Proc. Nat. Acad. Sci. USA Vol. 69, No. 12, pp. 3713-3717, December 1972

Synthesis of Plastoquinone Analogs and Inhibition of Photosynthetic and Mammalian Enzyme Systems* (chloroplasts/beef heart/coenzyme Q/electron transport) JAN BQLERt, RONALD PARDINIt, HANNA T. M\IUSTAFAt, KARL FOLKERSt, RICHARD A. DILLEYT, AND FREDERICK L. CRANE: t Stanford Research Institute, Menlo Park, California and Institute for Biomedical Research, The University of Texas at Austin, Texas 78712; and $ Purdue University, Lafayette, Indiana 47907 Contributed by Karl Folkers, September 25, 1972

ABSTRACT New 5-hydroxy- and 5-chloro-6-alkyl-1,4- energy (ATP) and the utilization of the hydrogen of water as benzoquinones with one or two methyl groups on the a source of reducing power (NADPH2). Data from nucleus were synthesized as potential antimetabolites of several plastoquinones for biological research on photosynthetic experimental approaches indicate that plastoquinone does and mammalian enzyme systems; the primary emphasis function in the electron transport system of chloroplasts. was on photosynthesis. The data stem from extraction-restoration studies, spectro- 2,3-Dimethyl-5 - hydroxy - 6 - phytyl - 1,4 - benzoquinone photometric and chemical determination of redox changes, completely inhibited in chloroplasts the water-de- pendent electron transport, but photosystem I was in- correlation with metabolic function, and changes in electron sensitive to this analog. The data are consistent with the transport in mutants, as well as its universal distribution in all interpretation that this analog inhibits electron transport oxygen-producing photosynthetic organisms (3). in the chain prior to the site of electron donation from the To study relationships between structure and activity in ascorbate-dichlorophenolindophenol couple. Concentra- respiration, Redfearn and Whittaker (4) evaluated the tions of 70 MM and 120 ,M of this analog caused about 50 and 100% inhibition, respectively, of cyclic photophos- inhibition of succinoxidase from a heart muscle preparation by phorylation. both coenzyme Q and l)lastoquinone analogs. They found that 2,3-Dimethyl-5-hydroxy-6-phytyl-1 ,4-benzoquinone is a inhibitory quinones have at least one unsubstituted position new type of inhibitor of photosynthetic electron transport in the nucleus, that the nature of the substituent ortho to the that specifically inhibits the rate-limiting step between photosystems I and II. Structurally related analogs caused free position was important, and that the inhibitory effect inhibitions in the range of 50-100% in chloroplasts. disappeared in the presence of sulfhydryl compounds. Qui- These analogs showed marginal inhibition in mito- nones with one unsubstituted position in the nucleus are known chondrial coenzyme Qjo-oxidase systems from beef heart. to react by a 1,4-addition with molecules containing a thiol group. This reaction might be involved in the mechanism of Plastoquinone-9 (Plastoquinone-9,I), a compound found inhibition by the compounds studied by these investigators. 