Biochimicu et Biophysics Acta, 456 (1976) 129-148

‘0 Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherlands

BBA 86031

UNCOUPLING OF OXIDATIVE PHOSPHORYLATION

WALTER G. HANSTEIN

Depurtmenr qf Biochemistry, Scripps Clinic and Research Foundation, La Jolla. Cal& 92037 ( U.S.A)

(Received November 19th. 1975)

CONTENTS

I. Introduction .................................

II. Phosphorylation efficiency ...........................

III. Respiratory control ..............................

IV. Types of uncoupling .............................

V. Interactions of uncouplers with other inhibitors. .................

VI. Interactions of uncouplers with the mitochondrial membrane and with membrane

components .................................

VII. Affinity labeling of mitochondria by uncouplers .................

VIII. The concept of stoichiometry. .........................

IX. Uncoupling by picrate. ............................

X. On the mechanism of uncoupling of oxidative phosphorylation ...........

Acknowledgements ................................. References ....................................

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I. INTRODUCTION

Food stuff such as lipids, carbohydrates and proteins are metastable in the

presence of oxygen. Were it not for the hurdles of high activation energies, all organic

material would react with oxygen and produce heat, carbon dioxide, water, nitrogen

and other inorganic compounds. Living things conserve the redox energy present in

organic materials and oxygen, and convert it into forms of energy which are more

useful than heat. In cells of higher organisms, this vital function is performed by

Abbreviations: CCCP, Cl-CCP, carbonyl cyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethylphenylhydrazone; HQNO, 2-heptyl-4-hydroxyquinoline N-oxide; S-6, 5-chloro-3-(p-chlorophenyl)-4’-chlorosalicylanilide ; S-13, 5-chloro-3-r-butyl-2’-chloro-4’-nitrosali- cylanilide; SF-6847, 3,5-di-r-butyl-4-hydroxybenzylidene malononitrile; TTFB, 4,5,6,7-tetrachloro-2- trifluoromethylbenzimidazole.

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mitochondria, which have therefore been called “the microscopic powerplants” of

the cells [I]. Specifically, several complex enzyme assemblies [2,3] located in the

inner mitochondrial membrane catalyze oxidative phosphorylation, a network of

reactions in which the oxidation of substrates is coupled to the synthesis of ATP by

the phosphorylation of ADP. ATP, a high-energy compound, serves as the general

source of chemical energy for a large variety of metabolic processes such as muscular

contraction, transport of ions and small molecules, and syntheses necessary for growth

and maintenance of the organism. In tightly coupled mitochondria, very little oxi-

dation occurs in the absence of ATP synthesis. This phenomenon is known as

respiratory control.

Uncouplers, a large class of relatively simple organic chemicals, have the ability

to abolish the coupling of substrate oxidation to ATP synthesis. As a consequence,

the latter reaction comes to a halt, because it is deprived of the necessary energy input,

whereas the former reaction, uninhibited by respiratory control, proceeds at maximal

rate and produces heat instead of ATP [4].

There is much detailed knowledge about the composition and the mode of

action of the electron transport system [2,5-71, and the ADP-phosphorylation system

[8-IO] in mitochondria. In contrast, the nature of the device which couples these two

systems is not known with certainty, nor is there a generally accepted mechanism of

uncoupling of oxidative phosphorylation.

Several general reviews on this topic covering the literature prior to 1974 are

available [I lLl5]. The scope of this article does not permit exhaustive treatment of

even the most recent literature. It will only be possible to introduce the problem, to

discuss selected aspects which have been most actively investigated in the last several

years, and to summarize what recent studies of uncouplers may have taught us about

the mechanism of oxidative phosphorylation.

II. PHOSPHORYLATION EFFICIENCY

Some basic facts, summarized in Fig. 1, are necessary to appreciate why it is

important to understand uncoupling of mitochondrial oxidative phosphorylation.

Electron transport from NADH or succinate to oxygen, via coenzyme Q and

cytochrome c, is catalyzed by a number of flavoproteins, cytochromes, nonheme

iron and copper proteins (not shown), and accompanied by the release of free energies

amounting to 52 or 36 kcal/mol, respectively [4]. The energy in each major step is

conserved in a common pool [16], designated by a squiggle sign (-). At present, it is

not known (and is a matter of heated debates) whether the form of potential energy

produced in the coupling events is best described as a chemical intermediate, as a

combination of membrane potential and pH-gradient, or as a conformational state.

The energy for the synthesis of just one molecule of ATP is generated by the

passage of two electrons through each of the coupling sites 1, II and III. Thus,

oxidation of one molecule of NADH supports the formation of no more than three

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amvtal

SUCCINATE/FLlhWRATE

lr aride hvdroxylamine .

rotenone Electron transport system

cyanide

(Flawproteins, nonheme iron proteins, cyto- chromes, copper, co- enzyme Q) SITE I SITE II SITE III

Coupling system (OSCP, B-type factors,

gua"i%$q+/&

ION TRANSPORT- .

UBP. DCCD-binding (161 HEAT

protein) II

ATP-synthesizing system (F,-ATPase)

oligomycin, DCCD

PHOSPMTE

arsenate

ADP

aurovertin it

ATP

Fig. I. Coupling of electron-transport and phosphorylation in mitochondria, and the sites of action of inhibitors and activators. CoQ, co-enzyme A: cyt. c, cytochrome c; DCCD, dicyclohexyl carbo- diimide; HQNO, 2-heptyl-4-hydroxyquinoline-N-oxide; OSCP, oligomycin-sensitivity conferring protein: UBP. uncoupler binding protein (see Section VII).

molecules of ATP, and the molar ratio of esterified phosphate to consumed oxygen

(P/O ratio) is maximally three. As seen in Fig. 1, the theoretical P/O ratio with

succinate as substrate is 2. Uncouplers lower the P/O ratio by converting essentially

all of the potential energy (-) into heat [4].

