Proceedings of the National Academy of SciencesVol. 67, No. 2, pp. 560-571, October 1970

Fluorescent Probe Environment and theStructural and Charge Changes

in Energy Coupling of Mitochondrial Membranes*

Britton ChanceJOHNSON RESEARCH FOUNDATION, SCHOOL OF MEDICINE, UNIVERSITY OF

PENNSYLVANIA, PHILADELPHIA

Read as an Invited Paper on the Conformational Basis of Energy Transduction, April 29, 1970

Abstract. The use of fluorescent probes to give continuous readouts of thestructural states of mitochondrial membranes during energy coupling seems alogical extension of their use in the study of protein structural changes. A cle 1trcorrelation of the probes' fluorescence characteristics with the acquisition ofenergy coupling can be demonstrated in fragmented and natural membraneusing 1-anilinonaphthalene-8-sulfonate (ANS) and ethidium bromide respec-tively. The present contribution attempts to bring together contemporaryviewpoints of this and other laboratories and the recent experimental data andgive some detailed information on probe environment and on the structural orcharge changes occurring upon energization.The energy-dependent region of the membrane is located at an aqueous inter-

face between an outer layer of proteins (presumably cytochromes) and the mem-brane permeability barrier; the aromatic portion of ANS appears to be locatedin the lipid phase and the sulfonic acid group in the aqueous phase. Theaqueous phase is probably a structured water region near paramagnetic mem-brane components such as cytochrome. Membrane energization arising fromaltered redox potential changes of cytochromes (bT) is communicated to thewater structure through altered structural states of, the hemoproteins, causinga decreased volume of the structured water region and increased interaction withthe paramagnetic components in the energized state. Attendant alterationsof protonic equilibria of membrane components induce both local and trans-membrane changes in charge distribution, with consequent movements of ions,including the probe molecules themselves.

In considering the role of protein structure in the control of enzyme activity,several workers have put forward hypotheses to explain the relationship be-tween membrane structure and energy coupling and the control of electronflow. Most of the information on membrane structure comes from techniquessuch as electron microscopy or low-angle x-ray scattering, in which the membraneis fixed in one of its many possible structural states. Thus the data obtainedgive a "single-frame" or "stop-motion" rather than a "dynamic" result.So far only two techniques-study of light-scattering changes and probe re-sponses-seen suitable for evaluation of structural changes during the course

560

VOL. 67, 1970 CONFORMATION AND ENERGY TRAN:VSDUCTION 561

of acquisition and release of energy coupling and control phenomena in mito-chondrial membranes.

Light scattering itself records a multiplicity of phenomena, ranging fromosmotic responses' to more subtle changes observed in membrane fragments.2However, electron microscopy seems to indicate that such changes are associated,at least int mitochondria, with large-scale alterations in the density of the matrixspace.' Greatly improved results may be expected from "laser Doppler" tech-niques where particle size distributions may be measured directly from the mem-brane suspension in the course of structural changes.4Another approach to achieve a more direct and continuous readout of mem-

brane structure changes has been introduction of the organic fluorochromes notedpreviously by Weber5 for their sensitivity to the changes of structure and con-formation of the protein molecules and initially employed by Newton6 to recordthe interaction of bacterial membranes with polymyxin.The greatest advantage of the fluorescent probe technique is the large number

of parameters that can be measured continuously under a variety of conditionsto which the native membranes have been exposed.7'8 The experiments includeexcitation and emission spectra, quantum yield, life time and polarization. Theinterpretation of these data gives information on (1) the membrane occupancyor the number of sites binding the probe, as provided by appropriate interpreta-tions of the equilibrium data, (2) the probe mobility in the membrane determinedfrom its polarization and life times, (3) the chemical nature of the membraneenvironment occupied by the probe (lipid and/or protein) evaluated by deletionor addition of either one or both of these components to natural or artificialmembranes, (4) in favorable cases, the proximity of the probe to natural orartificial membrane components, which may be determined by energy-transferand depolarization measurements as well as by the effect of other parameters suchas pH, temperature, and charge of the solvent, and (5) the energy-linked altera-tions in the nature and charge of the membrane environment as determined bychanges of all these parameters; In fact these results, when compared to theinformation content of other probe methods, makes the fluorescent approach offoremost importance in assessing the role of membrane conformation changesin energy coupling and control of electron flow.As has been elegantly summarized in the contribution of Waggoner and Stryer7

to this symposium, many aspects of the static structure of the membranes canbe explored with fluorescent probes.9 It is our purpose, however, to explorethe dynamic aspects of the membrane structure and to correlate them with theenergy-coupling events which in other contributions to this symposiums areidentified by different techniques. The correlation can be further explored bycomparing probe fluorescence changes with the kinetics of the energy-responsivecytochrome b (bT) upon addition of a pulse of exygen' I to anaerobic mitochondrialsuspension.