0 Catlin et al. (5) showed in studies of new inhibitors of co- enzyme Q enzyme systems that some of the analogs CH-3 H 5-hydroxy of coenzyme Q strongly inhibit succinoxidase and DPNH- Il Il CH:l oxidase in intact mitochondria or in mitochondria extracted CHEW (CH2CH= C-CH.,)9H to remove coenzyme Qio. Specifically, 2,3-dimethoxy-5- hydroxy-6-phytyl-1,4-benzoquinone completely inhibited suc- 0 I cinoxidase activity, and reduced by 80% DPNH-oxidase activity in the absence of supplementary coenzyme Qjo in CH30 CH3 intact enzyme preparations. These data of Catlin et al. (5) predict that similar analogs Il Il CH3 of plastoquinone might inhibit photosynthetic enzyme CH3O (CH29CH C-CH,)nH systems; consequently, synthesis and testing of such analogs of plastoquinone were undertaken. II The effort was focused on two main groups of potential predominately in the chloroplasts of higher plants, is a plastoquinone antagonists, namely, hydroxy (IIIa-e) and naturally occurring benzoquinone with an isoprenoid side chloro (IVa-c) analogs. The starting quinones without the chain. Its structure was determined (1), and it was synthesized hydroxy or chloro functions were known compounds. They (2). It was proposed that it has an important role in the elec- were converted to the corresponding new hydroxy- and tron transport system of photosynthesis that is analogous to chloroquinones by use of the Thiele-Winter acetylation that of coenzyme Q (II) in mitochondria (3) by acting as an procedure (6). In most cases, boron trifluoride etherate was oxidation-reduction carrier in the electron transport system used inl place of the more conventional sulfuric acid as the associated with the transformation of light into chemical acid catalyst; good yields of the triacetates were obtained. These acetates were hydrolyzed in refluxing acidic methanol * This is paper no. CLVI in the series, "Coenzyme Q." under nitrogen to the corresponding hydroxybeuizohydro- 3713 Downloaded by guest on October 2, 2021 3714 Chemistry: Boler et al. Proc. Nat. Acad. Sci. USA 69 (1972)

TABLE 1. Hydroxy- and chloroalkylquinones none. The decomposition produced free radicals, which reacted with the quinone (9). Generally, the hydroxyquinones were formed in low yields and were extremely unstable and difficult to purify. They partly decomposed on thin-layer plates, but the chloro com- pounds were formed in fairly good yields and were relatively stable. The structures of the products were assigned by their R2 R3 R5 R6 nuclear magnetic resonance (NMR) spectra (Table 2). For R6 = phytyl, the sidechain protons that were distinguished III a CH3 CH3 OH Phytyl were of the ring methylene (-6.9r) group, and the vinylic b OH CH3 CH3 Phytyl (-5.OT) and the protons of the methyl group at the double c OH Phytyl CH3 CH3 bond (-8.3T). For R6 = 8'-cyclohexyloctyl, the ring methyl- d H CH3 OH Phytyl enes were found at -7.66Tr remaining e CH3 CH3 OH 8'-Cyclohexyloctyl and the sidechain protons had two broad absorptions between 8.3r and 8.8T. IV a CH3 CH3 Cl Phytyl b Cl CH3 CH3 Phytyl EXPERIMENTAL c CH3 Cl CH3 Phytyl 2,3-Dimethyl-1 ,4,5-triacetoxybenzene. To a solution of o-xylo- quinone (5 g, 37 mmol) in acetic anhydride (20 ml), was added redistilled boron trifluoride- etherate (1 ml). The quinones, which were characterized by oxidation with silver mixture was stirred at room temperature for 24 hr and poured oxide to the corresponding hydroxyquinones. The chloro- into ice water; the crude triacetate was collected by filtra- quinones were prepared by a 1,4-addition of hydrogen chloride tion. Recrystallization from ether-hexane gave 7.2 g (70%) to the starting quinone to yield the chlorohydroquinone, of pure product, mp 87°-88° (mp 87°-88°, ref. 10). which was oxidized by dichromate in sulfuric acid. The following triacetates were prepared in a similar man- The alkyl quinones (III and IV, Table 1) were prepared ner in good yields: 2,5-dimethyl-1,3,4-triacetoxybenzene, from the hydroquinones or quinones by two alkylation mp 1070 (mp 106°-107°, ref. 10; 1080, ref. 11); 2,6-dimethyl- procedures. The phytyl derivatives were prepared by the 1,3,4-triacetoxybenzene, mp 102°-103° (mp 103°-104°, ref. conditions of acid-catalyzed alkylation of Daves et al. (7) of 12), sulfuric acid was used as the acid catalyst; and 2-methyl- the appropriate hydroquinone with an allylic alcohol (phytol). 