The site of inhibition of mitochondrial electron transfer by coenzyme Q analogues

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 191, No. 1, November, pp. 306-315, 1978

The Site of Inhibition of Mitochondrial Electron Transfer by Coenzyme Q Analogues

HENRY ROBERTS,* WAN MEE CHOO,* STUART C. SMITH,* SANGKOT MARZUKI,* ANTHONY W. LINNANE,* THOMAS H. PORTER,?

AND KARL FOLKERST

* Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia, and t Institute for Biomedical Research, University of Texas, Austin, Texas, 78712

Received April 10, 1978; revised June 30,1978

The effects of 33 quinone derivatives on mitochondrial electron transfer in yeast were examined. Twenty-two of the compounds were also tested for their effects on the growth of

yeast cells. Four strong inhibitors of electron transfer were identified: 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, 7-w-cyclohexyloctyl-6-hydroxy-5,8-quinolinequinone, 7-n- hexadecyl-mercapto-6-hydroxy-5,8-quinolinequinone, and 3-n-dodecyhnercapto-2-hydroxy- 1,4-naphthoquinone. They inhibit the growth of yeast with ethanol as an energy source, but not when glucose is the energy source. The NADH oxidase activity of isolated mitochondria is 50% inhibited by these quinone derivatives at about 10m8~, or 0.5 Fmol/g mitochondrisl protein; WOO-fold higher concentrations do not affect electron transfer from NADH or succinate to coenzyme Q2. The effects of the inhibitors on cytochrome spectra indicate that they block electron transfer between cytochromes b and cl. A possible antagonism between these compounds and coenzyme Q at a site between cytochromes b and c, is discussed in terms of Mitchell’s “protonmotive Q cycle” hypothesis (Mitchell, P. (1976) J. Theor. Biol. 62, 327-367). 6-P-naphthylmercapto-5-chloro-2,3-dimethoxy-l,4-benzoqu~one inhibits electron transfer between succinate and coenzyme Qe or phenazine methosulfate, suggesting

a site in the succinate-coenzyme Q reductase complex with a different qumone speciticity from that of the site in the cytochrome bcl complex. Seven of the quinone derivatives inhibit growth on both glucose and ethanol media, indicating that their effect is not the result of inhibition of respiration.

For many years, respiratory inhibitors have played an important role in the eluci- dation of the pathway of electron flow in mitochondria. More recently, information about mitochondrial function has been obtained from studies of the inheritance of mutations in eukaryotic microorganisms which are resistant to inhibitors of the electron transfer chain. Mutations conferring resistance to antibiotics have enabled the contributions of the nuclear and mitochondrial genetic systems to the biogenesis of mitochondria to be distinguished. Bio- chemical and genetic studies of oligomycin- resistant strains of Saccharomyces cerevi- siae in this laboratory have identified two mitochondrial genes coding for subunits of the oligomycin-sensitive ATPase (1).

Several mitochondrial mutants of S. cer- evisiae which are resistant to antimycin

and other antibiotics affecting coenzyme &Hz-cytochrome c reductase have been isolated (l-4). A complex picture of cross-resistance patterns and interactions with nuclear genes is emerging from genetic analysis of these mutants. Further analysis of the antibiotic-resistant mutants indicates that there are several loci determining the sensitivity of coenzyme QHz-cytochrome c reductase to inhibitors within a short region of the mitochondrial genome (5). In addition, there are at least two other loci within the same region of the genome, in which mutations result in the loss of cytochrome b (6-8). A number of new inhibitors affecting CoQ(H2)‘-cytochrome c reductase are,

‘Abbreviations used: CoQ(Hg), coenzyme Q (reduced form); PMS, phenazine methosulfate; SDS, sodium dodecyl sulfate.

306

0003-9861/78/1911-0306$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

INHIBITION OF ELECTRON TRANSFER BY COENZYME Q ANALOGUES 307

therefore, currently being studied in this laboratory in an attempt to establish the number of mitochondrial genes involved in the determination of the cytochrome bcl complex and to identify the corresponding gene products.

Several 2-hydroxy-1,4-naphthoquinones and 2,3-dimethoxy-6-hydroxy-1,4-benzoquinones have previously been shown to inhibit mitochondrial electron transfer. In many cases, inhibition at the level of cytochrome bcl was indicated (9-12). The ob- servation that the inhibition of electron transfer by certain of these naphtho- and benzo-quinones can be reversed by various coenzyme Qs (11-14) suggested interfer- ence with coenzyme Q function. This is supported by the fact that many of these inhibitors are structurally related to coenzyme Q. Some structural features conferring inhibitory properties on the compounds were tentatively identified.

