to change the J values of the spectral overlap t’o a significant extent. The results can be best explained by altered binding characteristics of cytochrome c t,o cardiolipin as a function of pH. In addition to pH dependence of cytochrome c binding, ionic conditions of the medium affect the binding. Cytochrome c quenching of AS fluorescence in the presence of cardiolipin vesicles as a function of MgCl, concentration is illustrated in Fig. 7. At 0.1 M Rig CIZ and 2 ~151cytochromc c concentration, AS fluorescence is quenched only 6 % compared with S7 % in the magnesium-free sample. Intermediate quenching values are obt’ained at 5 and 10 mM MgClz, consistent with the electrostatic nature of cytochrome c binding. Similar results mere obtained with I
224
VANDERKOOI,
ERECINSKA,
2.i -
AND
CHANCE
4.i -
F 3”nlsef --I 01 FIG. 5. The pH dependence of cytochrome c quenching of AS incorporated into cardiolipin. The mixture contained 10 mM PO, 10 ,UM AS, 1 FM oxidized cytochrome c (added at the arrow), and 1 mg cardiolipin/ml. The pH was adjusted by NaOH or HCI addition. Fluorescence intensity was read using 360 nm for excitation; 460 nm for emission. Inset refers to fluorescence intensity of AS in a solution containing 10 PM AS and 1 mg cardiolipin/ml. At the beginning of the flow, 0.1 pM cytochrome c was added. Procedure used is as described by Chance et al. (38). Upper trace records the mixing time (20 msec) of the apparatus while lower two traces record successive additions of cytochrome c.
i “,\ ‘i;--
E 0
IO- M MqCI,
2 LL a, .? z20j
5~1O-~b’ MgCI,
1
/
,
0
0
I -
c \-
r
2
IO-% MgCIZ
3
Minutes
FIG. 6. Effect
of a pH jump of AS fluorescence in the presence of cardiolipin and cytochrome c. The pH of a solution containing 0.5 PM oxidized cytochrome c, 10 mM POa , and 10 fin AS, and 1 mg cardiolipin/ml was adjusted by NaOH or HCl addition to pH values indicated on the figure. Excitation: 360 nm; emission 460 nm. our experiment additional lipid did not increase fluorescence intensity, indicating that all the AS was incorporated into the lipid matrix.
Cytochrome
c
Binding to Mitochondria
Pigeon
Heart
Addition of cytochrome c to cytochrome c-depleted mitochondria results in restora-
10-3M MgCI, i-1
ml” +
FIG. 7. MgCl, effect on cytochrome c quenching of AS fluorescence in the presence of cardiolipin vesicles. The mixture contained 0.06 mg cardiolipin/ml, 10 mM PO, buffer, pH 7.0, 2 pM oxidized cytochrome c and MgClz in concentrations indicated. Excitation was at 360 nm; emission at 440 nm.
tion of electron transport. In Fig. 8, 02 consumption, cytochrome c binding, and cytochrome c quenching of AS fluorescence are measured at various cytochrome c concentrations. In this particular experiment, maximal restoration of oxygen consumption occurs at 0.5 nmoles cytochrome c + cl/mg protein, or about one-half the amount of cytochrome c found in intact
BINDING
OF CY’I’OCFIItOMI~
225
c
Total Cyt c (,pM)
Frc. 8. Comparison of oxygen consumption, cytochrome c binding and AS fluorescence intensity. Mitochondria (1 tng protein/ml) were incubated in the presenc’e of 30 PHI AS, 0.225 M nlaw ttitol, 0.05 M sucrose, and 50 m&f MOPE, pH 7.2 for 2 hr at, 0°C. Cytochrome c was added followed by measuretnent of cyt,ochrome binding, AS fluoand 02 rottsutnption, as described in rescence Experimkt,al Procedures. A, cytochrome c bound/trig protein, determined as described in the Methods. 0, fluorescence intensity using 360 nm for excitat,iott and 460 nm for emission where PO is the relative fluorescence intettsity of AS prior to cyt,ochrome c: addit,ion and F is the relat,ive fluorescence intensity after csytochrome c addition. 0, OI c~onsumptiott ttsing 3 mki sucrinate, 3 mM glutamate, 10 mM PO, in the presence of 0.3 my ADP (open symbols) and absence (closed symbols) of ADP.
pM
Cytochrome
c Added
FIG. 0. Cyt,ochrome c binding to oxidized and reduced mitochondria. Cytochrome c-depleted mitochondria (0.6-l mg protein/ml) were incnbated in the presence of 30 JAM AS, 0.225 M tnannitol, 0.05 M sucrose, and 50 mM MOPS, pH 7.2, for 1 hr. Cytochrome c was added and after centrifugation, cytochrome c binding was determined as described in Methods. O---O, no additions; O--O, 3 mM succinate, 3 mM glutamate, and 1 mM KCN added. Inset: Relative fluorescence intensity using 360 nm for excitation and 480 nm for etnission. Medium same as above. Dott,ed line: arrow 1, 1 PM cytochrome c was added; arrow 2, 3 tnlx succitlate and 3 my glutamate were added; ‘arrow 3, 1 mM KCN added. Solid line: same as dotted line except at arrow l? no caytochrome c was added.
mitochondria (10). Cytochrome c binding, measured directly after centrifugation and characteristics of the oxidized and reduced indirectly by the quenching of AS fluorescytochromc, or to alt’ered binding characcence by bound cytochromc c parallel each teristics of t’he mitochondrial membranes other, and are not saturated at cvtochrome which may depend on the redox state of c concentrations where stimulation of O2 the electron carriers. The effect may also consumption is maximal. This result sug- be secondary to the different ionic condigests that at less than 0.25 nmolcs cytotions under which both experiments were chrome c/mg protein electron flux t’hrough carried out. cytochromc c is rate limiting; above t’his In order t’o eliminate the latter possibilamount another step in elect’ron transfer ity, clxperimcnts mere carried out in which becomes rate limiting (25). the subskate was present but the respiratory Comparison of cytochrome c binding to chain remained oxidized by the presence cytochrome c-depleted mitochondrial mem- of inhibitors. The results of all three cxbranes in the absence and prrsencr of pcriment’s are presented as double-reciprocal oxidizable substrate is shown in Fig. 9. plots in Fig. 10. The double-reciprocal Oxidized cytochromc c binds significantly plots appear linear (Kg. 10A) while sigmore to the mit’ochondrial membrane in the nificant’ variation from nonlinearity is “oxidized state” (i.e., in the absence of seen in the more sensitive Scatchard plot added substrate) than t,hr reduced cyto(Fig. 10B). At sat’uration 2.4-2.S nmoles chrome c to the membranes reduced by the of cytochromc c per mg protein wcrc found. addition of malatc + glutama,tc + KCX. ,4ffiriity constants are S.C, X 10” >I for This may be due: either to alt’crcd binding oxiclizcd cytochromc c bound to the mito-
226
VANDERKOOI,
cyt c. ,dK’
ERECIRSKA,
AND CHANCE
r
FIG. 10. Binding of cytochrome c to cytochrome c-depleted pigeon heart mitochondria. A. Oxidized cytochrome c was added to a mixture containing 0.67 mg cytochrome c-depleted mitochondria preincubated for 1 hr with 0.225 M mannitol, 0.05 M sucrose, 50 mM MOPS, pH 7.2, and 10 MM AS. Cytochrome c binding was determined as described in the Methods. Cytochrome c concentration given in the abscissa refers to unbound cytochrome c. A-A, no additions; O-O, 10 pM rotenone was added followed by cytochrome c and 3 mM each of malate and glutamate final concentration. 0% was bubbled through the sample prior to centrifugation. O---O, 3 mM each of malate and glutamate followed by 1 mM KCN addition. B. Same as A. Data are plotted according to the equation r/A = Kn - Kr, where P is given in nmoles cytochrome c/mg protein, A is by free cytochrome c, and K is the affinity constant (39).