1,4,5-triacetoxybenzene, mp 112°-114° (mp 114°-115°, ref. 6). The resulting phytyl hydroxyhydroquinones become air- oxidized during purification by thin-layer chromatography. 2,3-Dimethyl-1,4,5-trihydroxybenzene. To a solution of 2,3- The phytyl chlorohydroxyquinones are relatively stable, and dimethyl-1,4,5-triacetoxybenzene (5 g, 18 mmol) in methanol were treated with an oxidizing agent to obtain the quinones (30 ml), was added hydrochloric acid (5 ml). The mixture was before final purification by thin-layer chromatography. refluxed in an atmosphere of nitrogen. The hydrolysis was The 8'-cyclohexyloctylbenzoquinone (IIIe) was synthesized followed by thin-layer chromatography and was stopped by thermal decomposition of the appropriate diacyl peroxide when no more triacetate was detected in the reaction mixture (8) in the presence of 2,3-dimethyl-5-hydroxy-1,4-benzoqui- (after about 1 hr). The cold solution was then evaporated

TABLE 2. Spectral data for analogs of plastoquinone

NMRt

Compound xEtOH CH3 (1,4-benzoquinone) No. nm Ring-CH3 Ring- H Ring-CH2 Viny 1 R Alkyl 2,3-Dimethyl-5-hydroxy-6- IIa 276 8.05 (s) 6.96 (d) 4.98 (t) 8.32 (s) 8.5-9.3 (m) phytyl- 2,.5-Dimethyl-3-hydroxy-6- IIIb 276 8.05 (s) - 6.86 (d) - 8.32 (s) 8.6-9.3 (m) phytyl- 271 (sh) 8.15 (s) 2,6-Dimethyl-3-hydroxy-5- IIc 276 7.98 (s) 6.85 (d) 5.09 (t) 8.30 (s) 8.4-9.2 (m) phytyl- 8.12 (s) 2-Methyl-5-hydroxy-6-phytyl- IIId - 7.98 (d) 3.57 (q) 6.97 (d) 5.03 (t) 8.34 (s) 8.6-9.3 (m) 2,3-Dimethyl-5-hydroxy-6-(8'- IIMe 276 8.02 (s) - 7.66 (t) 8.2-9.0 (m) cyclohexyloctyl)- 269 (sh) 8.07 (s) 2,3-Dimethyl-5-chloro-6-phytyl- IVa 272 7.95 (s) - 6.73 (d) 5.02 (t) 8.29 (s) 8.6-9.3 (i) 265 (sh) 8.00 (s) 2,5-Dimethyl-3-chloro-6-phytyl- IVb 272 7.85 (s) 6.85 (d) 5.16 (t) 8.29 (s) 8.5-9.3 (m) 263 (sh) 7.94 (s) 2,6-Dimethyl-3-chloro-5-phytyl- IVc 272 7.85 (s) - 6.81 (d) 5.12 (t) 8.27 (s) 8.5-9.2 (i) 263 (sh) 7.97 (s)

* sh = shoulder. t Spectra were obtained on carbon tetrachloride solutions with a Varian Associates HR-100 spectrometer. Values are in r units. The letters in parentheses refer to peak shape: s = singlet, d = doublet, t = triplet, m = multiplet, -q = quartet. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Synthesis of Plastoquinone Analogs 3715

under reduced pressure, and the crude product was dried 0 100, briefly under reduced pressure. z til H2O- NADP 1 The following trihydroxybenzenes were prepared in a 0° 80 similar manner: 2,5-dimethyl-1,3,4-trihydroxybenzene; 2,6- o 60 dimethyl-1,3,4-trihydroxybenzene; and 2-methyl-1,4,5-tri- z 40 hydroxybenzene. Because of the instability of the trihydroxy- u 20 V~~~~~~~~'~0 benzenes, the products were used immediately in the sub- Ito ,--lo sequent alkylation reactions. Inhibitor4onc (M) Inhibitor Conc (M) 2,3-Dimethyl-5-chloro-1 ,4-benzoquinone. To chilled con- centrated hydrochloric acid (10 ml), o-xyloquinone (1.34 g, FIG. 1. Inhibition of NADP reduction by IIIa in spinach 10 was added. The mixture was stirred over- chloroplasts. The assay consisted of: 70 mM KCl, 3 mM