Ubiquinol:Cytochrome c Oxidoreductase (Complex III)

EFFECT OF INHIBITORS ON CYTOCHROME b REDUCTION IN SUBMITOCHONDRIAL PARTICLES AND THE ROLE OF UBIQUINONE IN COMPLEX III*

Akemi Matsuno-Yagi and Youssef HatefiDagger

From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

Received for publication, February 14, 2001, and in revised form, March 20, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c1) of complex III, addition of succinate reduced heme bH followed by a slow and partial reduction of heme bL. Similar results were obtained when SMP were treated only with KCN or NaN3, reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of bH reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both bH and bL were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c1 on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing <= 0.06 mol of Q/mol of complex III, and Q-replenished SMP. The results showed that Q was not required for electron transfer from ISP to b, a reaction that was inhibited by antimycin (also by myxothiazol or MOA-stilbene as reported elsewhere). It was also shown that antimycin did not inhibit electron transfer from b (bH) to Q, in clear contrast to the assumption of the Q-cycle hypothesis regarding the site of antimycin inhibition.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Quinol:cytochrome c (c2, plastocyanin) oxidoreductases are energy-transducing enzyme complexes found in the respiratory chains of all aerobic organisms as well as in the electron transfer systems of chloroplasts and photosynthetic bacteria (1-4). They differ in subunit composition and nature of their quinol electron donor and their electron acceptor, but they are all dimeric, contain a bis-heme cytochrome b, a unique binuclear iron-sulfur protein and cytochrome c1 (or f in chloroplasts), and pump protons with a H+/e stoichiometry of 2. The x-ray crystal structure of the mitochondrial enzyme (complex III, bc1 complex) from cow, chicken, and rabbit has been solved to about 3.0-Å resolution (5-7). These structures show a linear sequence of electron carriers as follows: Q---bH---bL---ISP---c1.1 The bis-heme cytochrome b is the only transmembranous redox protein of complex III, with heme bL located near the cytoplasmic surface of the enzyme, and heme bH located 21 Å away from bL toward the matrix side. ISP and c1 are largely extramembranous on the cytoplasmic side, where the extramembranous domain of ISP containing a [2Fe-2S] cluster oscillates between bL and c1 to facilitate rapid electron transfer. Ubiquinone (Q) binds near bH. In the Q-cycle hypothesis, first proposed by Mitchell in 1975 (8-10), two separate Q-binding sites are conceived, a Q reduction site (Qi or QN site) near bH and a QH2 oxidation site (QO or QP site) between bL and ISP. Intensive research in the intervening years has not produced any direct evidence for the presence of Q or QH2 between bL and ISP. The x-ray crystal structures of the complex III of three different species as reported by three different groups also have failed to locate Q at this site, even when excess Q6 or Q10 was diffused into bovine complex III crystals (11).2 However, kinetic results and EPR spectra of ISP in Q-extracted and Q-replenished preparations have been interpreted in favor of the presence of one or two molecules of Q (with 10-fold different binding affinities) functioning between bL and ISP (1-4, 12).

Our own results (13-15), which could not be reconciled with the Q-cycle hypothesis, are summarized below. All the experiments were done with SMP to avoid the use of detergents, and the electron donors used in forward electron transfer experiments were the respiratory chain substrates, NADH and succinate.

1) Electron transfer from bH to bL, and from bL to ISP, is inhibited by either antimycin or myxothiazol (or HQNO, MOA-stilbene, or stigmatellin). These inhibitions are incomplete. The bH and bL reoxidation rates through the leak in the antimycin block were, respectively, 0.012 and 0.116 s-1, and through the leak in the myxothiazol block were, respectively, 0.0033 and 0.046 s-1, indicating that myxothiazol is the stronger inhibitor and that electron transfer from bH to bL is the more sensitive step to either inhibitor (13). Because in these experiments reduced bL was oxidized through the leak by these inhibitors 10-12 times faster than reduced bH, the results suggested that forward electron transfer in complex III proceeds from bH to bL (13). There is, however, no experimental support for the Q-cycle view that forward electron transfer proceeds from bL to bH.

2) Consistent with the above results, it was shown that reverse electron transfer from ISP/c1 (reduced with ascorbate + TMPD) to b was inhibited not only by myxothiazol (or stigmatellin or MOA-stilbene) but also by antimycin (15). The Q-cycle hypothesis proposes that antimycin inhibits electron transfer from bH to Q, for which there has been no direct and unequivocal experimental support.

