Differential Effects of Antimycin on Ubisemiquinone Bound in Different Environments in Isolated Succinate Cytochrome c Reductase Complex*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 8, Issue of April 25. pp , 1980 Printed in U.S.A. Differential Effects of Antimycin on Ubisemiquinone Bound in Different Environments in Isolated Succinate Cytochrome c Reductase Complex* (Received for publication, October 12, 1979) Tomoko Ohnishi and Bernard L. rumpower$ From the Department of Biochemistry and Biophysics, University of Pehzsyluania, Philadelphia, Pennsylvania 19104, and the Department of Biochemistry, Dartmouth Medical School, Hanouer, New Hampshire Isolated succinate cytochrome c reductase complex from bovine heart mitochondria manifests a prominent g = 2.00 EPR signal attributable to ubisemiquinone. The ubisemiquinone signal can be formed by reduction of the complex with mixtures of succinate plus fumarate or by potentiometric titration in the presence of mediator dyes. The ubisemiquinone appears to be heterogeneous, consisting of at least two populations of stable semiquinone which can be distinguished on the basis of their different thermodynamic and EPR characteristics and their differential response to antimycin. The two different populations of ubisemiquinone contributing to the g = 2.00 EPR signal were selectively measured at two different levels of microwave power, one at 10 pw and the other at 100 mw at 50 K. At the high microwave power, addition of antimycin caused an increase in intensity of the g signal and a shift of the potentiometric titration curve toward a more positive potential. This antimycin-enhanced, rapidly relaxing signal is attributable to the stable ubisemiquinone pair which was reported to be proximal to ironsulfur center 5-3 of succinate dehydrogenase. In contrast, addition of antimycin caused the disappearance of the g = 2.00 signal which is selectively observed at the low microwave power. his differential response to antimycin of the g = 2.00 EPR signal is attributed to ubisemiquinone bound in two different environments, which stabilize the semiquinone to different extents. In addition to the ubisemiquinone in proximity to succinate dehydrogenase, it is proposed that a second site exists for binding of ubi- semiquinone, which gives rise to a more slowly relaxing EPR signal which is abolished by antimycin, and that this site is identical with or closely related to the antimycin binding site in the cytochrome b-cl segment. The values of p& and ~ KR of approximately 6.4 and 8.0, respectively, have been estimated for the ubiquinone species located in the cytochrome b-cl region of the respiratory chain. Thus this species of ubisemiquinone functions mostly in the anionic form in the physiological ph range, similar to the ubisemiquinones associated with succinate dehydrogenase. Prior to 1975 ubiquinone was generally viewed as a mobile carrier of the respiratory chain which shuttles reducing equiv- * This investigation was supported by National Institutes of Health Research Grants GM and GM and National Science Foundation Research Grant PCM The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Established Investigator of the American Heart Association dents from the dehydrogenase complexes to the cytochrome b-cl complex (1). Kroger and Klingenberg (2, 3) tested the mobile carrier function of ubiquinone by measuring the kinetics of ubiquinone oxidation and reduction and the oxidationreduction poise of ubiquinone in respiring mitochondria. These experiments indicated that approximately 85 to 90% of the total ubiquinone responds as a homogenous pool and that the oxidation-reduction behavior of ubiquinone is determined by the activities of a single donor and a single acceptor. As a result of the latter finding it was postulated that reduction of ubiquinone to ubiquinol occurs by a donor-catalyzed reduction of quinone to semiquinone which is then converted to ubiquinol (plus ubiquinone) by a noncatalyzed dismutation (2, 3). Likewise, it was postulated that oxidation of ubisemiquinone occurs by a noncatalyzed dismutation. In support of the view that oxidation and reduction of ubisemiquinone occur without the participation of acceptors and donors for ubisemiquinone Kroger has argued that the stability constant for durosemiquinone in aqueous solutions is so low that if this value is applied to ubisemiquinone in situ the molar ratio of ubisemiquinone to other oxidation-reduction components, such as succinate dehydrogenase, would be less than W3:l, which would make it unlikely that ubisemiquinone would be effective as a donor or acceptor of reducing equivalents (4). Although Backstrom and coworkers (5) first showed that EPR-detectable levels of ubisemiquinone are formed by respiring mitochondria, the possible importance of bound ubisemiquinone has come to be considered only recently. Wiks- trom and Berden (6) proposed that the oxidation-reduction poise of the ubisemiquinone couples (Q/QH. and QH./QH2) may determine the apparent potential of the b cytochromes. Subsequently Mitchell drew attention to the possible importance of bound ubisemiquinone in his formulation of the protonmotive Q cycle in which the vectorial movement of ubiquinone and ubiquinol (or the equivalent thereof) across the membrane transports protons for energy transduction at coupling sites I1 and I11 (7). An important feature of the Q cycle mechanism is that it predicts that there must be two segregated sites for binding of ubisemiquinone at the cytoplasmic and matrix regions of the membrane. In addition, these two sites, referred to as center o and center i, respectively, must stabilize ubisemiquinone to different extents in The abbreviations used are: Q, ubiquinone; TTFA, 2-thenoyltrifluoroacetone; DBH, ubiquinone having a decyl side chain; TES, N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; QH., ubisemiquinone; QY, ubisemiquinone anion; QHz, ubiquinol; SQ,, a stable form of ubisemiquinone which appears to be localized in the ubiquinonol-cytochrome c-reductase segment; SQ,, a stable form of ubisemiquinone associated with succinate-ubiquinone reductase segment; pw, microwatts; mw, milliwatts; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