In this report, several specific inhibitors of coenzyme Q(H&cytochrome c reductase in yeast mitochondria are identified, and a preliminary biochemical analysis is pre- sented to show that they are potentially useful for the study of mitochondrial biogenesis. The effects of some of these compounds on oxidase activities of beef heart mitochondria and on Plasmodium have been described previously (for a review, see 15).

MATERIALS AND METHODS

Materials. NADH, ADP, cytochrome c, phenazine methosulfate (PMS), and antimycin were obtained

from Sigma Chemical Co. (St. Louis, MO.). Yeast extract was from Difco Laboratories (Detroit, Mich.).

Coenzyme Q2 was a gift from Dr. S. Kijima, Eisai Company Ltd., Tokyo. All other materials were of the highest purity commercially available.

Yeast strains. The auxotrophic haploid strains of

S. cereuisiae used were L410 (a ural hi&) and J69-IB (a adel hid).

Growth of cells. Cells were grown in 2-liter fluted flasks on a rotary shaker in 600 ml of medium contain-

ing 1% ethanol, 1% yeast extract, and a mineral salts solution (16). The following growth supplements were also added: 50 pg/ml of uracil and 50 pg/ml of hi&dine for L410; 50 pg/ml of adenine and 50 pg/ml of hi&line for J69-1B. Cells were harvested in the mid-logarith- mic phase of growth at a cell density of 2-3 mg cell dry weight/ml.

In vivo sensitivity to inhibitors on ccgur plates.

Filter paper discs containing known amounts of inhib-

itor were placed on lawns of cells as described previ-

ously (17) except that the nutrient agar contained 1% yeast extract, mineral salts, and growth supplements

as in the liquid media; 2% ethanol or 2% glucose, and 40 8g/ml SDS were added where indicated. Zones where no growth occurred were recorded after 48-72

h incubation at 28°C. Isolation of mitochondriu. The cells were washed

once with water and once with resuspension buffer (13

mM Tris-HCl (pH 7.4) containing 0.33 M sorbitol, 0.27

M mannitol, and 0.7 mM EDTA) and were resuspended in an equal volume of the resuspension buffer. The

cells were disrupted by shaking with glass beads in a Braun cell homogenizer, model MSK (50 g of glass beads (diameter 0.5 mm) to 25 ml of suspension) for

two 15-s periods. The homogenate was diluted with

the resuspension buffer to four times the original packed cell volume and the pH brought back to 7.4 with 1 M KOH. Debris was removed by two or three

centrifugations at 1,500 g (maximum) for 5 min until no further pellet formed. Mitochondria were then sedimented by centrifugation at 17,500 g (maximum)

(Sorvall SS34 rotor) for 10 min and were taken up in the resuspension buffer. The mitochondria were

washed with the resuspension buffer by centrifuging the suspension for 5 mm at 1,500 g, then for 10 min at

17,500 g, and resuspending them in the resuspension buffer. The washing was repeated twice more.

Assay techniques. NADH oxidase activities of mitochondria were determined by following the absorb-

ance at 340 nm in 3 ml of medium containing 0.6 M

sorbitol, 10 mu KH,PO,, 12 mu maleic acid, and 1.2

mu EDTA. The pH was adjusted to 6.4 with KOH. ADP (0.2 mu), cytochrome c (3 PM), and NADH (0.6 mM) (final concentration) were then added, and the

reaction was started by addition of mitochondria (60 pg protein, unless otherwise indicated). NADH-coen-

zyme Q2 reductase and succinate-coenzyme QZ reductase activities were determined spectrophotometric-

ally by the methods of Sanadi et al. (18) and Ziegler and Rieske (19), respectively, except that the pH was

7.4 in both cases. Succinate-PMS reductase was as- sayed by the latter method, by substituting 1 mM PMS for CoQ2. Protein was determined by the method of

Lowry et al. (20). Cytochrome spectra. The absorption spectra of iso-

lated mitochondria were recorded using a Cary model 14 double beam spectrophotometer with O-O.1 slide wire, at room temperature. The effects of respiratory

inhibitors on the oxidation state of the cytochromes were measured as follows. Mitochondria were sus- pended at a protein concentration of 2 mg/ml in 1 ml

of the basic medium used for the NADH oxidase assays (pH 7.4) and added to the sample and reference cuvettes to record a base line. The mitochondria in the sample cuvette were reduced by the addition of

either NADH (4 pmol) or succinate (60 pmol). Inhibi- tors were added to the sample cuvette either before or

308 ROBERTS

after the addition of substrate, as indicated in the text.

The cuvette was thoroughly aerated before recording the spectrum against the untreated reference cuvette again.