3) Similar results as in point 2 above were obtained with Q-extracted SMP, in which the concentration of Q had been reduced 200-fold from 12.5 to <= 0.06 mol of Q/mol of cytochrome b or c1, and the rate of forward electron transfer from succinate and NADH to cytochrome c had been reduced by 97% and >99%, respectively (13, 15). These results, (i.e. antimycin-sensitive reverse electron transfer from ISP/c1 to b in the Q-extracted SMP), which suggested noninvolvement of Q as an obligatory electron carrier between ISP and b, predated the inability of the three different laboratories to find Q between ISP and b in the x-ray crystal structures of several different preparations of complex III from three different species (5-7, 11). The contention of the proponents of the Q-cycle hypothesis is that antimycin, which binds in complex III crystals near bH, and stigmatellin, myxothiazol, and MOA-stilbene, which bind in complex III near bL and ISP, are all structural analogues of Q (1-4, 16). There is, however, no clear experimental support for this contention, because none of these inhibitors have been shown to compete with Q for binding to complex III, whereas there is published evidence that the presence of Q favors antimycin binding (17).

This paper reports on the reduction in SMP of cytochrome b by succinate in the absence and the presence of various complex III inhibitors. It also reports on the features of reverse electron transfer from ascorbate + PMS via ISP to b in SMP, Q-extracted SMP, and Q-replenished SMP. The results are in full agreement with our previous findings and conclusions (13-15).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Antimycin, sodium succinate, PMS, and FCCP were obtained from Sigma; myxothiazol from Roche Molecular Biochemicals; and stigmatellin and ascorbic acid from Fluka. MOA-stilbene was a generous gift of Dr. G. von Jagow, Universitatsklinikum, Frankfurt, Germany. The sources of other chemicals were as indicated elsewhere (13-15).

Preparations of Bovine SMP, Q-depleted SMP, and Q-replenished SMP-- SMP were prepared from bovine heart mitochondria in the presence of 1.5 mM ATP (18) and Q-depleted SMP as described previously (13). Ubiquinone-replenished SMP were prepared as follows. To 52 mg of dry Q-depleted SMP were added 0.5 ml of spectrograde n-pentane and 1.0 µmol of Q10 in ~ 0.1 ml of n-pentane. The mixture was homogenized in a glass homogenizer on ice, transferred to a test tube, and centrifuged for 1-2 min at top speed in a tabletop clinical centrifuge. The supernatant was removed, and the pellet was dispersed in 0.5 ml of n-pentane and centrifuged as before. The pentane-washed pellet was dried under vacuum and suspended by homogenization in 0.5 ml of a buffer containing 0.25 M sucrose and 10 mM Tris acetate, pH 7.5. The readdition of Q10 in this manner restored >= 85% of the succinoxidase activity of the SMP. Protein concentration was determined by the method of Lowry et al. (19).

Assays-- Reduction of cytochrome b was monitored at 563 minus 575 nm in a buffer containing 0.25 M sucrose, 5 mM MgCl2, and 50 mM Tris-HCl, pH 8.0. Other conditions are given in the figure and table legends. Absorbance changes were monitored by an SLM DW-2000 dual wavelength spectrophotometer. The data shown were collected and stored in a computer on line to the spectrophotometer. Rapid mixing of SMP (plus additives) with succinate for the data of Fig. 1 and Table I was achieved using the stopped-flow attachment (MilliFlow) of Spectronic Instruments. The light path of the observation chamber was 10 mm, and the dead time, as specified, by MilliFlow was 2 ms. However, as stated below, the overall system did not allow a reliable monitoring of absorbance changes due to reduction of the chromophores under study below 0.1 s.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Inhibitors on Reduction of Cytochrome b by Succinate-- Fig. 1 shows spectrophotometric traces of the progress of cytochrome b reduction upon rapid mixing of succinate with SMP pretreated with antimycin (trace A), myxothiazol (trace B), and KCN (trace C). The extent of b reduction 0.2 s after mixing as estimated from these and similar experiments are given in Table I. The 0.2-s absorbance increases may represent the initial rates of cytochrome b reduction, but the limitations of the spectrophotometer did not allow the initial rates up to 0.1 s to be reliably recorded, and we prefer to report the results without any assumptions and extrapolations. The data are averages of three to six experiments, using three separate preparations of SMP. To avoid complications, the SMP were used as prepared without treatments for further activation of succinate dehydrogenase. The NADH oxidase activity of SMP is several times higher than that of their succinoxidase activity, but NADH could not be used in these experiments, because complex III inhibitors like myxothiazol and MOA-stilbene also inhibited complex I, albeit to a smaller extent. These limitations aside, the data of Table I contain the following significant features.