chondrial membrane in the absence of substrate, 1.8 X lo6 M for oxidized cytochrome c in the presence of substrate, and 0.5 X lo6 M for reduced cytochrome c in the presence of substrate and KCN (%cduced state” of the membrane). It is also seen from Fig. 9 that upon reduction of the respiratory chain carriers by substrates in the presence of cyanide AS fluorescence intensity increases both in the cytochrome c-depleted and cytochrome c-reconstituted membranes. The increase in fluorescence intensity is, however, much great’er in the reconstituted membranes due to the fact that not only oxidized coenzyme Q (26) but also reincorporated cytochrome c act as quenchers. Since reduced cytochrome c binds less than the oxidized cytochrome to the membranes, the quenching is smaller than that which occurs in the absence of oxidizable substrate upon the addition of oxidized cytochrome c. This result agrees with our analysis of the binding of cytochrome c under corresponding experimental conditions.
[KC’1 FIG. 11. Effect of KC1 on cytochrome c binding
and AS fluorescence intensity. Cytochrome c-depleted mitochondria (0.73 mg protein/ml) were incubated in a medium containing 50 mM MOPS, pH 7.2, KC1 in molar concentrations indicated on the abscissa and suhcient mannitol to make the solution 0.300 osmolar. Fluorescence intensity was measured using 360 nm excitat,ion and 480 nm emission. F,J and F are the relative fluorescence intensity of the AS mitochondrial suspension in the absence and presence of cytochrome c, respectively, and R is the nmoles cytochrome c bound/mg protein. Under
conditions
of high
ionic
strength
both cytochrome c binding to mitochondria and the quenching of the fluorescent ASmitochondrial complex by cytochrome c is diminished (Fig. 11). Half-maximal inhibition of both processes occurs at around 20 m&I KC1 concentration. The linear plot of inverse binding as a function of KC1 concentration (Fig. 11, inset) suggests competitive
inhibition
of
cytochrome
c
binding by KCl. This is indicative of the electrostatic nature of cytochrome c binding and is consistent with inhibition by monovalent and polyvalent cations ported in the literature (3, 27, 28).
rc-
DISCUSSION In order to quenching with
correlate binding
AS fluorescence of cytochrome
c
to the A&membrane complex, it is necessary to establish that quenching occurs by the distance-dependent nism and not by
energy transfer mechareabsorption of emitted
RINDING
OF CYTOCHROME
light, which is distance independent. The following results are consistent with energy transfer as being the mechanism for fluorescence quenching: (1) the emission spectra of quenched and unquenched AS fluorescence arc the same, although the fluorescent yield of the unquenched sample is one-third of the quenched one (Fig. 1) ; (2) the fluorescent lifetime of quenched AS is smaller than unquenched AS (Fig. 2) ; (3) under conditions which are unfavorable to binding, such as high ionic strength or high pH, the effectiveness of quenching by cytochrome c is greatly reduced (Figs. 4, 5, and 11) ; and (4) in the cast of mitochondria, fluorescence quenching and cyt’ochrome c binding parallel each other (Figs. 8 and 11). Thn amount of cytochromc c bound to artificial mcmbrancs varies with pH and ionic strength of the medium. At low salt conwntration, one molecule of cytochrome c was bound per 80 molecules of cardiolipin (P’ig. 3). Much less cytochrome c is bound to egg lecithin micclles. Das et al. reported t,hat cytochromc c-egg l&thin + phosphatidylethanolaminc complexes contained molar protein to lipid ratios of 24-41 (4) Purified cardiolipin forms a cytochromc c complex with a 8: 1 ratio, compared with 22 : 1 for phosphatidylethanolamine and 13O:l for lecithin (28). Iiimclberg et al. prcparcd mixed cardiolipin phosphatidylcholine (1:4) micclles and found a 70: 1 lipid to protcin ratio (22). A major diffcrCIKX: bctwcen tha expcrimcnts in the litwature and t’hosc described in this papor is that in the former case the lipid st’ructurcs were prepared in t,he presoncc of cytochrome c and are onion-like multilaycrs; in our case, cytochromc c was added to prcformcd sonicatcd vesicles, presumably resulting in binding predominantl,y t)o t’hr outside of t,lic vcsiclt:. The rate c~f cytochroma c binding to cardiolipin is c:hara&rized by two phases, a fast phase occurring wit’hin 20 msec after addit’ion, followed by a slower phase. The fast phase may bc duo to coulombic att’raction bct)wcen tho positively charged sites on t,hc protein and the negative: sitca on phosgholil)ids. The slower phase, which
c
227
takes up to a minute t’o complete (Fig. 4) may bc due to actual penetration of cytochrome c to a limited extent into t’hr rardiolipin lipid phase. The affinities of reduced and oxidized cytochrome c t,o both cardiolipin and mitochondrial membranes are different, consist& with a number of differences in the physiral and chemical properties of the carrier in both redox states. For example the optical rotatory dispersion spectra (29), as well as the nuclear magnetic resonance properties show dramatic differences between the oxidized and reduced states (30). The reduced form of cytochrome c is more resistant, to heat denaturation than is the oxidized form, while the oxidized form of yeast cyt’ochrome c is more susccptible to proteolytic at’tack than is the reduced form (31). These differences, if not accounted for by a change in t,he valency of iron, can be explained by a change in the struct’ure of the protein moiety. The same hypothesis has been put forward by RIargoliash et al. (32), and the change in the protein conformat’ion was confirmed recently by the X-ray crystallography studies of Dickerson and co-workers (33, 34). Changes in the conformation state of cytochrome c have also been suggested on the basis of a shift in the spectrum of cytochrome c aftcr its reduction with hydrated electrons (35). A difference in afinity of reduced and oxidized cytochrome c for the membrane is expcctcd on the basis of midpoint redox potential shift. The midpoint potential for soluble cytochrome c is 280 mV compared to 230 mV for rytochrome c bound inside phospholipid membranes in a succinatc-KC1 medium at pH 7.5 (36, 37). The relationship between midpoint potential and binding is formulated below:
where s refers to artificial or mitochondrial membranes, c+~ and c+~ reprcsent oxidized
228
VANDERKOOI,
ERECINSKA,
and reduced cytochrome c, respectively, and K, and K,. are the respective affinity constants of oxidized and reduced cytochrome c for the membrane. Using t’he principle of detailed balancing, the rat’io of the affinities of oxidized to reduced cytochrome c can be calculated. The midpoint potential difference between the bound and free forms is 50 mV, result’ing in a ratio of K,/K, equal t’o 7. The affinity of oxidized cytochrome c is 3.5 X lo6 M in the absence of substrate and 1.8 X lo6 RI in the presence of substrate. This compares with an affinity of 0.5 X lo6 nr for reduced cytochrome c. The difference in affinity of oxidized and reduced cytochrome c is a factor of 3.6 to 7.0, in agreement with midpoint potential shifts. Recent cxperiments have confirmed midpoint potential differences between free cytochrome c and cytochrome c added to cytochrome cdepleted mitochondria (J. G. Lindsay, personal communication). In view of the fact that the data presented in this paper are consistent with different affinities of cytochrome c depending on its redox state, Nicholls’ observation that the K, for cytochrome c reactivation of cytochrome c-deficient submitochondrial particles increases upon azide addition (25) can be interpreted as being due to different affinities of reduced and oxidized cytochrome c to the membrane. ACKNOWLEDGMENTS The authors thank Dr. David Wilson and Dr. George Radda for stimulating discussions. Thanks are due to Dr. Martin Pring for help in computer analysis and Mrs. E. Brocklehurst and Miss Keiko Sera for the preparation of mitochondria. 1. JACOBS, E. E., AND SANADI, D. R. (1960) J. Biol. Chem. 236, 531-534. 2. WIDMER, C., AND CRANE, F. L. (1958) Biochim. Biophys. Acta 27, 203-204. 3. DAS, M. L., AND CRANE, F. L. (1964) Biochemistry 3,696-700. 4. DAS, M. L., HAAK, E. D., AND CR~NIC, F. L. (1965) Biochemistry 4, 859-865. 5. REICH, M., AND WAINIO, W. W. (19Gl) J. Biol. Chem. 236,305&3061. 6. GREEN, D. E., AND FLEISCHER, S. (1963) Biochim. Biophys. Acta 70, 554-582. 7. FROMBERG, P. (1970) Fed. Eur. Biochem. Sot. Lett. 11, 205-208.