Fig. 1 also indicates that all partial reactions involving utilization of high

energy are also subject to inhibition by uncouplers. These include, among others,

ATP-32Pi exchange (reactions 17 and 18), ATP-driven reverse electron-flow from

succinate to NAD (reactions 3, 2, 17, 14), respiration-driven reverse electron-flow

from succinate to NAD (reactions 3, 2, 7, 9, 14), and ATP synthesis driven by an ion

gradient (reactions 1.5, 18). Indeed, the existence of a common high-energy inter-

mediate X - I (a precursor of the noncommittal -) was postulated to a large degree

on the basis of the effects of uncouplers [17].

III. RESPIRATORY CONTROL

In the presence of oxidizable substrate, oxygen, ADP and phosphate, the rates

of respiration and phosphorylation are fast, and mitochondria are in the active state

(state 3) [17]. In the controlled state (state 4), i.e., in the presence of substrate and

oxygen, but in the absence of either ADP or phosphate, there is little respiration in

tightly coupled mitochondria. The respiratory control ratio (RCR), the quotient of

the rates in the active state and the controlled state, is a measure of the degree of

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control imposed on oxidation by phosphorylation. Appropriate concentrations of

uncouplers raise the rate of respiration from the state 4 to the state 3 level, and

inhibit the rate of phosphorylation nearly completely, bringing the mitochondria to

the uncoupled state (state 3~) [I 81.

Respiratory control is not necessarily an integral part of oxidative phospho-

rylation. Aged mitochondria [l9], broken (light) mitochondria [20] and submito-

chondrial particles obtained by sonic disruption of mitochondria [2l] lack respiratory

control nearly completely, but retain the full capacity of phosphorylation coupled

to oxidation (loose coupling). Nevertheless, the release of respiratory control is a

useful test for uncoupling agents. This is in spite of the fact that there are agents

which release respiratory control without being uncouplers in the usual sense, (see

Section IV), and the existence of membrane-impermeable uncouplers which are unable

to affect respiratory control in intact mitochondria (see Section IX).

The stimulation of an oligomycin-sensitive ATPase by uncouplers (reactions

17, 19) in intact mitochondria is in many respects similar to the release of respiratory

control. This aspect of uncoupling, and the inhibitory effect of high concentrations

of uncouplers on electron transport and ATPase activity [22] will not be discussed in

this article.

IV. TYPES OF UNCOUPLING

Uncoupling in a broad sense may be brought about by a variety of agents or

treatments which have nothing more in common than their effect on respiratory control.

on mitochondrial ATPase and on the phosphorylation efficiency. For the study

of the mechanism of uncoupling it is therefore necessary to classify uncoupling

according to the agents and procedures which elicit this kind of response in mito-

chondria.

1. Strucfural uncoupfing. All procedures which impair the integrity of the inner

mitochondrial membrane also decrease respiratory control, increase the ATPase

activity, and often lower the phosphorylation efficiency. Such treatments include

mechanical disruptions (freezing and thawing, shearing forces, sonication), aging [ 191,

incubation with phospholipases [23], and addition of detergents [24].

2. Uncoupling by cations and ionophorrs. Cations such as ethidium bromide

[25] and K +, in the presence of the antibiotic ionophore valinomycin [26] are known

to uncouple oxidative phosphorylation, presumably by the use of potential energy for

ion transport in futile cycles (Fig. I, reaction 15). Under certain conditions, the

antibiotics Dio-9 [27] and A 20668 [28] act as uncouplers, possibly by a similar

mechanism, since they increase the proton permeability in liposomes [29] and mito-

chondria [28], respectively. The release of respiratory control by arsenate is oli-

gomycin-sensitive [30] (see Fig. I ), in contrast to the effects of cations and ionophores.

It appears that uncoupling by arsenate is due to direct, nonhydrolytic interference

with ATP synthesis [31].

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3. Uncoupling involving covalent binding. Alkylating agents of the mustard gas

type [32] and electrophiles such as isothiocyanates [33] have been reported to have

uncoupling effects on mitochondria. Although not much is known about the me-

chanism, the inhibitory effect of an alkylating uncoupler introduced by Wang [34]

demonstrates that alkylation of the membrane does not per se prompt a release of

respiratory control.

In mitochondria, uncoupling by CCCP and I, I ,3-tricyano-2-aminopropene is

prevented, but not reversed by aminothiols [35]. In isolated bacterial membrane

vesicles, however, uncoupling by CCCP is blocked as well as reversed by dithio-

threitol, cysteine and other thiols [36]. Furthermore, high concentrations of CCCP

diminish the reactivity of such vesicles toward N-ethyl maleimide, a powerful SH-

reagent. In contrast, none of the uncoupling effects of dinitrophenol is influenced by

thiols. These and other data [37] indicate that carbonyl cyanide hydrazones may react

in two different ways: as a sulfhydryl reagent, and as an uncoupler similar to dini-

trophenol. It is, of course, a very interesting question whether reactions with certain

SH-groups alone will result in uncoupling, as suggested by data obtained with

4-bromophenylisothiocyanate [33].