The cytochrome b concentration in mitochondria was determined using the reduced-minus-oxidized difference extinction coefficient AE (562-575 nm) = 25.6

mM-’ cm-’ (21).

RESULTS

The quinone derivatives studied were de- rived from the following compounds (Scheme I): 1,4-benzoquinone (I, Ia, Ib), 1,4naphthoquinone (II), f>,&quinolinequinone (III, IIIa), 5,8-quinoxalinequinone (IV), and 4,7-dioxobenzothiazole (V) by substitution at R1 and Rz. Derivatives of compound I are similar to coenzyme QG (VI), the natural ubiquinone in yeast.

Inhibition of mitochondrial NADH oxidase. Thirty-three quinone derivatives were screened for inhibitory action on mitochondrial respiration in yeast by means of NADH oxidase assays of mitochondria isolated from L410 cells (Table I). Com- pounds 1, 2, 3, and 4 inhibited the activity by 50% at concentrations around 10m8~, or 0.5 pmol/g mitochondrial protein. Much weaker inhibition of NADH oxidase (50% inhibition at concentrations around 10m6~) was observed with compounds 5, 6, and 7. A further 9 compounds (8-16) very weakly inhibited NADH oxidase (50% inhibition above 5 x 10e6~) and the remaining 17 compounds did not produce any effects on NADH oxidase at the highest concentrations tested.

In vivo sensitivity to quinone derivatives. The effects of the derivatives on the growth of whole cells on media containing

ET AL.

ethanol or glucose as an energy source was used to discriminate between those compounds which only affected mitochondrial respiration, and those that had other growth-inhibitory effects or which had no effect at all in vivo. Filter paper discs containing various quantities of each compound were placed on lawns of cells as described in Materials and Methods. The inhibitory effects of the compounds were quantitatively determined by observing the sizes of the zones where no growth occurred round the discs (Table II). The test was also carried out with 0.004% SDS in the agar medium on which the cells were grow- ing, as SDS is known to increase the perme- ability of cell membranes to some compounds (Trembath et al., in preparation).

In Table II, the 22 compounds tested are arranged according to the type of inhibition observed. Except where indicated in the table, the inhibition was similar for both strain I.410 and J69-lB, and in the presence or absence of SDS.

Seven compounds (Nos. 5,7,9,10,13,26, and 32 in Table II) inhibited growth on both glucose and ethanol media, most of them requiring SDS for maximal effect. The first five of these compounds weakly inhibited NADH oxidase in isolated mitochondria, but this is unlikely to be their main effect, in view of their strong inhibition of cell growth on glucose media, partic- ularly with compound 32.

Compounds 2, 3, and 4, which strongly inhibited NADH oxidase, also clearly inhibited the growth of yeast cells on ethanol media without having any detectable effect on glucose-supported growth in the quantities used. This indicates that the only


TABLE I

INHIBITION OF MITOCHONDRIAL NADH OXIDASE ACTIVITY BY QUINONE DERIVATIVES”

Quinone derivative COIlCeIl- tration

Inhibi-

NO. Parent COIlI-

pound”

RI

1 III OH 2 v OH

3 III OH 4 II OH

5 I OH

6 III OH 7 I OH

8 IIIa OH

9 I H

10 I Cl 11 I OH

12 Ia CHs 13 II H 14 III (CH&cyclohexyl

15 III NH(CHzl&Hz

16 I Cl

17 I CHJ 18 I H

19 IV H 20 II H

21 IV H

22 Ib CI-L 23 I H 24 I OH

25 I H

26 I

27 I 28 II

29 IV

30 II

31 II 32 III

33 III

H

H H H

H

H NH-cyclooctyl

NH(CH&&V-piperidine H

RJ

S(CHzl&H3 (CHz)&H3 (CH&-cyclohexyl

S(CHz)&Hz (CH&CHs

(CH~)&HJ (CH&-cyclohexyl (CH&cyclohexyl

S-/3-naphthyl S-P-naphthyl (CH&(CH=CHCH&(CH,)sCH.,

NH-cycloheptyl

NH(CH,,,N[(CH,,,CH,], OH

H

S(CHd&Hz S(CHd&Ha S(CHz)&Hz

S(CHP)ICHB

S(CHzluCHz (CHl)a-cyclohexyl

S(CHP)~CH~ S(CH&cyclohexyl [CH&H=C(CH&H&,H

SCHzCH=C(CHz)CHz .[CH,CH,CH(CH,,CH,],H

S-p-methoxyphenyl

S(CH&phenyl S(CH&-phenyl

NH(C&)zCHz

NH(CHkCH3 NH(CH&-cyclohexyl H

a The activity was measured in the presence of 20 yg/ml mitochondrial protein.