AND
CHANCE
8. BARTLETTE, G. R. (1959) J. Biol. Chem. 234, 466-468. 9. WAGGONEI~, A. S., AND STRYER, L. (1970) Proc. LVat. Acad. Sci. USA 6’7, 579-584. 10. CHBNCE, B., AND HAG1H.4n.4, B (1963) in Proc. Congr. Biochem. 5th Moscow 1961 (Sissakian, A.N.M., ed.) Vol. 5, pp. 3-37. 11. BOV~RIS, A., ERECINSKA, M., AND WAGNER, M. (1972) Biochim. Biophys. Acta 266, 223-242. 12. M~RGOLIASH, E., AND FROHWIRT, N. (1959) Biochem. J. 71, 570-572. 13. F~~RSTER, T. (1965) in Modern Quantum Chemistry, Istanbul Lectures (Sinanoglu, O., ed.), section III-B, pp. 93-137, Academic Press, New York. 14. BRAND, L., AND WITHOLT, B. (1967) in Methods, in Enzymology (C. H. W. Him, ed.), Vol. 11, pp. 776-856, Academic Press, New York. 15. Wu, C-W, AND STRYER, L. (1972) Proc. Nat. Acad. Sci. USA 69, 1104-1108. 16. STRYIQ~, L. (1965) J. Mol. Biol. 13, 482-495. 17. F~~RSTF,IL, T. (1959) Discuss. Faraday Sot. 27, 7-17. 18. DICKERSON, R. E., KOPKA, M. L., BORDERS, C. L., VARNUM, J., WEINZIERL, J. E., AND MARGOL~ASH, E. (1967) J. Mol. Biol. 29, 77-95. H., AND AKESSON, A. (1941) 19. THEORI”LL, J. Bmer. Chem. Sot. 63, 1804-1811. 20. BANGHAM, A. D. (1968) Progr. Biophys. Mol. Biol. 18, 29-93. Bio21. PAPAH.~DJOPOULOS, D. (1968) Biochim. phys. Acta 163, 240-254. 22. MACHINIST, J. M. DAS, M. L., AND CRANE, F. L. (1964) Biochemistry 3, 696-700. 23. KIMELBERG, H. K., LEE, C. P., Claude, A., AND MR~NA, L. (1970) J. Membran.e Biol. 2. 235-251. H., AND AKE:SSON, A. (1941) 24. THEORELL, J. Amer. Chem. Sot. 63, 1812-1871. 25. NICHOLLS, P. (1965) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S., and Morrison, M., eds) Vol. 2, pp. 764-783, J Wiley, New York. 26. CHANCE, B. (1972) in Coll. on Bioenergetics, Bressanone, 1971 (1972) Academic Press, New York. 27. MACHINIST, J. M., D.4s, M. L., CRANE, F. L., AND Jacor~s, E. E. (1961) Biochem. Biophys. Res Commun. 6, 475-478. 28. Das, M. L., HIRATSUKA, H., MACHINIST, J. M., AND CI~ANE, F. L. (1962) Biochim. Biophys. Acta 60, 435 434. 29. EICHHORN, G. L., AND CAIRNS, J. F. (1958) Nature (London) 181, 994. Chem. 237, 30. KOWALSKY, A. (1962) J. Biol. 1807-1819.
BINI)IN(;
OF CYTOCHROME
M., TOMON.\KI, T., HARRIS, T., AND K. (1957) -1. Hiochetx (‘l’okyo) 44, 453-458. 132. MARGOLIASH, E., FITCH, W. M., .\ND I)ICKI~X. SOS, R. E. (1968) Brookhaveu S’!/w~p. Rio/. 21, 359-305. 33. I)~~KERSOS, IL. I<., T.waNo, T., EISI~:NUERG, D., KALIAI, 0. B., hvlS@N, I,., COOPER, A.,
31. XOZUKI,
OKUNUKI,
AND &~.UlGOLIASH,
E.
(1971)
.I. &0l.
Che77~.
246, 1511-1535. 34. T.\KANO, .\ND
0. B., SprirLg 36, 397-401.
T., SWANSON, It., KALLAI, I~ICKERSON, R. IS. (1971) Cold
Harbor
Symp. QuatbL. Rid.
c
2.2. ‘Y
35. PECHT, I., .~ND FARAGGI, M. (19i2) Proc. Nat. Acad Sci. USA 69, 90%!&. 36. Dwrox,P.L., wlLSON,I). F., AND LFX,C.P. (1970) Biochemistry 9, 5077-5082. 37. KIMELBERG, H. K., AND LEE, C. P. (1970) J. Membra?ae Riol. 2, 252-262. 38. CHANCE, Is., l)EVAVL'I', I>., ~~I~;GALI.AIS, v., L., ASD YONETANI, T. (1967) in MEL-~, Fast Reactions and Primary Processes in Chem. Kinetics Nobel. Symp. V. (Claesson, S. ed) pp. 437-464, Interscience, Xew York. 39. HUGHES, T. R., ASD KLOTZ, I. M. (1956) Methods Biochem. Anal. 3, 2G5-299.
OF
BIOCHl?YISTRY
.IND
BIOPHYSICS
Cytochrome I. Use of a Fluorescent
c Interaction Chromophore
with Artificial JANE
VAn’DERI
Johnson
164, 219-229 (1973)
with
in the Study of Cytochrome
and Mitochondrial
RL4RIA
Membranes
ERECI%IiA,
c Interaction
Membranes’ .4ND
BRITTON
CHANCE
Research Foundation, Department of Biophysics and Physical Biochemistry, I’nirersity of Pennsylvania, Philadelphia, Pennsykjania I.9104 Received
August
21, 1972
Quenching of 12.(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS3 fluorescence is biphasic (tm below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids. Addition of cytochrome c to @ochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytoehrome c-depleted mitochondria is 1.8 X 106 M, while the affinity constant for reduced cytochrome c is 0.5 X lo6 M. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance wit,h midpoint potential differences between the bound and free forms.