2 Effects of Antimycin on Bound Ubisemiguinone 3279 order for the ubisemiquinone couples to function effectively in a more quantitative fashion, we have undertaken a theras oxidation-reduction components in the prescribed electron modynamic analysis of the spin-coupled semiquinone signals transfer mechanism (for a review see Refs. 7 and 8). There have been several reports describing specifically bound ubisemiquinone in the succinate-cytochrome c reduc- tase segment of the respiratory chain. Extensive EPR studies have been conducted on a ubisemiquinone species in the succinate-ubiquinone reductase segment which at low temperature (12 K) gives rise to a unique paired signal arising from spin coupling of two neighboring oxidation-reduction components, one of which was shown to be ubisemiquinone (9). Thermodynamic and quantitative EPR analysis of this spin-coupled signal allowed the assignment of the other interacting component to a second ubisemiquinone having equivalent oxidation-reduction properties (10, 11). The ubisemiquinone pair in the succinate-ubiquinone re- where (22). To avoid complications arising from the use of ethanol ductase segment exhibits a very stable intermediate oxidation- (23), stock solutions of antimycin, T'TFA, and water-insoluble oxidareduction state, indicating stabilization by the surrounding tion-reduction mediator dyes were dissolved in dimethyl sulfoxide. environment. Computer simulation studies of the spectra from Preparation of Succinate. Cytochrome c Reductase Complexthe spin-coupled ubisemiquinone pair suggested an edge-to- Succinate. cytochrome c reductase complex was prepared from phosedge location and separation of 7.7 A, assuming only dipolar interaction between the two ubisemiquinone molecules (9). Using oriented multilayer preparations of beef heart mitochondria, Salerno and coworkers (12, 13) showed that the was dialyzed to remove cholate (22), the membranous complex was semiquinone pair is highly ordered in the inner mitochondrial centrifugedfor 60 min at 37,000 X g, and the supernatant was membrane with the intramolecular axis being perpendicular to the membrane plane. On the basis of identical power saturation behavior of EPR signals from the spin-coupled ubisemiquinones and iron-sulfur center S-3 of succinate dehydrogenase, Ingledew and coworkers (10) proposed that at low temperature center S-3 functions as a magnetic relaxer to at least one ubisemiquinone of the pair via spin coupling, thus implying a close proximity of center S-3 to the ubisemiquinone pair. This cross-relaxation * J. C. Salerno and T. Ohnishi, submitted for publication. and the g = 2.00 signal in isolated succinate-cytochrome c reductase complex. The purpose of this paper is to describe the different effects of antimycin on the EPR signals arising from two or more forms of ubisemiquinone, which appear to differ in the extent to which they are stabilized by their environment and in their location with respect to other oxidation-reduction components in the succinate-cytochrome c reductase segment of the respiratory chain. EXPERIMENTAL PROCEDURES Materials-Cytochrome c (type 111) and antimycin were obtained from Sigma. TTFA was obtained from Fisher. DBH, an analogue of ubiquinone-2 having a decyl side chain, was obtained by chemical synthesis and reduced to the hydroquinone form as described else- phate-washed bovine heart mitochondria by fractionation with ammonium sulfate in the presence of cholate (22). A fresh preparation of reductase complex was used for each series of EPR experiments. After the last step of the preparation, in which the reductase complex discarded. The reductase was then suspended by homogenization in 150 mm NaC1, 20 mm TES, ph 7.4, to obtain a final concentration of approximately 40 mg per ml. For the EPR experiments performed at varying ph the reductase was suspended in 50mMNaC1, 100 mm TES, previously adjusted to the desired ph. A portion of the suspended reductase was withdrawn for measurements of cytochrome content and electron transfer activities, and the remainder was maintained at 4 C while it was transported to Philadelphia, Pennsylvania. Experiments with the reductase complex were conducted within 15 h of preparation. EPR Measurements-EPR measurements were performed using also gives rise to an extraordinarily rapid relaxation of the g a Varian E-109 EPR spectrophotometer. The samples were rapidly = 2.00 signal of this semiquinone population when measured frozen in EPR tubes by immersion in a 1:5 mixture of methylcycloat higher temperatures (14). TTFA, an inhibitor of electron hexane:isopentane (81 K). For obtaining spectra the sample tempertransfer in the succinate-ubiquinone reductase segment (15) ature was controlled by a variable temperature cryostat (Air Products which