Materials and Methods. Pigeon heart mitochondria were isolated by the methodof Chance and Hagihara'2; submitochondrial particles were prepared from beef heartmitochondria and from bacterial cells (M. denitrificans) by sonicationl4 and lysozyme-shock treatment." All three preparations were suspended in 0.225 M mannitol-0.075 M

562 N. A. S. SYMPOSIUM: B. CHANCE PROC. N. A. S.

sucrose, with either 40 mM Tris pH 7.4 or 40 mM morpholinopropane sulfonate (MOPS)pH 7.4 as a buffer. Membranes depleted of lipid or protein were prepared by S. Fleischer.'.The probes employed were obtained commercially and were recrystallized from appro-priate solvents. The 1-anilinonaphthalene 8-sulfonic acid (ANS) anion is used withmembrane fragments and the ethidium bromide cation is used with intact membranes.'6,'7The kinetics of the fluorescence change in response to oxygen pulses were measured inthe rapid flow apparatus'8 and fluorescence was excited from the side of the observationtube by light at 390-405 nm (ANS) or 546 nm (ethidium bromide). The fluorescenceemission was measured in the region of 520 nm and above 615 nm, respectively.

Results. Sonicated membrane fragments, in contrast to the intact membranesof mitochondria, show a large fluorescence increase upon the addition of theanionic probe ANS. An explanation is afforded by electron microscopic'9 andantigen-antibody studies20 and by our own data on ferricyanide-inaccessiblecytochromes of these membranes, which suggest that they are "inside-out"fragments of the native membranes. Fig. 1 shows the ANS fluorescence spec-

100

80 5 s+5mM Succinate

M 60 / 6 +4 M Succinote

0 5|oa

/

protein ANSml. Curve 2, + lO~uM ANS; Curve 3, + 10 ,uM + 4 M+I01M ANSaddition of25au+2.5,uMANS 3 s0.1mg Protein/mics b

20- 2-

0.43mg Protein/mI0 ~~~~~~~~~~00 460 500 540 580 420'460 500 540 560

X (nm) X(nm)

A ~~~~~~B

FIG. 1. Fluorescence emission spectra of ANS in the presence of sonicated membranefragments of beef heart mitochondria (A) and M. denitrificans (B). Conditions: (A) (1) 0.3M mannitol-sucrose-0.020 M Tris-HCI pH 7.4. 1, Oligomycin-supplemented beef heartsubmitochondrial particles, 0.1 mg protein per ml; 2, plus 2.5 /AM ANS; 3, plus 2.5 AMiVANS + 5 mnM succinate. (B) 0.5 M sucrose-0.010 M phosphate buffer pH 7.0, 0.43 mgprotein per ml. Curve 2, + 10 juM ANS; Curve 3, + 10,gM ANS + 4 mnM succinate. Incollaboration with J. K. Matsubara. Spectra were obtained in a Hitachi MPF-2A spectro-fluorometer, Excitation wavelength, 360 nmn.

trum obtained on such sonicated fragments of mammalian and bacterial mem-branes. Curve 1 illustrates mainly light-scattering changes and leakage throughthe secondary filter obtained in the presence of the membranes only. Theaddition of 2.5 and 10 JLM ANS, curves 2, causes small fluorescence increases,but in both cases a striking effect is caused by the addition of succinate. A very

VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 563

large emission peak characteristic of the fluorescence enhancement of the ener-gized state of these membranes emerges at 470 nm. The enhancement of fluores-cence is largely eliminated either by inhibition of electron transport or of energycoupling.

