CHAPTER 32
Uncouplers a n d Inhibitors of O x i d a t i v e Phosphorylation
E. C. Slater
I. Introduction: Definitions 503
II. Uncouplers 504 A. Historical 504 B. Characteristics of Uncouplers 505
C. Mechanism of Action of Uncouplers 506 D. Classification of Uncouplers 507
III. Inhibitors 512 References 513
I. INTRODUCTION: DEFINITIONS
The energy made available by intracellular respiration, catalyzed by the mitochondria, is largely utilized for the synthesis of ATP, brought about by a process known as oxidative phosphorylation.
All oxidative phosphorylation reactions can be described by Eq. (1).
AH2 + Β + ADP + Pi ^± A + B H2 + ATP (1) The energy required for the phosphorylation of A D P by P4 [Eq. ( l a ) ]
ADP + Pi ^± ATP - 8000 cal (AG</) (la) is supplied by the hydrogen or electron transfer from the A H2 ±^ A sys
tem at lower oxidation-reduction potential to B H2 ±^ Β at higher potential.
We distinguish between two types of oxidative phosphorylation:
(a) substrate-linked, where A H2 is one of the substrates—phosphoglycer- aldehyde, pyruvate or α-ketoglutarate; and (b) respiratory-chain, where A H2 and Β are both members of the respiratory chain.
503
504 Ε. C. SLATER An uncoupler of oxidative phosphorylation permits the oxidation of A H2 by Β to proceed without net phosphorylation [Eq. (2) ]
AH2 -f Β —> A + BHo (2)
Although the concept of uncoupling is quite general, the term will be restricted in this article to uncoupling of respiratory-chain phos
phorylation.
An inhibitor of oxidative phosphorylation acts on the link between the oxidation reaction and the phosphorylation. In consequence reaction
(1) is completely inhibited. Inhibition of the oxidation reaction can be relieved by addition of certain uncouplers.
II. UNCOUPLERS
A. Historical
The stimulatory effects of nitrophenols on the over-all metabolism of the animal were known to the pharmacologists as long ago as 1885, when Cazeneuve and Lepine (1) found that ingestion of dinitro-a-naphthol by the dog caused a high fever. This finding was confirmed and extended to other species and to different dinitrophenols by a number of workers, notably Mathews and Longfellow (2), Heymans and Bouchaert (3), Magne, Mayer, and Plantefol (4, 5), and Plantefol (6). Heymans and Bouchaert (3) and Magne et al. (4, 5) clearly demonstrated that the dinitrophenols acted directly on the tissues and not by stimulating the heat-regulating center. They showed, moreover, that the increased heat production was not due to increased muscular activity.
The first detailed biochemical study of the mechanism of action of these compounds was made by Van Uytvanck ( 7 ) , who found that injection of dinitro-a-naphthol increased the amount of phosphate (presumably inorganic phosphate) in the blood and muscle of pigeons and increased the lactate content of the muscle. Cahn (8) obtained similar results with dinitrophenol. Isolated tissues were first used in the important work of Euler (9,10), Ehrenfest and Ronzoni (11), Dodds and Greville (12), and Ronzoni and Ehrenfest (13), which showed that nitrophenols caused a marked stimulation of the respiration of a number of tissues [Ehrenfest and Ronzoni (11) obtained a sevenfold stimulation with frog muscle].
In addition, Dodds and Greville (14) found that 4,6-dinitro-o-cresol increased aerobic glycolysis, while Ronzoni and Ehrenfest (13) showed
32. U N C O U P L E R S A N D I N H I B I T O R S 505 that 2,4-dinitrophenol increased the rate of hydrolysis of creatine phos
phate. Further studies led to the generalization that nitrophenols stimu
lated intracellular respiration, whereas energy-requiring functions, such as cell division in sea urchin eggs (15,16), growth of yeast (17), assimila
tion in microorganisms (18), sperm motility (19), and phosphate uptake by yeast (20), were inhibited.
