©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Ubiquinol-Cytochrome c Oxidoreductase
THE REDOX REACTIONS OF THE BIS-HEME CYTOCHROME b IN UBIQUINONE-SUFFICIENT AND UBIQUINONE-DEFICIENT SYSTEMS (*)

(Received for publication, October 6, 1995; and in revised form, December 19, 1995)

Akemi Matsuno-Yagi Youssef Hatefi (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Antimycin and myxothiazol are stoichiometric inhibitors of complex III (ubiquinol-cytochrome c oxidoreductase), exerting their highest degree of inhibition at 1 mol each/mol of complex III monomer. Phenomenologically, however, they each inhibit three steps in the redox reaction of the bis-heme cytochrome b in submitochondrial particles (SMP), and all three inhibitions are incomplete to various extents. (i) In SMP, reduction of hemes b(H) and b(L) by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol. Each inhibitor alone allows reduced b(H) and b(L) to accumulate, indicating that each inhibits the reoxidation of these hemes. (E)-Methyl-3-methoxy-2-(4`-trans-stilbenyl)acrylate in combination with antimycin or 2-n-heptyl-4-hydroxyquinoline-N-oxide in combination with myxothiazol causes less inhibition of b reduction than the combination of antimycin and myxothiazol. (ii) Reoxidation of reduced b(L) is inhibited by either antimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline-N-oxide, (E)-methyl-3-methoxy-2-(4`-trans-stilbenyl)acrylate, or stigmatellin). (iii) Reoxidation of reduced b(H) is also inhibited by any one of these reagents. These inhibitions are also incomplete, and reduced b(L) is oxidized through the leaks allowed by these inhibitors at least 10 times faster than reduced b(H). Heme b(H) can be reduced in SMP via cytochrome c(1) and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N`,N`-tetramethyl-p-phenylenediamine). Energization of SMP by the addition of ATP affords reduction of b(L) as well. Reverse electron transfer to b(H) and b(L) is inhibited partially by myxothiazol, much more by antimycin. Ascorbate + TMPD also reduce b(H) in ubiquinone-extracted SMP in which the molar ratio of ubiquinone to cytochrome b has been reduced 200-fold from 12.5 to 0.06. Reconstitution of the extracted particles with ubiquinone-10 restores substrate oxidation but does not improve the rate or the extent of b(H) reduction by ascorbate + TMPD. These reagents also partially reduce cytochrome b in SMP from a ubiquinone-deficient yeast mutant. The above results are discussed in relation to the Q-cycle hypothesis.


INTRODUCTION

Ubiquinol-cytochrome c oxidoreductase (complex III, bc(1) complex) was discovered and purified from bovine heart mitochondria in 1961(1, 2) . This enzyme complex is now known to be a member of a class of analogous protonmotive quinol-cytochrome c (c(2), plastocyanin) oxidoreductases, which are present in the respiratory chains of all aerobic organisms as well as in the plant and bacterial photosynthetic electron transfer systems(3, 4, 5, 6) . Quinol-cytochrome c (c(2), plastocyanin) oxidoreductases are multiprotein-lipid complexes, the simplest of which are the enzymes from Paracoccus denitrificans, Rhodobacter capsulatus (three subunits each) (4, 7) and Rhodobacter sphaeroides (four subunits)(8, 9) , and the most complex are those of mitochondria (at least 11 subunits)(10) . However, they all have in common three redox proteins: a bis-heme cytochrome b, a binuclear iron-sulfur protein, and cytochrome c(1) (or cytochrome f in chloroplasts). In mitochondria, the bis-heme cytochrome b is largely membrane-intercalated and contains a low potential heme b(L) (E(M) -90 mV) near the outside surface of the inner membrane and a high potential heme b(H) (E(M) +40 mV) at the center of the membrane about 20 Å away from heme b(L)(5, 6, 11) . The binuclear iron-sulfur protein (often referred to as the Rieske iron-sulfur protein and here abbreviated as ISP) (^1)has a high E(M) (+280 mV) with a unique EPR spectrum (g = 1.76, 1.90, 2.023), and its two iron atoms are liganded to the protein via two cysteine sulfurs and two imidazole nitrogens of histidine residues(6, 12) . Cytochrome c(1) has an E = +230 mV(12) . ISP and cytochrome c(1) are largely extramembranous on the cytosolic side of the mitochondrial inner membrane. ISP is bound to the complex by a hydrophobic helix, and cytochrome c(1) is anchored to the membrane via a single hydrophobic helix at its COOH terminus(5) . Thus complex III can be thought of as being composed of a low potential center (hemes b(L) and b(H)) and a high potential center (ISP and cytochrome c(1)).

