©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Potential Induced Redox Reactions in Mitochondrial and Bacterial Cytochrome b-c Complexes (*)

(Received for publication, February 7, 1996; and in revised form, March 8, 1996)

Dmitri Tolkatchev Linda Yu Chang-An Yu

From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Purified cytochrome b-c(1) complexes from beef heart mitochondria and Rhodobacter sphaeroides were reconstituted into potassium-loaded asolectin liposomes for studies of the energy-dependent electron transfer reactions within the complexes. Both complexes in a ubiquinone-sufficient state exhibit antimycin-sensitive reduction of cytochromes b (both low and high potential ones) upon induction of a diffusion potential by valinomycin in the presence of ascorbate. Addition of N,N,N`,N`-tetramethyl-p-phenylenediamine (TMPD) to the ascorbate-reduced potassium-loaded asolectin proteoliposomes resulted in reduction of cytochrome b. Upon addition of valinomycin, the induced diffusion potential caused a partial reoxidation of cytochrome b and partial reduction of cytochrome b in beef heart cytochrome b-c(1) complex in the presence of antimycin and/or myxothiazol. Surprisingly, when ubiquinone-depleted beef heart cytochrome b-c(1) complex liposomes were treated under the same conditions, no cytochrome b reduction was observed but only the oxidation of cytochrome b, and the oxidation was not oxygen-dependent. We explain this effect by b, iron-sulfur protein short-circuiting under these conditions, assuming that both antimycin and myxothiazol markedly affect subunit b conformation. The electrochemical midpoint potential of heme b appears to be significantly higher than that of heme b in the presence of myxothiazol, which cannot be accounted for only by the potential-driven electron transfer between these two hemes plus the shift in chemical midpoint potentials caused by myxothiazol. A model for energy coupling consistent with structural findings by Ohnishi et al. (Ohnishi, T., Schagger, H., Meinhardt, S. W., LoBrutto, R., Link, T. A., and von Jagow, G.(1989) J. Biol. Chem. 264, 735-744) is presented. This model is a compromise between pure ``redox-loop'' and pure ``proton-pump'' mechanisms. Reoxidation of high potential heme b is observed in an antimycin- or antimycin plus myxothiazol-inhibited, ascorbate plus TMPD-prereduced R. sphaeroides b-c(1) complex, upon membrane potential development, suggesting that a similar electron transfer mechanism is also operating in the bacterial complex.


INTRODUCTION

Ubiquinol-cytochrome c oxidoreductase (b-c(1) complex or complex III) accomplishes vectorial proton translocation across the mitochondrial inner membrane coupled to electron flow from ubiquinol to cytochrome c. To date, experimental data on electron-transfer mechanisms are more or less consistent with Mitchell's Q(^1)-cycle hypothesis(1, 2, 3, 4) . However, the details of membrane energization resulting from charge separation in the b-c(1) complex reactions remain unclear.

Electron transfer between the low and high potential cytochromes b (b and b respectively) contributes about 40% to the total electrogenic span of mitochondrial complex III(5, 6, 7) . The remaining 60% of the membrane potential is believed to be associated with vectorial proton translocation(1, 6, 8, 9) . This 60% portion of the total electrogenicity is closely related to the structural arrangement of b-c(1) across the membrane. On the basis of electrochemical and structural studies(5, 6, 7, 8, 9, 10, 11, 12, 13) , different authors place the b heme near the outer (P) side of the mitochondrial membrane and b heme in the middle(5, 7, 11) , or place b in the middle of the membrane with b on the inner (N) side(6, 8, 9, 12) , or place the hemes at approximately equal distance from the outer and inner sides(13) , respectively.

Important data on electron pathways and energy transduction has been obtained with the help of the specific inhibitors myxothiazol and antimycin(2, 3, 4, 5, 6, 7) . Although it is generally believed that the binding sites of these two inhibitors have essentially different locations (antimycin blocks electron transfer between high potential b and quinone and myxothiazol inhibits quinol oxidation by ISP and low potential b(2, 3, 4) ), the specific action of the inhibitors remains somewhat ambiguous. To reconcile contradictory data on the transmembrane arrangement of the b-c(1) complex, Konstantinov (9) proposed that antimycin also arrests redox-linked proton uptake and release in center o by affecting low potential b and changing its E/pH dependence (where E indicates midpoint redox potential)(14) . Furthermore, some spectroscopic and potentiometric studies suggest the proximity of myxothiazol to both center o and i(15, 16, 17) .

Most of the electrochemical data for the cytochrome b-c(1) complex were obtained from submitochondrial particles, mitochondria or chromatophores. For studying electron transfer within the complex, several advantages can be gained by the use of the purified cytochrome b-c(1) complex, reconstituted in asolectin liposomes. The influence of other components of the electron transfer chain is eliminated, and the effect of quinone can be studied using quinone-depleted and -replenished b-c(1) complexes. Recently Miki et al.(13) performed an elegant study of reverse electron transport in purified cytochrome b-c(1) complexes reconstituted into potassium-loaded vesicles. Potential induced reduction of b, without apparent b reduction, was observed in the presence of ascorbate. When high potential b is prereduced by an ascorbate and TMPD mixture, an induced membrane potential causes low potential b heme reduction at the expense of high potential b heme. The important role of quinone in b reduction was shown, as was the inhibitory effect of antimycin and myxothiazol on reverse electron transport(13) . However, a detailed study of the effects of inhibitors on energy-linked electron transfer in the mitochondrial cytochrome b-c(1) complex, in the presence and absence of quinone, was not performed(13) . We have examined potential induced reduction of cytochromes b in intact and Q-depleted preparations of beef heart b-c(1) complex at saturating concentrations of antimycin and myxothiazol. Additionally, we describe the energy-linked cytochrome b reduction in the Rhodobacter sphaeroides cytochrome b-c(1) complex reconstituted into liposomes.