Greville (21, 22) showed that the stimulation of respiration by dinitro
phenol was not due to its acting as an artificial hydrogen carrier like methylene blue. A new idea came from D e Meio and Barron (28), who suggested that dinitrophenol acts by combining with some of the sub
stances acting as agents for the control of the speed of cellular oxidations, thus increasing the activity of oxidizing enzymes. Lardy and Elvehjem
(24) suggested that dinitrophenol and other uncoupling compounds acted either by allowing oxidation to occur without phosphorylation or by catalyzing the hydrolysis of an intermediate phosphate compound. In either case, oxidation would proceed without phosphorylation. This idea was first given direct experimental support by Loomis and Lipmann (25) and Cross, Taggart, Covo, and Green (26) when they found that 2,4- dinitrophenol and a number of other compounds inhibited the synthesis of A T P by mitochondrial preparations without affecting respiration. Since ATP is required for many energy-utilizing functions of the cell, the earlier findings were explained.
B. Characteristics of Uncouplers
Uncouplers have the following properties:
(1) They stimulate the respiration of isolated mitochondria suspended in a medium deficient in phosphate acceptor1 (27-32) or deficient in phosphate1 (25, 33), or sometimes even in a medium containing phosphate and acceptor (34).
(2) They completely abolish the synthesis of A T P normally coupled with mitochondrial respiration, except the substrate-linked phosphoryla
tion in the oxidation step α-ketoglutarate -> succinate.
(3) They promote the hydrolysis of A T P added to mitochondria (24, 27,30,35).
(4) They inhibit the PrA T P (86) and the A D P - A T P exchange re
actions (37) and the exchange of oxygen atoms of Pi and water (38) and of A T P and water (39), catalyzed by mitochondria in the absence of added substrate.
1 Except when the rate of respiration is limited by the rate of the subtrate- linked phosphorylation associated with succinyl CoA.
506 Ε . C. SLATER
C. Mechanism of Action of Uncouplers
No final decision can be made between the two possible mechanisms of action suggested by Lardy and Elvehjem (21+). Ernster and his co-workers
{40-42)
favor the view that dinitrophenol acts by allowing the oxidation reaction to proceed without phosphorylation. Most workers favor the view that dinitrophenol promotes the hydrolysis of an intermediate high- energy compound. However, it is now clear that this cannot be a phosphate compound, as suggested by Lardy and Wellman {29, 80), since dinitrophenol abolishes the need for phosphate for maximal rates of respiration {25,88).
Ernster's theory is described by Eqs. (3) and (4)
D P N H + fp + Pi — DPN+ + fpH ~ Ρ + OH" (3) fpH ~ Ρ + 2 F e3 + + ADP ;=± fρ + 2 Fe2* + ATP + 2 H+ (4) Sum: D P N H + 2 F e3 + + ADP + Pi ;=± D P N+ + 2 Fe2+ + ATP + H+
where fp stands for the DPNH-oxidizing flavoprotein, and F e3 + and F e2 + for the oxidized and reduced carrier next to the flavoprotein in the cyto
chrome region. The dinitrophenol-induced ATPase is explained by Eqs.
( 5 ) - ( 7 ) .
ATP + fpH2 ^± ADP + fpH — Ρ (5)
fpH ~ Ρ + D P N+ + OH" ^ fp + Pi + D P N H (6)
(D initrophenol)
D P N H + fp + H+ • D P N+ + fpH2 (7)
(Dinitrophenol)
Sum: ATP + H20 • ADP + Pj
The alternative theory can be formulated {48) by the reaction scheme Respiration - > - , ^ - 2 ^ - P ^ ATP
(Dinitrophenol)
where ~x is the dinitrophenol-sensitive high-energy compound, ~2 is a second high-energy compound which is sensitive to oligomycin (see later), while ^ P is a high-energy phosphate compound. One possibility (but not the only one), written in the form of chemical equations, is shown in Eqs. ( 8 ) - ( 1 3 ) .
32. U N C O U P L E R S A N D I N H I B I T O R S 507
AH2 + Β + I ^ A ~ I + B H2 (8)
A ~ I + X ^ X ~ I + A (9) X ~ I + Pi — Χ ~ Ρ + I (10) Χ ~ Ρ + ADP ^± X + ATP (11)
(Dinitrophenol)
A ~ I + H20 > A + I (12)
X ~ I + oligomycin —> X ~ I — oligomycin (13) where A H2 and Β are components of the respiratory chain, and I and X
are unknown intermediates. The dinitrophenol-induced ATPase is here given by the reverse of Eqs. (11), (10), and (9), followed by Eq. (12), the PrA T P exchange reactions by Eqs. (10) and (11), and the A D P - A T P exchange reaction by Eq. (11).