Early work on the mechanisms of electron transfer and proton translocation by complex III revealed certain peculiar features that generated various hypothetical mechanisms(12) . Among these, the protonmotive Q-cycle, originally proposed by Mitchell(13, 14, 15) , has become the generally accepted hypothesis for the mechanisms of electron transfer and proton translocation by complex III. Fig. 1shows the Q-cycle scheme as presented by Trumpower(3, 5) . It consists of four principal steps: (i) single electron transfer from QH(2) to ISP and then to cytochromes c(1) and c; (ii) a second electron transfer from the one-electron oxidized quinol (Q) to b(L) (oxidation of QH(2) at this site (Q) involves the translocation of 2H outward); (iii) electron transfer from b(L) to b(H); (iv) electron transfer from b(H) to Q. This site of Q reduction is referred to as the Q site. A second cycle of one-electron Q reduction at the Q site together with acquisition of 2H from the inside reforms QH(2). The net result in two cycles is transfer of two electrons from QH(2) out to cytochrome c and translocation of 4H across the membrane. Two sets of inhibitors are known, which are classified as Qand Q site inhibitors(5, 16, 17) . Myxothiazol (and other inhibitors containing a beta-methoxyacrylate moiety), stigmatellin, mucidin, and undecylhydroxydioxobenzothiazole are considered inhibitors of QH(2) oxidation at the Q site, and antimycin, diuron, and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) are considered inhibitors of Q reduction at the Q site (5, 16, 17) . The Q cycle scheme incorporates the peculiar features of complex III, which were considered incompatible with a linear electron transfer pathway. For example (i) all of the inhibitors mentioned above phenomenologically interrupt electron transfer between the low and the high potential centers, resulting in the reduction of the b and the oxidation of the c cytochromes. However, if a Q site inhibitor (e.g. antimycin) and a Qsite inhibitor (e.g. myxothiazol) are added together, then the reduction of b(H) and b(L) by respiratory substrates would also be inhibited(3, 5) . The Q cycle shows that addition of either a Q or a Q site inhibitor leaves an uninhibited path open for b reduction, but when both sites are blocked, the b reduction is inhibited. (ii) A second early observation was that in antimycin and QH(2)-treated preparations, oxidation of the high potential center by potassium ferricyanide resulted in a transitory extra reduction of cytochrome b(18, 19) . The Q cycle shows that the high potential center must be oxidized in order to accept an electron from QH(2) and yield Q, which is the electron donor to b(L) at the Q site. (iii) The proton stoichiometry of 4H/2e determined for complex III is accounted for by the Q cycle, which shows that for every electron transferred from QH(2) to the high potential center, two protons are translocated from QH(2) to the outside (for other experimental observations in support of the Q cycle, see the excellent recent review by Brandt and Trumpower(5) ).


Figure 1: Schematic representation of the Q-cycle hypothesis (after Trumpower, (3) ). Heme b is b(H) and heme b is b(L).



However, we showed in 1982 that in SMP the antimycin block leaks at a rate of 20-40 nmol of substrate (NADH or succinate) oxidized per min/mg of protein and that the post-steady-state oxidation of the b hemes through the leak in the antimycin block is biphasic, with reduced b(L) undergoing oxidation 10 times faster than reduced b(H)(20) . These results are difficult to reconcile with the Q cycle, because antimycin appears to inhibit the oxidation of both b(L) and b(H), and the faster oxidation of b(L) than b(H) requires that the path of electrons from these hemes through the leak in the antimycin block to be either separate or b(H) b(L) acceptor, with the first step being considerably slower than the second. Neither possibility agrees antimycin with the path being b(L) b(H)

Q, as specified by the Q cycle. This paper reports the results of experiments, which shed new light on the mechanism of electron transfer by complex III, as well as on the effects of antimycin, myxothiazol, and other related inhibitors on the reactions catalyzed by this enzyme.


EXPERIMENTAL PROCEDURES

Chemicals

Antimycin A, HQNO, and FCCP were obtained from Sigma. Myxothiazol was from Boeringer Mannheim. Stigmatellin, ascorbic acid, and TMPD were from Fluka. ATP was from Pharmacia Biotech Inc. MOA-stilbene was a generous gift of Dr. G. von Jagow, Universitatsklinikum, Frankfurt, Germany. The sources of other chemicals were as indicated elsewhere(21, 22) .

Preparation of Bovine SMP

SMP were prepared from bovine heart mitochondria in the presence of 1.5 mM ATP during sonication as described previously(22) . Protein concentration was determined by the method of Lowry et al.(23) .