prevents reoxidation of iron-sulfur center S-3 (16), and Chemicals, Inc., LTD-3-110). The temperature was monitored destabilizes the semiquinone state of the rapidly relaxing g = with an Allen-Bradley Co. carbon resistor directly below the sample for temperatures below 50 K. Higher temperature experiments (>I signal' and concomitantly causes the disappearance of K) were conducted using a JEOL liquid nitrogen flow system, and the the dipolar coupled signals detectable at low temperature (17, temperature was measured by a thermocouple (Chromel-gold at 0.07% la), which shows that the rapidly relaxing semiquinone signal iron). Double integrations of the EPR spectra were performed on a and the spin-coupled signals arise from the same semiquinone Nicolet Industries signals averager (NIC-1024). The free radical signal population. of Peptostreptococcus elsdenii flavodoxin (24) was used as a standard There have also been several reports indicating the exist- for ubisemiquinone spin quantitation, and Cu(1I).EDTA complex ence of a second population of stable ubisemiquinone, possibly was employed for spin quantitation of iron-sulfur clusters. Potentiometric titrations (25, 26) were conducted anaerobically in bound in the cytochrome b-cl segment. The power saturation the presence of mediator dyes including 1,4-naphthoquinone, 1,2- behavior of the g = 2.00 semiquinone signal in submitochon- naphthoquinone, 1,4-naphthoquinone-2-sulfonate, duroquinone,indrial particles at intermediate oxidation-reduction potentials digodisulfonate, 2-OH-1,4-naphthoquinone, and pyocyanine. The meis biphasic and can be resolved into two component curves, diators wereadded to a final concentration of 40 PM, exceptfor one of which starts to be saturated at approximately 0.1 mw, pyocyanine, which was added at 20 p. and the other is nonsaturable even at 200 mw at 200 K (18). Assays-The methods for measurement of cytochrome content by differencespectroscopyand the characteristicspectra havebeen Siedow et al. (19) conducted reductive titrations of the g = described elsewhere (8). Succinate-ubiquinone reductase and succi signal in isolated ubiquinol. cytochrome c reductase com- nate-cytochrome c reductase activities were measured by previously plex from yeast mitochondria and calculated that the maximal described procedures (22). Ubiquinol-cytochrome c reductase activity semiquinone signal accounted for 10% of the chemically de- was measured in a mixture containing 50 p~ cytochrome c, 40 m~ termined ubiquinone, which indicates a much higher stability sodium phosphate, 20 mm sodium malonate, 0.5 m~ EDTA, 0.25 m~ constant of ubisemiquinone in these experiments than that KCN, ph 7.5. The nonenzymic rate of cytochrome c reduction was obtained by adding DBH (60 p ~ ) allowing, the reaction to proceed reported for durosemiquinone in aqueous solution (20). An for 5 s, then adding reductase complex. The rates of cytochrome c even higher stability constant was reported from a potentio- reduction were calculated from the initial rate of absorbance increase metric titration of the g = 2.00 signal in mammalian cyto- at 550 nm using A E ~ = ~ rn"' ~ cm". chrome b-c, complex (21). For measurement of ubiquinone content 10-mg portions of reduc- In order to study the bound ubisemiquinones in the succi- tase complex were lyophilized in Pyrex centrifuge tubes (13 x 175 nate-cytochrome c reductase region of the respiratory chain mm). The lyophilized powder was then dispersed in 2 ml of anhydrous ethanol by placing the tubes in a bath type sonicator. One milliliter of chloroform was then added to the samples, after which they were mixed in a Vortex shaker, and the insoluble residue was separated by

3 3280 Effects of Antimycin on Bound Ubisemiquinone centrifugation. The residual pellets were extracted two more times with chloroform-ethanol (Ll), and the combined extracts were evaporated to dryness under NP. The extracted lipid was then dissolved in ethanol and centrifuged to remove any insoluble materials. The extracted ubiquinone-io was measured from the absorption difference spectrum uponaddition of potassiumborohydride (27) using an extinctioncoefficient of Aeox.red = 13 cm at 275 nm (28). RESULTS Characterization of Isolated Succinate. Cytochrome c Reductase Complex-The preparations of isolated reductase complex had succinate-ubiquinone reductase activities of 9 to 11 units per mg, antimycin-sensitive ubiquinol-cytochrome c reductase activities of 23 to 25 units per mg, and succinatecytochrome c reductase activities of 5 to 7 units per mg. As noted elsewhere (8) the reductase complex contains both cytochromes b-562 and b-566, and approximately 75% of the total cytochrome b is rapidly reduced by succinate (29). The concentration of succinate dehydrogenase was estimated by spin quantitation of center S-3, assuming one center S-3 per dehydrogenase molecule, and the concentration of cytochrome c1 was measured spectrophotometrically (8) and used as an indication of the amount of b-cl complex. These Eh hv) measurements indicated that the amount of succinate dehy- FIG. 1. Potentiometric titrations showing the effect of antidrogenase relative to b-cl complex in the succinate-cytomycin on the g = 2.00 EPR signals of two different species of chrome c reductase used for these experiments vaned from bound ubisemiquinone and on the low temperature EPR sig- 0.3:l to 1:l. nals from the spin-coupled ubisemiquinone pair in isolated The preparations of isolated reductase complex contained succinate