EXPERIMENTAL PROCEDURES

Asolectin (crude soybean phospholipids) was obtained from Associate Concentrates (Woodside, Long Island, NY) and partially purified according to Sone et al.(18) . Horse heart cytochrome c, valinomycin, nigericin, MOPS, and sodium ascorbate were purchased from Sigma. N,N,N`,N`-Tetramethyl-p-phenylenediamine (TMPD) and safranine O were purchased from Aldrich. Myxothiazol was a product of Boehringer Mannheim (Mannheim, Germany). Antimycin A was a product of U. S. Biochemical Corp., and 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one hydrochloride (MCLA) was a gift from Dr. Anraku (University of Tokyo).

The mitochondrial cytochrome b-c(1) complex was purified as described earlier(19) . The R. sphaeroides cytochrome b-c(1) complex was prepared by a method involving Triton X-100 solubilization followed by DEAE-Bio-Gel A and DEAE-Sepharose 6B column chromatography in the presence of 0.01% dodecyl maltoside. This method (17) was developed by adaptation of those of Yu and Yu (20) and Ljungdahl et al.(21) . Quinone-depleted mitochondrial cytochrome b-c(1) complex was prepared by repeated ammonium sulfate precipitation, removing 90% of the phospholipids and quinone, and subsequent asolectin replenishment(22) . The specific activity of mitochondrial b-c(1) complex at room temperature was better than 10 µmol of cytochrome c reduced per min/nmol of b heme. Whereas the activity of quinone- and phospholipid-depleted preparations was less than 10% that of the intact mitochondrial b-c(1) complex, the activity of the phospholipid-replenished preparation was better than 90%. The activity of the bacterial b-c(1) complex was more than 2 µmol of cytochrome c reduced per min/nmol of b heme. Activity assay mixtures contained 50 mM sodium/potassium phosphate buffer, pH 7.0, 0.3 mM EDTA, 50 µmol of cytochrome c and 25 µmol of Q(0)CH(2).

Reconstitution of potassium-loaded proteoliposomes was performed by the cholate dialysis method(13, 23, 24) . To prepare mitochondrial b-c(1) complex proteoliposomes, acetone-washed phospholipids at 40 mg/ml were sonicated at 0 °C until clear, in buffer containing 10 mM MOPS, pH 7.4, 100 mM KCl, and 1.65% sodium cholate. Intact or quinone-depleted b-c(1) complex was added to give a protein/asolectin (w/w) ratio of 1:40. The phospholipid/protein mixture was incubated for 30 min at 0 °C and dialyzed at 4 °C against 10 mM MOPS buffer containing 100 mM KCl for 36 h with 2 buffer changes. To remove external potassium, proteoliposomes were dialyzed against 10 mM MOPS buffer, pH 7.4, containing 100 mM NaCl, for 10 h with 4 buffer changes. The procedure was modified for the bacterial b-c(1) complex to provide better stability and reconstitution. Phospholipids were sonicated in buffer containing 10 mM MOPS, pH 7.9, containing 300 mM KCl and 1.65% sodium cholate. The protein/asolectin (w/w) ratio in the final mixture was 1:45-1:50. Sodium cholate was removed by dialysis against 10 mM MOPS buffer, pH 7.9, containing 300 mM KCl for 24 h with 3 buffer changes. Replacement of external potassium with sodium was done by dialyzing the proteoliposomes against 10 mM MOPS buffer, pH 7.9, containing 300 mM NaCl, for 10 h with 4 buffer changes.

The oxidation control ratio, defined as the ratio of the specific activities in the presence and absence of valinomycin plus nigericin (23) , was more than 10 for proteoliposomes with mitochondrial b-c(1) complexes and about 2 for reconstituted bacterial b-c(1) complexes. In the presence of ionophores, reconstituted beef heart b-c(1) complex exhibited a specific activity close to that of dispersed complex. Stimulation of reconstituted bacterial b-c(1) complex by valinomycin and nigericin gives a specific activity of about 0.9 µmol of cytochrome c reduced per min/nmol of b heme, less than 50% of the dispersed complex. Attempts to use other detergents to improve the oxidation control of the bacterial b-c(1) complex were not successful. 0.5 or 1% sodium cholate or 0.5-1.5% dodecyl maltoside or decanoyl-N-methylglucamide did not improve the specific activity or the oxidation control ratio.

Spectral measurements were carried out on Shimadzu UV-2101PC and SLM-AMINCO DW-2000 spectrophotometers. The presence of a potassium-diffusion potential was confirmed by the safranine method (13, 25) . Five µl of proteoliposomes were added to 1 ml of medium, containing 5 µM safranine, 10 mM MOPS, pH 7.4, and 100 mM NaCl. The reaction was initiated by adding 0.3 µg of valinomycin. To collapse the gradient 1 µg of nigericin was added. The absorbance was followed at 525 nm.