4. Uncoupling by phenols and other anionic aromatic compounds. Many of the

effects of the classical uncoupler 2,4-dinitrophenol were known, in a general way [l 11,

long before uncoupling as a phenomenon associated with mitochondrial oxidative

phosphorylation had been recognized by Lardy and Elvehjem [38] and demonstrated

by Loomis and Lipmann [39]. The overall physiological effects of uncouplers are

profound and dramatic [40]: “Dinitrophenol exerts a remarkable stimulating effect

on fat metabolism, and the metabolism is sufficient to produce hyperthermia. Dini-

trophenol has been tried extensively for clinical reduction of obesity; it is very effective.

Unfortunately, its action is not always reliable and toxic manifestations, frequently

with fatal results, appear unexpectedly”. The radical, biocidal effects of uncouplers

have made them useful as herbicides and fungicides [4l].

Phenols are the largest group (about 40 different compounds have been de-

scribed in the literature) in a class of uncouplers which includes other groups such as

salicylanilides (S-6, S-1 3), carbonyl cyanide hydrazones (FCCP, CCCP), benzimi-

dazoles (TTFB), and similar heterocyclic systems. A common characteristic among

the groups in this class of uncouplers is a phenolic or anilinic configuration, electro-

negatively substituted at the benzene ring and on the nitrogen in position X:

0 / \ - XH (X= NH,O,S) -

Extensive qualitative and quantitative structure-function relationship studies

have been published which stress the importance of lipophilicity and acidity for the

ability to uncouple effectively [41l481. Nevertheless, these criteria do not fully

define the effectiveness of uncouplers: 3,5-dibromo-4-cyanophenol is 6-9 times more

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effective than 2,4_dinitrophenol, even though their acidity and lipophilicity are nearly

identical [44].

There is evidence that all uncouplers in this class of anionic aromates uncouple

in the same or a very similar fashion, and the remainder of this article will concentrate

on the discussion of the properties and interactions of these compounds and on the

mechanism by which they act as uncouplers.

V. INTERACTIONS OF UNCOUPLERS WITH OTHER INHIBITORS

In the controlled state of tightly coupled mitochondria (state 4), electron

transport is inhibited, either as a result of direct interactions between components

of the oxidative phosphorylation system, or indirectly through an electrochemical

proton gradient (see Section X). Other, similar controls of electron transport by the

energy conservation system are apparent in the effects of uncouplers on the potencies

of respiratory inhibitors. These include (a) amytal and guanidine, (b) HQNO and

(c) azide and hydroxylamine, which inhibit electron transport at phosphorylation

sites I, II, and III, respectively (see Fig. 1).

Chance and Hollunger [I81 showed that, in the presence of uncouplers, amytal

and guanidine inhibit electron transport through site I much less efficiently than in

the presence of ADP and phosphate (state 3). Similarly, Howland [49] demonstrated

that concentrations of HQNO capable of inhibiting more than 90”/,, of state 3 res-

piration decreased the rate only by 50:;, in the presence of 2,4_dinitrophenol as

uncoupler (state 3~). This corresponds to a difference by a factor of about 10 between

the Ki values in state 3u and in state 3. As shown by Wilson and Chance [50], azide

inhibits respiration in the presence of ADP and phosphate (state 3) or of Ca’+ much

more than in their absence (state 4) or in the presence of uncouplers (state 3~).

Uncouplers such as 2,4_dinitrophenol, pentachlorophenol, TTFB, and FCCP

increase the apparent Ki of azide (0. I mM) by factors ranging from I .3 to about 10.

Similar results have been obtained with hydroxylamine [.51,52], with the important

difference that Ca’+ releases effectively the state 3 inhibition by hydroxylamine, but

not by azide. Azide [53], and to a certain degree hydroxylamine [52], are uncouplers

at higher concentrations than those necessary for the inhibition of electron transport

(see Table I).

These data show that the degree of energization in the energy conservation

system not only influences electron transport per se (respiratory control) but also

the effectiveness of respiratory inhibitors. In addition, it appears that these effects

are not independent from the means by which deenergization is achieved, e.g., by

ADP + phosphate, uncouplers or calcium uptake. Differential effects of this kind

are very difficult to rationalize without invoking direct and multiple interactions

between components of the oxidative phosphorylation system.

VI. INTERACTIONS OF UNCOUPLERS WITH THE MITOCHONDRIAL

AND WITH MEMBRANE COMPONENTS

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MEMBRANE

As expected from relatively small molecules with both hydrophobic and hydro-

philic structural characteristics, uncouplers interact with a variety of soluble proteins,

lipids and biomembranes. Several enzymes such as kinases, the soluble F,-ATPase

and pyridine nucleotide-dependent dehydrogenases, which are all specific for adenine-

containing substrates, are inhibited by uncouplers [55-571. Serum albumin binds

uncouplers strongly enough to reverse uncoupling of mitochondria completely [58].

The interactions of uncouplers with phospholipid liposomes and bilayers

manifest themselves in increased swelling (decreased light-scattering) [59,60] and

increased electric conductance [32,59-651, respectively. Apparently, these effects are

brought about by complexes consisting of pairs of uncharged uncoupler acids and

anions [64-661 present in the lipophilic phase, and also by uncoupler acids and anions

imbedded in the polar-unpolar interphase of the phospholipid assembly [64,65,67,68].