cytochrome c reductase complex. The reductase com- 3.8 to 5.8 mol of ubiquinone per mol of cytochrome cl. Al- plex was suspended at 43 p ~ based, on cytochrome CI content, and though it is not possible to ascertain whether all or only a potentiometric titrations conducted as described under Experimental Procedures. ---, show the results of titrations in the absence of portion of this ubiquinone contributes to the formation of antimycin; -, show the results in the presence of 1.1 mol of stable ubisemiquinone as described below, it is obvious that antimycin per mol of c,. The titration curves in A and B show the isolated succinate. cytochrome c reductase complex contains peak to peak amplitude of the g = 2.00 signal measured at 10 pw and a molar excess of ubiquinone relative to cytochromes and thus 100 mw, respectively, at 50 K. The curues in C show the amplitudes differs in this regard from preparations of complex I11 (see of the EPR signals arising from the ubisemiquinone pair, which were Table 10 in Ref. 30), which contain 11 mol of ubiquinone per measured as shown in Fig. 2. The spectra were obtained under the mol of complex. following EPR conditions: microwave frequency, 9.1 GHz; modulation amplitude, 6.3 G; input microwave power, 5 mw sample temperature, EPR Measurements-The dashed line in Fig. 1A shows 10 K. the results of a potentiometric titration at ph 7.4 of the g = 2.00 EPR signal measured at 10 pw and 50 K in the absence reported for semiquinones in aqueous solutions as discussed of antimycin. The succinate-cytochrome c reductase used for below. this titration contained succinate dehydrogenase in an amount equivalent to the b-cl complex. These EPR conditions selec- The dashed line in Fig. 1B shows the potentiometric titratively monitor a novel ubisemiquinone signal, which appears tion of the g = 2.00 EPR signal measured at 100 mw and 50 K in the absence of antimycin. As shown below, this EPR to arise from ubisemiquinone in the b-cl segment, as will be signal observed at very high power appears to arise primarily described later. We have tentatively designated this ubisemifrom the ubisemiquinone associated with succinate dehydroquinone signal as SQc to indicate that this signal appears to genase (9-13), which we have designated here as SQ.. The arise from ubisemiquinone in the b-cl segment. titration curve shows a maximum at 70 mv, and the maximum The titration curve shows that the maximum signal intenconcentration of the g = 2.00 signal attributable to SQ, is 0.3 sity of SQ, is obtained at a potential of approximately 60 mv. spin per cytochrome cl. Thus SQs appears to be a more stable Double integration of the semiquinone signal at this potential form of ubisemiquinone than SQc. If SQs arises from one pair gave the maximum semiquinone approximately 0.1 spin per of ubiquinone per c1 the two ubisemiquinone oxidation-reduccytochrome ci. If it is assumed that this amount of semiquition couples would have potentials corresponding to E, (Q/ none arises from 1 mol of ubiquinone per mole of c1, the QH.) = +43 mv and E2 (QH. /QH2) = +97 mv, and a stability resulting stability constant of the ubisemiquinone radical constant of 1.2 X 10. Alternatively, if SQs arises from a 5- would be approximately 4.9 X and the bell-shaped - titrafold M excess of ubiquinone relative to iron-sulfur center S-3, tion curve would fit reasonably well to the theoretical curve the values of El and Ez would be +17 mv and +123 mv, E1 Ez for the oxidation-reduction reactions Q- QH. respectively, with a stability constant of 1.6 X lo-. QH2 having El (Q/QH.) = +21 mv and E2 (QH./QH2) +99 A potentiometric titration of the signals arising from the mv. If this ubisemiquinone is derived from a 5-fold M excess spin coupling of SQs observed at much lower temperature (12 of ubiquinone relative to ci (see above), the stability constant K) is shown in Fig. 1C. The titration of the spin-coupled would be 1.7 X and El and E2 would be -24 and +144, ubisemiquinone pair, which was monitored as shown below respectively. Although it is not possible, as noted above, to (Fig. 2), shows an E, value almost identical with that of the determine whether the entire content of ubiquinone contrib- rapidly relaxing g = 2.00 signal. The bell-shaped titration utes to the formation of this ubisemiquinone these results curve is steeper than that of the free radical signal from SQ,; clearly indicate that ubisemiquinone in the isolated reductase the half-width of the titration curve of the spin-coupled ubicomplex has a much greater stability constant than has been semiquinone signal is approximately 60 mv while that of the

4 Effects of Antimycin on Bound Ubisemiquinone 3281 g = 2.00 signal of SQ. is about 85 mv. These observations are in agreement with previous studies of spin-coupled signals from the SQs pair using beef heart mitochondria and submitochondrial particles (10, 11). The solid lines in Fig. 1, A to C, show the effect of antimycin on the two species of ubisemiquinone which contribute to the g = 2.00 signal and on the spin-coupled ubisemiquinone pair. Antimycin appears to extensively destabilize SQo as indicated by the decline in maximal signal intensity observed at low power (Fig. 1A). The shift of the titration curve toward higher potentials is due to an increased contribution from SQ, to the g = 2.00 signal under these conditions. It is difficult to estimate the maximal spin concentration of the SQc species in the antimycin-treated system, since the residual signal includes a