To prepare samples for the recording of potential induced cytochrome spectra, 100 µl of proteoliposomes were diluted four times in 0.5-ml cuvette with the corresponding NaCl-containing dialysis buffer and, if not otherwise stated, reduced for 5 min with 10 mM ascorbate or for 30 min with 10 mM ascorbate plus 100 µM TMPD. Inhibitors and uncouplers solutions were made in ethanol. Unless otherwise stated, the antimycin- and myxothiazol-to-cytochrome b ratios were 4:1 and 8:1, respectively. Transmembrane diffusion potentials were induced by adding 0.3 µg of valinomycin to 0.4 ml of proteoliposomes. Spectra were taken every 10 s in the 540-580-nm region. The time necessary to record one spectrum was 6 s; the slit width was 2 nm; and the sampling interval varied from 0.05 to 0.2 nm. Deconvolution of the spectra was performed using extinction coefficients from West et al.(7) . The ethanol concentration in samples was kept at less than 1%.


RESULTS

Response of Mitochondrial Cytochrome b-c(1) Complex Reconstituted in Liposomes to an Induced Diffusion Potential

The mitochondrial cytochrome b-c(1) complex reconstituted in liposomes had a high oxidation control ratio, 10:15, and in the presence of the uncouplers valinomycin plus nigericin, the specific activity of the reconstituted system was close to that of the detergent-dispersed complex. The effect of uncouplers is to dissipate the membrane potential that otherwise inhibits the energy-coupled electron transfer activities of redox enzyme complexes(23) . Since it was possible to restore full activity using uncouplers without completely disrupting the membrane, and the inner part of the membrane is not accessible to the cytochrome c used as electron acceptor in the activity assay, most of the reconstituted b-c(1) complex must have been oriented as it is in the inner mitochondrial membrane.

In the absence of ion exchange, both sides of the proteoliposome membrane have approximately equal electric potential. This was tested by safranine, an optical probe for the membrane potential (13, 25) (see ``Experimental Procedures''). Valinomycin acts selectively as a potassium cation uniportor. Rapid diffusion of cations through the membrane (along the concentration gradient) creates an excess of positive charges on the outer side of the membrane and an excess of negative charges on the inner side of the membrane. A valinomycin-induced electrical potential (diffusion potential), negative on the inside, shifts the equilibrium in those reactions in which charges are moved across the membrane. The diffusion potential caused reduction of cytochrome b in vesicles pretreated with ascorbate (Fig. 1). The direct cytochrome b reduction by ascorbate in the absence of induced diffusion potential was negligible, less than 5% of cytochrome b was reduced after a 30-min incubation of vesicles with 10 mM ascorbate. In agreement with Miki et al.(13) , nigericin or excess valinomycin collapsed the diffusion potential and reversed the cytochrome b reduction.


Figure 1: Energy-dependent reduction of cytochrome b in reconstituted mitochondrial b-c complex. 0.3 µg of valinomycin was added to 0.4 ml of proteoliposomes in 10 mM MOPS/NaOH, pH 7.4, 100 mM NaCl, containing 1.23 µM cytochrome b and 5 mM ascorbate, at room temperature; curves a-d represent difference spectra between valinomycin added and valinomycin-free ascorbate-reduced proteoliposomes. a, 1 min after valinomycin addition; b, 3 min after valinomycin addition; c, after reduction is completed (6 min after valinomycin addition); d, after reduction by solid sodium dithionate. The curves were vertically shifted to avoid overlapping. The upper inset is an enlargement of curve a in the lower panel.



Contrary to the report of Miki et al.(13) , we observed reduction of both hemes b and b. At the beginning of the reaction, reduction of b was more obvious, the absorption peak being at 563 nm, with a characteristic shoulder at 558 nm. As reduction proceeds, the peak position shifts to a shorter wavelength, indicating an increasing contribution of reduced b heme. After potential induced b reduction was complete (about 40-45% of total dithionite-reducible cytochrome b), the peak maximum was 562.3 nm. Deconvolution of this final absorption using extinction coefficients given by West et al.(7) gives a composition of 75% b and 25% b. Reduction of both cytochromes b is completely inhibited by antimycin, as observed by Miki et al.(13) . This suggests that the electron is input from center i. The observed ``transient'' character of low potential b heme reduction is apparently caused by the gradual loss of the diffusion potential.

The presence of low concentrations (up to 2 Q/cytochrome b) of exogenous quinone (Q(0)C) enhances cytochrome b reduction to 60-65% of the dithionite-reducible cytochrome b (Fig. 2). The reduction rate is significantly greater than in the absence of exogenous Q. At Q/cytochrome b ratios greater than 1, transient energy-dependent cytochrome c(1) partial reoxidation is clearly seen ( Fig. 3a), and cytochrome b reduction is more obvious (Fig. 3). Cytochrome b reduction, as well as transient cytochrome c(1) reoxidation, is totally abolished by the addition of antimycin (not shown). Cytochrome c(1) oxidation is an indirect indication for the quinone being reduced at center o. The purified cytochrome b-c(1) complex contains less than 1 mol of quinone per mol of cytochrome c(1), most of the quinone molecules may be located at center i (or associated with cytochrome b) and little in center o. Added exogenous Q will bind to center o (or to cytochrome b) and this Q may be able to accept the first electron from cytochrome b via b and the second electron from ISP-cytochrome c(1) upon induction of diffusion potential.


Figure 2: Energy-dependent reduction of cytochrome b in potassium-loaded reconstituted proteoliposomes in the presence and absence of exogenous quinone. The traces illustrate the time course of absorption at the 562-575 wavelength pair after valinomycin addition in the absence added Q(0)C (a) and presence of 0.87 µM (b) and 3.85 µM Q(0)C (c). The cytochrome b concentration was 1.09 µM. Other conditions were as in Fig. 1.