There is often [32], but not always [60,62,63] a moderately good correlation be-

tween the ability of uncoupling agents to release respiratory control in mitochondria

and the capacity to increase electric conductance in phospholipid bilayers. The

correlation between the efficiencies of uncouplers to induce swelling in liposomes

and to uncouple mitochondria is better [60] and appears to be more significant in

view of similar parallelities between mitochondrial swelling and respiratory stimu-

lation [28,69]. As seen in Fig. 2, the latter correlation accomodates not only un-

TBA .

/

. DOC.

TPB

Fig. 2. Correlation between uncoupler induced swelling and respiratory stimulation in mitochondria. DNP, dinitrophenol; BTZ, SH-benzothiazole; SA, thiosalicylic acid; FCCP, carbonyl cyanide-p-tri- fluoromethoxyphenyl hydrazone; CL-CCP, carbonyl cyanide m-chlorophenyl hydrazone; 1799, 2,2’-bis (hexafluoroacetonyl )acetone: Dicum, dicumarol; 6847, SF-6847; TTFB, 2-trifluoromethyl- tetrachlorobenzimidazole: S13, 5-chloro-3-t-butyl-2’-chloro-4’-nitro-salicylanilide; PCP, penta- chlorophenol; TPB, tetraphenyl boron; TBA, tributylamine; O.A., oleic acid; M.A., myristic acid; DOC, deoxycholate; ARS, arsenate. From Cunarro and Weiner [69].

136

couplers of the anionic aromatic type, but also detergents, amines and arsenate (see

Section IV). The type of mitochondrial swelling used in these experiments is assumed

to be due to an uncoupler-mediated increase in proton permeability. The correlation

shown in Fig. 2 has therefore been taken as evidence that the release of respiratory

control by uncouplers is the result of enhanced proton permeability.

Early studies of uncoupler binding by mitochondria and mitochondrial proteins

[70] have led Weinbach and Garbus to conclude that uncoupler-induced conformatio-

nal changes in mitochondrial proteins are the basis of uncoupling [71]. It was

already clear from their work that mitochondria can bind lipophilic uncouplers in

amounts which are orders of magnitudes higher than the minimum necessary for

uncoupling. Later studies by Wang et al. [72] and Bakker et al. [73] confirmed that,

in direct binding studies, uncouplers such as CCCP, pentachlorophenol and TTFB

appear to bind predominantly in a non-specific, partition-like manner.

With the use of a new, largely hydrophilic uncoupler, 2-azido-4-nitrophenol

Hanstein and Hatefi have demonstrated the existence of specific uncoupler binding

in addition to unspecific partitioning [37] (Fig. 3). The specific, high-affinity un-

Fig. 3. Concentration dependence of uncoupler equilibrium binding by mitochondria. Curve A:

total binding; Curve B: specific binding (derived from Curve A by graphical or computational means);

NPA, 2-azido-4-nitrophenol. From Hanstein and Hatefi [37]

coupler binding site in mitochondria has many of the properties which one would

expect from a component involved in uncoupling of oxidative phosphorylation : (I)

It is specific for uncouplers of the anionic aromatic type. (2) Specific binding is not

affected by other types of uncouplers or inhibitors such as ionophores and arsenate,

or rotenone, antimycin, cyanide and oligomycin (see Fig. 1). (3) It is independent

of the energy state of mitochondria, e.g.. the presence or absence of ATP or substrate,

137

and partial or full deenergization by arsenate or valinomycin-K+ (& picrate). (4) The

number of binding sites, about 0.6 nmol/mg protein in beef heart mitochondria and

0.3-0.4 nmol/mg protein in rat liver mitochondria, is comparable to the concentration

of other components of the oxidative phosphorylation system [74]. (5) The uncoupler

binding site is apparently ubiquitous in mitochondria, e.g., beef heart [37], rat liver

[54], yeast (Hanstein, W. G. and Griffiths, D. E., unpublished observations) and

Eugha gracilis mitochondria (Hanstein, W. G. and Kahn, J. S., unpublished ob-

servations), but not in other membrane systems such as erythrocyte ghosts [75].

Other important characteristics of specific uncoupler binding include the pH-in-

dependence of the dissociation constant, a Hill-slope of 1.0 and the almost entirely

enthalpic nature of the free energy of binding, and suggest that uncouplers bind as

single, anionic molecules in a non-cooperative fashion, without inducing net con-

formational changes. The experimental difficulties in determining the specific binding

parameters of uncouplers which are much more lipophilic than 2-azido-4-nitrophenol

and 2,4-dinitrophenol can be overcome in indirect, competitive binding experiments

(see Fig. 5 in ref. 37), which allow the calculation of a competitive dissociation

constant and of the extent of specific and unspecific binding [54]. This technique has

made it possible to determine the dissociation constants of pentachlorophenol and

S-13 in addition to azide and, in submitochondrial particles, to picrate. Table I shows

dissociation constants of uncouplers obtained by direct or competitive binding studies

at 3 “C, together with the ranges of concentrations (up) in which those uncouplers

abolish 50 y(‘, of oxidative phosphorylation or respiratory control at 30 ‘C. This table

also includes dissociation constants and ‘p,, values in terms of concentrations of

uncouplers present in the mitochondrial phase. It is seen that, over a range of more

than three orders of magnitude, there is a close correlation (r > 0.99) between the

kinetic (y,,) and thermodynamic (I&) values. After application of the appropriate

temperature corrections for specific binding ( 1 H = -8 kcal and -4.6 kcal [54], for

the aqueous and the mitochondrial phase, respectively), one arrives at the relation

which applies to p,</K, values at 30 “C, both as ratios of concentrations in the

aqueous and in the mitochondrial phase. Together with the characteristics of specific

binding enumerated above, data such as those in Table I have been taken as indication

that interactions of uncouplers with the uncoupler binding site play an important

and possibly crucial role in the process of uncoupling.