In sonicated fragments of beef heart mitochondria it is possible to distinguishelectron flow and its coupling to energy conservation, since the electron flowdoes not result in energy coupling unless the membranes are supplemented witholigomycin.22 The consequent ANS responses (excitation at 390 nm and emis-sion at 470 nm) are presented as a function of time, Fig. 2A. The addition of

0.7MM S-13(Deenergization)

21pM PCP(Deenergizotion)150sec~~~~~~~10s

390-'470nm 45~2nFluorescence Fluorescence -°Increase Ices

1i+_1-4m 4mM Succinote- 7Lg Oligomnycin/mI (Energization)a(Et|e tion

/ml j- X 30O M NADH

- ~~~~~~~~~~~~~~~(Energization)6.7mM Succinote 8MMLlv ANS

3MM ANSA B

FIG. 2. Energization characteristics of membrane fragments prepared from beef heartmitochondria (A) and M. denitrificans (B). The traces were obtained in a Hitachi MPF-2Aspectrofluorometer. The excitation wavelength and the concentrations of various compoundsadded are indicated on the figure (Fig. 2959-4 and JKM-24). PCP, pentachlorophenol.

succinate to the membrane suspension supplemented with ANS causes a 1.5-foldincrease in the fluorescence, although independent controls show that the electronflow proceeds at the maximal rate appropriate to this substrate. However, theaddition of succinate and oligomycin cause a five-fold increase of ANS fluores-cence. Decrease of the respiration rate and increase in cytochrome b reductionrecorded simultaneously show that oligomycin has evoked an energy couplingand control in the membrane fragments. Thus the steady state of fluorescencereached on addition of oligomycin depends upon the efficiency of energy coupling.In support of this, when uncoupler (S-13) which causes a rapid deenergizationof the membrane, is added, there is an abrupt fluorescence decrease to a levelnearly approaching that recorded prior to the addition of succinate; when theANS fluorescence is taken as a basis for calculation the ratio of deflection re-corded in the presence of succinate plus oligomycin to that in the presence ofsuccinate, oligomycin, and 8-13 equals 21; therefore essentially all of the ANSfluorescence changes are associated with the membrane energization. The factthat the addition of succinate in the absence of uncoupler causes an increasein fluorescence indicates that even in the absence of oligomycin the membranesshow some, although a very slight, energy coupling. The remarkably selectiveresponse of the probe to energy coupling together with the great simplicity andsensitivity of the fluorescence readout have made it one of the most useful newtools in the study of the energy coupling phenomena. 2

564 N. A. S. SYMPOSIUM: B. CHANCE PROC. N. A. S.

Bacterial membranes'3 do not exhibit oligomycin dependence of their energy-linked functions but they respond with an increase in fluorescence upon theaddition of respiratory substrates in many ways similar to that obtained inmammalian membranes. When limited concentrations of NADH (30 AM,Fig. 2B, excitation 405 nm, emission 520 nm) are used to activate the electrontransfer, the four-fold increase in fluorescence disappears on the exhaustion of thesubstrate and the fluorescent trace declines to its initial level. Addition ofsuccinate to a concentration of 14 mM causes a 6.5-fold fluorescence increase,to a stable level because of a continuing oxidation of succinate. Uncouplingby pentachlorophenol (PCP) or addition of cyanide (not shown) causes a diminu-tion of fluorescence; the trace, however, does not return to the original level;the remaining portion of ANS fluorescence is due to the electron flow. Thus thebacterial membranes show an increase in ANS fluorescence due to both theelectron flow alone (3.6-fold) and electron flow-linked energy coupling (6.5-fold).Similar succinate-induced fluorescence changes, not related to energy coupling,were observed in mammalian submitochondrial membrane fragments by Dattaand Penefsky24 using N-methyl-2-anilino-6-naphthalene-sulfonate (MNS) as aprobe.Probe environment: The results of a large number of quantitative studies

of the membrane response to ANS summarized recently (Table 1)25 consider-

TABLE 1. Parameters of ANS response in energized and nonenergized membranes of sub-mitochondrial particles.

BindingKD sites Rel. Life

dissoc. (nmol/mg quantum time, Depolar-Xmax const. prot.) yield nsec ization

State (nm) (M) (n) (4) (r) (P)Nonenergized 470 35 X 10-6 80 1 5 (9) 0.22Energized 470 20 X 10-6 80 2.5 (5) 9 0.18