‘See Scheme I. ’ The uninhibited specific activity was 0.643 pmol/min/mg protein.

(PM)

0.0086 0.0089

0.020 0.023 0.749

0.786 1.47

6.45

19.6 20.2

23.1

44.6 77.4

9.0

30 25 15

18

18 37 21

18

55 20

20

10

10 10

123

9

21 40

64

tion PC)’

50 50

50 50

50 50

50 50

50 50

50

50 50 34

12.5 8.9 0

0

0 0

0 0

0 0

0

growth inhibitory effect of these compounds is on mitochondrial electron transfer. Inhibition of growth on ethanol was also observed with compounds 14, 15, and 16, which were very weak inhibitors of NADH oxidase, and with compounds 19, 22, and 29, which had no effect on NADH oxidase, suggesting that these compounds act at a site other than the respiratory chain.

Six other compounds had no detectable effects on ethanol- or glucose-supported

growth even in the presence of SDS, although two of these, compounds 6 and 8, weakly inhibited NADH oxidase in uitro.

Effects on coenzyme Q and phenazine methosulfate reductase activities. The effects of several of the derivatives on other mitochondrial electron transfer activities were studied to determine the site of action of the inhibitors (Table III). Compounds 2, 3, and 4 did not affect NADH-CoQ reductase activities significantly, even at much higher concentrations than those which in-

310 ROBERTS ET AL

TABLE II

EFFECT OF QUINONE DERIVATIVES ON THE GROWTH

OF YEAST CELL.C?

Quinone Sensitivity level (pgY deriva-

tive Carbon source: Glucose Ethanol

5 20’ G5 7 50’ 20

9 G5’ 10

10 10’ 10 13 s5’3 d 10 26 20’ G20

32 <5 <5 2

3 4

14

<

c5 c5’

20 C5’

15 65’

16 60’.d

19 No inhibition <lo

22 up to at least s20d

29 100 Pis GlOd

6 8

17 No inhibition

18 up to at least

30 100 PLg

33

‘Plates were seeded with a lawn of cells (strain L410 or J69-1B) in nutrient agar containing either 2%

glucose (k40 pg/mI SDS), or 2% ethanol (k40 ag/mI SDS). Paper discs impregnated with 5 pg to 100 pg of

quinone derivative were placed on the agar. After 2 or 3 days, the widths of the zones where no growth had

occurred around the discs were measured. *The table gives the level of the derivatives to

which strain IA10 is sensitive. The sensitivity level is defined for convenience as the minimum quantity of

drug which gives a zone of no growth 1 mm wide. ’ In presence of SDS. No effect without SDS. d Strain J69-1B. No effect on strain L410.

hibited NADH oxidase. Their primary site of action, therefore, lies on the oxygen side of the site where coenzyme Q is reduced by NADH dehydrogenase.

Table III also shows the effects of some of the compounds on enzyme activities uti- lizing succinate as an electron donor. The only compound that had any effect was compound 10, which inhibited succinate- CoQ reductase and succinate-phenazine methosulfate reductase activities by more than 50% at 5.5 FM, a considerably lower concentration than the 20.2 PM required to inhibit NADH oxidase by 50% (Table I).

Effects on cytochrome reduction. The reduction and oxidation of mitochondrial cytochromes in the presence of the inhibitors was measured spectrally to locate the sites of inhibition. Mitochontia were reduced by excess NADH or succinate. Fig. 1, A and B show that when compound 2 was added, cytochrome b remained reduced, whereas cytochromes cl, c, and (21613 became oxidized. Conversely, when the inhibitor was added to oxidized mitochondria, the subsequent addition of substrate caused reduction of cytochrome b, but not of cytochromes cl, c, or ~3, (Fig. lC), thus defining the site of action as being between cytochromes b and cl in the classical linear electron transfer chain. Identical results were obtained using compounds 3 or 4 (not shown) or antimycin (Fig. 1D) as the inhibitor.

Compound 10, which appears to be more specific for succinate-CoQ reductase (Table III) prevented the reduction of the cytochromes by succinate, as shown by the loss of the absorption peaks in Fig. lE, spectrum 3. The altered shape of this spectrum is due to the intense absorption of the inhibitor at shorter wavelengths. This compound did not inhibit the reduction of the cytochromes by NADH (Fig. 1F).

Compound 5, which inhibited NADH oxidase activity only at relatively high concentrations, did not inhibit the reduction of any of the cytochromes below 500 PM.

Higher concentrations of the inhibitor could not be tested owing to its intense absorption.