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Fig. 1.   Effects of inhibitors on the reduction of cytochrome b by succinate. SMP were treated with antimycin (trace A), myxothiazol (trace B), KCN (trace C), or antimycin and myxothiazol (trace D), then mixed with sodium succinate in a stopped-flow attachment of the SLM DW-2000 dual wavelength spectrophotometer. The progress of cytochrome b reduction was monitored at 563 minus 575 nm. For other details, see Table I and "Results"

                              
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Table I
Summary of the effects of inhibitors on the reduction by succinate of cytochrome b in SMP
Final SMP concentration was in the range 1.87-2.25 mg/ml, and the data given are averages of three to six experiments, using three different SMP preparations. SMP in 0.25 M sucrose, 5 mM MgCl2, and 50 mM Tris-HCl, pH 8.0, were preincubated for 5 min at room temperature with the reagents indicated then mixed by the stopped-flow attachment (MilliFlow, Spectronic Instruments) of the SLM DW-2000 dual wavelength spectrophotometer with an equal volume of sodium succinate in the above buffer. Absorbance changes were monitored at 563 minus 575 nm as indicated under "Experimental Procedures." The final concentrations of the reagents after mixing were: sodium succinate, 6 mM; KCN, 5 mM; NaN3, 20 mM; myxothiazol, 4 µM; MOA-stilbene, 4 µM; antimycin, 4 µM; ascorbic acid (neutralized with NaOH), 1.0 mM; and FCCP, 3 µM. The concentration of ethanol added as inhibitor solvent was <= 1% by volume. The SMP concentration of cytochrome b, as estimated from the absorbance difference at 563 minus 575 nm of Na2S2O4 reduced minus oxidized samples, using an varepsilon  = 20 mM-1 b heme (20), was 0.40-0.43 nmol/mg of protein. Delta A0.2 s, absorbance increase at 563 minus 575 nm due to cytochrome b reduction 0.2 s after rapid mixing of succinate with SMP ± inhibitors (see Fig. 1).

(i) It is seen that when SMP were treated with myxothiazol or MOA-stilbene, or with antimycin plus ascorbate and KCN (where complete prereduction of c + c1 was achieved), succinate was still able to reduce b but to a smaller extent than when they were treated with antimycin alone. As will be seen below, antimycin, unlike the other inhibitors used in Table I, affords complete reduction of both hemes bH and bL.

(ii) It is seen that in the presence of KCN or NaN3, which inhibit cytochrome oxidase, not complex III, the 0.2-s extent of cytochrome b reduction was somewhat less than, or roughly in the same range as, the extent of b reduction in the presence of myxothiazol or MOA-stilbene. The first entry in Table I also shows that the steady-state extent of b reduction by our coupled SMP in the absence of any inhibitor was in the same range as those mentioned above and that addition of the uncoupler FCCP nearly completely abolished this steady-state level of b reduction.

The results with KCN (or NaN3) as the inhibitor are particularly interesting, because the pattern of b reduction in the presence of KCN (or NaN3) is essentially the same as that in the presence of the complex III inhibitors that bind between bL and ISP. These results are shown in Fig. 2. The spectra of Fig. 2 show in the range 540-580 nm the reduction of SMP chromophores by Na2S2O4 minus the reduction of those chromophores by succinate in SMP treated with antimycin (trace A), MOA-stilbene (trace B), stigmatellin (trace C), and KCN (trace D). As seen in trace A (antimycin), the chromophores not reduced by succinate and subsequently reduced by Na2S2O4, were c + c1. In traces B (MOA-stilbene) and C (stigmatellin) the chromophores not reduced by succinate were c + c1 as expected, but also bL with an alpha  peak at ~566 nm. In trace D (KCN, which inhibits cytochrome oxidase) succinate had reduced a major portion of c + c1. However, as in traces B and C, heme bL was not reduced by succinate but by the subsequence addition of Na2S2O4. As was shown elsewhere, the same pattern of b reduction was obtained when succinate was added to SMP pretreated with antimycin plus ascorbate and KCN (14). In this and in conditions of traces B, C, and D of Fig. 2, bH was reduced by succinate as shown in Fig. 1 and Table I, and the reduction of bH was followed by a much slower and partial reduction of bL (14, 15). Indeed, in traces B and C of Fig. 1, the reduction of bH was largely completed in <0.2 s., and the subsequent slow absorbance increase was due mainly to the partial reduction of bL. What these biphasic b reductions have in common is the absence of oxidized ISP/c1 in electronic communication with bL. MOA-stilbene, myxothiazol, and stigmatellin inhibit electronic communication between bL and ISP; KCN does not inhibit complex III, but by inhibiting cytochrome oxidase allows ISP/c1 to become reduced (the 0.2-s extent of c + c1 reduction by succinate in the presence of KCN was about the same as that shown for b reduction in Table I for the experiment in the presence of NaN3). Treatment of SMP with antimycin alone allows both b hemes to become reduced by succinate, but prereduction of ISP/c1 in antimycin-treated SMP by ascorbate plus KCN inhibits rapid bL reduction, allowing only bH to be initially reduced (Table I).