MgCl2, mmol) reaction 17 mM tricine-KOH (pH 8.0), 1 mM NaDP, saturating amounts night at room temperature and then poured into ice water. of ferrodoxin, and 33 ug of chlorophyll per ml. 5 mM NH4C1 The product was extracted with chloroform. The extract, was added to obtain maximal rates of electron transport (i.e., containing 2,3-dimethyl-5-chloro-1,4-benzohydroquinone, was uncoupled electron flow). The control rate of NADP reduction oxidized with potassium dichromate-sulfuric acid. The was 204 umol of electron equivalents per hr per mg chlorophyll. product was extracted with several portions of chloroform. Electron transport was assayed by following 02 evolution with The yield of purified chloroquinone was 1.4 g (82%), mp 700. a Clark-type 02 electrode. In a similar manner, 2,5-dimethyl-3-chloro-1,4-benzoquinone, mp 46°-47° (mp 480, ref. 13); 2,6-dimethyl-3-chloro-1,4- benzoquinone, mp 56° (mp 55.5°-57°, ref. 14) were prepared. and photophosphorylation in isolated spinach chloroplasts. The chloroquinones were dissolved in ether and reduced to The isolation of chloroplasts and techniques for assaying the corresponding chlorohydroquinones with an aqueous electron transport and photophosphorylation have been solution of sodium dithionite immediately before acid- described (15). Electron transport was measured by following catalyzed alkylation. either NADP reduction spectrophotometrically or methyl viologen reduction with a Clark-type oxygen electrode to Alkylation of the hydroxy- and chloroberizoquinones measure the autoxidation of reduced methyl viologen. Photo- was Acid-Catalyzed Alkylation. To a solution of the benzo- phosphorylation catalyzed by phenozine methosulfate. The data in Fig. 1 demonstrate that the analog I11a, at hydroquinone (10 mmol) in dry dioxane (25 ml), an equivalent about 140 inhibits NADP reduction of was added. To the well-stirred redistilled 4M, completely by phytol solution, In this NADP was boron trifluoride etherate (1.5 ml) was added dropwise; the spinach chloroplasts. assay, reduction reaction mixture was stirred for 2 hr at room temperature. The dependent upon water as the ultimate source of electrons. The current schemes for electron transport in mixture was poured into ice water (100 ml), and extracted with chloroplasts two in water several of ether. The ether solution was dried over depict photosystems that act series to oxidize portions and transfer electrons NADP Plasto- sodium sulfate and under reduced pressure. The ultimately to (16). evaporated seems to act as an transport in product was purified by thin-layer chromatography on 1-mm quinone-9 electron carrier the chain that connects photosystem II (the water-oxidizing thick silica-gel plates; 5-15% ether in hexane was used as developing solvent. The dark brown-violet band was scraped photosystem) with photosystem I (the photosystem that accepts electrons from a and eluted with ether. The alkyl hydroxyhydroquinones were photosystem II) via series of electron air-oxidized to the this The transport carriers (16). Arnon and Horton (17) showed that quinone during purification. alkyl the of NADP chlorohydroquinones were oxidized with silver oxide in ether reduction by the artificial electron donor system before such consisting of ascorbate and dichlorophenolindophenol occurs purification by thin-layer chromatography. in Quinones IIIa-d and IVa-c (Table 1) were prepared in chloroplasts from which plastoquinone-9 has been com- the described manner. pletely