Determination of inhibitor binding pa- rameters. Preliminary experiments showed that the binding of compound 4 to the mitochondrion was very tight, and therefore a significant proportion of inhibitor molecules would be bound under the assay conditions. As there is no means of measur- ing the concentration of bound inhibitor, information about the number of binding sites could not be obtained by varying the concentration of the inhibitor. The binding of compound 4 was therefore analyzed as described in the Appendix by varying the concentration of mitochondria used in the NADH oxidase assay in the absence of inhibitor and in the presence of a fixed concentration of inhibitor (Fig. 2).


TABLE III

EFFECT OF QUINONE DERIVATIVES ON ENZYME ACTIVITIES OF ISOLATED MITOCHONDRIA

Inhibitor NADH-CoQ Succinate-CoQ Succinate-PMS reductase” reductaseb reductase’

Concentration Inhibition Concentration Inhibition Concentration Inhibition (PM) (%) (PM) (%) (FM) (W -

2 9.9 0 11.9 0 3 9.0 9 5.4 0

4 8.9 0 5.3 0 8.0 0

10 18.5 0 5.5 88 5.5 69

Antimycin 0.15 0 1.0 0 0.60 0

Malonate lo5 100

LI Uninhibited specific activity was 2.65 ymol NADH/min/mg protein. Protein concentration was 0.017

mg/ml. b Uninhibited specific activity was 0.063 -01 succinate/min/mg protein. Protein concentration was 0.20

m&ml. ’ Uninhibited specific activity was 0.021 pmol succinate/min/mg protein. Protein concentration was 0.20

mg/ml.

550 575 600 625 7

I

550 575 6oc 625

WAVELENGTH (nm)

FIG. 1. Effect of quinone derivatives on redox state of mitochondrial cytochromes. In each case, spectrum 1 was a base-line recorded with 1 ml of 2 mg/ml mitochondria in the sample and reference cuvettes. The following additions were then made to the sample cuvette: A, 4 pmol

NADH (2) and 30 nmol compound 2 (3); B, 60 pmol succinate (2) and 30 nmol compound 2 (3); C, 30 nmol compound 2 (2) followed by 4 pmol NADH (3) and a few grains of sodium dithionite (4); D, 4 pmol NADH (2) and 0.4 nmol antimycin (3); E, 60 pmol succinate (2), 140 nmol compound 10

(3) and sodium dithionite (4); F, 4 pmol NADH (2) and 190 nmol compound 10 (3).

In Fig. 2, the horizontal displacement of Fig. 3, the data of Fig. 2 are plotted accord- any point on the curve in the presence of ing to Eq. 6 (Appendix). As expected, the the inhibitor from the uninhibited curve plot is linear within experimental error. represents the concentration of mitochon- From the plot, the values of KD and n are drial protein which is inhibited, or Ci. In 5 X ~O-‘M and 1.1 nmol/mg protein, respec-

312 ROBERTS ET AL.

MlTOCHONORlAL PROTEIN (~glml)

FIG. 2. Dependence of NADH oxidase rate on concentration of mitochondrial protein. Assays were car-

ried out as in Materials and Methods. 0, no inhibitor added. l ,0.021 PM compound 4 added.

zoo7

Ci/[LI* (g/pmd)

FIG. 3. Data of Fig. 2 plotted as described in the

Appendix. Concentration of inactive protein was esti- mated from the horizontal distance of each point on the inhibited curve in Fig. 2 from the uninhibited curve.

tively. The binding is, therefore, very tight, and the number of binding sites is approx- imately double the cytochrome b content of the mitochondria (0.5 nmol/mg protein; data not shown).

DISCUSSION

Previous studies of several 1,4-naphtho- and 1,4-benzoquinones as inhibitors of electron transfer in mitochondria from a variety of organisms have suggested that they act between cytochromes b and cl (9, 10, 12). On the other hand, certain 1,4-benzoquinones were shown to inhibit succinate oxidase more strongly than NADH oxidase (13, 14), apparently inconsistent with a common site of inhibition in the two activities. The demonstration of the reversal of the inhibition by coenzyme Q in some cases (11, 14) led to the idea that the quinones acted as antagonists of coenzyme Q, which occupied separate pools in the NADH-CoQ reductase and succinate-CoQ reductase complexes.

For biochemical and genetic analysis of the cytochrome b-cl region of yeast mitochondria, it is essential to use inhibitors which act only on this part of the electron transfer chain, and which do so in both whole cells and isolated mitochondria. The present work was concerned with selecting from a large number of quinones, those which fulfil these requirements.