ably elaborate our preliminary data.'6 Interpretations of these data give usdeeper insight into the location and structure of membrane regions in whichthe phenomena of electron transfer and energy coupling occur. In our previouscommunications we compared the enhancement of probe fluorescence in themembrane with that observed in ethanol and concluded that the membraneenvironment approximates that of ethanol-water 4:1. This is further sup-ported by the peaks of the ANS emission spectrum (Xmax) in the energized stateat 470 nm, a value equal to that for ethanol-water 4: 1 (the Xmax value in wateris 515 nm and in hexane 454 nm26). Furthermore Waggoner and Stryer7 on thebasis of the response of octadecylnaphthylamine sulfonic acid (ONS) responsein phosphatidylcholine bilayers concluded27 that ONS is located in an environ-ment with a polarity corresponding to that of methanol-water 7:3. In supportof this conclusion, studies carried out in this laboratory'8 on oriented magnesiumstearate multilayers and phospholipid dispersion by means of low-angle x-raydiffraction indicate the ANS to be located with its aromatic moiety in the hydro-phobic region of the bimolecular leaflet, with the sulfonic acid projecting intothe hydrophilic medium. Assuming that the data on the artificial membranes

VOL. 67, 1970 CONFORMATIONAL AND ENERGY TRANASDLTCTION 565

are relevant in interpreting the ANS interactions with the biological membranes,we may visualize ANS to occupy an aqueous interphase region of the membrane.7A novel approach to the studies of the ANS environment in the membrane isafforded by preliminary studies of ANS proton resonances using high-resolution(220 MHz) nmr.29 Proton resonances identified with H atoms attached toC-2 (adjacent to the sulfonate group) and to C-5 (para to the sulfonate group)of ANS are clearly delineated at 1770 and 1830 Hz in Fig. 3, trace A. Theseresonances are no longer detectable when mem-branes are added, as evidenced by a single scan B(trace B, or by 100 computer-averaged scans(data not shown). Further studies of this phe-nomenon30 show the water and ethanol reso- Anances to be unaffected by the membrane con-centration employed, while the proton reso-nances of 50 mM\ ANS in water are 50%0 dimin- 2345 2095 1845 1515 1345ished by about 2 mg/ml of membrane protein. HzThe overall titration curve of the ANS pro-ton resonance versus membrane protein con- FIG. 3. Singlescan 220 MHzcentrationexhibitsa b character, exnmr proton resonance spectracentration exhibits a biphasic character, ex- of ANS. (A) 70 mM ANS in

pected from the previously reported existence ethanol (B) 12 mM in 17%of two types of binding sites. While many ethanol with membrane frag-

poorlyunderstood phenomena may affect 'm. - ments (60 mg protein per ml)poorly understood phenomena may affect mag- prepared from beef heart mito-netic resonance spectra in such complicated chondria.systems,3' two simple explanations can be putforward to account for the diminished ANS proton resonances in themembrane. Probe immobilization affords one explanation which, however,seems unlikely in view of the fact that glycerol-water mixtures covering the samerange of fluorescence depolarization do not cause the proton resonance to dis-appear. A preferred explanation is the existence of a rapid exchange of ANSwith a membrane environment containing paramagnetic centers, some of whichmay be cytochromes in the oxidized state or iron-sulfur proteins in the reducedstate. The broadenings observed would correspond to distances of <10 A be-tween the probe protons and paramagnetic centers. The cytochrome moleculescapable of interaction with ANS have to be located on the outside of the mem-brane permeability barrier since ANS does not penetrate lipid bilayers or thepermeability barrier of the sonicated membrane fragments. The existence ofsuch sets of cytochromes has recently been reported on the basis of 40% accessi-bility of these carriers to the highly charged anion ferricyanide."1 (These sets ofcytochromes are not capable of energy coupling and for this reason were omittedfrom diagram 5 of reference 11.) Tentative evidence for an adequately rapidANS exchange reaction with the membrane fragments is afforded by a rapidcombination reaction (compare Fig. 5) and computed dissociation velocityconstant of >20 sec' (compare Table 1). (The nmr line-broadening ob-served places this limit above 103 sec'.)

Kinetics of the ANS reaction: The relative contribution of protein and lipidto the ANS binding in the intact membrane has been studied by using the

566 N. A. S. SYMPOSIUM: B. CHANCE PROC. N. A. S.