VII. AFFINITY LABELING OF MITOCHONDRIA BY UNCOUPLERS

Covalent labeling of mitochondrial proteins by reactive uncouplers appears to

be the only practical way presently available to identify directly components other

than lipids that may be involved in uncoupling and coupling of oxidative phospho-

rylation.

TABLE I

BINDING AFFINITIES AND UNCOUPLING POTENCIES OF HYDROPHILIC AND HYDROPHOBIC UNCOUPLERS”

Uncoupler Aqueous phase Mitochondrial phaseb

KnC 31id ‘i ~,/&I &I’ 3JJd P2,lK,

2-Azido-4-nitrophenol (NPA) 6 2 3’ 5-10’ 1.3 110 I 60 50 - 100 0.7

2,4-Dinitrophenol (DNP) 19 _m 5’ 15-20’ 0.9 180 ~. 50 80-I 10 0.5

2,4,6_Trinitrophenol (TNP) 28 I 5’,” 40-90’ 2.3 1900 = 400 1600-3600 1.4

Pentachlorophenol (PCP) I.8 _’ 0.4”,” t-2e.h 0.8 1700 m* 400 500-l 000 0.4

5-Chloro-3-butyl-2’-chloro- 170 1 50’ I oo-300’ I.2

4’-nitrosalicylanilide (S-13)

Azide 3500 5OOL 3000-4000 I .o

Average pat/K,: 1.33 = 0.47 0.84 _: 0.44

’ In beef heart mitochondria or submitochondrial particles (2,4,6-dinitrophenol). All concentrations KD and it, are in /rM. b K, and qIj values for NPA, DNP, TNP and PCP in the mitochondrial phase were calculated from the corresponding values in the aqueous phase

by multiplication with the respective partition coefficients, assuming the equivalence of 1 mg mitochondrial protein with a volume of I /II. Partition coeffi-

cients (unspecific binding) were determined from binding curves such as shown in Fig. 3 (NPA, DNP, TNP), or from data published in ref. 70 (PCP). Partition coefficients for TNP at 3- C, and for DNP and PCP at 30 ‘C were calculated from values at 30 -C and 3 ‘C, respectively, using a value of 4H = 3.4 kcal determined for NPA [54] for all uncouplers. The partition coefficients (nmol uncoupler per g protein/nmol uncoupler per ml aqueous solution) used are (uncoupler in parentheses): at 3 ‘C, I7 (NPA), 9.5 (DNP), 70 (TNP), 920 (PCP): at 30 ‘C, 10 (NPA), 5.4 (DNP), 40 (TNP), 530 (PCP).

At 3 ‘C: determined directly or by inhibition studies (see text). At 30 “C.

[371. [761. Data are corrected for bound uncoupler.

[751. Calculated from data published in ref. 37. Estimated from data published in refs. 77 and 78.

1541.

139

Wang et al. [34] found that an alkylating uncoupler, 2,4-dinitro-S(bromo-

acetoxyethoxy)phenol (DNBP) labeled a variety of proteins in rat liver mitochondria,

most prominently polypeptides of a mol. wt. of 43000 and 52000. More selective

labeling by DNBP was achieved in beef heart mitochondria, where about 90%, of the

label was found in two polypeptide bands of mol. wt. of 44000 and 30000 (Wang,

J. H., personal communication).

Exploratory studies by Hanstein and Hatefi [37,75] with 2-azido-4-nitrophenol

as a photo-affinity label have shown that photo-activated 2-azido-4-nitrophenol

binds covalently to the uncoupler equilibrium binding site, resulting mainly in

decreased state 3 respiration and ATPase activity, without much decrease in phospho-

rylation efficiency or increase in state 4 respiration. Thus, covalent labeling by un-

couplers does not induce permanent uncoupling, but rather appears to freeze mito-

chondria to a certain degree in a permanent state 4 not amenable to stimulation by

ADP or uncouplers. Further studies by Hanstein (unpublished observations) have

shown that photo-activated 2-azido-4-nitrophenol labels two polypeptide bands to

a major degree: (I ) a band at a mol. wt. of 56000, which has been identified as subunit

I of F,-ATPase; and (2) a band at a mol. wt. of 31000. The latter peptide (the

uncoupler binding protein) is the only protein component essential for specific un-

coupler equilibrium binding by the inner mitochondrial membrane (Hanstein, W. Cr.,

unpublished observation), while the former may be involved in uncoupler binding by

soluble F,-ATPase [81]. It is possible therefore, as visualized in Fig. 4, to speculate

that in mitochondria the uncoupler binding site is made up by both the uncoupler

binding protein and subunit I of F,.

i

Fig. 4. Hypothetical structure of the uncoupler binding site. Uncoupler binding protein and sub- unit 1 of F,-ATPase are the two peptides most prominently labeled by photo-activatedNPA (Hanstein, W. G., unpublished). A chloroform/methanol extractable protein (possibly the DCCD-binding pro- tein, see ref. 8) and a protein of a mol. wt. of 42000-46000 (possibly F, [79], a B-type factor, ref. 80) are labeled to a minor degree [54] and may be in the vicinity of the uncoupler binding site. The uncoupler binding protein is assumed to be close to the electron transport system because of the effects of uncouplers on the action of respiratory inhibitors (Section V), and the effects of photo-

affinity labeling by 2-azido-4-nitrophenol on state 3 respiration (Section VII).