extracted with organic solvents; the water-dependent NADP reduction is reduced in such extracted chloroplasts and Radical Alkylation. The diacyl peroxide of 8'-cyclohexyl- can be normalized by the restoration of plastoquinone-9 to nonanoic acid was prepared by treatment of an ethereal the extracted chloroplasts. solution of the acid chloride with hydrogen peroxide and If the analog IIIa of plastoquinone inhibits NADP re- pyridine (8). To 2,3-dimethyl-5-hydroxy-1 ,4-benzoquinone duction from water by interacting close to or at the site of the (3.2 g, 20 mmol), the diacyl peroxide (10 mmol) in glacial endogenous plastoquinone-9, one might expect that the acetic acid (100 ml) was added. The mixture was heated on a ascorbate plus dichlorophenolindophenol-dependent NADP steam bath overnight. The acetic acid was removed under reduction might be insensitive to the inhibitor's activity. reduced pressure, and a hexane extract was purified by Experiments were performed to test this hypothesis. As the column chromatography on silica gel and was eluted with data in Table 3 show, electron transport from the donor increasing fractions of ether in hexane. The product, 2,3- system to methyl viologen (a synthetic electron acceptor that dimethyl-5-hydroxy-6-(8'-cyclohexyloctyl)-1,4-benzoquinone, can substitute for NADP) was indeed insensitive to the was eluted with 3% ether in hexane. After evaporation of the presence of concentrations of inhibitor that completely in- solvent, it was obtained as a pure yellowish-red solid melting hibited the water-dependent electron transport (Table 3). below room temperature. These results are consistent with the hypothesis that the quinone analog lila inhibits electron transport by interacting RESULTS AND DISCUSSION with some part of the electron transport chain prior to the 2,3-Dimethyl-5-hydroxy-6-phytyl-1 ,4-benzoquinone, MIla, was site of electron donation from the ascorbate-dichlorophenol- tested for inhibitory action by its effect on electron transport indophenol couple. Downloaded by guest on October 2, 2021 3716 Chemistry: Boler et al. Proc. Nat. Acad. Sci. USA 69 (1972)

TABLE 3. Effect of 2,3-dimethyl-5-hydroxy-6-phytyl-1 ,4-benzo- TABLE 5. Plastoquinone analogs in DPNH-oxidase and quinone on electron transport and phosphorylation in succinoxidase systems, intact mitochondrial systems spinach chloroplasts from beef heart

Electron Phospho- DPNH-oxidase Succinoxidase Assay transport* rylationt Spec. Spec. Control ascorbate + DPIP _ Addition* act.t % act.t % methyl viologen 2040 Control + IIIa 2040 None 0.251 57 0.419 72 120pyM 100 100 Control H20 -- methyl viologen 560 Qio 0.436 0.576 Control + 40 pM Illa 270 Q'o + MIIa 0.344 78 0.496 86 Control + 120 /M lIla 0 Qia + IIIb 0.422 96 0.580 100 Control pheiazine methosulfate 346 Qjo + IIId 0.454 104 0.510 88 Control + 70MuM IIIa 170 Qio + IVa 0.440 100 0.529 91 Control 120 MM IIIa 0 Qjo + IVb 0.484 111 0.562 97

The assays for electron transport consisted of the following: When enzyme is extracted, any residual respiration is assumed to to unextracted coenzyme The blank is subtracted H20 -` methyl viologen; 100 mM KC1, 5 mM MgC12, 0.8 mM be due Qio. ADP, 3 mM K2HPO4, 50 nmM tricine-KOH at pH 8.5, 20,g of from each value obtained. The assays were conducted according chlorophyll per ml) 0.4 mM methyl viologen, and 0.5 mM sodium to Arntzen et al. (19) and Jeng et al. (24). azide. 