membranes made either lipid-deficient or protein-deficient.15'30 To ensuresimilar fluorescence changes in both types of the preparations, a constant concen-tration of ANS but different amounts of protein for each preparation (0.5 mg/ml and 0.3 mg/ml) have been used. In both cases (Fig. 4) the ANS responseis biphasic. A fast phase, two-thirds completed during the flow interval (asindicated by the arrow) has a half-time of 15 msec, as calculated from a firstorder velocity constant of 45 sec-'. The slow phase, which follows the fastphase, exhibits a half-time of 200 msec. The magnitudes of the fluorescencechanges, appropriately extrapolated to the same protein concentration, arenearly identical for the rapid change in the lipid-depleted as well as protein-depleted membranes, while the slow fluorescence change is much smaller in thelipid-depleted preparation. On the basis of these and the nmr experimentsdescribed above, we identify the fast reaction of ANS with interaction of the

FIG. 4. Effect of depleting membranes oflipid (A) and protein (B) upon the kinetics

Flow of ANS reaction. In (A), heavy beef heartStarts Velocity mitochondria were extracted to the extent

|Stops aT Stops of 10% residual lipids (0.5 mg protein per___~____A.* ml.) In (B), vesicles were treated with urea,- ~~~~~~~~~~sothat '-~40% of the protein was extracted

436-520nmFluorescence (the ratio of jeg P to mg protein increases from

Increase 18 in the unextracted vesicles to 31 in the,A_/t\ __ __ 18 _<-Fresidue, indicating that only proteins haveE___ been extracted by urea treatment). Final

I-. f-lO0msec 1--iOOmsec protein concentration 0.3 mg/ml. Reaction88MuM ANS 88ytM ANS kinetics carried out in the pulse flow ap

A B paratus, 88 MAM ANS added at each dis-charge. Time during flow mixing is 20 msec.(Figure courtesy of J. M. Tager, S. Papa,E. Quagliarello and E. C. Slater.)

probe with protein-presumably the cytochrome coating of the membrane.The slow reaction, which is dependent on the amount of phospholipid in themembrane,'5 reflects a slow penetration of ANS molecules to the hydrophobicinterior of the membrane. The aqueous interface occupied by ANS may residebetween the protein (cytochrome) coating of the membrane and the lipid perme-ability barrier. It is, however, possible that a discrete discontinuity does notexist and that the lipid and protein are somewhat heterogeneously distributed.Membrane occupancy by ANS: Table 1 indicates 80 nmol/mg protein of

"high-affinity" binding sites for ANS. This value is very large, over 50 times theamount of cytochrome c. The number of binding sites, however, does not in-crease upon energization, which provides further evidence for the lack of grossstructural changes such as a membrane "opening" or reorganization phenomenonin the membrane upon energization.

Fluorescence depolarization: Although in preliminary experiments no changeof the value of fluorescence depolarization was found, more recent data show adrop from 0.22 to 0.18 in the transition from the nonenergized to the energizedstate.'6'25 One of the possible causes is an enhanced energy transfer betweenANS molecules due to their greater clumping in the energized state. 16

VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 567

Life time: The probe lifetime is found to have two populations of 5 and 9nsec.25 Membrane energization increases the population of the longer lifetime,and thus is qualitatively, although not yet quantitatively, consistent with theincreased quantum efficiency. These lifetimes and the fluorescence depolariza-tions give rotational relaxation times of the probe in the membrane environmentof 20 and 12 nsec in the nonenergized and energized states. This suggests agreater mobility of the probe in the energized environment.Quantum yield: The quantum yield in the membrane is"6 approximately

0.1 as compared to 0.004 for ANS in water and 0.98 in a hexane-like environ-ment. The quantum yield increases 2.5-fold in the energized state, while thepeak of the emission spectrum is unaltered. One might expect the quantumyield and the peak shift of the probe to be related, as indeed they are for solventpolarity and Z value changes.33 However, they appear to be unrelated in thecase of membrane energization. But the quantum yield given in Table 1 is aresult of an extrapolation to infinite protein:ANS concentration (where allANS molecules are accounted for), whereas the peak shift in our experiments hasbeen read out under conditions where the membrane occupancy varies. Thus,more weight should be given to the well-defined increase of quantum yield thanto the position of the emission maximum.

Values of To, the radiative lifetime (as calculated from the observed quantumyield and lifetimes), are 12.5 and 9.0 nsec for the nonenergized and the energizedstate respectively. This decrease of radiative lifetime is consistent with anincreased strength of binding in the energized state, and thus is in accord with thedecreased KD of Table 1. In order to account for the decreased radiative life-time and the increased quantum efficiency at the same time, it is necessary toconsider not only collisional perturbation, a relatively minor effect, but alsoparamagnetic perturbation of the activated state of the probe, which appears tobe far more important. This conclusion is consistent with the explanation ofparamagnetic perturbations for the diminution of the ANS proton resonancesas detected by nmr.A physical picture which can be deduced from these data might be useful