Figure 3: Energy-dependent absorption spectra of reconstituted proteoliposomes in the presence of exogenous quinone. The reaction mixture contained 1.33 µM cytochrome b, 15 mM ascorbate, and 2 µM Q(0)C. Other conditions were as in Fig. 1. Difference spectra between the current system state and the initial ascorbate reduced state were taken after 0.5 min (a), 2 min (b), and after reduction is completed (after 3 min) (c).



At higher Q/cytochrome b ratios (more than 2), a decrease of the potential induced cytochrome b reduction was observed (Fig. 4). Miki et al.(13) , who first reported the effect, ascribed it to direct reoxidation of reduced cytochrome b by oxidized quinone in center i. We think that this effect is more likely caused by a limited rate of electron transfer from ascorbate to cytochrome c(1) or ISP. As the excess exogenous Q in center o may accept more electrons from cytochromes b upon induction of diffusion potential, it acts as an electron sink and thus decreases the extent of cytochrome b reduction. Since to complete the Q reduction at the Qo site it is necessary to receive an electron from ISP/cytochrome c(1), and reduction of ISP/cytochrome c(1) by ascorbate is not rapid enough, a transient oxidation of cytochrome c(1) is expected. This factor, together with the limited lifetime of the diffusion potential, causes the apparent inverse dependence of potential induced cytochrome b reducibility upon quinone concentration at high Q/cytochrome b ratios.


Figure 4: Effect of exogenous quinone concentration on the potential induced cytochrome b reduction. box, in the presence of 5 mM ascorbate; circle, 10 mM ascorbate; up triangle, 20 mM ascorbate. Total cytochrome b (100%) was 1.16 µM, and 1-µl aliquots of Q(0)C were added in ethanol to give indicated quinone/cytochrome b ratio. Other conditions were as in Fig. 1.



Unfortunately the transient nature of cytochrome c(1) reoxidation makes correlation of the increase of c(1) reoxidation with the decrease of cytochrome b reduction difficult. To further investigate the reason for diminished cytochrome b reduction in the presence of an excess of quinone, we studied the dependence of cytochrome b reduction on the quinone/cytochrome b ratio, at different ascorbate concentrations. The results are shown in Fig. 4. At high quinone concentrations, cytochrome b reduction was proportional to the ascorbate concentration. At high ascorbate concentrations, maximal cytochrome b reduction persists to higher quinone/cytochrome b ratios, supporting our explanation of this phenomenon.

Influence of Electron Transfer Inhibitors

If quinone reduction in center o during reverse electron transfer is an energy-dependent step, then one proton must come from the P side while one electron comes from an intramembrane reductant, heme b. If both the proton and the electron were to come from the P side, their charges would compensate each other, and the reaction would not be energy-dependent. Thus it is important to establish the presence or absence of b center o electron transfer in the reconstituted cytochrome b-c(1) complex while center i is blocked by antimycin. On the other hand, study of center i b electron transfer with center o blocked (by myxothiazol) is important to establish whether this step is energy-linked. In an attempt to monitor these electron transfer reactions, we studied the response of inhibited proteoliposomes to induced membrane potential.

Ascorbate is able to reduce b(27) in the presence of TMPD, and addition of valinomycin to this system resulted in a reduction of b as described by Miki et al.(13) , without the reoxidation of b. Addition of antimycin to ascorbate plus TMPD-reduced and reconstituted b-c(1) complex liposomes caused a slow partial reoxidation of prereduced b and the well-known red shift of the reduced b maximum (15) . The rate of the reoxidation is increased when an excess of exogenous quinone was added to the system. In the absence of exogenous quinone, the reoxidation is slow enough to allow identification of the potential dependent reaction. Fig. 5illustrates difference spectra recorded immediately after the addition of valinomycin (a) and after the subsequent addition of nigericin (b). The nigericin-treated sample serves as a control of potential dependent reactions. Contrary to Gopher and Gutman (5) and Konstantinov et al.(6) , who independently reported partial oxidation of prereduced b with no reduction of b, we observed partial reoxidation of high potential heme b together with partial reduction of low potential heme b. The studies of Gopher and Gutman (5) and of Konstantinov et al.(6) were performed on submitochondrial particles (SMP) poised with succinate/fumarate. Therefore, we compared potential dependent reactions of antimycin-inhibited proteoliposomes in the presence and absence of a 10 mM fumarate/1 mM succinate poising medium containing a catalytic amount of succinate-ubiquinone reductase. We found that the energy-dependent partial reoxidation of high potential heme b together with partial reduction of low potential heme b in the reconstituted proteoliposomes were not changed upon addition of 10 mM fumarate/1 mM succinate to the medium. Deconvolution of difference spectra using the extinction coefficients from West et al.(7) gives a b oxidized/b reduced ratio about 1.15 (average of three experiments).


Figure 5: Energy-dependent intracytochrome b electron transfer in antimycin- and/or myxothiazol-inhibited proteoliposomes. Proteoliposomes containing 1.12 µM cytochrome b were prereduced with 10 mM ascorbate, 0.1 mM TMPD for 20 min. Then the appropriate inhibitor was added, and the resulting spectrum taken as a base line. Spectra were recorded immediately after potential development. a, antimycin-inhibited proteoliposomes, antimycin/cytochrome b ratio = 4; b, nigericin was added to valinomycin-treated sample a spectra; c, myxothiazol-inhibited proteoliposomes, myxothiazol/cytochrome b ratio = 8; d, combination of myxothiazol and antimycin inhibition.