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Fig. 2.   Difference spectra of chromophores reduced by Na2S2O4 minus those reduced by succinate in SMP treated with inhibitors. SMP at 1.08 mg/ml were treated with 2 µM antimycin (A), 4 µM MOA-stilbene (B), 4 µM stigmatellin (C), or 10 mM KCN (D). Then sodium succinate at a final concentration of 6 mM was added, and the absorbance of the sample was recorded in the wavelength range shown at a scan rate of 1.0 nm/s. The data were stored in a computer on line to the spectrophotometer. Then a few grains of Na2S2O4 were thoroughly mixed into each reaction mixture and the absorbance of the mixture recorded as before. The traces shown are the difference absorbencies of the Na2S2O4 reduced minus the succinate reduced spectra. Assay temperature was 30 °C here and in the experiments of Figs. 3 and 4.

Role of Ubiquinone in the Redox Reactions of Complex III-- In Fig. 3, traces A and C show the reduction of cytochrome b, respectively, in SMP and Q-extracted SMP by reverse electron transfer from ISP, which was reduced by ascorbate + PMS. As mentioned above, the SMP and the Q-extracted SMP used, contained per mole of cytochrome b or c1, respectively, 12.5 and <=  0.06 mol of Q, and as compared with SMP the NADH- and succinate-cytochrome c reductase activities of the Q-extracted SMP, had been reduced by 99 and 97%, respectively (13). It should also be mentioned here that Daldal and co-workers (21) have shown that when added to chromatophore membranes of a Rhodobacter capsulatus mutant lacking ISP, ascorbate + PMS reduced cytochrome c1, but not cytochrome b. Furthermore, as was shown elsewhere with ascorbate + TMPD, the b heme reduced by reverse electron transfer is bH (Em sime  +90 mV), whose Em is 120 mV more positive than that of bL (Em sime  -30 mV) (22, 23). Here, we have used PMS instead of TMPD, because PMS affords a faster reduction of ISP by ascorbate; as a result the extent and the rate of bH reduction via ISP are somewhat greater.


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Fig. 3.   Reduction of cytochrome b by reverse electron transfer from reduced ISP and the inhibition of this process by antimycin in SMP (traces A and B) and in Q-depleted SMP (traces C and D). SMP and Q-depleted SMP in sucrose-Tris buffer were treated with 10 mM KCN, 1.0 mM ascorbic acid (neutralized with NaOH), 2 µM antimycin where indicated, and 0.05 µM PMS. Because the extraction of Q from SMP involved removal of sucrose by washing of the particles, lyophilization, and eight extractions with n-pentane, it was felt that instead of protein concentration, the relative SMP concentrations here and in Fig. 4 should be stated as their Na2S2O4 reducible cytochrome b levels (see Table I). These concentrations were 0.59 µM cytochrome b in traces A and B and 0.50 µM cytochrome b in traces C and D.