in considering the nature of the energized state. It is possible, however, thatthis is only one of several possible physical representations consistent with theavailable data. In accordance with Radda, we postulate the probe to be locatedin pockets of structured water of restricted volume which account for the lowfluorescence depolarization and for the excimer emission.8'23 Furthermore, inorder to explain the paramagnetic affect, these pockets of water structure arelocated near the paramagnetic centers in the membranes such as cytochromes.Membrane energization involves movement of water molecules in the mem-brane out of these pockets to cause decreased collisional perturbations of theprobe. In addition, these pockets move nearer to the paramagnetic centers ofthe membrane (or vice versa), or indeed the paramagnetic centers in the mem-brane increase in intensity so that paramagnetic effects are enhanced.Membrane charge: Changes of membrane charge, which were not con-

sidered in detail in our initial publication, have now been investigated in extensoby Azzi34 from comparison studies of various probes and membranes with op-

568 N. A. S. SYMPOSIUM: B. CHANCE PROC. N. A. S.

posite charge separations. The general conclusion that membrane charge playsa large role in the ANS binding in energized-deenergized transitions, is docu-mented by the decrease of dissociation constant occurring upon the transition(Table 1).Relation of water structure and charge changes: A number of attempts have

been made to determine whether the change in water structure postulated aboveto explain the quantum yield and lifetime data precedes the change in membranecharge, as evidenced by the altered dissociation constant, or vice versa-a topicof some importance in considering basic mechanisms for energy coupling. Ithas been possible to demonstrated that a considerable degree of membrane de-energization occurred before a significant amount of ANS was able to bindadded bovine serum albumin molecules exterior to the protein coat of the mem-brane. This result suggests that the change in water structure precedes themovement of significant amounts of ANS. However, according to Azzi,35 thistransition time depends on the nature of the probe (1,8-ANS responds threetimes faster than 2,6-ANS) and thus at present this vexing question cannot bepositively resolved. The model of membrane structure that illustrated bothtypes of changes (Fig. 4 of ref. 11) seems appropriate.Membrane energization time in mitochondria: In membrane fragments, the

half-time for membrane energization is approximately 2 sec36 (see Fig. 1 of ref. 16),although the slowest electron-transport reaction has reached its steady state inapproximately 200 msec.37 We have interpreted this result to indicate that theconfiguration of the activated state of the membrane is achieved only aftersteady-state electron transport has been established. In order to afford a morefavorable situaton for comparing probe responses with membrane energizationtimes, we can utilize the recent report that in intact pigeon heart mitochondria,energization of the membrane causes a transition of cytochrome bT from its low-to its high-potential form, in 200 msec, and results in considerable decrease ofits oxidation rate." It appears desirable to compare cytochrome bT kineticswith probe responses under identical experimental conditions. Kinetics ofcytochrome b in response to a pulse of oxygen are recorded at 566-575 nm, theabsorbancy maximum for cytochrome bT'1 (Fig. 5). The initial fast phase iscompleted within 200 msec; thereafter cytochrome bi, whose potential has beenshifted to + 245 mV, enters its second, slow phase in which it equilibrates slowlywith its oxidant, cytochrome cl, at +220 mV. This reaction occupies the largerportion of 2 sec. If indeed a fluorescence probe accurately depicts membraneenergization, it would be expected to have responded in the first 200 msec. Theappropriate probe for measuring the energization phenomenon of the intactmembrane is the ethidium bromide cation.'6 Ethidium bromide has the desir-able property of exhibiting the same dissociation constant (0.7-1.0 /1M) in theenergized and de-energized states of the mitochondrial membrane and a rel-atively low membrane occupancy, 2 nmol/mg protein.38 Thus conditions areunusually favorable for choosing a probe: protein ratio (16 nmol/mg protein)at which occupancy changes are negligible. The experimental trace obtainedupon energization of the membrane in the presence of ethidium bromide (excita-tion 546 nm, emission 615 nm) suggests a definite parallelism between the probe

VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 569

FlowStarts Velocity StartsIFStops Trace h-Stops

546- 615nm00026 OD. 566- 575nm luorenceseI

AbsorbanceIncrease

Cytochrome b 27%Reduction

A f4500msec j-14500msec17pM 02 17pM 02

A B

FIG. 5. A comparison of the kinetics of (A) the transformation of cytochromebT from the low-potential, quickly-reacting form to the high-potential, slowly-reacting form with (B) the fluorescence increase observed with ethidium bromide-supplemented mitochondrial membranes. The excitation and emission conditionsfor ethidium bromide are indicated in the figure. Medium: 0.3 M mannitol-sucrose-0.040 M morpholinopropane sulfonate buffer, pH 6.7. 6.0 mM succinate, 1.2 mMglutamate, 3.0 mM malonate, 5.0 ,uM rotenone, 2.3 mg protein per ml pigeon heartmitochondria. Ethidium bromide in B was 14 MM.

response and the initial phase of the kinetics of cytochrome bT. Thereafter thereis a further phase of probe response which ends before cytochrome bT is completelyoxidized. Thus the initial phases of the probe and the cytochrome bT responseare in good agreement. Further slow changes in the probe response occur duringthe equilibration of cytochrome bT with cytochrome cl.Comparison of ethidium bromide and ANS responses during energization:

The experiments were further carried out to determine whether the time courseof increased fluorescence of ethidium bromide would correspond to the decreasedfluorescence of ANS, as found earlier. A comparison of the two traces in-dicates the two to be parallel over the first 400 msec of the fluorescence change.This result is of particular interest because the energy-independent dissociationconstant and a small number of binding sites for ethidium bromide suggest thatin this case the environmental responses are being observed, while for ANSthe relatively large dissociation constant and large number of binding sitessuggest that a probe redistribution is occurring. The fact that the kinetics areat present indistinguishable suggests that even in the intact mitochondria it isnot yet possible to resolve a structural and a charge event in membrane energiza-tion.

Discussion. Cationic and anionic probes, used under appropriate conditionswith appropriate intact or fragmented'membranes, characterize the structuraland charge properties of the energized state of the membrane and allow crucialcomparisons of these properties with the time course of the cytochrome energiza-tion reaction. It is apparent that the two components of membrane energiza-tion cannot yet be resolved temporally. This situation is reminiscent of theBohr effect in the oxygen liganding of hemoglobin, where the acquisition of thegaseous ligand (oxygen or carbon monoxide) on the ferrous iron of the hemo-protein is accompanied by the shift in dissociation constant of a histidine towardsa higher value, causing the ejection of a proton from protein and alteration of the

570 N. A. S. SYMPOSIUM: B. CHANCE PRoc. N. A. S.

protein charge. Application of this idea to membrane configuration seems areasonable hypothesis, and indeed has been described in some detail in connectionwith the fast membrane protonations in photosynthetic systems that accompanythe light activation of electron transport.39 Radda has suggested that the de-creased degree of water structure that is characteristic of the energized membranewill cause pK to increase, causing H+ binding and a greater affinity for the anionicprobes.

Since the probes appear to reside at the aqueous region of the membrane andnot to penetrate deeply into or through it, transport of the probe molecules isprobably not involved. On the other hand, membrane fragments are observedto have a valinomycin-stimulated potassium movement in the energized state.40The driving force for this potassium movement might well be due to an asym-metric distribution of membrane components of altered pK in the energizedstate, which causes the membrane as a whole to acquire a net charge and a con-sequent transmembrane flux of ions. We choose to term the general propertyinvolving the linkage of structural changes to membrane charge as a "membraneBohr effect" by analogy with the hemoglobin reaction.