For comparison, we examined the effect of myxothiazol plus antimycin, on ascorbate plus TMPD-prereduced b-c(1) complexes. Antimycin and myxothiazol block electron input and output to both centers i and o, and according to West et al.(7, 26) , development of a diffusion potential causes electron transfer from b to b (Fig. 5d). In this reaction b undergoes partial reoxidation and b undergoes partial reduction. The average of the b oxidized/b reduced ratio was 1.1 (three experiments), which is close to that in the presence of merely antimycin. Apparently, no or very slow b center o electron transfer occurs in antimycin-inhibited proteoliposomes.

In the succinate/fumarate poised SMP, Konstantinov et al.(6) observed an energy-linked reduction of cytochrome b via center i, induced by the addition of ATP, with no further reduction of b. Later West (26) showed that membrane potential, created by aeration of a suspension of mitochondria in the presence of ascorbate and hexammineruthenium chloride, forces electrons from the Q pool through center i into b with no change in the redox state of b in mitochondria equilibrated with succinate/fumarate in the presence of myxothiazol. Under such conditions, high potential cytochrome b is significantly reduced while electron input from center o is blocked.

In the myxothiazol-inhibited and ascorbate plus TMPD-reduced proteoliposomes, the high potential cytochrome b was reduced. In this system an induced diffusion potential caused a reduction of b at the expense of heme b. The ratio b-oxidized/b-reduced was 1.0 (Fig. 5c). If we assume that the partial reoxidation of b resulted from the lack of an electron donating system (succinate/fumarate/succinate-ubiquinone reductase) in the reconstituted liposomes, the data obtained from the myxothiazol-inhibited proteoliposomes are consistent with the data obtained with SMP and mitochondria(6, 26) .

In the absence of TMPD, the b was not reduced, and addition of valinomycin to myxothiazol-inhibited proteoliposomes caused a AA-sensitive reduction of b, while b remained oxidized (Fig. 6). Under similar conditions, reduction of cytochrome b was observed by Miki et al.(13) , but these authors did not analyze the species of cytochromes b reduced and erroneously assumed the reduced cytochrome to be b. This low potential heme b reduction was readily abolished by adding nigericin. An excess of exogenous quinone, which serves as ``redox buffer,'' increases the level of cytochrome b reduction to 20-25% of the cytochrome with no observable b reduction. Hence, myxothiazol inhibits the potential dependent b reduction but not the potential dependent b reduction.


Figure 6: Difference spectrum between energized and nonenergized myxothiazol-inhibited proteoliposomes. Proteoliposomes containing 1.12 µM cytochrome b and indicated amount of quinone were prereduced with 10 mM ascorbate for 10 min. Then myxothiazol was added, and the resulting spectrum taken as a base line. Spectra were recorded immediately after potential development. Myxothiazol concentration was 7 µM. a, in the absence of Q(0)C; b, in the presence of 2.93 µM Q(0)C.



Quinone-depleted Cytochrome b-c(1) Complex

Removal of phospholipids from the cytochrome b-c(1) complex has been shown to abolish the enzymatic activity and significantly decrease the stability(22) . Delipidation also removes quinone from the complex. However, immediate replenishment of phospholipids to the complex restores almost all activity. The reversibility of b-c(1) complex deactivation suggests that the relipidated complex is similar to the intact complex. Although the ubiquinol-cytochrome c reductase activity of quinone-depleted complexes, after reconstituting into liposomes and using Q(0)CH(2) as substrate, was more than 80% of that of the intact complex, the degree of the reduction of cytochrome b by the induced diffusion potential was only about 60-65% of that observed in the intact proteoliposomes in the presence of equimolar amounts of Q(0)C. This less than 100% potential induced reduction of the Q-replenished system can be explained by the decreased efficiency of Q(0)C, compared with natural Q. Proteoliposomes of quinone-depleted cytochrome b-c(1) complex, in the absence of added quinone, show only small potential induced cytochrome b reduction, about 10% of that observed in intact liposomes under the same conditions(13, 22) . This is consistent with the 10-15% residual quinone content of the depleted preparation(22) . That Q-depleted cytochrome b-c(1) complex liposomes do not have the diffusion potential induced cytochrome b reduction is simply due to the lack of reducing species.

Ascorbate plus TMPD reduced the cytochrome b in quinone-depleted liposomes much like in the intact liposomes. Potential development in Q-depleted cytochrome bc(1) liposomes showed potential dependent electron movement, from high to low potential hemes (Fig. 7, a and b). The reduction pattern, in the absence of quinone, is similar to that of the antimycin- and myxothiazol-treated Q-sufficient complex. The average b-oxidized/b-reduced ratio is 1.1. According to the Q-cycle model(1, 2, 3, 4) , since quinone depletion of the b-c(1) complex blocks electron transfer in both centers i and o, no additional influence of antimycin and myxothiazol on Q-depleted preparation should be expected.


Figure 7: Energy-dependent intracytochrome b electron transfer in antimycin- and/or myxothiazol-inhibited quinone-deficient proteoliposomes. Proteoliposomes containing 1.05 µM cytochrome b were prereduced with 10 mM ascorbate, 0.1 mM TMPD for 20 min. Then the appropriate inhibitor was added and the resulting spectrum taken as a base line. Spectra were recorded immediately after potential development. a, no inhibitor added; b, nigericin added to spectrum a; c, antimycin added; d, myxothiazol added; e, combined effect of antimycin and myxothiazol.