D. Classification of Uncouplers
Very many different types of compounds have the property of uncou
pling phosphorylation from respiration [see, e.g., list given by Lehninger (44)]- In fact, any substance which disrupts the mitochondrial structure, such as deoxycholate or C a + + , will have this effect. The most useful uncouplers, however, are those which act more specifically without dis
rupting the mitochondrial structure. The two types can be roughly distin
guished by their effect on the ATPase in the absence of added M g + + . More specific compounds such as dinitrophenol stimulate the ATPase maximally without the addition of M g + + . On the other hand, M g + + is required to elicit maximum ATPase induced by deoxycholate. Uncouplers can be classified into six groups: (1) nitrophenols, (2) halophenols,
(3) dicoumarol and related compounds, (4) antibiotics, (5) unsaturated fatty acids, (6) arsenate.
1. N I T R O P H E N O L S
The relative effectiveness as uncouplers of various nitrophenols has been studied by Cross et al. (26), Parker (45), and Gladtke and Liss (46).
Parker emphasized the role of pK and Gladtke and Liss that of the lipid solubility in determining the activity of an uncoupler. Hemker and Hulsmann (47) have shown that both factors are important. The optimal concentrations of different nitrophenols at various pH's for stimulation of the ATPase activity of rat liver mitochondria are given in Table I.
Accurate determinations of the form of the curve relating ATPase activity to concentration of dinitrophenol have revealed a double maxi-
508 Ε . C. SLATER
T A B L E I
COMPARISON OF OPTIMAL CONCENTRATIONS OF DIFFERENT NITROPHENOLS AT VARIOUS P H ' S FOR INDUCTION OF A T P A S E ACTIVITY OF RAT LIVER MITOCHONDRIA"
p Co p t c at pH's
Compound pK pQb
p-Nitrophenol 7.2 + 0 . 4 5 3.30 3.37 3.51 3.39 — 2,6-Dinitrophenol 3.7 - 2 . 3 5 4.77 4.22 3.68 3.12 — 2,6-Dinitro-3,4-dimethylphenol d d 4.92 4.51 4.36 3.95 3.60 2,6-Dinitro-4-isobutylphenol 4.3 - 3 . 5 4 5.16 4.88 4.71 4.48 4.26 2,6-Dinitro-4-isoamylphenol 4.1 - 4 . 1 5 5.55 5.42 5.18 4.92 4.71 2,6-Dinitro-4-isooctylphenol 4.05 - 4 . 7 5 6.08 5.89 5.64 5.41 5.24
α From Hemker and Hiilsmann (47).
B Q γ concentration of undissociated phenol in xylene concentration of undissociated phenol in water For the dinitrophenols at pH's > 5,
^ ^ j j ^ concentration of undissociated phenol in xylene concentration of total phenol in water
0 Ρ Copt = —log io C0p t ; CO Pt = concentration of phenol giving maximum ATPase activity.
d pK - pQ = 7.44.
mum, indicating that there may be at least two different dinitrophenol- induced ATPases, differing in the concentration of dinitrophenol giving maximum activity. Amytal appears to inhibit preferentially the ATPase activated by the higher concentrations of dinitrophenol, and antimycin at the lower concentrations (Hemker, unpublished).
When allowance is made for the small differences in pK and the large differences in lipid solubility, no difference is found among four alkyl- substituted 2,6-dinitrophenols (3,4-dimethyl, 4-isobutyl, 4-isoamyl, and 4-isooctyl) with respect to their ability to induce ATPase in rat liver mitochondria, if the concentrations are calculated on the basis of the amounts of undissociated phenol dissolved in the mitochondrial lipid
(Table I I ) .
From Table I, it can be calculated that maximum ATPase in rat liver mitochondria at pH 7 is obtained with 310 μΜ p-nitrophenol, 210 μΜ 2,6-dinitrophenol,2 44 μΜ 2,6-dinitro-3,4-dimethylphenol, 19 μΜ 2,6-
2 About the same concentration of 2,4-dinitrophenol is also necessary for maximal A T P a s e activity, which in this case is about 33% higher than that obtained with the 2,6-dinitrophenols.