Cytochrome c is unique among the mitochondrial respiratory eleckon carriers in its extractability and ability to restore both electron transport and ATP synthesis in the deficient mit’ochondria (1). i(‘urthermore, cytochrome c forms lipid complexes which provide an excellent model system for the st’udy of interactions between lipid and protein components of membranes (2-6). Two methods have been generally used to measure cytochromct c binding to phospholipids. In one, binding of cytochrome c is measured by extraction of the proteinlipid complex into an organic phase (3, 4); in the other, cyt’ochromc c is incorporated 1 This work was supported by USPHS GM12202. Computer time was supported by NIH Grant No. RR-15, using the Digital Equipment Corporation PDP-6 Computer of the University of Pennsylvania Medical School Computer Facility. * To whom correspondence should be addressed. 3 Abbreviations used: AS, 12.(9-anthrogl) stearic acid; MOPS, rnorpholilioprop:~rle sulfonate. 219 Copyright .\I1 rights
0 1973 by Academic Press, of reproduction in an>- funn
Inc. rcscrved
into multilayered phospholipid vesicles, and the complex is separated from the excess of cyt80chrome c by centrifugation (5, 6). n‘either of these methods yields information on the dynamics of cytochrome c interaction with membranes, and trapping of cytochrome c between the multilayered vesicles rather than binding to the lipid may be the actual parameter measured. This report describes a method to measure cyt!ochrome c binding to sonicated phospholipid vesicles and to mitochondrial membranes using resonance energy transfer from the excited state of a fluorescent chromophore to cytochrome c with subsequent quenching of fluorescence. The parameters of cytochrome c binding to artificial phospholipid membranes and to mitochondria are compared and discussed in terms of the mode of action of cytochrome c in the mitochondrial respiratory chain. A similar approach has been used by Fromberg to measure the distances between cytochrome
220
VANDERKOOI,
ERECIr;JSKA,
c and fluorescent chromophores in oriented lipid multilayers (7). MATERIALS Horse heart cytochrome c type VI was obtained from Sigma Chemical Company (St. Louis, MO). Bovine cardiolipin was obtained from Nutritional Biochemical Company (Cleveland, OH), and egg lecithin was a gift of Dr. T. Gulik-Krzywicki (Lab. de Biochimie, C.N.R.S., Gif-sur-Yvette, France). The purity of the lipids was checked by thinlayer chromatography on Silica Gel G plate (A. H. Thomas Company, Philadelphia, PA) using chloroform: methanol: ammonia (65: 25:4) as solvent followed by staining with iodine vapor. The cardiolipin appeared as one spot. Lecithin contained about 10% contamination, tentatively identified as diglyceride and lysolecithin. Lipid concentration was determined by weighing or by analyzing for phosphate (8). 12-(9-anthroyl) stearic acid (AS), obtained as a gift from Drs. A. S. Waggoner and L. Stryer (9), appeared as one spot on thin-layer chromatography under uv illumination. EXPERIMENTAL
PROCEDURES
Aqueous suspensions of phospholipids were prepared by sonicating the lipids in the presence of AS with a Branson sonifier for 5 min at 50 W followed by centrifugation at 100,OOOgfor 15 min to remove undispersed lipid. Fluorescence was measured at 90” to the exciting beam using a Hitachi MPF-2A fluorescence spectrometer. The sample compartment was maintained at 22-24°C with the use of circulating water. Pigeon heart mitochondria were prepared according to the method of Chance and Hagihara (10). Cytochrome c was extracted from mitochondria essentially by the method of Jacobs and Sanadi (1) with the modification described by Boveris et al. (11). Cytochrome c concentration was determined by reduced minus oxidized difference spectra using 19.7 as extinction coefficient (12). Oxygen uptake was (ESSO-540mM-l cm-l) measured at 24°C using a Clark oxygen electrode in 0.2 M mannitol-0.05 M sucrose-O.040 M morphoM phosphate medium, linopropane sulfonate-0.010 pH 7.0, with succinate plus glutamate as substrate. Cytochrome c binding was determined as the amount of cytochrome c found in the mitochondrial pellet after centrifuging down the mitochondria (10 min at SOOOgat 4°C) from the suspending medium in which the fluorescence assays had been done. The pellet was suspended in 0.05 M phosphate buffer containing 1% Triton X-100. The concentration of cytochrome c was estimated from
AND
CHANCE
the difference between total oxidized (+5 mM ferricyanide) - total reduced (+ dithionite). The measurements were done using a Johnson Foundation dual-wavelength spectrophotometer. The concentration of free cytochrome c was determined by measuring the amount found in the supernatant fluid from the 8OOOgcentrifugation. The recoveries were 95-100yc under all conditions. Dat,a concerning the quenching of AS fluorescence by cytochrome c were analyzed according to Fiister’s theory of dipole-dipole energy transfer (13). RO , the distance in Angstroms at which transfer efliciency is 5Ooj, was calculated by
Ro = (JKZQor~-4)1’6(9.79x 103), factor for dipole-dipole where K2 is the orientation transfer, Qo is the unquenched quantum yield of AS, n is the refractive index of the medium, and J is the spectral overlap integral (14). In our calculations KZ is taken to be 35, 1~ is assumed to be 1.4, and J was calculated by
J=
s
F(x)&)V dX . F(X) clX J
where F(A) is the AS fluorescence intensity in absence of quencher and e(x) is the cytochrome c extinction coefficient at wavelength (X) (15). For quenching of AS fluorescence by cytochrome c we calculate a J value of 5.55 X lo-l4 cm3 M-I for oxidized cytochrome c and 5.76 X lo-l4 cm3 M-I for reduced cytochrome c. The quantum yield &a of AS in cardiolipin was calculated to be 0.4, using 1-anilino-S-naphthalene sulfonate as a standard (16). Ro , the distance at which AS fluorescence is half-maximally quenched is calculated to be 49 .i for oxidized and 49.2 A for reduced cytochrome c. These values depend upon assumptions made in the calculations with regard to both K, the orientation factor and n, the refractive index, and must be regarded as tentative in the absence of precise data. The AS fluorescence lifetime was measured on an Ortec photon counting fluorescent lifetime instrument equipped with a Corning 760 filter for excitation, and a Corning 373 filter for emission. The oscilloscope tracings were photographed and read into the computer via an automatic graphic record digitizer, and the data analyzed by nonlinear regression to a variable number of exponentials. Normally a single exponential was tried first, and if the deviations showed good evidence of two species, two exponentials were tried and always found sufficient to fit the data.