relatively extensive contribution from the SQ. signal, and the spectrum has a low signal to noise ratio (see Fig. 4B). In contrast to its effect on the SQ, signal, antimycin significantly stabilized the intermediate oxidation-reduction state (0.45 spin/c,) of SQs as shown in Fig. 1B. By comparison, in the absence of antimycin, SQs attained a maximum concentration of 0.3 spin/cl as noted above. In similar fashion antimycin stabilized the spin-coupled ubisemiquinone signal as shown in Fig. 1 C and caused a similar positive shift in midpoint potential of approximately 40 mv and broadening of the bell-shaped titration curves for both the SQs g = 2.00 signal (Fig. 1 B) and signals from the spin-coupled ubisemiquinone pair (Fig. 1 C). If it is assumed that the SQs species which attains a maximum concentration of 0.45 spin in the presence of antimycin arises from a pair of ubiquinone molecules without interaction between them, the oxidation-reduction reactions of the individual semiquinone oxidation-reduction couples would have E1 (Q/QH.) = +96 mv and EP (QH./QH2) = +124 mv, respectively, and a semiquinone stability constant of 3.4 X 10". And if we assume that in the presence of antimycin the SQ, signal arises from 5 ubiquinones per cl, we obtain values for El and E2 of +68 mv and +152 mv, respectively, with a stability constant of 3.9 X lo-'. Although the titration curves are broader than theoretical curves, comparison of these E, and EP values with those of the control system (Fig. lb, dashed line) indicates that antimycin stabilizes the semiquinone state of SQs relative to its oxidized state rather than to the fully reduced state. Several low temperature EPR spectra at selected potentials from the experiment of Fig. 1C are shown in Fig. 2. As reported previously (9-11), these spectra display signals due to ubisemiquinone interaction at g = 2.05, 2.02, 1.99, and 1.97 partially overlapped with the signal from center S-3 of succi- 64 mv IllmV FIG. 2. Representative EPR spectra of isolated succinatecytochrome c reductase complex poised at various oxidationreduction potentials in the absence (A to C) and presence (D to F) of antimycin. The spectra were selected from the experiment shown in Fig. 1. The distances marked a and b were used to measure the relative signal amplitude arising from the spin-coupled ubisemiquinone pair in the control and antimycin-treated preparation. I I I IO 100 MicrowovePower (mw) FIG. 3. Power saturation behavior of the g = 2.00 EPR signals of isolated succinate.cytochrome c reductase complex, monitored at 50 K, in the absence (0) and presence (0) of antimycin. The control sample was poised at Eh = 78 mv and the antimycin-treated sample at E,, = 87 mv. The biphasic saturation curve obtained in the absence of antimycin (0-0) was resolved into two component curves (- - -). nate dehydrogenase. As illustrated by the heights marked a and b, the peak to peak amplitudes of the g = 1.99 signal were monitored as a parameter to give the relative signal size of the interacting ubisemiquinone pair shown in Fig. IC. An intensified signal amplitude from the spin-coupled semiquinones is clearly seen in the antimycin-treated system. The power saturation behavior of the g = 2.00 signal of isolated succinate. cytochrome c reductase complex in the absence and presence of antimycin is shown in Fig. 3. In the absence of antimycin (open circles) the power saturation curve is biphasic. This biphasic curve can be resolved into two components, as shown by the dashed lines, attributable to two species of ubisemiquinone. One of these ubisemiquinones (SQ,) starts to show saturation at approximately 0.2 mw while the amplitude of the other signal (SQJ is proportional to the square root of the input power up to 100 mw at 50 K. From the resolved curves it can be calculated that at 10 pw the relative contribution to the g = 2.00 signal from SQc and SQs is in a 3 to 1 ratio, while at 100 mw the ratio is 1 to 10. In the presence of antimycin (solid circles) the portion of the g = 2.00 signal due to the slower relaxing species is absent. The rapidly relaxing species (SQ.) exhibits an enhanced signal amplitude and is not saturable with input microwave power throughout the entire power range examined. These results are consistent with potentiometric titrations performed at low and high power presented in Fig. 1. EPR spectra of the g = 2.00 signals arising from the two different ubisemiquinone species are shown in Fig. 4. The overlapped spectra were resolved by the application of two extremely different levels of microwave power. The spectra shown in Fig. 4A are attributable to SQs. This signal has a somewhat broader (12 G) peak to peak width, and addition of antimycin causes a pronounced intensification of the g = 2.00 signal from this rapidly relaxing component. The spectra of the slowly relaxing species, SQc, are shown in Fig. 4B. This spectrum has a peak to peak width of only 10 G and, in contrast to the signal arising from SQs, this signal is almost completely abolished by antimycin. The residual signal which is observed at 10 pw in the presence of antimycin (Fig. 4B, solid line) is mostly due to the contribution from the overlapped SQs signal as described in the preceding section. In addition to their different responses to antimycin, the spectrum of SQs (Fig. 4A) exhibits a more Lorentzian line shape while the spectrum of SQ, is a more Gaussian shape. Fig. 5 shows the EPR spectra at low temperature (12 K) of isolated succinate. cytochrome c reductase complex poised at 67 mv with a succinate/fumarate couple in the absence (dashed line) and presence (solid line) of antimycin. Similar to the observations obtained in the potentiometric titrations,