Returning to traces A and C of Fig. 3, it is seen that there was essentially no difference in the rate and the extent of bH reduction via ISP between the SMP containing 12.5 mol of Q/mol of complex III and the Q-extracted SMP containing less than 0.06 mol of Q/mol of complex III. Traces B and D of Fig. 3 show that pretreatment of the particles with antimycin inhibited the rate and the extent of bH reduction by reverse electron transfer from ISP, fully consistent with our previous finding that in forward electron transfer antimycin inhibits electron transfer from bH to bL, and much less effectively from bL to ISP (13). It is also seen that antimycin inhibited the Q-extracted SMP (trace D) less than the normal SMP, which is consistent with the finding of Tsai and Palmer (17) that addition of Q to Q-extracted yeast complex III increased the affinity of the enzyme for antimycin. In Fig. 3, traces A and C show that addition of antimycin after bH reduction had little or no effect. This will be discussed in connection with Fig. 4 below.


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Fig. 4.   Reduction of cytochrome b by reverse electron transfer from reduced ISP in stigmatellin treated SMP (trace A), Q-depleted SMP (trace B), and Q-replenished SMP (trace C). Concentrations of KCN, ascorbate, PMS, and antimycin were the same as in Fig. 3. Stigmatellin concentration was 3 µM. Concentrations of SMP, Q-depleted SMP, and Q-replenished SMP were in terms of cytochrome b, respectively, 0.58 µM, 0.60 µM, and 0.76 µM.

Fig. 4 shows the reduction of bH by ISP reduced by ascorbate + PMS in SMP (trace A), Q-extracted SMP (trace B), and Q-extracted SMP replenished with Q10 (trace C). Before addition of ascorbate + PMS, the particles were treated with 3 µM stigmatellin. By comparison with traces A and C of Fig. 3, it is seen that treatment of the particles with stigmatellin decreased the rate and enhanced the extent of bH reduction. As pointed out elsewhere, the increase in the extent of b reduction in the presence of stigmatellin is surprising, because it has been reported that in isolated complex III stigmatellin increases the Em of ISP by 250 mV (24, 25). This increased reduction of bH in the presence of stigmatellin does not happen when the inhibitor is myxothiazol, MOA-stilbene, or ethoxyformic anhydride, which ethoxyformylates a histidine residue of ISP (15, 26, 27).3 However, the important point is that the slower and the greater extent of bH reduction in the presence of stigmatellin clearly show the absence of any difference in the progress of bH reduction between SMP (trace A), Q-extracted SMP (trace B), and Q-replenished SMP (trace C).

A second point of interest in Fig. 4 is what happened when antimycin was added to the reaction mixtures after the reduction of bH had reached near completion. It is seen in trace A that addition of antimycin resulted in near complete oxidation of bH. This antimycin-induced reoxidation of bH happened only to a small extent in trace B with Q-extracted SMP, but was restored to a considerable extent with the Q-replenished SMP (trace C). The Q-cycle hypothesis specifies that antimycin inhibits electron transfer from bH to Q (1-4). There has been no experimental proof of this assumption. However, the results of Fig. 4 clearly demonstrate that antimycin does not inhibit electron transfer from bH to Q. Rather it promotes the oxidation of bH by Q, possibly because antimycin lowers the Em of bH by 20-40 mV (23). The reason that in Fig. 3 addition of antimycin did not cause bH reoxidation is that under those conditions bH was kept reduced via the leak in the antimycin block. In Fig. 4, the combination of stigmatellin and antimycin caused a severe inhibition of bH reduction. This combined with the lowering of the Em of bH by antimycin resulted in its oxidation by the Q pool.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Conditions Affecting Cytochrome b Reduction-- An examination of Table I shows essentially three sets of data on the effect of inhibitors on cytochrome b reduction by succinate. (i) In SMP treated with antimycin alone, both hemes bH and bL were reduced with a combined Delta A0.2 s of 28-30. (ii) In SMP treated with antimycin plus myxothiazol (or antimycin plus MOA-stilbene), the reduction of both b hemes by succinate was strongly inhibited, in agreement with previous findings (1-4, 13). (iii) In SMP treated with myxothiazol, MOA-stilbene, KCN, or NaN3, or in ISP/c1 prereduced SMP treated with antimycin, only heme bH was initially reduced with an average Delta A0.2 s of 8 ± 1.5.

The results with antimycin, myxothiazol, or MOA-stilbene as the sole complex III inhibitor, or with antimycin + myxothiazol, can be rationalized in terms of the Q-cycle, but the results with KCN or NaN3, or with antimycin added to SMP containing prereduced ISP/c1, cannot be. If the Q-cycle mechanism were operative in the latter condition, one would expect (a) reduced ISP/c1 to disallow the single-electron oxidation of QH2 at the QP site of the Q-cycle and the formation of Q&cjs1138; to effect the reduction of the b hemes and (b) antimycin to block the back reduction of the b hemes by Q&cjs1138; via the QN site. Yet, our results show that under these conditions bH was reduced with a Delta A0.2 s similar to when the inhibitor was myxothiazol.