140

VIII. THE CONCEPT OF STOICHIOMET‘KY

implicit in considerations of specific uncoupler binding is the concept of

stoichiometry in uncoupling, of discrete molecular interactions which manifest

themselves in experimental facts such as uncoupler titers [53,78,82-851 and the number

of uncoupler binding sites [34,37]. These parameters are expected to be similar in

magnitude to the components in the oxidative phosphorylation system. The im-

portance of stoichiometric aspects has been questioned on the basis of direct binding

experiments with uncouplers such as S-13 [73], on kinetic grounds [87] and on grounds

of “substoichiometry” [85]. The data discussed in Section VI suggest indeed that

specific binding of highly lipophilic uncouplers such as S-13 may be overshadowed

by extensive unspecific, partition-like binding, and therefore not amenable to accurate

determination by direct binding studies. However, as seen in Table I, it is possible

to demonstrate specific binding of S-l 3 by measurement of the competition between

2-azido-4-nitrophenol and S-13 for the uncoupler binding site. Objections against

concepts of stoichiometry on the basis of substoichiometry are arguments of a mixed

kinetic-thermodynamic nature which stem from the often reported observation

Fig. 5. Relationship between uncoupling and uncoupler binding in a kinetic model of respiratory

control. In the reaction cycle A . B . C - A. A is a high-energy state, and B and C are low-

energy states of the same carrier in the electron transport system. A and B are reduced, and C is

oxidized, or vice versa. The deenergization step A f B is assumed to be rate limiting (k, k” -:Z k~.

X,) in the absence of uncouplers or ADP. Progressive uncoupler binding increases the rate constant

/I, from k, to a maximum of k,, - h,. The steady-state rate at unity carrier concentration, ktk&J (k,kz k,k,, k,h,). has been calculated as a function of the free uncoupler concentration for

two sets of rate constants: in one set, the rate constant for the reaction A T B in the presence of

excess uncoupler is larger than those for reactions B C and C A (left curve); and in a second

set, all rate constants are about equal under these conditions (middle curve). The corresponding

respiratory control ratios are 49 and 34. respectively.

141

[78,82,84,85], that in many cases less than I mol of uncoupler per mol of respiratory

chain is sufficient for complete uncoupling. The assumption of stoichiometric un-

coupler binding as an important step in uncoupling is therefore believed to be invalid.

Similarly, the findings of several laboratories [78,82,85,87] that the uncoupler titer

(the amount of uncoupler necessary for complete uncoupling) is not invariant of the

rate of electron transport and of the number of coupling sites involved, have been

thought to be incompatible with the concept of stoichiometry.

The question underlying these arguments can be stated in a more general form :

is the concept of stoichiometric uncoupler binding as the crucial step in uncoupling

compatible with ratios of yIL/KD which are smaller, may be much smaller than unity?

The kinetics of a simple model of uncoupling, shown in Fig. 5, demonstrate that this

is indeed possible. In the kinetic scheme in Fig. 5, A, B, and C stand for the reduced

energized, reduced-deenergized and oxidized forms, respectively, of one component

of the electron transport system such as cytochrome a. In the steady state, the rate-

limiting step A+ B may be accelerated by equilibrium binding of uncouplers up to a

point at which this step becomes not rate-limiting (left curve) or not alone rate-limiting

(middle curve). Uncoupling as measured by increased turnover rates has been

calculated for these two cases, and it may be seen in Fig. 5 that in either case 9 ,l/K, is

considerably smaller than unity. It is interesting that in the case where all rates are

equal in the presence of saturating amounts of uncoupler (middle curve), the ye ,,/K,

value (0.34) is about the same as the experimental value (0.3) deduced from Table I.

It would appear therefore that the simple model shown in Fig. 5 has some validity.

It certainly shows the possibility that substoichiometry in uncoupling is a direct

consequence of the kind of kinetic situation which in all probability exists in the steady

states of oxidative phosphorylation.

IX. UNCOUPLING BY PICRATE

Examination of the binding and uncoupling properties of picrate (2,4,6-trini-

trophenol), as compared to other uncouplers such as 2,4-dinitrophenol and 2-azido-4-

nitrophenol, has provided data of crucial importance for the understanding of the

mechanism of uncoupling and of oxidative phosphorylation. It has been known for

many years that in mitochondria picrate does not uncouple oxidative phosphorylation

[41,70,76,88,89] in concentrations up to 2 mM (Fig. 6). Similarly, as shown by

Hanstein and Hatefi, picrate does not bind to the uncoupler binding site in intact

mitochondria [76], in contrast to 2,4-dinitrophenol and 2-azido-4-nitrophenol, and

despite the obviously close structural similarities between picrate and the latter two

uncouplers. That absence of both uncoupling and specific uncoupler binding are due

to the nearly complete inability of picrate to permeate the inner mitochondrial mem-

brane has been demonstrated by the use of submitochondrial particles obtained by

sonication of mitochondria. These particles have a predominantly inside-out orien-

tation as compared to mitochondria and have retained the capacity to catalyze oxi-