Preparation of SMP from Wild-type and Ubiquinone-deficient Yeast (Saccharomyces cerevisiae)

The wild-type yeast strain (D273-10B) and the ubiquinone-deficient strain E3-24 (24) were generous gifts of Dr. Diana Beattie, West Virginia University, Morgantown, WV. Each strain was spread on an agar plate, and the cells were grown from a single colony in a medium containing galactose as carbon source as described in the literature(25) . Mitochondria were prepared in a medium containing 0.6 M sorbitol and 10 mM potassium phosphate, pH 7.4, after treatment of the cells with zymolyase according to Daum et al.(26) . SMP were prepared in a medium containing 50 mM potassium phosphate, 120 mM KCl, and 1 mM EDTA at pH 7.5, as reported by Clejan et al.(27) .

Assays

Reduction of cytochrome b was monitored spectrophotometrically at 563 - 575 nm in a buffer containing 0.25 M sucrose, 5 mM MgCl(2), and 50 mM Tris HCl, pH 8.0. SMP concentration was 1-1.5 mg/ml. Results were essentially the same at 565 - 575 nm. KCN, ascorbic acid (neutralized with NaOH), and TMPD were added at 10 mM, 10 mM, and 0.2 mM, respectively. Inhibitors of complex III were added from an ethanolic solution, and the concentrations used are indicated in the figure legends. Ethanol concentration never exceeded 1%. Absorbance changes and spectra were recorded using an SLM DW2000 dual wavelength spectrophotometer. The data shown were collected and stored in a computer on-line to the spectrophotometer. Assay temperature was 30 °C throughout.

Determination of the Q Content of SMP

Ubiquinone was extracted from saponified SMP as described previously(28) . Briefly, bovine or yeast SMP (10-50 mg) were saponified with 10% ethanolic KOH in the presence of pyrogallol to prevent Q destruction. Then Q was extracted with spectrograde cyclohexane. After washing with water, cyclohexane was removed under vacuum, and the extracted Q dissolved in a small volume of ethanol. The concentration of Q was determined from its oxidized minus KBH(4)-reduced absorption spectrum at 275 nm, using an extinction coefficient of 12.26 mM cm(29) . In our experience, this procedure is more reliable than direct solvent extraction for correct determination of the Q content of particles.


RESULTS

The studies reported here were performed on SMP from bovine and yeast mitochondria rather than on purified complex III for the following reasons: (i) to avoid possible complications due to the presence of detergents in purified complex III; (ii) to be able to use respiratory substrates such as NADH and succinate, rather than added quinols, as electron donors to complex III; and (iii) to take advantage of the energy-coupling capability of bovine SMP. However, because the bovine SMP used in these studies were well coupled, it was necessary in certain experiments to add an uncoupler to the reaction mixture in order to release the kinetics of electron transfer from coupling constraints. As controls, experiments were carried out in the absence of uncouplers, using a different type of bovine SMP preparation that is inherently uncoupled. It was thus ascertained that other than releasing respiratory control, the added uncoupler had no effect on the redox reactions studies. It should also be added here that all the complex III inhibitors used (except HQNO, see below) were separately checked to determine the concentration at which they exerted their maximal inhibitory effects. They were then used at slightly or several times higher than their maximal effective concentration (or mol/mol of complex III in SMP) in the experiments reported below.

Effect of Inhibitors on the Pre-steady-state Reduction of b(H) and b(L) in Bovine SMP

Most inhibitors of complex III are generally divided into two groups, the ``Q(i) site'' and the ``Q(o) site'' inhibitors, as explained in the Introduction. However, for reasons that will become apparent, the functional connotation of these designations is apt to confuse this report. Therefore, we will simply refer here to the two groups of inhibitors, respectively, as I-side inhibitors (as represented by antimycin) and O-side inhibitors (as represented by myxothiazol), in conformity with the positions of point mutations on the cytochrome b molecule of R. capsulatus and R. sphaeroides that result in resistance to one or another set of inhibitors(4) .

It is well known that an I-side or an O-side inhibitor blocks the oxidation, but not the substrate-induced reduction, of b(H) and b(L). However when the two types of inhibitors are added together, the reduction of these hemes by respiratory substrates is also inhibited(3, 5) . For the purpose at hand, we have used here antimycin and HQNO as examples of I-side inhibitors and myxothiazol and MOA-stilbene as examples of O-side inhibitors. Antimycin, myxothiazol, and MOA-stilbene exert their maximal inhibitory effects at a concentration comparable with that of cytochrome b or c(1) of complex III. The concentration of HQNO required for maximal inhibition is 20-40 µM(20) . However, even at concentrations greater than that required for maximal inhibition, none of these inhibitors caused complete inhibition. Consequently, when an I-side and an O-side inhibitor were added together, b(H) and b(L) could still be reduced by succinate or NADH, albeit at a slow rate (Fig. 2). The combination of antimycin and myxothiazol caused the greatest inhibition of b reduction, MOA-stilbene was not as effective as myxothiazol when used together with antimycin, and HQNO at 20-40 µM caused the least inhibition of b reduction when used in combination with either myxothiazol or MOA-stilbene (Fig. 2, traces A-D, respectively). Traces E and F in Fig. 2show the extent of b reduction by succinate when a single inhibitor (antimycin in E, myxothiazol in F) was added. The heavy bars show the extent of b reduction at 563 - 575 nm after the addition of Na(2)S(2)O(4). In agreement with previous results(3, 5, 30) , it is clear from Fig. 2that there appear to be two pathways for reduction of b(H) and b(L) by respiratory substrates, one pathway sensitive to I-side and the other to O-side inhibitors. However, these data also show that the available inhibitors (including diuron and stigmatellin) do not cause complete inhibition of either pathway, and depending on the electron leak allowed by the combination of an I-side and an O-side inhibitor at their maximal inhibitory concentrations, the b hemes could be reduced at a faster or a slower rate. As will be seen below, inhibition of the reoxidation of reduced b(H) and b(L) by the above inhibitors also leaks. However, the fact that in the experiments of Fig. 2reduced b accumulated in the presence of an I-side and an O-side inhibitor indicates that these reagents inhibit the oxidation of the b hemes more than their reduction.