6 mM ascorbate plus 0.6 mM dichlorophenolindophenol * In each case, 100 nmol of the quinone were added. (DPIP) were used as an alternative source of reducing power in t Microatoms of oxygen per minute per milligram of protein. some experiments. A Clark-type O2 electrode was used to measure electron transport. were also inhibited by the analog lIIa The phosphorylation assays were as follows. 100 mM KCl, 5 cyanide reduction, mM MgCl2, 0.8 mM ADP, 3 mM K2HPO4, 0.03 mM phenazine (19). methosulfate, and 20,Mg chlorophyll per ml at pH 8.5. In Table 4 are tabulated the inhibitory effects of additional The analog was dissolved in methanol; the equivalent addition plastoquinone analogs: 2-methyl-5-hydroxy-6-phytyl-1,4-ben- of pure methanol had no inhibitory effect. zoquinone (hIId), 2,3-dimethyl-5-chloro-6-phytyl-1,4-benzo- * Mmol of methyl viologen reduced per mg of chlorophyll per hr. quinone (IVa), and 2,5-dimethyl-3-chloro-6-phytyl-1,4-benzo- t ymol of ATP formed per mg of chlorophyll per hr. quinone (IVb). These three analogs showed inhibitions of 50-100%. These results are consistent with the interpretation that the dimethylhydroxy analog of plastoquinone inhibits electron This dimethylhydroxy analog IlIa is also a potent in- transport and photophosphorylation in chloroplasts by hibitor of cyclic photophosphorylation (Table 3). The data interacting at the sites that normally function with the indicate that 70 MM lila caused about 50% inhibition, and endogenous plastoquinone-9. Presumably, the analog must 120 MM MIIa completely inhibited cyclic photophosphoryla- inhibit either the reduction or the oxidation of plastoquinone-9. tion. The inhibitor II1a has been previously studied at two These results are also consistent with the study by Bohme sites of ATP formation in subchloroplast particles (18). and Cramer (20) that showed inhibition at plastoquinone Reactions in the photosystem II system, such as ferri- sites by 2,5-dibromo-3-tnethyl-6-isopropyl-1,4-benzoquinone (21) except that IIIa seems to be a better inhibitor. Representatives of these plastoquinone analogs have also been tested in succinoxidase and DPNH-oxidase systems of mammalian mitochondria (22), and corresponding systems TABLE 4. Inhibitory effects of plastoquinone analogs in photosynthesis TABLE 6. Plastoquinone analogs in DPNH-oxidase and Amount % succinoxidase systems, extracted mitochondrial systems used Inhibi- from beef heart Compound No. (mg) Acceptor tion 2-Methyl-5-hydroxy- IIId 0.3 Indophenol 80 DPNH-oxidase Succinoxidase 6-phytyl-1,4-ben- 0.5 Indophenol 100 Spec. Spec. zoquinone 0.3 Methyl 50 Addition* act.t % act.t % viologen 2,3-Dimethyl-5- IVa 0.6 Methyl 80 None 0.056 0 0.112 0 chloro-6-phytyl- viologen Qio 0.160 100 0.332 100 1,4-benzoquinone Qo + IIIa 0.133 74 0.328 98 2,5-Dimethyl-3- IVb 0.6 Methyl 50 Qjo + IIIb 0.266 201 0.352 109 chloro-6-phytyl- viologen Qjo + IIId 0.276 211 0.286 79 1,4-benzoquinone Qio + IVa 0.297 231 0.338 102 Qio + IVb 0.292 226 0.342 104 All assays were with chloroplasts using 60 Mg of chlorophyll. The experimental conditions were similar to those described for Refer to footnotes of Table 5. Extraction for removal of co- Table 3. enzyme Qjo was by the method of Szarkowska (23). Downloaded by guest on October 2, 2021 Proc. Nat. Aca. Sci. USA 69 (1972) Synthesis of Plastoquinone Analogs 3717