32. UNCOUPLERS AND INHIBITORS 509
T A B L E II
CALCULATION FROM DATA IN T A B L E I OF CONCENTRATION IN MITOCHONDRIAL LIPID (CL) OF DIFFERENT NITROPHENOLS NECESSARY FOR OPTIMUM A T P A S E ACTIVITY
AT VARIOUS P H ' S0
p CL 6 at pH's
Compound pK pQ 5 6 7 8 9
p-Nitrophenol 7.2 + 0 . 4 5 3.55 3.62 4.06 4.62 2,4-Dinitrophenolc 4.1 - 2 . 0 6 3.61 4.06 4.52 4.96 —
2,6-Dinitrophenol 3.7 - 2 . 3 5 3.72 4.17 4.63 5.07 —
2,6-Dinitro-3,4-dimethylphenol d d 2.48 3.07 3.82 4.51 5.16 2,6-Dinitro-4-isobutylphenol 4.3 - 3 . 5 4 2.33 3.05 3.88 4.65 5.43 2,6-Dinitro-4-isoamylphenol 4.1 - 4 . 1 5 2.30 3.04 3.93 4.67 5.56 2,6-Dinitro-4-isooctylphenol 4.05 - 4 . 7 5 2.28 3.09 3.84 4.61 5.44
α From Hemker and Hiilsmann (47).
b p CL = p Co p t + pQ + pH + \og{K + [H+]).
c This compound was not so extensively investigated as the 2,6-dinitrcphenol, but no significant difference was found between the values of p Co p t for the two compounds.
For purposes of calculation, it has been assumed that they were identical.
d pK - pQ = 7.44.
dinitro-4-isobutylphenol, 6.6 μΜ 2,6-dinitro-4-isoamylphenol, and 2.3 μΜ 2,6-dinitro-4-isooctylphenol. Virtually complete inhibition of phosphate esterification is, however, obtained at considerably lower concentrations.
For most experiments it is immaterial which compound is used. However, the nitrophenols are strongly yellow in neutral solution, which can be a disadvantage if spectrophotometric measurements are also to be under
taken. Under these circumstances, the highly lipid-soluble isooctyl com
pound is to be preferred.
The amount required for complete uncoupling depends not only on the lipid solubility and the pK of the uncoupler, and on the pH of the medium, but also on the nature of the mitochondria (e.g., less 2,4-dinitro- phenol was required with the preparations of blowfly sarcosomes used by Slater and Lewis (48) than with rat heart sarcosomes) and on the concentration of the mitochondria and of extraneous material. Jongejans- Sickler et al. (49) have measured the relative affinities of uncoupling agents for protein by filtering a solution of each compound in ascites fluid through cellophane under pressure, and measuring the concentration of the uncoupling agent in the protein-free filtrate. Under these conditions the following values were obtained for the percentage of uncoupling agent bound by the ascites fluid: p-nitrophenol, 57%; 2,4-dinitrophenol, 76%;
5 1 0 Ε . C. SLATER
2 - methyl - 4 , 6 - dinitro - 5 - isopropylphenol ( 4 , 6 - dinitrocarvacrol), 9 5 % ; 2-isopropyl-4,6-dinitro-5-methylphenol (2,4-dinitrothymol), 9 8 % . In con
sequence of this binding, the activities of all four compounds in ascites fluid in vitro are of the same order of magnitude, whereas their activities differ markedly when tested in Krebs-Ringer bicarbonate. It has also been shown that serum albumin can protect against uncoupling by 2,4-dinitro
phenol (50, 51).
2 . H A L O P H E N O L S
Loomis (52) showed that di- and trihalophenols uncouple oxidative phosphorylation. Weinbach (53, 54) has made a special study of the uncoupling action of pentachlorophenol. Since it has a high pK, this com
pound (as well as the other halophenols) has the advantage of being colorless at neutral pH's. It has the serious disadvantage, however, that, at concentrations only a little higher than necessary for complete un
coupling, it inhibits many of the reactions catalyzed by the mitochondria and, at somewhat higher concentrations, solubilizes the mitochondria.