BINDING
OF CYTOCIIROMIS
RESULTS
Quenching of AS Fluorescence in the Preserxe of Phospholipid Dispersions by Cytochrome c Addition of cytochrome c to a dispersion of cardiolipin-AS results in decreased izS fluorcscencc intensity. The fluorescence excitation and emission spectra of AScardiolipin dispersion in the presence and absence of cytochrome c are presented in Fig. 1. For comparison, t)he spectra mcasurcd in the presence of cyt’ochrome c have been recorded using an instrumental gain t,hree t’imes higher than that of the unquenched AS. Although the fluorescence yield of the AS-cardiolipin dispersion is sizably quenched by t’he presence of 1 PM cytochromc c, the fluorescence spectrum remains unchanged. This result eliminates a trivial reabsorption mechanism and suggests t’hat a nontrivial transfer of electronic excitation energy between the molecular electronic system of AS to cytochrome c is the mechanism of quenching (17). Consistent with this interpret’at’ion is a decrease in the lifetime of the excited state under conditions of fluorescence quenching. In Fig. 2, the decay curves of AS fluorescence in the presence and absence of cyt80chrome c are compared. The
c
221
fluorescence decay of 10 ~L\I AS in 1 mg cardiolipin/ml can be represented by a single exponential with a lifetime of 5.9 nsec, indicative of one population of AS molecules. The lifetime of the quenched sample is best fitted by two decay curves, with lifetimes of 5.5 nsec and 1.7 nsec contributing 30 and 70 %, respect,ively, to the signal. The existence of two lifet,imes indicates that there are two populations of AS molecults which are located at different distances from the quencher. AS located in the out’er layer of t’he bimolecular leaflet composing the cardiolipin vesicle is quenched upon cytochrome c binding and result’s in a shortened lifetime (less than 2 nsec). AS located on the inner surface of the bimolecular leaflet is not quenched osince it lies at a distance greater than 49 A which is t)he calculated distance for half maximum quenching (cf. Experiment’al Procedures). (The AS chromophore located on the inner surface is calculated to be about 75 ,& from the heme assuming a diameter of 30 8 for cytochromc c (lS), a t,hickncss of 60 d for the bimolecular leaflet, and the position of t’he A! chromophore in the membrane to be 15 A from the aqueous interface (9).) In the absence of cytochrome c the fluorescence intensity of AS-cardiolipin dispersions is linearly proportional to both t,he
FIG. 1. Uncorrected excitation and emission spectra of AS-cardiolipin suspensions in t,he presence and absence of cytochrome c. The medium contained 1 mM PO, buffer, pH 7.0, 1 mg/ml cardiolipin and 30 PM AS. Excitation wavelength for emission curve: 360 nm; emission wavelength for excitation curve: 460 nm. -, no additions; -----, 1 PM ferricytochrome c added. Instrumental gain was three times that of the unquenched sample.
222
VANDERKOOI,
ERECIfiSKS,
AND CHANCE
016
012
020
FIG. 3. Fluorescence of AS-cardiolipin mixed vesicles in the presence of cytochrome C. Medium contained 10 rnM PO, , pH 4.5. Cardiolipin was added from a stock solution containing 1 mM cardiolipin and 20 ~RI ilS. n , no cytochrome C; 0, 0.2 ~ZVX oxidized cytochrome c; A, 1.0 PM oxidized cytochrome c.
Factors Injluencing the Fluorescence Quenching of BS-phospholipid Dispersions by Cytoch rome c “XC
“WC
FIG. 2. Fluorescent lifetime of AS in cardiolipin micelles. A and B. Medium contained 10 mM PO 1, pH 6.5,l mg cardiolipin/ml, and 10b~ AS. Squares refer to experimental values. The line is a theoretical curve for exponential decay assuming alifetime of 5.9 nsec. C and D. Medium contained 10 mM PO1, pH 6.5, and 1 mg cardiolipin/ml, 10 pM AS, and 2 PM ferricytochrome c. The line is a theoretical curve for exponential decay assumingalifetime of 3.0 nsec. E and F. Same as C and D, assuming two lifetimes of 1.7 and 5.5 nsec. Inset A. Photograph of the oscilloscope tracing of A (upper) and C (lower).
The composit8ion of t)he phospholipid dispersion profoundly affects cytochrome c quenching of AS fluorescence. Figure 4 shows t’hat quenching of AS fluorescence by oxidized cytochrome c is more extensive in t,he presence of negatively charged cardiolipin dispersions t’han in zwitterionic egg lecithin dispersions. In mixed micelles of egg lecithin and cardiolipin the quenching of AS fluorescence is int,ermediate to that observed with either
cardiolipin
cytochrome
or egg lecithin.
c is somewhat
Reduced
less effective
than the oxidized form; this is due to a lower affinity of t,he reduced cytochrome for the
lipid dispersions, as demonstrated by double chromophore and lipid concentration (Fig. 3). In the presence of 0.2 PM and 1.0 PCIRI reciprocal plot’s (Fig. 4B). Consistent with the coulombic nature of oxidized cytochrome c, the fluorescence cytochromc c binding, the degree of quenchintensity of AS is quenched at low lipid ing of AS fluorescence is dependent upon concentration; however, at higher lipid of the medium concentrations, it is linear to the concen- the pH and salt concentration as well as the ionic character of artificial tration of AS and cardiolipin. Extrapolatmembranes. The magnitude ing the linear portion of the curve to zero phospholipid of quenching, and by extrapolation, the fluorescence intensity yields a value which degree of binding, is greatest at acidic is an ‘expression of the binding of cytopH values while at high pH values cytochrome c to cardiolipin. Using the conditions chrome c has litt’lc effect’ on the AS fluoresdescribed in the legend of Fig. 3, it was found cence (Fig. a). The isoelectric point of cytothat 1 molecule of cytochrome c was bound chrome c is reported to be 10.7 (19) and the per 80 molecules of cardiolipin.
BINDING
pM
02;
B
Cyt
OF CYTOCHROME
c
r)
’ .
FIG. 4. Effect of cytochrome c on AS fluorescence in cardiolipin egg lecithin mixed micelles. A. Oxidized cytochrome c was added to a medium containing 10 mM PO, , pH 6.9, and phospholipid vesicles prepared in the presence of AS to give 5 PM final concentration and the following concentrations of lipid: 0-0, 0.5 mg cardiolipin/ml; H, 0.25 mg cardiolipin/ml and 0.25 mg egg lecithin/ml; A----A, 0.5 mg egg lecithin/ml. Excitation: 360 nm; emission 460 nm. B. Doublereciprocal plot of the percentage quenching of AS fluorescence in the presence of oxidized (0) and reduced (0) cytochrome c. Conditions the same as above. Medium contained 0.25 mg cardiolipin and 0.25 mg egg lecithin/ml.