that were extracted for removal of endogenous coenzyme Qio 3. Crane, F. L. & Henniger, M. D. (1966) Vitam. Horm. 24, (23) (Tables 5 and 6). None of the compounds tested showed 489-517. 4. Redfearn, E. R. & Whittaker, P. A. (1962) Biochim. Bio- marked inhibition in these systems at the levels tested. phys. Acta 56, 440-444. However, in the DPNH-oxidase system, analog lIla, 2,3- 5. Catlin, J. C., Pardini, R. S., Daves, G. D., Jr., Heidker, J. dimethyl-5-hydroxy-6-phytyl-1,4-benzoquinone, showed some C. & Folkers, K. (1968) J. Amer. Chem. Soc. 90, 3572-3574. inhibition in both intact and extracted mitochondria. Notably, 6. Thiele, J. & Winter, E. (1900) Annaler Der Chemie 311, 341-362. analogs IMIb, MId, IVa, and IVb exhibited stimulation in the 7. Daves, G. D., Jr., Moore, H. W., Schwab, D. E., Olsen, extracted mitochondrial DPNH-oxidase system from beef R. K., Wilczynski J. J. & Folkers, K. (1967) J. Org. Chem. heart. 32, 1414-1417. A similar stimulation by 6-phytyl- and 6-farnesyl-2,3- 8. Silbert, L. S. & Swern, D. (1959) J. Amer. Chem. Soc. 81, dimethoxy-5-hydroxy-1,4-benzoquinones has been observed 2364-2367. 9. Pettit, G. R., Fleming, W. C. & Paull, K. D. (1968) J. Org. for the DPNH-oxidase system in yeast mitochondria from Chem. 33, 1089-1092. which coenzyme Q6 was extracted (22). The significance of the 10. Fieser, L. F. & Ardao, M. I. (1956) J. Amer. Chem. Soc. 78, higher specific activities in the presence of the analogs IMIb, 774-781. hId, IVa, and IVb in respect to the control activity is being 11. Asahina, Y. & Ishibashi, E. (1929) Ber. Deut. Chem. Gesell- further studied. the to a shaft 62, 1207-1208. Possibly, analogs that appear elicit 12. Erdtman, H. (1932) Sv. Kem. Tidskr. 44, 135-148 [from stimulation are substituting for coenzyme Qio or are acting in Chem. Abstr. 26, 4803 (1932)]. a synergistic manner with it. In succinoxidase, both lila and 13. Carstanjen, E. (1881) J. Prakt. Chem. (2), 23, 421-435. MId (2-methyl-5-hydroxy6-phytyl-1,4-benzoquinone) in- 14. Smith, L. I. & Irwin, W. B. (1941) J. Amer. Chem. Soc. 63, hibited coenzyme Qjo to some extent in the intact system, but 1036-1043. 15. Dilley, R. A. (1970) Arch. Biochem. Biophys. 137, 270-283. only IIId inhibited the extracted mitochondrial oxidase. 16. Boardman, N. K. (1968) Advan. Enzymol. 30, 1-79. 17. Arnon, D. I. & Horton, A. A. (1963) Acta Chim. Scand. 17, We thank the Merck, Sharp and Dohme Research Laborato- Suppl. 1, 135-139. ries, Rahway, N.J., the Nutrition Foundation, Inc., and the Nor- 18. Neumann, J., Arntzen, C. J. & Dilley, R. A. (171) Bio- wegian Research Council for their partial support of this research. chemistry 10, 866-873. 19. Arntzen, C. J., Neumann, J. & Dilley, R. A. (1971) Bio- 1. Trenner, N. R., Arison, B. H., Erickson, R. E., Schunk, C. energetics 2, 73-83. H., Wolfe, D. E. & Folkers, K. (1959) J. Amer. Chem. Soc. 20. Bohme, H. & Cramer, W. A. (1971) FEBS Lett. 15, 349-351. 81, 2026-2027; Kofler, M., Langemann, A., Ruegg, R., 21. Trebst, A., Harth, E. & Draber, W. (1970) Zeitschrift ftur Chopard-dit-Jean, L. H., Rayround, A. & Isler, 0. (1959) Naturforschung., 25b, 1157-1159. Helv. Chim. Acta 42, 1283-1292. 22. Castelli, A., Bertoli, E., Littarru, G. P., Lenaz, G. & Folkers, 2. Kofler, M., Langemann, A., Ruegg, R., Gloor, U., Schwieter, K. (1971) Biochem. Biophys. Res. Commun. 42, 806-812. U., Wursch, J., Wiss, 0. & Isler, 0. (1959) Helv. Chim. Acta 23. Szarkowska, L. (1966) Arch. Biochem. Biophys. 113, 519-525. 42, 2252-2254; Shunk, C. H., Erickson, R. E., Wong, E. L. 24. Jeng, M., Hall, C., Crane, F. L., Takahashi, N., Tamura, & Folkers, K. (1959) J. Amer. Chem. Soc. 81, 5000. S. & Folkers, K. (1968) Biochemistry 7, 1311-1322. Downloaded by guest on October 2, 2021