Cytochrome c interaction with membranes

.kRCHIVk:S OF BIOCHl?YISTRY .IND BIOPHYSICS 164, 219-229 (1973)

Cytochrome c Interaction with Membranes

I. Use of a Fluorescent Chromophore in the Study of Cytochrome c Interaction

with Artificial and Mitochondrial Membranes’

JANE VAn’DERI<OOI,2 RL4RIA ERECI%IiA, .4ND BRITTON CHANCE

Johnson 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 di- minishes 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(‘urther- more, 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 protein- lipid complex into an organic phase (3, 4); in the other, cyt’ochromc c is incorporated

1 This work was supported by USPHS GM- 12202. 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) ste-

aric acid; MOPS, rnorpholilioprop:~rle sulfonate.

into multilayered phospholipid vesicles, and the complex is separated from the ex- cess of cyt80chrome c by centrifugation (5, 6). n‘either of these methods yields in- formation 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

219

Copyright 0 1973 by Academic Press, Inc. .\I1 rights of reproduction in an>- funn rcscrved

220 VANDERKOOI, ERECIr;JSKA, AND CHANCE

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 thin- layer chromatography on Silica Gel G plate (A. H. Thomas Company, Philadelphia, PA) using chlo- roform: 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 iden- tified 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 illumina- tion.

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,OOOg for 15 min to remove undispersed lipid.

Fluorescence was measured at 90” to the excit- ing beam using a Hitachi MPF-2A fluorescence spectrometer. The sample compartment was main- tained 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 (ESSO-540 mM-l cm-l) (12). Oxygen uptake was measured at 24°C using a Clark oxygen electrode in 0.2 M mannitol-0.05 M sucrose-O.040 M morpho- linopropane sulfonate-0.010 M phosphate medium, 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 SOOOg at 4°C) from the sus- pending 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

the difference between total oxidized (+5 mM ferricyanide) - total reduced (+ dithionite). The measurements were done using a Johnson Founda- tion dual-wavelength spectrophotometer. The concentration of free cytochrome c was determined by measuring the amount found in the supernatant fluid from the 8OOOg centrifugation. The recoveries were 95-100yc under all conditions. Dat,a concern- ing 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 efli- ciency is 5Ooj, was calculated by

Ro = (JKZQor~-4)1’6(9.79 x 103),

where K2 is the orientation factor for dipole-dipole 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

s F(x)&)V dX

J= .

J F(X) clX

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 emis-

sion. 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 c 221

RESULTS

Quenching of AS Fluorescence in the Preserxe of Phospholipid Dispersions by Cyto-

chrome c

Addition of cytochrome c to a dispersion of cardiolipin-AS results in decreased izS fluorcscencc intensity. The fluorescence excitation and emission spectra of AS- cardiolipin dispersion in the presence and absence of cytochrome c are presented in Fig. 1. For comparison, t)he spectra mcas- urcd 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

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 contribut- ing 30 and 70 %, respect,ively, to the signal. The existence of two lifet,imes indicates that there are two populations of AS mole- cults 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

“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 theoreti- cal 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 theoreti- cal 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. Photo- graph of the oscilloscope tracing of A (upper) and C (lower).

chromophore and lipid concentration (Fig. 3). In the presence of 0.2 PM and 1.0 PCIRI oxidized cytochrome c, the fluorescence intensity of AS is quenched at low lipid concentration; however, at higher lipid concentrations, it is linear to the concentration of AS and cardiolipin. Extrapolat- ing the linear portion of the curve to zero fluorescence intensity yields a value which is an ‘expression of the binding of cytochrome c to cardiolipin. Using the conditions described in the legend of Fig. 3, it was found that 1 molecule of cytochrome c was bound per 80 molecules of cardiolipin.

012 016 020

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 Quench- ing of BS-phospholipid Dispersions by

Cytoch rome c

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 or egg lecithin. Reduced cytochrome c is somewhat 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 reciprocal plot’s (Fig. 4B).

Consistent with the coulombic nature of cytochromc c binding, the degree of quenching of AS fluorescence is dependent upon the pH and salt concentration of the medium as well as the ionic character of artificial phospholipid membranes. The magnitude of quenching, and by extrapolation, the degree of binding, is greatest at acidic pH values while at high pH values cytochrome c has litt’lc effect’ on the AS fluorescence (Fig. a). The isoelectric point of cytochrome c is reported to be 10.7 (19) and the

BINDING OF CYTOCHROME c 223

pM Cyt c

02; B 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. Ex- citation: 360 nm; emission 460 nm. B. Double- reciprocal 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 charac- terized 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

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 AS- cardiolipin 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 ~151 cytochromc c concentration, AS fluorescence is quenched only 6 % compared with S7 % in the mag- nesium-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<Cl, although at higher concentrat’ions (not shown).