Four potent inhibitors of NADH oxidase in isolated mitochondria were identified. Three of these compounds (5+undecyl- 6-hydroxy-4,7-dioxobenzothiazole (compound 2), 7-w-cyclohexyloctyl-6-hydroxy- 5&quinolinequinone (compound 3)) and 3-n-dodecyhnercapto-2-hydroxy -1,4-aaph- thoquinone (compound 4)), were found to inhibit electron transfer from NADH or succinate to cytochrome cl, but not to cytochrome b or coenzyme Q. Their site of action is therefore between cytochromes b and cl. They were effective at lower concentrations than previously reported for other benzo- and naphthoquinones. They also strongly inhibited the growth of yeast cells on ethanol media but not on glucose media, indicating that they have no other growth-inhibitory effects.

Most of the other compounds tested inhibited NADH oxidase much more weakly or not at all, and no inhibition of electron transfer from NADH to CoQ, cytochrome b, and cytochrome cl could be demon- strated. Six of these compounds (Nos. 14, 15, 16, 19, 22, and 29) specifically inhibited


the growth of yeast cells on ethanol media, and their small effects in vitro suggest either that the mitochondria lost their sensitivity during isolation or that the inhibition of electron transfer is not their primary effect. One possible site of action is the CoQ biosynthetic pathway.

One of the benzoquinones, compound 10, inhibited electron transfer from succinate to CoQ and from succinate to PMS, but had little effect on electron transfer between NADH, CoQ, cytochrome b, and cytochrome cl, suggesting that it acts on succinate dehydrogenase. This compound pro- duced very small (~1 mm) zones of inhibition in uiuo on both glucose and ethanol media. This indicates either that the cells are impermeable to the inhibitor even in the presence of SDS or that the inhibitor is so insoluble in the medium (owing to the naphthylmercapto side chain) that it does not diffuse. It also indicates that a process required for growth on glucose is blocked.

The wide range of compounds employed in the present work permits some conclu- sions to be drawn about the influence of structure on the inhibitory effects of the compounds. Those which strongly inhibit electron transfer in the cytochrome b-cl region have in common two fused aromatic rings, a hydroxyl group ortho to one of the quinone oxygens, and a long aliphatic side chain adjacent to the other oxygen. The most potent inhibitors, compounds 1 and 2, have a quinolinequinone and a benzothia- zole nucleus, respectively, and the change to a naphthalene nucleus (compound 4) slightly decreased the inhibitory effect in vitro and the permeation of the cells in uiuo. Since compound 1 has a very similar structure to compounds 3 and 4, and inhibits NADH oxidase as strongly as compound 2, it is likely that this compound also inhibits electron transfer between cytochromes b and cl, although this was not tested. Compound 6 is an anomaly, since its structure is similar to the above compounds, but it is a much weaker inhibitor in vitro and has no effect in uiuo. This may be due to its C15 aIky1 chain, which makes it very insoluble in aqueous media. Com- pounds 1, 3, 4, and 6 have been shown to inhibit strongly NADH and succinate oxidase in beef heart mitochondria (23, 24).

Further modifications of the ring system result in weaker inhibition of NADH oxidase. For example, compound 14, in which the position of the nitrogen atom in the quinoline nucleus is altered relative to the hydroxyl substituent, and compound 8, in which the ring containing the nitrogen atom is saturated, inhibit NADH oxidase only at concentrations lOOO-fold higher than compound 1. The replacement of this ring by two methoxy groups (in 5 and 7) produces an 80- to lOO-fold decrease in ef- fectiveness. A similar difference between compound 7 and the related naphthoquinone was reported by Phelps and Crane (12).

The replacement of the ortho-hydroxyl substituent on the quinone ring by H, Cl, or CH3 causes the loss of most or all of the inhibitory effect (compare compounds 16, 17, and 18 with 5; 19 and 20 with 4; and 21 with 3). Modifications of the substituent adjacent to the other quinone oxygen atom have much more diverse effects. The introduction of a thioether link between the alkyl chain and the nucleus (in 1 and 4), or the addition of a cyclohexane ring to the end of the alkyl chain (in 3) apparently has little effect. The introduction of unsaturated linkages in the alkyl chain reduces the inhibitory effects markedly (compare 11 and 24 with 5). Castelli et al. (14) also found that 2,3-dimethoxy-5-hydroxy-1,4- benzoquinones with unsaturated side chains were required in relatively high quantities to inhibit succinate oxidase. Other reports have indicated that 2-hydroxy-1,4-naphthoquinones with shorter side chains in the 3 position were less effective inhibitors than those with eight or more carbons (9,25). Side chains with sub- stituted amino groups have a variable influence. Compounds 12,13, and 15, which lack the essential hydroxyl substituent, but contain alkylamino side chains, are more effective than the corresponding compounds 18, 20, and 22, which contain alkyhnercapto side chains. However, the remaining alkylamino compounds have no effect on NADH oxidase.