142

P:O = 2.4 P:O = 2.4

RCR = 7.5 RCR = 6.1

+TNP P:O = 2.3

RCR = 8.5

P:O = 2.2

RCR = 4.8

Fig. 6. Effect of picrate on respiratory control ratio and phosphorylation efficiency in intact mito-

chondria. From Hanstein and Hatefi [6].

dative phosphorylation. In contrast to mitochondria, submitochondrial particles

bind picrate in a specific manner (see Table I), and energy-dependent reactions cata-

lyzed by submitochondrial particles such as oxidative phosphorylation, ATP-driven

reverse electron-flow, oligomycin-dependent respiratory control, and respiration-

driven transhydrogenation may be effectively uncoupled by picrate [76]. It is im-

portant to note that specific binding of picrate represents true equilibrium binding and

is not the result of an energy-dependent accumulation driven by a membrane potential,

as envisioned by Skulachev for a variety of ions including picrate [32]. This is because

specific binding of picrate by submitochondrial particles has been determined by

r 50 100 150 ---2&C+%

DNP 0, TNP [uMJ

Fig. 7. Effects of picrate and 2,4-dinitrophenol on the proton permeability of submitochondrial

particles. From Hanstein and Hatefi [76].

143

competition experiments in the presence of 2-azido-4-nitrophenol concentrations

sufficient for nearly complete uncoupling (see Fig. 7 in ref. 76). Another important

finding is that picrate, in contrast to other uncouplers, does not increase the proton

permeability in submitochondrial particles more than marginally, as seen in Fig. 7.

These results suggest that the uncoupler binding site is located at the inside of

the inner mitochondrial membrane, and demonstrate again the direct relationship

between uncoupling and specific uncoupler binding. Equally important, neither

membrane permeability nor protonophoric property appear to be essential for the

ability of a compound to act as an uncoupler. The results and conclusions of Hanstein

and Hatefi [76] are in agreement with data obtained by Leader and Whitehouse [89]

who demonstrated that picryl acetate, but not picrate, uncouples intact mitochondria.

These authors proposed that picryl acetate enters mitochondria, is hydrolyzed and

yields picrate which then acts from the inside as the uncoupling agent. Competitive

binding studies with picryl acetate, 2-azido-4-nitrophenol and mitochondria will be

necessary to fully confirm this interpretation. The scheme in Fig. 8 summarizes these

findings and emphasizes the secondary role of membrane permeability in the un-

coupling process catalyzed by uncouplers of the anionic aromatic type.

IRlll Mitochondrial

Membrane

lJutridc I I Inside

Fig. 8. Relationship between membrane permeability and ability to uncouple. Dinitrophenol is membrane-permeable, uncouples and binds to the uncoupler binding site in mitochondria (outside- out) and submitochondrial particles (inside-out). Picrate is membrane-impermeable, and uncouples and binds to the uncoupler binding site only if present at the inside of the inner mitochondrial membrane. Consequently, picrate is effective in uncoupling and specific uncoupler binding in sub- mitochondrial, inside-out particles. Picrate uncouples mitochondria only if carried inside as mem- brane-permeable picryl acetate, which then undergoes hydrolysis.

X. ON THE MECHANISM OF UNCOUPLING OF OXIDATIVE PHOSPHORYLATION

Most hypotheses of the mechanism of oxidative phosphorylation differ mainly

by what they assume to be the nature of the primary intermediate, the energy of which

is derived directly from the redox energy and subsequently used for ATP synthesis and

other energy-dependent processes [ 111.

144

Chemical hypotheses envision the coupling process as the transformation of

redox energy into chemical energy which is stored in a few reactive bonds of a chemical

intermediate. In conformational hypotheses, it is assumed that as the result of the

coupling event, potential energy is stored in a conformational state of a protein and

therefore distributed over many bonds which are distorted with respect to their

geometrical ground state. The chemiosmotic hypothesis of Mitchell [90,91] proposes

the establishment of an electrochemical potential gradient of protons, a combination

of a pH-gradient and a transmembrane potential, as the immediate result of the

coupling process.

It is evident that every one of these hypotheses implies a different mechanism

of uncoupling if it is assumed that uncouplers interact directly with the primary

coupling product. Thus, uncoupling by stoichiometric, molecular interactions between

uncouplers and proteins, by conformational transitions induced by uncoupler binding,

or by short-circuiting of a pH-gradient are natural extensions of chemical, confor-

mational or chemiosmotic hypotheses, respectively. Direct correlations between

protonophoric and uncoupling properties (Fig. 2) have been taken as evidence [28,60,

691 for Mitchell’s proposal that uncouplers dissipate the intermediate high energy

state (w) by catalyzing the collapse of the proton electrochemical gradient through

their ability to carry protons across the mitochondrial membrane [91]. The results

obtained with picrate (Section IX) indicate however, that this is not the only mech-

anism of uncoupling. Inspection of Table 1 shows that y,,/K,, of picrate is only

moderately higher than the values of other uncouplers which have the ability to act

as proton carriers as well as to bind to the mitochondrial uncoupler binding site.

Thus, it appears that protonophoric properties of uncouplers do not contribute very

much to their effectiveness as uncouplers. This is in agreement with the conclusion of

Padan and Rottenberg [92] that the phosphorylation reaction controls the rate of

respiration directly, and not through a proton electrochemical gradient. It is there-

fore not surprising to iind that many bona tide uncouplers (pentachlorophenol, TTFB,

thiosalicylate), in addition to arsenate (see Section IV), do not fit well in correlations

between proton-carrying and uncoupling properties such as shown in Fig. 2. The

differences between expected and observed values are one to two orders of magnitude

with these uncouplers, and may be compared with a maximal deviation of about 70’!,,

in the correlation between uncoupling and uncoupler binding (Table I).