The results with KCN and NaN3 are even more at variance with expectations from the Q-cycle hypothesis. Here there were no complex III inhibitors, and there were several high potential electron sinks (SMP contain twice as much cytochrome oxidase as complex III) to afford multiple single-electron oxidations of QH2 at the QP site, thereby producing multiple Q&cjs1138; molecules for rapid reduction of the b hemes. Yet, as seen in Table I and Fig. 2, this did not happen. Only bH was initially reduced under these conditions, with a Delta A0.2 s at best in the same range as when the SMP were treated with a QP site inhibitor such as myxothiazol or MOA-stilbene. As stated above, the common feature among the conditions that greatly inhibit bL reduction, but allow bH reduction (apparently at a rate lower than that of bH reduction by SMP treated with only antimycin), is the absence of oxidized ISP/c1 in electronic communication with bL. Trumpower and co-workers (28, 29) have shown recently that lowering the Em of ISP by mutation in yeast decreases the initial rate of cytochrome b reduction by menaquinol in purified complex III. These results together with the data of Table I suggest that the redox state of bL, which would be affected by the Em of ISP, influences the reduction of bH by the nearby QH2. In other words, for rapid reduction of bH by QH2, bL must be maintained at a high oxidation state, which ISP with an Em of +280 mV can achieve. As the Em of ISP is lowered, the reduced level of bL increases, and the rate of bH reduction by QH2 drops. The same result is obtained when ISP is prereduced, its electronic communication with bL is largely inhibited by myxothiazol (or MOA-stilbene), or its oxidation is prevented by inhibition of cytochrome oxidase with KCN or NaN3.

Role of Ubiquinone in Complex III-- Figs. 3 and 4 show clearly that the reduction of cytochrome b by reverse electron transfer from reduced ISP is unaffected by extraction of Q from SMP to the extent that its concentration in SMP is lowered from 12.5 to <=  0.06 mol/mol of complex III. As stated above, Daldal and co-workers (21) have shown that in chromatophore membranes from a R. capsulatus mutant lacking ISP, ascorbate + PMS (which were used in our studies here as electron donors in the experiments of Figs. 3 and 4) reduced cytochrome c1, but not cytochrome b. Furthermore, Fig. 3 shows that 2 µM antimycin inhibited reverse electron transfer from ISP to bH, which agrees with our finding that antimycin strongly inhibits electron transfer between bL and bH (13, 30). The inhibition of this reaction by antimycin, as well as by myxothiazol and ethoxyformic anhydride, which modifies ISP (27), further supports the contention that in the experiments of Figs. 3 and 4 heme bH is reduced via bL by reverse electron transfer from reduced ISP. The fact that Q is not required for this process agrees with the inability of three independent groups to find Q between ISP and bL in the x-ray diffraction patterns of complex III from three different species (5-7). It has been stated, without any experimental support, that after oxidation of QH2 at the QP site of the Q-cycle the oxidized Q leaves this site (1). This hypothetical explanation rationalizes the inability of investigators to find Q between ISP and bL in the x-ray diffraction patterns of complex III, even when Q6 or Q10 was diffused into the crystals (5-7, 11). However, the assumption that oxidized Q leaves the QP site is totally inconsistent with the data of Figs. 3 and 4, and with the well documented earlier results regarding ATP-driven reverse electron transfer in mitochondria from the level of cytochrome c to NAD (Ref. 31 and see also Ref. 32). Clearly, if Q were an obligatory electron carrier between ISP and cytochrome b and absent in the oxidized form at this location, such reverse electron transfer via complexes III and I to NAD could not take place.

As mentioned above, the Q-cycle hypothesis states that antimycin inhibits electron transfer from bH to Q at the QN site, for which there has been no direct experimental proof. That this assumption is also incorrect is clear from the results of Fig. 4, which show that addition of antimycin promotes the oxidation of bH by Q. The reason for the antimycin effect is that it lowers the Em of bH by 20-40 mV (23) and, in combination with stigmatellin, inhibits further bH reduction by reverse electron transfer via ISP. As a result reduced bH is oxidized by Q, which binds near this heme. That the electron acceptor from reduced bH is Q is supported by the data of traces B and C of Fig. 4, which show that this antimycin-promoted bH oxidation happens only to a very small extent in Q-extracted SMP (trace B), but to a considerable extent when Q10 is added to the Q-extracted SMP.