However, in the presence of antimycin, ascorbate plus TMPD-reduced cytochrome b underwent partial reoxidation upon potential development in the Q-depleted preparation (Fig. 7c). Difference spectra between valinomycin-treated and valinomycin-free samples show a negative absorption peak at 562.5 nm, with no b reduction. The oxidation of high potential b heme occurred rapidly, within the time required to obtain a spectrum. Addition of nigericin restored the initial cytochrome b spectrum. Surprisingly, myxothiazol had a similar effect on energized, quinone-depleted vesicles. In the presence of myxothiazol, oxidation of b, with no apparent effect on b, was observed with a negative absorption peak at 561.5 (Fig. 7d). The combined effect of myxothiazol and antimycin was the same as that of antimycin alone (Fig. 7e). A possible explanation is that, at least in Q-depleted preparations, there exists an electron pathway, sensitive to antimycin and myxothiazol, that is not compatible with the Q-cycle model. However, it is not known whether or not this hypothetical pathway only appears in the presence of antibiotic under the altered phospholipid environment.

Using MCLA, an indicator of superoxide(28) , did not result in a change in luminescence upon adding valinomycin to the sample containing either Q-depleted or Q-sufficient proteoliposomes that were prereduced by ascorbate plus TMPD and antimycin-inhibited (data not shown). Preliminary flushing of ascorbate plus TMPD-reduced, Q-depleted proteoliposomes with argon did not change their response to potential development in the presence of antimycin or myxothiazol. Thus generation of O(2) in the system can be excluded and oxygen cannot be considered as possible electron acceptor.

The remaining quinone in Q-depleted preparations is not likely to account for the observed phenomenon since it is not likely to be moving rapidly between Q-depleted and Q-sufficient complexes. The b-c(1) complex is roughly a cylinder, one-third part of which is buried in the phospholipid bilayer(11) . If we neglect differences in density between the complex and the membrane, the average distance between neighboring complexes is approximately 11 complex diameters, much greater than the distance between hypothetical centers o and i. Besides, in reconstituted mixtures of intact and quinone-deficient b-c(1) complexes, cytochrome b reducibility is linearly proportional to the intact complex percentage (data not shown).

R. sphaeroides Cytochrome b-c(1) Complex

Since differences in the spatial organization of the mitochondrial and bacterial cytochrome b-c(1) complexes remain unclear, it is appropriate to try to reconstitute the latter in asolectin potassium-loaded vesicles and investigate the possibility of energy-linked cytochrome b reduction in the bacterial system. Dialysis in the presence of high salt concentrations made it possible to observe antimycin-sensitive cytochrome b reduction after potential development (Fig. 8a). The maximum of potential-reduced cytochrome b absorption was at 560 nm. The simultaneous reduction of low potential heme b was clearly seen in the difference spectrum between potential and ascorbate plus TMPD-reduced complexes (Fig. 8b). Potential induced cytochrome b reduction was approximately 18% of the total. Reduction of both cytochromes b was completely inhibited by antimycin and is not observed in 0.3 M potassium buffer.


Figure 8: Energy-dependent reactions in reconstituted bacterial b-c complex. 0.3 µg of valinomycin was added to 0.4 ml of potassium-loaded proteoliposomes (0.95 µM cytochrome b) prereduced by 10 mM ascorbate (a and b) or 10 mM ascorbate plus 0.1 mM TMPD (c and d) in 10 mM MOPS/NaOH, pH 8.0, 300 mM NaCl, at room temperature. After the potential induced reaction is completed, a difference spectrum is recorded with the valinomycin-free sample as a reference; a, diffusion potential reduced cytochrome b; b, difference spectrum between potential reduced and ascorbate + TMPD-reduced proteoliposomes; c, potential induced reoxidation of b in antimycin-inhibited reconstituted bacterial b-c(1); d, potential induced reoxidation of b in antimycin plus myxothiazol-inhibited, reconstituted bacterial b-c(1).



In the presence of myxothiazol and antimycin, or antimycin alone, b in bacterial cytochrome b-c(1) proteoliposomes was reduced by ascorbate plus TMPD as in the case of the mitochondrial complex. The prereduced cytochrome b was oxidized upon potential development (Fig. 8, c and d). Although no measurement of heme b reduction was carried out, because of high turbidity, the reduction of cytochrome b was expected. The electron transfer sequence within the bacterial complex appears to be identical to that of the mitochondrial system.


DISCUSSION

In the lack of detailed direct structural data on the spatial organization of the b-c(1) complex, the study of the potential dependent reactions plays an important role for clarification of not only energy transduction mechanisms, but also of the closely related problem of the geometric arrangement of the complex across the membrane. According to Gopher and Gutman (5) and Konstantinov et al.(6) , energy-linked oxidation of prereduced b in antimycin-inhibited submitochondrial particles occurs through center o, while b is in equilibrium with the succinate/fumarate pair. This is consistent with a P side location for b. On the other hand, based on the facts that high potential heme reduction in center i is energy-independent (6, 26) and that high potential heme b was reduced by membrane-impermeable redox agents from the N side(8, 12) , Konstantinov (9) places b electrically on the inner side and b in the middle of the membrane. According to his model, antimycin A, an inhibitor of electron transfer between Qi (center i) and high potential b, also inhibits proton extrusion in center o. This can explain the energy-independent redox state of heme b in antimycin-inhibited SMP(5, 6) .