5 Effects of Antimycin on Bound Ubisemiquinone A (50" K1 SQs was intensified also in the substrate poised system. The ph dependence of oxidation-reduction potentials provides information regarding the protonation of semiquinones and hydroquinones. The ph dependence of E1 (Q/QH.), EP (QH- /QHz), and E,,, (Q/QH2) is shown in Fig. 6. The ph range examined was relatively narrow because of the limited availability of mediator dyes which give sufficient buffering activity in the required potential range and which do not interfere with the measurements of the g = 2.00 signal of ubisemiquinones. The E,,, values were obtained from the ambient oxidation-reduction potential (EA) required to obtain the maximum signal height in potentiometric titrations analogous to that in Fig. 1, except the potentiometric titrations were performed at multiple ph values. At a given ph, values of E, and Et were then calculated from the E,,, value and the SQ, concentration at the peak of theg = 2.00 signal titration curve I++ 10 gauso FIG. 4. EPR spectra of the g = 2.00 signals arising from ubisemiquinone at two different microwave power levels. The spectra shown in A are of the rapidly relaxing signal, SQs, and those in B are of the slowly relaxing component, SQ, , the spectra in the absence of antimycin; "-, spectra in the presence of antimycin. I "" control - + ant A t t I t FIG. 5. EPR spectra at 12 K of isolated succinate cytochrome c reductase complex poised at Eh = 67 mv with succinate/ fumarate couple in the absence (- - -) and presence (-) of antimycin. EPR conditions were the same as described in the legend to Fig. 1. signals from the spin-coupled ubisemiquinone pair were intensified by addition of antimycin. Attempts on computer simulation of the spectra offered a satisfactory fit assuming an increase of approximately %fold of the signals from the ubisemiquinone spin-spin interaction relative to the center S-3 signal in the antimycin-treated system (results not shown). The g = 2.00 signal generated by the succinate/fumarate couple showed biphasic power saturation behavior in the K temperature range (results not shown) in similar fashion to the potentiometrically poised system described above. Likewise, the maximum SQc signal concentration was diminished by antimycin, while the rapidly relaxing broader spectrum of I I OH FIG. 6. Effect of ph on the midpoint potentials of the two successive one-electron transfer reductions, namely quinone to semiquinone (E,) and semiquinone to hydroquinone (Ea), and on the midpoint potentials (E,,,) of the two-electron transfer reduction (quinone to quinol) for the ubiquinone pool located in the cytochrome b-cl region of the isolated succinatecytochrome c reductase complex. Preparations of reductase cornplex were suspended in 0.15 M NaCl at final concentrations of 30 to 50 p~ cytochrome CI, and the ph of the suspension was adjusted to appropriate values using either TES or TES plus Hepes buffer at final concentrations of 50 rnm. Potentiometric titrations were performed as described under "Experimental Procedures," and the g = 2.00 signal amplitude of SQ. monitored at 10 pw power was plotted, as exemplified in Fig. 1A curves, except at various ph values. The E, values were obtained directly from the titration peaks as explained in the text. The values of E1 and EZ were then calculated from the E, values and the maximum semiquinone spin concentrations obtainable in potentiometric titrations performed at various ph values as presented in Fig. 7. I 0, PH FIG. 7. The ph dependence of the maximum SQ, concentrations of the potentiometric titrations. Spin quantitation of the ubiserniquinone g = 2.00 signals was conducted on a Nicolet Signal Averager, using the free radical signal of P. elsdenii flavodoxin as a standard. Other experimental conditions are same as in Fig. 6.

6 Effects of Antimycin on Bound Ubisemiquinone 3283 relative to the total pool size of Qc which was assumed to be in one to one ratio to that of cytochrome cl. The ph-independent nature of E, and the 120 mv per ph unit dependence of E2 within the range of ph 6.6 to 7.9 reveal that in the physiological ph range SQc and the corresponding hydroquinone are mostly in the anionic form (8;) and protonated form (QH,), respectively. The ph dependence of the maximum free radical concentration of the potentiometric titration curves is shown in Fig. 7. The relative peak spin concentration of the g = 2.00 signal was expressed as a percentage of the cytochrome c1 concentration measured spectrophotometrically in each succinatecytochrome c reductase preparation. The line drawn in the figure is a theoretical curve obtained by assuming pk values for the H' dissociation of semiquinone and hydroquinone as 6.4 and 8.0, respectively. DISCUSSION In the present study we have obtained evidence supporting the existence of at least two different populations of ubisemiquinone, SQ, and SQc, in isolated succinate-cytochrome c reductase complex. Although the rapidly relaxing species, SQs, has been extensively characterized in submitochondrial particles (9-14) and there have been suggestions of a second ubisemiquinone species in these more complex systems (18), this is the first instance in which evidence has been reported for multiple forms of ubisemiquinone in an isolated respiratory