It should be pointed out that t’he fluorescence intensity of the AS-cardiolipin dispersion prior to the addition of cytochrome c is about 30 % higher at high salt’ concentrations (s-100 rnlr RlgC1.J than in a salt- free solution. Possible interpretations of this phenomenon include a more favorable partit,ion of AS in the phospholipid vesicle at high ionic strcngt’h, or a change in t*he vesicle structure as a function of ionic strength of the medium which may result in increased quantum efficiency of the chromophore. The second possibility is more likely since under the conditions of

224 VANDERKOOI, ERECINSKA, AND CHANCE

2.i -

4.i -

01 --I F 3”nlsef

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.

E 0 2 LL

a, .?

1

z20j

0

,

I - r

0 2 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 Pigeon Heart Mitochondria

Addition of cytochrome c to cytochrome c-depleted mitochondria results in restora-

i “, ‘i;-- IO- M MqCI, \ /

5~1O-~b’ MgCI,

c \- 10-3M MgCI,

IO-% MgCIZ 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 oxi-

dized 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~ c 225

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 fluorescence and 02 rottsutnption, as described in 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.

mitochondria (10). Cytochrome c binding, measured directly after centrifugation and indirectly by the quenching of AS fluorescence by bound cytochromc c parallel each other, and are not saturated at cvtochrome c concentrations where stimulation of O2 consumption is maximal. This result suggests that at less than 0.25 nmolcs cytochrome c/mg protein electron flux t’hrough cytochromc c is rate limiting; above t’his amount another step in elect’ron transfer becomes rate limiting (25).

Comparison of cytochrome c binding to cytochrome c-depleted mitochondrial membranes in the absence and prrsencr of oxidizable substrate is shown in Fig. 9. Oxidized cytochromc c binds significantly more to the mit’ochondrial membrane in the “oxidized state” (i.e., in the absence of added substrate) than t,hr reduced cytochrome c to the membranes reduced by the addition of malatc + glutama,tc + KCX. This may be due: either to alt’crcd binding

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 incn- bated in the presence of 30 JAM AS, 0.225 M tnan- nitol, 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.

characteristics of the oxidized and reduced cytochromc, or to alt’ered binding characteristics of t’he mitochondrial membranes which may depend on the redox state of the electron carriers. The effect may also be secondary to the different ionic conditions under which both experiments were carried out.

In order t’o eliminate the latter possibility, clxperimcnts mere carried out in which the subskate was present but the respiratory chain remained oxidized by the presence of inhibitors. The results of all three cx- pcriment’s are presented as double-reciprocal plots in Fig. 10. The double-reciprocal plots appear linear (Kg. 10A) while significant’ variation from nonlinearity is seen in the more sensitive Scatchard plot (Fig. 10B). At sat’uration 2.4-2.S nmoles of cytochromc c per mg protein wcrc found. ,4ffiriity constants are S.C, X 10” >I for oxiclizcd cytochromc c bound to the mito-

226 VANDERKOOI, ERECIRSKA, AND CHANCE

cyt c. ,dK’ 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 pre- incubated 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 (%c- duced 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 AS- mitochondrial 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 rc- ported in the literature (3, 27, 28).

DISCUSSION

In order to correlate AS fluorescence quenching with binding of cytochrome c to the A&membrane complex, it is necessary to establish that quenching occurs by the distance-dependent energy transfer mechanism and not by reabsorption of emitted

RINDING OF CYTOCHROME c 227

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 unfavor- able 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’o- chrome 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 + phos- phatidylethanolaminc 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 phosphatidyl- choline (1:4) micclles and found a 70: 1 lipid to protcin ratio (22). A major diffcr- CIKX: bctwcen tha expcrimcnts in the litwa- ture 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’rac- tion bct)wcen tho positively charged sites on t,hc protein and the negative: sitca on phosgholil)ids. The slower phase, which

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 rardio- lipin lipid phase.

The affinities of reduced and oxidized cytochrome c t,o both cardiolipin and mitochondrial membranes are different, con- sist& 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 sus- ccptible 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 RIar- goliash 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 suc- cinatc-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 CHANCE

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 cxperi- ments have confirmed midpoint potential differences between free cytochrome c and cytochrome c added to cytochrome c- depleted 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 par- ticles 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.

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Cytochrome c interaction with membranes

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