3 . D I C O U M A R O L
Martius and Nitz-Litzow (55) showed that dicoumarol [3,3'-methyl- enebis(4-hydroxycoumarin)] and related compounds are active uncou
plers. Dicoumarol is also a powerful inhibitor of a flavoprotein, which has been called variously menadione reductase (56), phylloquinone reductase (57), vitamin Κ reductase (58), and D T diaphorase (59). It appears, however, that this inhibition is unrelated to the uncoupling activity.
Furthermore, there is little evidence that the uncoupling activity is related to the well-known antiblood-clotting activity of dicoumarol.
4 . A N T I B I O T I C S
Many antibiotics, both phenols and polypeptides, are active uncou
plers. This was first shown by Hotchkiss (60) and Cross et al. (26) for gramicidin. Aureomycin (61) and valinomycin (62) are also uncouplers.
5 . U N S A T U R A T E D F A T T Y A C I D S
The uncoupling activity of long-chain fatty acids was discovered by Pressman and Lardy (63-65). Hiilsmann et al. (66-68) showed that the active principle of a protein fraction isolated from disintegrated, aged mitochondria by Polis and Shmukler (69) and Pullman and Racker (70), called mitochrome by the former workers, consisted of a mixture of long-
32. U Ν COUPLERS A N D I N H I B I T O R S 511 chain unsaturated fatty acids, which could be separated from the hemo- protein by extraction with isooctane. Borst and Loos (71) showed that unsaturated fatty acids are much more potent uncouplers than the saturated. The degree of unsaturation is less important, e.g., oleic, linoleic, linolenic, and elaidic acids are all about equally effective. Lehninger and Remmert (72) found also that an uncoupling factor (called U factor) could be extracted with isooctane from aged mitochondria or mitochon
drial fragments and suggested that this factor may be a long-chain fatty acid.
Unsaturated fatty acids produced by enzymic hydrolysis of neutral lipids or phospholipids in the mitochondrial preparation are probably at least partly responsible for the high ATPase activity of aged mitochon
dria. The formation of uncoupling fatty acids during incubation of mitochondria can be inhibited by E D T A , ATP, or D F P (68), or by ATP + CoA (72).
Two characteristic properties of fatty acids make it easy to test whether an uncoupling preparation isolated from natural materials owes its activity to unsaturated fatty acids. Firstly, the uncoupling activity is extracted into isooctane; secondly, the activity is counteracted by serum albumin, which binds unsaturated fatty acids very strongly, especially oleic acid (73). The beneficial effects of serum albumin on the oxidative phosphorylation of isolated insect sarcosomes (74-76) has been shown to be due to the presence of unsaturated fatty acids in these preparations (77, 78). This is also probably the reason why albumin increases the P / O ratio of mitochondria isolated from tumors (79, 80). Unsaturated fatty acids are also the active principles in the inhibitor produced by Tetra- hymena pyriformis (81).
Uncoupling by unsaturated fatty acids may be distinguished from that brought about by other anionic detergents in that it is readily reversible by the subsequent addition of serum albumin.
6. A R S E N A T E
Arsenate differs from the uncoupling agents mentioned above in that it also uncouples the substrate-linked phosphorylation linked with the oxidation of phosphoglyceraldehyde (82) and α-ketoglutarate (83). Crane and Lipmann (84) showed that it also uncoupled respiratory chain phos
phorylation. Like other such uncouplers it induces an ATPase (85, 86) and inhibits the PrA T P exchange reaction (86} 87), the A D P - A T P exchange reaction (85), and the exchange of oxygen atoms between Pi and water (87).
As an uncoupler of respiratory chain phosphorylation, arsenate is much
512 Ε. C. SLATER less useful than the compounds mentioned above. High concentrations are necessary, the degree of uncoupling increases with time (84, 86), and uncoupling is usually incomplete. The main use of arsenate is in com
bination with dinitrophenol when it is necessary to uncouple substrate- linked as well as respiratory chain phosphorylation.