chain complex. Thus our findings lend credence to the possibility that multiple forms of bound ubisemiquinone may function in the segment of the respiratory chain between succinate and cytochrome c. These two populations of ubisemiquinone differ in their relaxation behavior, the line shape of the EPR spectra, and their response to inhibitors of respiration. The g = 2.00 signal from SQ,, which has previously been shown to be abolished by TTFA (17, IS), is enhanced by antimycin, indicating that this ubisemiquinone is further stabilized by this inhibitor. The signal from SQ, shows very rapid relaxation behavior, such that it is not saturated even at 200 mw at 50 K, probably due to cross-relaxation of this ubisemiquinone with iron-sulfur center S-3 of succinate dehydrogenase as previously proposed by Ingledew and coworkers (10). The identical response to antimycin of the g = 2.00 signal of SQ. and the signals from spin coupling of the ubisemiquinone pair, namely a simultaneous positive shift of midpoint potentials and stabilization of the semiquinone state, further strengthens the notion that these two different EPR signals arise from the same population of bound ubisemiquinone. The g = 2.00 EPR signal of SQ, arises from either a pair of semiquinone anion and quinone (QTQ) or a semiquinone anion hydroquinone (Q;QH2), while the split signals observed at low temperature are due to the semiquinone anion pair (QYQ;) located in proximity to iron-sulfur center S-3.2 The stability constant ofsq, is lower and the spin-spin interaction of the paired semiquinone signals is less prominent in the isolated succinate-cytochrome c reductase in comparison to the intact mitochondrial membrane system (10-12). This difference is probably due to some slight modification to the environment of the binding site of SQ. during the isolation. Likewise, modification of the SQ, binding site may explain why reductase prepared by another procedure showed almost no spin-spin interaction. At low microwave power (510 pw) the g = 2.00 signal appears to arise primarily from a second species of ubisemiquinone, which we have designated SQ,. This slowly relaxing signal is abolished by antimycin, indicating destabilization of this semiquinone by the inhibitor. The g = 2.00 signal from SQ, also differs from that of SQ, in that the former has a peak to peak width of approximately 10 G, compared to 12 G for the latter. Comparison of the relative spin concentrations of the two ubisemiquinone g = 2.00 signals at 50 K indicates that SQs has a stability constant one order of magnitude greater than that of SQc, assuming that the two semiquinone species arise from the same total pool of ubiquinone in the isolated reductase complex. Based on the intensity of the g = 2.00 EPR signals arising from SQs and SQc it is obvious that both of these ubisemiquinones must be localized in environments which significantly stabilize the radical compared to that of semiquinones in free solution. Estimates of the stability constants of SQ, and SQ.. range from 1.7 X to 1.3 X lo", depending on what portion of the total endogenous ubiquinone contributes to the semiquinones. Although directly measured values for the stability constant of ubisemiquinone in free solution at neutral ph are not available, Mitchell (7) has estimated that ubisemiquinone in a hydrophobic environment would have a stability constant of approximately 10"". Thus our results indicate that binding of ubisemiquinone in isolated succinate. cytochrome c reductase complex results in stabilization by as much as 7 to 9 orders of magnitude. The results infigs. 1, 2, and 4 indicate that antimycin increases the stability of the ubisemiquinone species associated with succinate dehydrogenase. Previously Ohnishi and coworkers (31) reported that SQ. is mostly in the anionic form at physiological ph. Also, Salerno and Ohnishi (32) observed intensified g = 2.00 and spin-spin interaction signals in antimycin-treated beef heart mitochondria poised with succinate and fumarate in the presence of ferricyanide or ascorbate, which is shown here more clearly with the isolated reductase complex. These investigators (32) suggested that the observed stabilization of SQs may be due to hindrance of proton uptake in the reduction of semiquinone anion to hydroquinone as in the case ofh& in the bacterial chromatophore system (33, 34). Direct potentiometric titration of SQ- in the antimycintreated reductase complex shown in Fig. lb, however, demonstrated that antimycin stabilizes SQs relative to Q- rather than to QsH2. The increased level of SQs in the presence of antimycin also provides independent evidence in support of the previous suggestion (22,35) that the decreased efficacy of TTFA observed in the presence of antimycin is due to competition between TTFA and ubisemiquinone (SQ.) associated with iron-sulfur center 5-3. On the basis of the destabilization of the slowly relaxing g = 2.00 EPR signal by antimycin we suggest that SQc is a novel form of ubisemiquinone which is bound in the cytochrome b- CI segment. This suggestion is consistent with the observed formation of ubisemiquinone in isolated preparations of b-cl complex from yeast (19) and bovine heart mitochondria (21). (SQ; + 2H' + e -+ QSHB), The identity of the site(s) in the b-cl segment which bind and stabilize ubisemiquinone is not known. Previously it was suggested (8) that cytochrome b-562 might bind ubisemiquinone and that antimycin might inhibit electron transfer by displacing ubisemiquinone from this acceptor/donor site. This possibility would be consistent with the effect of antimycin on the EPR signal which we attribute to SQ,. and with evidence that antimycin binds to one of the b cytochromes (36). Likewise the estimated potentials of the SQc semiquinone couples would be consistent with a protonmotive Q cycle (7, 37) in which ubisemiquinone at center i is partitioned between center S-3 of succinate dehydrogenase and cytochome b-562. At the same time we cannot exclude the possibility that SQ,. may be bound at a site distal to the antimycin binding site and that the effects of antimycin on the EPR signal may be