In summary, the results presented here are in complete agreement with our previous reports and conclusions as depicted in Fig. 5, which shows a linear path of electron transfer from QH2 right-arrow bH right-arrow bL right-arrow ISP right-arrow c1. Electron transfer between bH and bL is strongly inhibited by antimycin, which binds to the N side of cytochrome b near heme bH, and by myxothiazol, MOA-stilbene, and stigmatellin, which bind to the P side of cytochrome b between bL and ISP (13, 30). In agreement with the lack of any direct and unequivocal literature evidence to the contrary, our results show the absence of a requirement for Q for electron transfer between ISP and cytochrome b. Obligatory single electron transfer from QH2 on the N side to bH results in the formation of Q&cjs1138;, which would accumulate if electron withdrawal from bH should be slower than its reduction by QH2. This possibility agrees with the fact that a stable ubisemiquinone radical has been shown by electron paramagnetic resonance spectroscopy (EPR) to be present at this site and that the EPR signal due to ubisemiquinone radical is sensitive to treatment of the enzyme with antimycin (33-36). This effect of antimycin is understandable in terms of the results shown in Fig. 4. Thus, antimycin would block the forward oxidation of reduced bH and promote reverse electron transfer from reduced bH to Q&cjs1138;.


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Fig. 5.   The electron transfer pathway of complex III. Curved arrows indicate the oscillation of the extramembranous domain of ISP between bL and c1 (1, 6). Wavy lines show the reactions inhibited by P side (myxothiazol, MOA-stilbene, stigmatellin) and N side (antimycin, HQNO) inhibitors (13). Dashed arrows are hypothetical proton translocation steps. Hollow N and P indicate the negative (matrix) and the positive (cytoplasmic) sides of complex III, respectively.

Finally, the electron transfer pathway of Fig. 5 agrees with recent rapid kinetic measurements of cytochromes b and c1 reduction. Using purified bovine complex III and a photoreleasable decylubiquinol as substrate, Chan and co-workers (37) have shown that cytochrome b is reduced with a rate constant of 270 s-1, and cytochrome c1 with a rate constant of 60 s-1, 4.5 times slower. In addition, using purified yeast complex III and menaquinol as substrate, Trumpower and co-workers (28, 29) have shown that cytochrome b is reduced at an initial rate of 58 s-1, but cytochrome c1 at a rate of 8.4 s-1, 7 times slower. Attempting to rationalize their unexpected results in terms of the Q-cycle hypothesis, Chan and co-workers (37) state that the much slower rate of cytochrome c1 reduction relative to cytochrome b reduction indicates the slow rate of electron transfer from ISP to c1. However, Millett and co-workers (38) have shown that in purified bovine complex III electron transfer from ISP to cytochrome c1 occurs with a rate constant of 16,000 s-1.

    ACKNOWLEDGEMENTS

We thank Calixto Munoz for the preparation of bovine heart mitochondria and Thu Ho for expert technical assistance.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant DK08126. This is publication number 13697-MEM from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 858-784-8092; Fax: 858-784-2054; E-mail: hatefi@scripps.edu.

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101446200

2 Kim et al. (11) stated that the QO pocket was empty in their complex III crystals. Nevertheless, difference density maps indicated that diffused Q6 or Q10 was localized only near bH.

3 Preliminary experiments have suggested that in SMP from Saccharomyces cerevisiae, the extent of cytochrome b reduction by ascorbate + PMS was also little affected by stigmatellin (A. Matsuno-Yagi and Y. Hatefi, unpublished data).

    ABBREVIATIONS

The abbreviations used are: Q and QH2, respectively, oxidized and reduced ubiquinone; Q10, ubiquinone containing 10 isoprenoid units at position 6 of the benzoquinone ring; bH and bL, respectively, the high potential and the low potential b hemes of complex III cytochrome b; ISP, iron-sulfur protein; c1, cytochrome c1; SMP, bovine-heart submitochondrial particles; HQNO, 2-n-heptyl-4-hydroxyquinoline-N-oxide; MOA-stilbene, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; PMS, phenazine methosulfate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; EPR, electron paramagnetic resonance; Asc, ascorbate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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