Some structural data on the arrangement of the b hemes became available after the study of the spin-spin interactions of paramagnetic probes with redox active sites of reconstituted b-c(1) complex(11) . The data obtained by Ohnishi et al. (11) exclude the location of the low potential b heme in the middle of the membrane and the location of the high potential b heme close to the matrix side surface. Nevertheless, the low potential b heme is somewhat buried within the b-c(1) complex, the effective distance from the heme to the outside surface being approximately 1.6 nm.

Recently Miki et al.(13) studied the energy-linked reduction of cytochrome b in the reconstituted potassium-loaded proteoliposome system. They proposed an alternative model of potential-linked reversed electron transfer in the beef heart cytochrome b-c(1) complex reconstituted into potassium-loaded phospholipid vesicles, which can be described in terms of the redox-loop mechanism. The conclusions made by these authors imply that the high and low potential hemes are at approximately equal distances from N and P sides of the membrane, respectively. However, no study of potential dependent reactions in antimycin- and/or myxothiazol-inhibited reconstituted proteoliposomes was performed to confirm this model.

Our results described in this paper support a proton motive mechanism that is a compromise between two extremes, a pure redox-loop mechanism and a pure proton-pump mechanism. The possibility of this compromise was stressed before(1, 6, 9, 29) , and the influence of antimycin on the electrogenic reactions within the frame of the corresponding model was discussed(9, 12) .

We assume that both antimycin and myxothiazol not only inhibit the electron transfer in the centers i and o, but also change the conformation of the cytochrome b subunit comprising both high potential and low potential b hemes, thus affecting redox-linked electrogenic proton movement. The ability of antimycin to modify the E(m)/pH dependence of cytochrome b and to affect the electrogenic proton extrusion from center o has been already discussed(9, 12, 14) . We obtained some insight concerning the effect of myxothiazol on the properties of cytochrome b when EPR spectra of inhibited and uninhibited R. sphaeroides cytochrome b-c(1) complexes were studied. Both b and b EPR signals (g = 3.49 and g = 3.76 respectively) were shifted upfield in the presence of myxothiazol, suggesting that the inhibitor strongly affects both b cytochromes(17) .

The evidence for a conformation change caused by antimycin and/or myxothiazol is also provided by our observation of energy-dependent b oxidation in the presence of myxothiazol and/or antimycin in Q-depleted b-c(1) complexes. According to the Q-cycle model (2, 3, 4) , the first electron of quinol is passed to iron-sulfur protein (ISP) and then passed to cytochrome c(1). The resulting semiubiquinone is able to reduce the low potential cytochrome b. The low potential cytochrome b transfers the electron to high potential cytochrome b, which reduces quinone in center i. Low potential cytochrome b is not able to directly exchange electrons with ISP. If it did, the electron would not recycle through the high potential cytochrome b but would transfer from b to the iron-sulfur protein which has a more positive midpoint potential. According to this model, in the absence of quinone there should be no communication between the cytochromes b and other parts of the electron transfer chain. The only possible electron movement is between transmembranous b hemes.

Contrary to the Q-cycle model, we observed potential-linked b reoxidation, with no change in the b redox state, in Q-depleted antimycin- and/or myxothiazol-inhibited proteoliposomes. A possible explanation is b-ISP short-circuiting. If the high potential cytochrome b is rapidly equilibrating with an external redox medium through b and ISP, an electron will move from reduced heme b, along the electric diffusion potential, to b located near the P side, which immediately passes the electron to an external acceptor. The short-circuiting is a consequence of conformational changes caused by the presence of inhibitors (antimycin and/or myxothiazol) and Q deficiency, and it is observed only if both factors are present. Conformational change affecting both cytochromes b, in Q-depleted preparations, has been clearly demonstrated by EPR studies (30) .

The electron pathway and redox coupling to proton translocation in the Q-sufficient b-c(1) complex can be illustrated as follows (Fig. 9). The model includes some of the features proposed by Konstantinov and co-workers(6, 9, 12) , and it is consistent with the structural findings made by Ohnishi et al.(11) . This model implies the existence of at least two redox-linked ionizable groups(1, 12, 14, 29) , one closer to the outside and another closer to the inside, the pK values of which depend on the redox states of both hemes b and, possibly, on the redox states of quinone in centers i and o. We assume that myxothiazol and antimycin significantly change the responses of these groups to the redox processes in the b-c(1) complex.


Figure 9: A model for reverse electron transfer coupled to potential induced proton translocation in reconstituted b-c complex. The energy-dependent steps are shown by bold arrows; dashed lines represent coupling of electron transfer to proton translocation steps in uninhibited (A), antimycin-inhibited (B), and myxothiazol-inhibited b-c(1) complex (C). Horizontal arrows represent nonelectrogenic electron and proton transfers. See text for further description.