It is now possible to reconcile the basic features of chemical coupling mech-anisms which now find a firm foundation in the kinetic spectroscopic and thermo-dynamic properties of cyochrome bT for one of the three sites of energy con-servation in the chain,'0 with the experimental observations that the membranecan at the same time undergo a structural and charge change. The energycoupling seems to reside at the level of the electronic state of the heme of cyto-chrome bT which undergoes the essential redox potential changes. Structuralchanges in the heme environment act to create an asymmetric membrane Bohreffect and thereby evoke a consequent charge and potential change across themembrane, which explains its ion-transporting properties. This viewpointembraces the most useful concepts of current theories of energy coupling andion transport in mitochondrial membranes.

Many thanks are due to collaborators in these experiments (particularly G. Radda, A. Azzi,A. R. Caswell, R. B. Freedman, C. P. Lee, and M. Erecinska), to G. Radda for many discus-sions and unpublished data, and to G. Ballard and J. S. Leigh for discussion on some points.

Abbreviation: ANS, 1-anilinonaphthalene 8-sulfonic acid.

* Supported by USPHS grant 12202.

'Tedeschi, H., J. Cell Biol., 25, 229 (1965).2 Chance, B., and C-P. Lee, FEBS Letters, 4, 181 (1969).3 Hackenbrock, C. R., J. Cell Biol., 37, 345 (1968).4Ford, N., and R. Hershberg, personal communication.6Weber, G., Advan. Protein Chem., 8, 415 (1953).6 Newton, B. A., J. Gen. Microbiol., 10, 491 (1954).7Waggoner, A. S., and L. Stryer, Proc. Nat. Acad. Sci. USA, 67, 579 (1970).8 Radda, G. K., Bioenergetics, in press.9 Kasai, M., T. R. Podleski, and J-P. Changeux, FEBS Letters, 7, 13 (1970).10 Young, J. H., G. A. Blondin, G. Vanderkooi, and D. E. Green, Proc. Nat. Acad. Sci. USA,

67, 550 (1970).11 Chance, B., D. F. Wilson, P. L. Dutton, and M. Ereciaiska, Proc. Nat. Acad. Sci. USA,

66, 1175 (1970).12 Chance, B., and B. Hagihara, Proceedings of the Fifth International Congress of Biochemis-

try, Moscow, 1961, ed. A.N.M. Sissakian (New York: Pergamon Press, 1963), vol. 5, p. 3.

VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 571

13Matsubara, J. K., and B. Chance, in preparation.14Lee, C-P, and L. Ernster, in Methods in Enzymology, eds. R. W. Estabrook and M. E.

Pullman (1967), vol. 10, p. 543.16 Azzi, A., S. Fleischer, and B. Chance submitted to Biochim. Biophys. Acta.16 Azzi, A., B. Chance, G. K. Radda, and C-P. Lee, Proc. Nat. Acad. Sci. USA, 62, 612

(1969).17 Gitler, C., B. Rubalcava, and A. Caswell, Biochim. Biophys. Acta, 133, 479 (1969).18 Chance, B., D. DeVault, V. Legallais, L. Mela, and T. Yonetani, in Fast Reactions and

Primary Processes in Chem. Kinetics, ed. S. Claesson, Nobel Symp. V (New York: Inter-science, 1967), p. 437.

19 Racker, E., D. D. Tyler, R. W. Estabrook T. E. Conover, D. F. Parsons, and B. Chance,in Oxidases and related redox systems, eds. T. E. King, H. S. Mason and M. Morrison (NewYork: T. Wiley, 1965), p. 1077.

10 Fessenden, J. M., and E. Racker, J. Biol. Chem., 241, 2483 (1966).21 Chance, B., M. Erecisiska and C-P. Lee, Proc. Nat. Acad. Sci. USA, 66, 928 (1970).22Lee, C-P., L. Ernster, and B. Chance, Eur. J. Biochem., 8, 153 (1969).23 Nordenbrand, K., and L. Ernster, Abstracts VIII International Congress of Biochem.,

Switzerland, 1970.24 Datta, A., and H. S. Penefsky, J. Biol. Chem., 245, 1537 (1970).2 Brocklehurst, J. R, R. B. Freedman, D. J. Hancock, and G. K. Radda, Biochem. J., 116,

721 (1970).26 Stryer, L., J. Mol. Biol., 13, 482 (1965).27 Stryer, L., personal communication.28 Lesslauer, W., personal communication.29 Phillips, W. D., M. Poe, and C-P. Lee, personal communication.30 Leigh, J. F., and R. Hershberg, personal communication.31 Metcalfe, J. C., P. Seeman, and A. S. V. Burger, Mol. Pharmacol., 4, 87 (1968).32Chance, B., H. Wohlrab, A. Azzi, S. Fleischer, H. Drott, and C-P. Lee, Round Table Dis-

cussion on Electron Transport and Energy Conservation, eds. J. M. Tager, S. Papa, E. Quag-liarello and E. C. Slater, in press, p. 206.

33 Brand, L., J. R. Gohlke, and D. S. Rao, Biochemistry, 6, 3510 (1967).34Azzi, A., Round Table Discussion on Electron Transport and Energy Conservation, eds.

J. M. Tager, S. Papa, E. Quagliarello and E. C. Slater, in press.35 Azzi, A., Fed. Proc., 29, 2751 (1970).36 Actually the time for the ANS fluorescence increase is somewhat underestimated in that

experiment because of the short duration of the steady state, and as seen in Fig. 2, 25-30 secmay be required to complete the response.

37 Chance, B., G. K. Radda, and C-P. Lee, Round Table Discussion on Electron Transportand Energy Conservation, eds. J. M. Tager, S. Papa, E. Quagliarello and E. C. Slater, in press,p. 19.

3 Raboul, A., and A. Cheruy, personal communication.39 Chance, B., A. R. Crofts, M. Nishimura, and B. Price, Eur. J. Biochem., 13, 364 (1970).40 Montal, M., B. Chance, C-P. Lee, and A. Azzi, Biochem. Biophys. Res. Commun., 34, 104

(1969).