pK of cardiolipin is expected to be below 3 (20) ; thus, the positively charged protein is able to bind to the negatively charged lipid. Interestingly, there is little quenching of AS fluorescence by cytochrome c in the presence of egg lecithin, which is zwitterionic between pH values of 3.5-11.5 (al), suggesting that cytochrome c cannot bind to the highly charged surface of egg lecithin. These results are consistent with those reported by other workers (5, 22, 23). The fluorescence quenching of the AS-cardiolipin complex by cytochrome c is characterized by a fast’ phase, followed by a slower phase. An attempt was made t’o resolve the kinetics of the fast phase with use of stopped-flow mixing apparatus (inset
c
223
of Fig. 5). The fast phase of quenching is completed within the 20-msec mixing time of the instrument., while the slower phase is undetecbable in the time scale of this recording. The quenching of the fluorescence of AScardiolipin mixtures by cytochrome c is reversed by altering the pH. In Fig. 6 we see the results of an experiment in which the pH was rapidly changed from 2.0 to 11.3 by addition of concentrated NaOH, resulting in increased fluorescence. This could be reversed by the addition of HCI. The marked dependence of AS fluorescence quenching by cytochrome c on the pH of the medium cannot be explained by the shifts in absorpbion spectrum of cytochrome c it’self (24) since t’hey are not large enough to change the J values of the spectral overlap t’o a significant extent. The results can be best explained by altered binding characteristics of cytochrome c t,o cardiolipin as a function of pH. In addition to pH dependence of cytochrome c binding, ionic conditions of the medium affect the binding. Cytochrome c quenching of AS fluorescence in the presence of cardiolipin vesicles as a function of MgCl, concentration is illustrated in Fig. 7. At 0.1 M Rig CIZ and 2 ~151cytochromc c concentration, AS fluorescence is quenched only 6 % compared with S7 % in the magnesium-free sample. Intermediate quenching values are obt’ained at 5 and 10 mM MgClz, consistent with the electrostatic nature of cytochrome c binding. Similar results mere obtained with I
224
VANDERKOOI,
ERECINSKA,
2.i -
AND
CHANCE
4.i -
F 3”nlsef --I 01 FIG. 5. The pH dependence of cytochrome c quenching of AS incorporated into cardiolipin. The mixture contained 10 mM PO, 10 ,UM AS, 1 FM oxidized cytochrome c (added at the arrow), and 1 mg cardiolipin/ml. The pH was adjusted by NaOH or HCI addition. Fluorescence intensity was read using 360 nm for excitation; 460 nm for emission. Inset refers to fluorescence intensity of AS in a solution containing 10 PM AS and 1 mg cardiolipin/ml. At the beginning of the flow, 0.1 pM cytochrome c was added. Procedure used is as described by Chance et al. (38). Upper trace records the mixing time (20 msec) of the apparatus while lower two traces record successive additions of cytochrome c.
i “,\ ‘i;--
E 0
IO- M MqCI,
2 LL a, .? z20j
5~1O-~b’ MgCI,
1
/
,
0
0
I -
c \-
r
2
IO-% MgCIZ
3
Minutes
FIG. 6. Effect
of a pH jump of AS fluorescence in the presence of cardiolipin and cytochrome c. The pH of a solution containing 0.5 PM oxidized cytochrome c, 10 mM POa , and 10 fin AS, and 1 mg cardiolipin/ml was adjusted by NaOH or HCl addition to pH values indicated on the figure. Excitation: 360 nm; emission 460 nm. our experiment additional lipid did not increase fluorescence intensity, indicating that all the AS was incorporated into the lipid matrix.
Cytochrome
c
Binding to Mitochondria
Pigeon
Heart
Addition of cytochrome c to cytochrome c-depleted mitochondria results in restora-
10-3M MgCI, i-1
ml” +
FIG. 7. MgCl, effect on cytochrome c quenching of AS fluorescence in the presence of cardiolipin vesicles. The mixture contained 0.06 mg cardiolipin/ml, 10 mM PO, buffer, pH 7.0, 2 pM oxidized cytochrome c and MgClz in concentrations indicated. Excitation was at 360 nm; emission at 440 nm.
tion of electron transport. In Fig. 8, 02 consumption, cytochrome c binding, and cytochrome c quenching of AS fluorescence are measured at various cytochrome c concentrations. In this particular experiment, maximal restoration of oxygen consumption occurs at 0.5 nmoles cytochrome c + cl/mg protein, or about one-half the amount of cytochrome c found in intact
BINDING
OF CY’I’OCFIItOMI~
225
c
Total Cyt c (,pM)
Frc. 8. Comparison of oxygen consumption, cytochrome c binding and AS fluorescence intensity. Mitochondria (1 tng protein/ml) were incubated in the presenc’e of 30 PHI AS, 0.225 M nlaw ttitol, 0.05 M sucrose, and 50 m&f MOPE, pH 7.2 for 2 hr at, 0°C. Cytochrome c was added followed by measuretnent of cyt,ochrome binding, AS fluoand 02 rottsutnption, as described in rescence Experimkt,al Procedures. A, cytochrome c bound/trig protein, determined as described in the Methods. 0, fluorescence intensity using 360 nm for excitat,iott and 460 nm for emission where PO is the relative fluorescence intettsity of AS prior to cyt,ochrome c: addit,ion and F is the relat,ive fluorescence intensity after csytochrome c addition. 0, OI c~onsumptiott ttsing 3 mki sucrinate, 3 mM glutamate, 10 mM PO, in the presence of 0.3 my ADP (open symbols) and absence (closed symbols) of ADP.
pM
Cytochrome
c Added
FIG. 0. Cyt,ochrome c binding to oxidized and reduced mitochondria. Cytochrome c-depleted mitochondria (0.6-l mg protein/ml) were incnbated in the presence of 30 JAM AS, 0.225 M tnannitol, 0.05 M sucrose, and 50 mM MOPS, pH 7.2, for 1 hr. Cytochrome c was added and after centrifugation, cytochrome c binding was determined as described in Methods. O---O, no additions; O--O, 3 mM succinate, 3 mM glutamate, and 1 mM KCN added. Inset: Relative fluorescence intensity using 360 nm for excitation and 480 nm for etnission. Medium same as above. Dott,ed line: arrow 1, 1 PM cytochrome c was added; arrow 2, 3 tnlx succitlate and 3 my glutamate were added; ‘arrow 3, 1 mM KCN added. Solid line: same as dotted line except at arrow l? no caytochrome c was added.
mitochondria (10). Cytochrome c binding, measured directly after centrifugation and characteristics of the oxidized and reduced indirectly by the quenching of AS fluorescytochromc, or to alt’ered binding characcence by bound cytochromc c parallel each teristics of t’he mitochondrial membranes other, and are not saturated at cvtochrome which may depend on the redox state of c concentrations where stimulation of O2 the electron carriers. The effect may also consumption is maximal. This result sug- be secondary to the different ionic condigests that at less than 0.25 nmolcs cytotions under which both experiments were chrome c/mg protein electron flux t’hrough carried out. cytochromc c is rate limiting; above t’his In order t’o eliminate the latter possibilamount another step in elect’ron transfer ity, clxperimcnts mere carried out in which becomes rate limiting (25). the subskate was present but the respiratory Comparison of cytochrome c binding to chain remained oxidized by the presence cytochrome c-depleted mitochondrial mem- of inhibitors. The results of all three cxbranes in the absence and prrsencr of pcriment’s are presented as double-reciprocal oxidizable substrate is shown in Fig. 9. plots in Fig. 10. The double-reciprocal Oxidized cytochromc c binds significantly plots appear linear (Kg. 10A) while sigmore to the mit’ochondrial membrane in the nificant’ variation from nonlinearity is “oxidized state” (i.e., in the absence of seen in the more sensitive Scatchard plot added substrate) than t,hr reduced cyto(Fig. 10B). At sat’uration 2.4-2.S nmoles chrome c to the membranes reduced by the of cytochromc c per mg protein wcrc found. addition of malatc + glutama,tc + KCX. ,4ffiriity constants are S.C, X 10” >I for This may be due: either to alt’crcd binding oxiclizcd cytochromc c bound to the mito-
226
VANDERKOOI,
cyt c. ,dK’
ERECIRSKA,
AND CHANCE
r
FIG. 10. Binding of cytochrome c to cytochrome c-depleted pigeon heart mitochondria. A. Oxidized cytochrome c was added to a mixture containing 0.67 mg cytochrome c-depleted mitochondria preincubated for 1 hr with 0.225 M mannitol, 0.05 M sucrose, 50 mM MOPS, pH 7.2, and 10 MM AS. Cytochrome c binding was determined as described in the Methods. Cytochrome c concentration given in the abscissa refers to unbound cytochrome c. A-A, no additions; O-O, 10 pM rotenone was added followed by cytochrome c and 3 mM each of malate and glutamate final concentration. 0% was bubbled through the sample prior to centrifugation. O---O, 3 mM each of malate and glutamate followed by 1 mM KCN addition. B. Same as A. Data are plotted according to the equation r/A = Kn - Kr, where P is given in nmoles cytochrome c/mg protein, A is by free cytochrome c, and K is the affinity constant (39).