6 - p- Naphthyhnercapto - 5 - chloro - 2,3 - dimethoxy-1,4-benzoquinone (compound 10) is apparently a specific inhibitor of the succinate-CoQ reductase complex, for

314 ROBERTS ET AL.

which a 5-hydroxyl substituent is evidently not required. Several other halogenated benzoquinones have been shown to act at this site (26) which has a different structural specificity from that of the site in the cytochrome b-cl region reported here. Pre- vious reports have implied a site for CoQ in succinate dehydrogenase (27, 28).

The results of this study indicate that the three compounds, 2,3, and 4, with the possible addition of compound 1, are likely to be useful in investigations of the cytochrome b-cl complex, since they block electron transfer between cytochromes b and cl. If these compounds act as antagonists of coenzyme Q, the data strongly suggest that coenzyme Q is involved in electron transfer from cytochrome b to cytochrome cl, as well as from substrates to cytochrome b. An analogous suggestion was made recently by Downie and Cox (29) for the electron transfer system of Escherichia coli.

Wikstrom and Berden (30) proposed that the CoQ(H)/CoQ couple passes electrons to cytochrome cl, while the CoQ(Hz)/ Co&(H) couple reduces cytochrome cl via cytochrome b in a parallel pathway. This hypothesis was extended by Mitchell (31, 32) as a “protonmotive Q cycle,” in which coenzyme Q is oxidized and reduced at two Q-reactive centers denoted by o and i, respectively (see Fig. 2 of reference 32). At center o, one of the two electrons from coenzyme Q(H2) is transferred to cytochrome cl, and the other is transferred to cytochrome(s) b. The oxidized coenzyme Q is proposed to migrate to center i, where it accepts electrons from cytochrome(s) b and from the dehydrogenases. The reduced coenzyme Q then carries two protons and two electrons back to center o. This model is consistent with the observed inhibition of the oxidation of cytochrome b by coenzyme Q analogues if it is proposed that the analogues inhibit the oxidation of cytochrome b by coenzyme Q at center i. Alter- natively, coenzyme Q analogues might inhibit the reduction of cytochrome cl at the Q-reactive center o, provided that they do not block the reduction of cytochrome b at this center. It is possible that coenzyme Q analogues act at both of these centers, as Mitchell (32) suggested for 2-n-heptyl-4-hy- droxyquinoline-N-oxide. A distinction be-

tween these alternatives may be possible after an examination of the effects of coenzyme Q analogues on the oxidant-induced reduction of cytochrome b in the presence of antimycin, a phenomenon which is thought to be mediated by the Q-reactive center o (32).

APPENDIX

The binding of a ligand (inhibitor), L, to a site, E, on a macromolecule,

L+E=EL

is described by a simple dissociation con- stant

KD = [EI[Ll [ELI

if the binding sites are homogeneous and independent. The total concentration of the ligand is given by

[L]” = [L] + [EL]. PI By analogy with Scatchard’s analysis (22), Equations [l] and [2] give

[ELI 1 [ELI -=--- [Ll”[El KD KD[L]“’

131

If it is assumed that the activity is propor- tional to the concentration of free sites, it follows that the percentage of inhibition corresponds to the fraction of ligand sites which are occupied, provided that inhibition is complete at high concentrations of inhibitor when all binding sites are occupied. Therefore,

[EL] u” -u -=- [El u

[41

where u” and u are the activities in the absence and presence of the inhibitor, respectively, at a given protein concentration. Furthermore, if the total number of binding sites per unit mass of protein is n, then the concentration of occupied binding sites is related to the concentration of protein which is inactive, Ci, by

[EL] = nCi. [51

Substituting Equations [4] and [5] into

INHIBITION OF ELECTRON TRANE

Equation [3],

u”-u 1 TlCi -=--- [Ll”u KD KD[L]“’

[61

0 C, Thus a plot of yLi: against E

is analogous to a Scatchard p&-and has a gradient of -n/KD and an intercept on the abscissa of l/n.

REFERENCES

1. GROOT OBBINK, D. J., HALL, R. M., LINNANE, A. W., LUKINS, H. B., MONK, B. C., SPITHILL, T.

W., AND TREMBATH, M. K. (1976) in The Ge- netic Function of Mitochondrial DNA (Saccone,

C. and Kroon, A. M., eds.), pp. 163-173, Elsevier, Amsterdam.

2. BURGER, G., LANG, B., BANDLOW, W., SCHWEYEN,

R. J., BACKHAUS, B., AND KAUDEWITZ, F. (1976) Biochem. Biophys. Res. Commun. 72, 1201-

1208. 3. SC’BIK, J., KOVACOVA, V., AND TAKACSOVA, G.