The relationships between uncoupling and specific uncoupler binding (see

Sections VI and IX) are intuitively easiest to rationalize in the framework of a chemical

hypothesis. More severe restrictions on the mechanism of uncoupling are the result

of studies of the uncoupling effect of picrate. As discussed in the previous section, it

appears that in the absence of membrane permeability and protonophoric properties,

binding to the specific uncoupler binding site is a necessary and possibly sufficient

condition for uncoupling. The consequences of these results go well beyond the

particulars of uncoupling by picrate and may be summarized as shown in Fig. 9.

This scheme is similar to the one proposed by Slater [93] and by others, and its

general features are based on the fact that in the steps leading directly from electron

145

AiP

Fig. 9. Reactions and equilibria in oxidative phosphorylation IH, enthalpy; 1pH proton gradient

across the inner mitochondrial membrane: -, hypothetical high-energy state or intermediate. The

rates in reactions indicated by solid arrows are about equal in state 4, and faster than those indicated

by broken arrows.

transport to ATP synthesis, the rates of the forward and the back reactions are of

about equal magnitude, i.e., that the oxidative phosphorylation system in state 4 is

close to thermodynamic equilibrium [93,94]. In other words, the free energy of

electron transport is equal to the free energy required for ATP synthesis and to the

free energy available from ATP. [n the absence of uncouplers or protonophores, there

is very little loss of energy in the form of heat (AH). As proposed by Slater [9.5] and

demonstrated by Thayer and Hinkle [96,97] for submitochondrial particles, a pH-

gradient ( IpH) is in reversible equilibrium with a high energy state (-), either

directly as shown, or indirectly through the electron transport system. Protonophores

such as amines and many uncouplers, and antibiotics such as nigericin and valino-

mycin-K+ catalyze the dissipation of potential energy as heat (AH). The important

new finding is that picrate, an uncoupler with marginal protonophoric properties, is

able to convert the energy stored in (-) into heat (, 1H) with little effect on the pH-

gradient. Since the latter is in equilibrium with all other intermediates in the energy-

conserving reactions, picrate, even though it is not a protonophore, should have

induced the collapse of the pH-gradient, if km, were of the same order of magnitude

as k, in Fig. 9.

The thermodynamic consequence of the fact that the energy-dependent build-up

of the pH-gradient is fast (k, w 30/s, calculated from data in ref. 97), whereas the

reverse reaction is slow (k_, = 0.1/s at fully uncoupling concentrations of picrate,

ref. 76) is straight-forward: the free energy stored in the pH-gradient under the

conditions of these experiments is lower than the free energy in the high-energy state

(-), by pRTln-(k_,/k,) = 3.4 kcal. Combined with the above-mentioned fact that

very little, if any, free energy is lost during oxidative phosphorylation, this indicates

that the pH-gradient cannot be part of or identical with the high energy state indicated

by the squiggle sign in Fig. 9.

Recently, Thayer and Hinkle [96,97] demonstrated that a combination of a

pH-gradient and a membrane potential is capable of driving the synthesis of ATP at

rates comparable to those observed in oxidative phosphorylation. These data are not

necessarily in conflict with the results discussed above. This is because it is possible

146

to balance out an unfavorable energy situation by a proper choice of reactant con-

centrations, and it is difficult to evaluate rates of ATP synthesis driven by an artifically

imposed proton electrochemical gradient if comparisons are based on initial instead

of steady-states rates. Moreover. the experimental data used for kinetic comparisons

were obtained under conditions where ATP synthesis never exceeded 0.7 nmol

ATP/mg protein [97], an amount which is below the total of adenine nucleotides

bound to F,-ATPase and, more important, below the amount of bound ADP,

0.8 nmol/mg protein [98]. Thus, the phosphorylation of free ADP may not have been

measured in these experiments.

The conclusions based on the study of uncoupling by picrate are in agreement

with the results of Nicholls [99] obtained from more direct measurements. These

data indicate that the proton electrochemical gradient is always 50-90 mV lower than

the phosphate potential calculated on the basis of 2 protons/ATP, i.e., the energy

available from the proton electrochemical gradient is 2-4 kcal short of the energy

required for ATP synthesis. Discrepancies of this kind have been noted before (see ref.

99) and have not found a fully satisfactory explanation. For instance, it is possible that

in intact mitochondria the proton/ATP ratio is 3 rather than 2, the additional proton

being used for the transport of either phosphate, ADP or ATP [ 121. Indeed, measure-

ments of maximal phosphate potentials in submitochondrial, inside-out particles

(ETP,) indicate that the free energy necessary for ATP synthesis in the absence of

oligatory transport of phosphate components is about I I kcal/mol (Hinkle, P. C.,

personal communication) i.e., 4-6 kcal lower than in mitochondria [IOO]. However,

this explanation requires a P/O ratio of 2 rather than 3 for NADH-linked substrates

in state 4 [ 121. Furthermore, the thermodynamic conclusions derived from the

properties of picrate as an uncoupler are based on results obtained with submito-

chondrial particles and are therefore independent from the transport considerations

discussed above.

ACKNOWLEDGEMENTS

Preparation of this manuscript and unpublished results reported herein were

supported by Grant GM 19734 and by Research Career Development Award 5-K4-GM

38291 from the U.S. Public Health Service.

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