chondrial membrane in the absence of substrate, 1.8 X lo6 M for oxidized cytochrome c in the presence of substrate, and 0.5 X lo6 M for reduced cytochrome c in the presence of substrate and KCN (%cduced state” of the membrane). It is also seen from Fig. 9 that upon reduction of the respiratory chain carriers by substrates in the presence of cyanide AS fluorescence intensity increases both in the cytochrome c-depleted and cytochrome c-reconstituted membranes. The increase in fluorescence intensity is, however, much great’er in the reconstituted membranes due to the fact that not only oxidized coenzyme Q (26) but also reincorporated cytochrome c act as quenchers. Since reduced cytochrome c binds less than the oxidized cytochrome to the membranes, the quenching is smaller than that which occurs in the absence of oxidizable substrate upon the addition of oxidized cytochrome c. This result agrees with our analysis of the binding of cytochrome c under corresponding experimental conditions.
[KC’1 FIG. 11. Effect of KC1 on cytochrome c binding
and AS fluorescence intensity. Cytochrome c-depleted mitochondria (0.73 mg protein/ml) were incubated in a medium containing 50 mM MOPS, pH 7.2, KC1 in molar concentrations indicated on the abscissa and suhcient mannitol to make the solution 0.300 osmolar. Fluorescence intensity was measured using 360 nm excitat,ion and 480 nm emission. F,J and F are the relative fluorescence intensity of the AS mitochondrial suspension in the absence and presence of cytochrome c, respectively, and R is the nmoles cytochrome c bound/mg protein. Under
conditions
of high
ionic
strength
both cytochrome c binding to mitochondria and the quenching of the fluorescent ASmitochondrial complex by cytochrome c is diminished (Fig. 11). Half-maximal inhibition of both processes occurs at around 20 m&I KC1 concentration. The linear plot of inverse binding as a function of KC1 concentration (Fig. 11, inset) suggests competitive
inhibition
of
cytochrome
c
binding by KCl. This is indicative of the electrostatic nature of cytochrome c binding and is consistent with inhibition by monovalent and polyvalent cations ported in the literature (3, 27, 28).
rc-
DISCUSSION In order to quenching with
correlate binding
AS fluorescence of cytochrome
c
to the A&membrane complex, it is necessary to establish that quenching occurs by the distance-dependent nism and not by
energy transfer mechareabsorption of emitted
RINDING
OF CYTOCHROME
light, which is distance independent. The following results are consistent with energy transfer as being the mechanism for fluorescence quenching: (1) the emission spectra of quenched and unquenched AS fluorescence arc the same, although the fluorescent yield of the unquenched sample is one-third of the quenched one (Fig. 1) ; (2) the fluorescent lifetime of quenched AS is smaller than unquenched AS (Fig. 2) ; (3) under conditions which are unfavorable to binding, such as high ionic strength or high pH, the effectiveness of quenching by cytochrome c is greatly reduced (Figs. 4, 5, and 11) ; and (4) in the cast of mitochondria, fluorescence quenching and cyt’ochrome c binding parallel each other (Figs. 8 and 11). Thn amount of cytochromc c bound to artificial mcmbrancs varies with pH and ionic strength of the medium. At low salt conwntration, one molecule of cytochrome c was bound per 80 molecules of cardiolipin (P’ig. 3). Much less cytochrome c is bound to egg lecithin micclles. Das et al. reported t,hat cytochromc c-egg l&thin + phosphatidylethanolaminc complexes contained molar protein to lipid ratios of 24-41 (4) Purified cardiolipin forms a cytochromc c complex with a 8: 1 ratio, compared with 22 : 1 for phosphatidylethanolamine and 13O:l for lecithin (28). Iiimclberg et al. prcparcd mixed cardiolipin phosphatidylcholine (1:4) micclles and found a 70: 1 lipid to protcin ratio (22). A major diffcrCIKX: bctwcen tha expcrimcnts in the litwature and t’hosc described in this papor is that in the former case the lipid st’ructurcs were prepared in t,he presoncc of cytochrome c and are onion-like multilaycrs; in our case, cytochromc c was added to prcformcd sonicatcd vesicles, presumably resulting in binding predominantl,y t)o t’hr outside of t,lic vcsiclt:. The rate c~f cytochroma c binding to cardiolipin is c:hara&rized by two phases, a fast phase occurring wit’hin 20 msec after addit’ion, followed by a slower phase. The fast phase may bc duo to coulombic att’raction bct)wcen tho positively charged sites on t,hc protein and the negative: sitca on phosgholil)ids. The slower phase, which
c
227
takes up to a minute t’o complete (Fig. 4) may bc due to actual penetration of cytochrome c to a limited extent into t’hr rardiolipin lipid phase. The affinities of reduced and oxidized cytochrome c t,o both cardiolipin and mitochondrial membranes are different, consist& with a number of differences in the physiral and chemical properties of the carrier in both redox states. For example the optical rotatory dispersion spectra (29), as well as the nuclear magnetic resonance properties show dramatic differences between the oxidized and reduced states (30). The reduced form of cytochrome c is more resistant, to heat denaturation than is the oxidized form, while the oxidized form of yeast cyt’ochrome c is more susccptible to proteolytic at’tack than is the reduced form (31). These differences, if not accounted for by a change in t,he valency of iron, can be explained by a change in the struct’ure of the protein moiety. The same hypothesis has been put forward by RIargoliash et al. (32), and the change in the protein conformat’ion was confirmed recently by the X-ray crystallography studies of Dickerson and co-workers (33, 34). Changes in the conformation state of cytochrome c have also been suggested on the basis of a shift in the spectrum of cytochrome c aftcr its reduction with hydrated electrons (35). A difference in afinity of reduced and oxidized cytochrome c for the membrane is expcctcd on the basis of midpoint redox potential shift. The midpoint potential for soluble cytochrome c is 280 mV compared to 230 mV for rytochrome c bound inside phospholipid membranes in a succinatc-KC1 medium at pH 7.5 (36, 37). The relationship between midpoint potential and binding is formulated below:
where s refers to artificial or mitochondrial membranes, c+~ and c+~ reprcsent oxidized
228
VANDERKOOI,
ERECINSKA,
and reduced cytochrome c, respectively, and K, and K,. are the respective affinity constants of oxidized and reduced cytochrome c for the membrane. Using t’he principle of detailed balancing, the rat’io of the affinities of oxidized to reduced cytochrome c can be calculated. The midpoint potential difference between the bound and free forms is 50 mV, result’ing in a ratio of K,/K, equal t’o 7. The affinity of oxidized cytochrome c is 3.5 X lo6 M in the absence of substrate and 1.8 X lo6 RI in the presence of substrate. This compares with an affinity of 0.5 X lo6 nr for reduced cytochrome c. The difference in affinity of oxidized and reduced cytochrome c is a factor of 3.6 to 7.0, in agreement with midpoint potential shifts. Recent cxperiments have confirmed midpoint potential differences between free cytochrome c and cytochrome c added to cytochrome cdepleted mitochondria (J. G. Lindsay, personal communication). In view of the fact that the data presented in this paper are consistent with different affinities of cytochrome c depending on its redox state, Nicholls’ observation that the K, for cytochrome c reactivation of cytochrome c-deficient submitochondrial particles increases upon azide addition (25) can be interpreted as being due to different affinities of reduced and oxidized cytochrome c to the membrane. ACKNOWLEDGMENTS The authors thank Dr. David Wilson and Dr. George Radda for stimulating discussions. Thanks are due to Dr. Martin Pring for help in computer analysis and Mrs. E. Brocklehurst and Miss Keiko Sera for the preparation of mitochondria. 1. JACOBS, E. E., AND SANADI, D. R. (1960) J. Biol. Chem. 236, 531-534. 2. WIDMER, C., AND CRANE, F. L. (1958) Biochim. Biophys. Acta 27, 203-204. 3. DAS, M. L., AND CRANE, F. L. (1964) Biochemistry 3,696-700. 4. DAS, M. L., HAAK, E. D., AND CR~NIC, F. L. (1965) Biochemistry 4, 859-865. 5. REICH, M., AND WAINIO, W. W. (19Gl) J. Biol. Chem. 236,305&3061. 6. GREEN, D. E., AND FLEISCHER, S. (1963) Biochim. Biophys. Acta 70, 554-582. 7. FROMBERG, P. (1970) Fed. Eur. Biochem. Sot. Lett. 11, 205-208.
AND
CHANCE
8. BARTLETTE, G. R. (1959) J. Biol. Chem. 234, 466-468. 9. WAGGONEI~, A. S., AND STRYER, L. (1970) Proc. LVat. Acad. Sci. USA 6’7, 579-584. 10. CHBNCE, B., AND HAG1H.4n.4, B (1963) in Proc. Congr. Biochem. 5th Moscow 1961 (Sissakian, A.N.M., ed.) Vol. 5, pp. 3-37. 11. BOV~RIS, A., ERECINSKA, M., AND WAGNER, M. (1972) Biochim. Biophys. Acta 266, 223-242. 12. M~RGOLIASH, E., AND FROHWIRT, N. (1959) Biochem. J. 71, 570-572. 13. F~~RSTER, T. (1965) in Modern Quantum Chemistry, Istanbul Lectures (Sinanoglu, O., ed.), section III-B, pp. 93-137, Academic Press, New York. 14. BRAND, L., AND WITHOLT, B. (1967) in Methods, in Enzymology (C. H. W. Him, ed.), Vol. 11, pp. 776-856, Academic Press, New York. 15. Wu, C-W, AND STRYER, L. (1972) Proc. Nat. Acad. Sci. USA 69, 1104-1108. 16. STRYIQ~, L. (1965) J. Mol. Biol. 13, 482-495. 17. F~~RSTF,IL, T. (1959) Discuss. Faraday Sot. 27, 7-17. 18. DICKERSON, R. E., KOPKA, M. L., BORDERS, C. L., VARNUM, J., WEINZIERL, J. E., AND MARGOL~ASH, E. (1967) J. Mol. Biol. 29, 77-95. H., AND AKESSON, A. (1941) 19. THEORI”LL, J. Bmer. Chem. Sot. 63, 1804-1811. 20. BANGHAM, A. D. (1968) Progr. Biophys. Mol. Biol. 18, 29-93. Bio21. PAPAH.~DJOPOULOS, D. (1968) Biochim. phys. Acta 163, 240-254. 22. MACHINIST, J. M. DAS, M. L., AND CRANE, F. L. (1964) Biochemistry 3, 696-700. 23. KIMELBERG, H. K., LEE, C. P., Claude, A., AND MR~NA, L. (1970) J. Membran.e Biol. 2. 235-251. H., AND AKE:SSON, A. (1941) 24. THEORELL, J. Amer. Chem. Sot. 63, 1812-1871. 25. NICHOLLS, P. (1965) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S., and Morrison, M., eds) Vol. 2, pp. 764-783, J Wiley, New York. 26. CHANCE, B. (1972) in Coll. on Bioenergetics, Bressanone, 1971 (1972) Academic Press, New York. 27. MACHINIST, J. M., D.4s, M. L., CRANE, F. L., AND Jacor~s, E. E. (1961) Biochem. Biophys. Res Commun. 6, 475-478. 28. Das, M. L., HIRATSUKA, H., MACHINIST, J. M., AND CI~ANE, F. L. (1962) Biochim. Biophys. Acta 60, 435 434. 29. EICHHORN, G. L., AND CAIRNS, J. F. (1958) Nature (London) 181, 994. Chem. 237, 30. KOWALSKY, A. (1962) J. Biol. 1807-1819.
BINI)IN(;
OF CYTOCHROME
M., TOMON.\KI, T., HARRIS, T., AND K. (1957) -1. Hiochetx (‘l’okyo) 44, 453-458. 132. MARGOLIASH, E., FITCH, W. M., .\ND I)ICKI~X. SOS, R. E. (1968) Brookhaveu S’!/w~p. Rio/. 21, 359-305. 33. I)~~KERSOS, IL. I<., T.waNo, T., EISI~:NUERG, D., KALIAI, 0. B., hvlS@N, I,., COOPER, A.,
31. XOZUKI,
OKUNUKI,
AND &~.UlGOLIASH,
E.
(1971)
.I. &0l.
Che77~.
246, 1511-1535. 34. T.\KANO, .\ND
0. B., SprirLg 36, 397-401.
T., SWANSON, It., KALLAI, I~ICKERSON, R. IS. (1971) Cold
Harbor
Symp. QuatbL. Rid.
c
2.2. ‘Y
35. PECHT, I., .~ND FARAGGI, M. (19i2) Proc. Nat. Acad Sci. USA 69, 90%!&. 36. Dwrox,P.L., wlLSON,I). F., AND LFX,C.P. (1970) Biochemistry 9, 5077-5082. 37. KIMELBERG, H. K., AND LEE, C. P. (1970) J. Membra?ae Riol. 2, 252-262. 38. CHANCE, Is., l)EVAVL'I', I>., ~~I~;GALI.AIS, v., L., ASD YONETANI, T. (1967) in MEL-~, Fast Reactions and Primary Processes in Chem. Kinetics Nobel. Symp. V. (Claesson, S. ed) pp. 437-464, Interscience, Xew York. 39. HUGHES, T. R., ASD KLOTZ, I. M. (1956) Methods Biochem. Anal. 3, 2G5-299.