7 3284 Effects of on Antimycin Bound Ubisemiquinone secondary to its inhibitory activity in the respiratory chain. It should be pointed out that our results do not exclude the possibility that ubiquinone may be mobile in the membrane and fulfii a pool function in the respiratory chain as has been proposed (1-3). Alternatively it is possible that ubiquinone is restricted to the respiratory chain complexes, where it may be tightly bound, and that the pool function kinetics may result from lateral mobility of the respiratory complexes themselves (38-40). However, the results presented here and previous studies (5,9-11, 14, 19, 21,22) demonstrate the feasibility that ubisemiquinone may attain sufficient concentrations relative to other oxidation-reduction components to function as an acceptor/donor in the respiratory chain, thus obviating the necessity of postulating a noncatalyzed dismutation (4) as part of the electron transport process within the respiratory complexes. Acknoudedgrnents-We wish to thank Mr. Takamitsu Maida and Mr. Mark Lampson for their expert technical assistance. We are also grateful to Dr. H. Blum for performing computer simulations, to Dr. J. C. Salerno for stimulating discussions, to Dr. Vincent Massey for the gift of P. elsdenii flavodoxin, and to Dr. Helen Davies, who kindly transported multiple Thermos bottles between Hanover and Philadelphia. 14. Konstantinov, A.A., and Ruuge, E. K. (1977) FEBS Lett. 81, Tappel, A. L. (1960) Biochem. Phermacol. 3, Ackrell, B. A. C., Kearney, E. E., Coles, C. J., Singer, T. P., Beinert, H., Wan, Y., and Folkers, K. (1977) Arch. Biochem. Biophys. 182, Ingledew, W. J., and Ohnishi, T. (1977) Biochem. J. 164, Konstantinov, A. A., and Ruuge, E. K. (1977) Bioorg. Chem. 3, Siedow, J. N., Power, S., de la Rosa, F. F., and Palmer, G. (1978) J. Biol. Chem. 253, Bensasson, R., and Land, E. J. (1973) Biochim. Biophys. Acta 325, Yu, C. A,, Nagaoka, S., Yu, L., and King, T. E. (1978) Biochem. Biophys. Res. Commun. 82, Trumpower, B. L., and Simmons, Z. (1979) J. Biol. Chem. 254, Salerno, J. C., and Ohnishi, T. (1976) Arch. Biochem. Biophys. 176, Massey, V., and Palmer, G. (1966) Biochemistry 5, Dutton, P. L. (1971) Biochim. Biophys. Acta Wilson, P. D., Erecinska, M., Dutton, P. L., and Tzudsuki, T. (1970) Biochem. Biophys. Res. Commun. 41, Trumpower, B. L., Aiyar, A. S., Opliger, C. E., and Olson, R. E. (1972) J. Biol. Chem. 247, Morton, R. A. (1965) in Biochemistry of Quinones (Morton, R. A., ed) pp , Academic Press, New York 29. Trumpower, B. L., and Katki, A. (1975) Biochem. Biophys. Res. Commun. 65, Hatefi, Y. (1976) in The Enzymes of Biological Membranes (Martonosi, A., ed) Vol. 4, pp. 3-41, Plenum Press, New York REFERENCES 1. Green, D. E. (1962) Comp. Biochem. Physiol. 4,81-I22 2. Kroger, A., and Klingenberg, M. (1973) Eur. J. Biochem. 34, Kroger, A,, and Klingenberg, M. (1973) Eur. J. Biochem. 39, Ohnishi, T., Salerno, J. C., Maida, T., Yu, C. A., Nagaoka, S., and 323 King, T. E. (1978) in Frontiers of Biological Energetics (Dut- 4. Kroger, A. (1976) FEBS Lett. 65, ton, L., Leigh, J., and Scarpa, A,, eds) Vol. 1, pp , 5. Backstrom, D., Norling, B., Ehrenberg, A., and Ernster, L. (1970) Academic Press, New York Biochim. Biophys. Acta 197, Salerno, J. C., and Ohnishi, T. (1978) in Frontiers of Biological 6. Wikstrom, M. K. F., and Berden, J. A. (1972) Biochim. Biophys. Energetics (Dutton, L., Leigh, J., and Scarpa, A., eds) Vol. 1, Acta 283, pp , Academic Press, New York 7. Mitchell, P. (1976) J. Theor. Biol. 62, Petty, K. M., Jackson, J. B., and Dutton, P. L. (1977) FEBSLett. 8. Trumpower, B., and Katki, A. (1979) in Membrane Proteins in 84, Energy Transduction (Capaldl, R. A., ed) pp , Marcel 34. Cogdall, R. J., Jackson, J. B., and Crofts, A. R. (1972) Bioener- Dekker, Inc., New York getics 4, Ruzicka, F. J., Beinert, H., Schepler, K. L., Dunham, W. K., and 35. Trumpower, B. L. (1978) in Frontiers of Biological Energetics Sands, R. H. (1975) Proc. Natl. Acad. Sei. U. S. A. 72, (Dutton, L., Leigh, J., and Scarpa, A., eds) Vol. 2, pp , 2890 Academic Press, New York 10. Ingledew, W. J., Salerno, J. C., and Ohnishi, T. (1976) Arch. 36. Slater, E. C. (1973) Biochim. Biophys. Acta 301, Biochem. Biophys. 177, Trumpower, B. L. (1976) Biochem. Biophys. Res. Commun. 70, 11. Ohnishi, T., Salerno, J. C., Blum, H., Leigh, J. S., and Ingledew, W. J. (1977) in Bioenergetics of Membranes (Packer, L., Pa- 38. Hochli. M.. and Hackenbrock. C. R. (1976) Proc. Natl. Acad.. sei. pageorgiou, G. C., and Trebst, A,, eds) pp , Elsevier- U. SI A. 73, North Holland, Amsterdam 39. Hochli, M., and Hackenbrock, C. R. (1977) J. Cell Bid. 72, Salerno, J. C., Harmon, J. J., Blum, H., Leigh, J. S., and Ohnishl, 291 T. (1977) FEBS Lett. 82, Heron, C., Ragan, C. I., and Trumpower, B. L. (1978) Biochem. J. 13. Salerno, J. C., Blum, H., and Ohnishi, T. (1979) Biochim. Biophys. 174, Acta 547,