III. INHIBITORS
In 1955, Hollunger (88) introduced a new type of respiratory inhibitor, guanidine, which inhibits respiration coupled with phosphorylation, with
out having any effect on the nonphosphorylating respiration of mitochon
drial fragments or of mitochondria in the presence of dinitrophenol.
Guanidine was not widely used, probably because the mitochondria must be preincubated with rather high concentrations for complete inhibition.
In 1958, Lardy et al. (89) introduced the fungicide oligomycin which, in very low concentrations, has effects similar to those brought about by guanidine. Oligomycin is a neutral, unsaturated, optically active alcohol which may also contain ketone groups (90, 91). N o elements besides carbon, hydrogen, and oxygen are present. It is a very useful inhibitor of oxidative phosphorylation.
Oligomycin has no effect on the respiration of rat liver mitochondria in the absence of either phosphate or phosphate acceptor, but inhibits completely the increment of the respiration brought about by the addition of phosphate or phosphate acceptor, respectively (43). The inhibition of the respiration of mitochondria, measured in the presence of both phos
phate and phosphate acceptor, is usually not complete, amounting to about 90% with glutamate as substrate and 60% with succinate (4%, 89).
In both cases, however, phosphorylation is completely inhibited, except for the substrate-linked phosphorylation step of α-ketoglutarate oxida
tion. The residual respiration is due to the fact that, in isolated mitochon
dria, respiration is not completely coupled to phosphorylation, the degree of "loose coupling" being greater for succinate than for glutamate. The degree of inhibition by oligomycin is a good measure of the tightness of the coupling. Inhibition of respiration by oligomycin is completely re
lieved by dinitrophenol (43, 89) but not by arsenate (43, 92). The respira
tion of nonphosphorylating mitochondrial fragments is not inhibited by oligomycin (43, 89).
Oligomycin preparations used are a mixture of three structurally related compounds (A, B, and C) of molecular weights 424, 394, and 478, respec
tively (90, 91). The relative proportions of the three components can be
32. UNCOUPLERS AND INHIBITORS 513 determined by paper chromatography (91). Oligomycin is usually added as an ethanolic solution.
Using a preparation consisting mainly of oligomycin B, 0.75 μg oligo- mycin/ml (1.4 ^moles/mg mitochondrial protein) was found sufficient for maximal inhibition of respiration (48). The amount required is pro
portional to the protein concentration (unpublished).
Oligomycin also inhibits completely the dinitrophenol-induced ATPase of mitochondria and also the ATPase of mitochondrial fragments (89).
Chappell and Greville (98) have used oligomycin to prevent the hydroly
sis of A T P induced by dinitrophenol, while still retaining its uncoupling activity. Since it is without effect on the ATPase of microsomes, oligo
mycin can be used to measure the degree of contamination of mitochondria by microsomes (43, 94).
Although low concentrations of oligomycin (about 1 μ-g/mg protein) are sufficient to inhibit the dinitrophenol-induced ATPase of mitochondria by about 50%, very high concentrations (about 50 /*g/mg protein) are required for complete inhibition (unpublished). Apparently, part of the dinitrophenol-induced ATPase is much more resistant to oligomycin than the rest.
Oligomycin inhibits the Pt ±=z ATP reaction (89). According to Lardy (89) and Huijing and Slater (43), it has no effect on the A D P ±^ A T P reaction, but Cooper (95) and Wadkins (96) have found extensive inhibi
tion.
The action of oligomycin can be explained by Eqs. ( 8 ) - ( 1 3 ) , shown above, if it is further assumed that the noncoupled oxidation, in the pres
ence of oligomycin, proceeds by hydrolysis of A ^ I . If the concentration of X does not exceed that of I, the inhibition of the A D P - A T P exchange reaction [Eq. (11)] is explained.
It should be noted that, according to this scheme, oligomycin does not inhibit the formation of all high-energy compounds but only the con
version of these compounds to ATP. This is in contrast to uncouplers which lead to the complete discharge of all high-energy compounds. Thus, oligomycin is a useful reagent for testing whether the high-energy inter
mediates of oxidative phosphorylation can be directly utilized for energy- requiring reactions in the mitochondria, without having first to be con
verted to ATP. An example is the succinate-induced reduction of D P N + , which is inhibited by dinitrophenol, but not by oligomycin (97).
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