Fig. 9A shows the events postulated to take place during the potential induced reverse electron transfer in uninhibited proteoliposomes. The reduced quinone in center i reduces heme b, and an ionizable group in the vicinity of the heme accepts a proton from the quinone molecule. Simultaneously, proton uptake from the P side by another redox-linked ionizable group occurs, coupled to the heme b reduction by quinol in center i. The diffusion potential drives an electron from high potential heme b to low potential heme b. The low potential heme passes the electron and the proton from the redox-linked ionizable group to the quinone molecule, and proton release to the N side, coupled to the low potential heme oxidation in center o by quinone, occurs. The second electron and the second proton necessary to completely reduce Qo come from the P side through cytochrome c(1)-ISP chain. The quinone reduced in center o freely diffuses to center i. As a result of multiple turnovers of the Q-b-b-Q loop with three electrogenic processes involved, proton uptake from the P side, proton release to the N side, and electron movement from heme b to heme b, the overall Q-pool and cytochrome b redox potential lowers, and the cytochrome b reduction induced by the diffusion potential is observed. The electrogenic proton uptake from P side is included on the picture because a marked distance (1.6 nm) between the low potential heme and the outside surface was reported(11) .

In the antimycin-inhibited proteoliposomes, electron transfer from center i to b is blocked, and proton uptake from the P side is inhibited. Apparently, proton release to the N side is also inhibited by antimycin (Fig. 9B). Heme b rapidly exchanges electron with Q/QH pair in center o. However, since significant components of the electrogenic span are lost, the oxidation of low potential heme b by Q to yield QH does not occur to a measurable extent due to the more negative midpoint redox potential of the Q/QH pair, and we observe reduction of low potential heme together with partial oxidation of high potential heme b after the diffusion potential development. In SMP the Q/QH pair is rapidly equilibrated with the Q-pool by stoichiometric amounts of succinate-ubiquinone reductase, which forms a supercomplex with the b-c(1) complex (31) . Since the electron transfer from b to Q/QH is not accompanied by proton translocation, the redox potential of the heme b in energized SMP is equal to that established by the succinate/fumarate pair(5, 6) .

We observed that b remains oxidized while cytochrome b is partially reduced after potential development in the myxothiazol-inhibited ascorbate-reduced proteoliposomes. Since the electron from donor (ISP) is blocked by the inhibitor, the reducing power is apparently supplied by the Q/QH(2) pair. The excess exogenous quinone creates a ``redox buffer,'' and the chemical potential of the Q/QH(2) pair will remain essentially constant during the course of the reaction. In the presence of exogenous quinone, 20-25% of cytochrome b can be reduced with little or no b reduction. A rough estimate of the apparent electric potential difference between the two b hemes is 160 mV or more, with b heme being more reducible than b heme in the presence of a diffusion potential. On the other hand, other experiments with antimycin- or myxothiazol- plus antimycin-inhibited, or quinone-depleted preparations, show that the induced potential difference between the two hemes is less than 100 mV, with b heme being less reducible than b heme. The difference cannot be accounted for by a myxothiazol influence on the b heme chemical midpoint potentials because both b cytochromes titrate in the presence of myxothiazol with E(m) values 15-30 mV more positive than in its absence(16) .

Our explanation for the effect is illustrated in Fig. 9C. In the myxothiazol-inhibited b-c(1) complex, the electron transfer from Qi to high potential heme is electroneutral. The electron is passed to the high potential heme together with a proton, which binds to a redox-linked ionizable group in the vicinity of the b heme. It is likely that myxothiazol affects a redox-linked ionizable group, located in the vicinity of center o, and thus no proton is taken up from the P side. Even if the proton uptake from P side is not inhibited by myxothiazol, its contribution to the total electrogenic span may be rather small, since the low potential heme is close to the P side. On the next step, b heme is oxidized by b heme. In the uninhibited b-c1 complex, the proton that came from the Qi remains with the b heme and is released to the N side only on the subsequent step, when b heme is reoxidized by Qo (Fig. 9A). The electron transfer from b heme to b heme is not electrogenic enough to reduce b heme to a greater extent than b heme (see experiments with antimycin- plus myxothiazol-inhibited proteoliposomes and Q-depleted proteoliposomes). Probably, due to the influence of myxothiazol, the proton bound near the b heme is released to the N side during the electron transfer from high potential to low potential heme. This proton movement from the deeply buried b heme to the N side accounts for more than 60 mV of apparent electric potential difference between the b hemes and contributes to low potential heme reduction with b heme remaining significantly oxidized.

The important aspect of this work was to analyze the influence of antimycin and myxothiazol on the potential dependent reactions in the b-c(1) complex. The results obtained show that these inhibitors not only block electron conductivity between the Q-pool and cytochromes b, but they affect the conformation of the cytochrome b subunit and, possibly, the coupling of proton translocation to electron transfer in the b-c(1) complex.

The observation of a potential induced cytochrome b reduction, in reconstituted bacterial b-c(1) complexes, is consistent with there being significant structural similarity between mitochondrial and bacterial ubiquinol-cytochrome c oxidoreductases. Further detailed study of the bacterial b-c(1) response to membrane potential in reconstituted proteoliposomes is in progress in our lab.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM30721. This publication is approved by the director of the Agricultural Experiment Station, Oklahoma State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: Q, ubiquinone; QH(2), ubiquinol; Q(0)C, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; Q(0)CH(2), 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol; TMPD, N,N,N`,N`-tetramethyl-p-phenylenediamine; MOPS, 3-(N-morpholino)propanesulfonic acid; MCLA, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one hydrochloride; ISP, iron-sulfur protein; SMP, submitochondrial particles; EPR, electron paramagnetic resonance; AA, antimycin A; myx, myxothiazol.


ACKNOWLEDGEMENTS

We are grateful to Dr. A. A. Konstantinov for his valuable suggestions and fruitful discussion of the experimental results. We would like to thank Dr. Roger Koeppe and Dr. Michael Mather for critical reading of this manuscript.


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