(1977) Eur. J. Biochem. 73,275-286. 4. PRATJE, E., AND MICHAELIS, G. (1977) Mol. Gen.

Genet. 152, 167-174. 5. LINNANE, A. W., AND HALL, R. M. (1978) in

Molecular Biology of Mitochondrial Mem-

branes (Tzagoloff, A., ed.), pp. 321-335, Plenum Press, New York.

6. COBON, G. S., GROOT OBBINK, D. J., HALL, R. M.,

MAXWELL, R., MURPHY, M., RYTKA, J., AND LINNANE, A. W. (1976) in Genetics and Biogen-

esis of Chloroplests and Mitochondria (Biicher, T., Neupert, W., SebaId, W. and Werner, S.,

eds.), pp. 453-460, Elsevier, Amsterdam. 7. TZAGOLOFF, A., FOURY, F., AND AKAI, A.(1976)

Mol. Gen. Genet. 149,33-42.

8. KOTYLAK, Z., AND SLONIMSKI, P. P. (1976) in The Genetic Function of Mitochondrial DNA (Sac- cone, C., end Kroon, A. M., eds.), pp. 143-154,

Elsevier, Amsterdam. 9. BALL, E. G., ANFINSEN, C. B., AND COOPER, 0.

(1947) J. Biol. Chem. 168,257-270. 10. ESTABROOK, R. W. (1958) J. Biol. Chem. 230,

735-750.

11. TAKEMORI, S., AND KING, T. E. (1964) J. Biol. Chem. 239, 3546-3558.

iFER BY COENZYME Q ANALOGUES 315

12. PHELPS, D. C., AND CRANE, F. L. (1975) Biochem- istry 14, 116-122.

13. CATLIN, J. C., PARDINI, R. S., DAVES, G. D., HEIDKER, J. C., AND FOLKERS, K. (1968) J. Amer. Chem. Sot. 90, 3572-3574.

14. CASTELLI, A., BERTOLI, E., LI’ITARRU, G. P., LENAZ, G., AND FOLKERS, K. (1971) Biochem. Biophys. Res. Commun. 42,806-812.

15. PORTER, T. H., AND FOLKERS, K. (1974) Angelu. Chem. 86.635-645.

16. WALLACE, P. G., HUANG, M., AND LINNANE, A. W. (1968) J. Cell Biol. 37,207-220.

17. GROOT OBBINK, D. J., SPITHILL, T. W., MAXWELL,

R. J., AND LINNANE, A. W. (1977) Mol. Gen. Genet. X1,127-136.

18. SANADI, D. R., PHARO, R. L., AND SORDAHL, L. A. (1967) in Methods in Enzymology (Estabrook,

R. W., end P&man, M. E., eds.), Vol. 10, pp. 297-302, Academic Press, New York.

19. ZIEGLER, D., AND RIESKE, J. S. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 231-235, Academic

Press, New York.

20. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.

21. BERDEN, J. A., AND SLATER, E. C. (1970) Biochim. Biophys. Acta 216,237-249.

22. SCATCHARD, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672.

23. PORTER, T. H., BOWMAN, C. M., AND FOLKERS, K. (1973) J. Med. Chem. 16,115-118.

24. BOWMAN, C. M., SKELTON, F. S., PORTER, T. H., AND FOLKERS, K. (1973) J. Med. Chem. 16, 206-209.

25. HOWLAND, J. L. (1965) Biochim. Biophys. Acta 105,205-213.

26. HOUGHTON, R. L., FISHER, R. J., AND SANADI, D. R. (1976) FEBS Lett. 68.95-98.

27. DE KOK, J. AND SLATER, E. C. (1975) Biochim. Biophys. Acta 376.27-41.

28. ROSSI, E., NORLING, B., PERSSON, B., AND ERNS- TER, L. (1970) Eur. J. Biochem. 16,508-513.

29. DOWNIE, J. A., AND Cox, G. B. (1978) J. Bacterial. 133.477-484.

30. WIKSTR~~M, M. K. F., AND BERDEN, J. A. (1972) Biochim. Biophys. Actu 283,403-420.

31. MITCHELL, P. (1975) FEBS Lett. 59, 137-139.

32. MITCHELL, P. (1976) J. Theor. Biol. 62, 327-367.

The site of inhibition of mitochondrial electron transfer by coenzyme Q analogues

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