Figure 2: Reduction of the b hemes by succinate in SMP. The reaction mixture contained 0.25 M sucrose, 50 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 2 µM FCCP, and 1.38 mg of SMP/ml. Where indicated, 2 µM antimycin A (Ant A), 4 µM myxothiazol (Myxo), 5 µM MOA-stilbene (MOA), 40 µM HQNO, and 5 mM succinate were added. Reduction of b was monitored at 563 - 575 nm. The horizontal bar to the right of each trace indicates full reduction of the b hemes achieved by addition of a few grains of Na(2)S(2)O(4). The assay temperature was 30 °C in this and subsequent experiments.



Effect of Inhibitors on the Post-steady-state Oxidation of Reduced b(H) and b(L) in Bovine SMP

As was shown earlier(20) , the post-steady-state reoxidation of the b hemes of complex III through the leak in the antimycin block is biphasic, with b(L) undergoing reoxidation 10 times faster than b(H). Consistent with these results, we also showed that in antimycin-treated SMP, the attenuation of the rate of electron flow from complex I or II to complex III (by using as respiratory substrate NADH in the presence of 2 mM Seconal, succinate at a concentration below its K(m), or NADPH that is slowly oxidized by complex I) resulted in the appearance mainly of reduced b(H), presumably because the faster oxidation rate of b(L) through the leak in the antimycin block kept its steady-state level highly oxidized(20) . An example of these results is shown in Fig. 3A as a point of reference. It is seen that in antimycin-treated SMP, addition of NADH (solid trace) resulted in roughly twice as much cytochrome b reduction as the addition of NADPH (dashed trace). After near exhaustion of NADH, b was reoxidized at a fast initial rate followed by a slower rate. When the slow NADPH oxidation was inhibited at the level of complex I by the addition of rotenone, the species of b reduced by NADPH was oxidized at a rate comparable with the rate of the slow reoxidation phase of the NADH-treated sample. Fig. 3B shows the absorption spectrum of the alpha peak of the slow oxidizing b heme species reduced by NADPH (mainly b(H), (max) = 563 nm) and the remainder of b reduced in addition to b(H) by NADH (mainly b(L), (max) = 565 nm).


Figure 3: Reduction and reoxidation of the b hemes in SMP. The experimental conditions were the same as in Fig. 2. Concentrations of the inhibitors added were 2 µM antimycin A (Ant A), 4 µM myxothiazol (Myxo), and 12 µM rotenone (Rot), unless otherwise indicated. In A, 100 µM NADH (solid trace) or 100 µM NADPH (dashed trace) was added. In B, spectra of the alpha peaks of the b hemes are shown as reduced with NADPH (dashed trace) or with NADH minus the NADPH-reduced spectrum (solid trace). The NADH concentration was 50 µM in C, D, and E and 200 µM in F.



In SMP, the inhibition of reoxidation of b(H) and b(L) by HQNO, stigmatellin, myxothiazol, or MOA-stilbene also leaked. The reoxidation pattern of the b hemes in the presence of 20-40 µM HQNO was similar to that in the presence of antimycin, except that the leak through the HQNO block was greater (Fig. 3C). (^2)The reoxidation of b through the leak in the stigmatellin block showed two distinct phases, with the decay of reduced b(L) beginning to plateau before the reoxidation of b(H) started (data not shown). In this case, the second reoxidation phase was much faster than that shown in Fig. 3A. The patterns of b reduction and reoxidation in the presence of myxothiazol and MOA-stilbene were similar. Heme b(L) was only partially reduced with either succinate (Fig. 2F) or NADH (Fig. 3, D and E), and when NADH was the substrate, myxothiazol and MOA-stilbene also caused partial inhibition of complex I at the concentrations these inhibitors were used to effect maximal inhibition of complex III. This is shown in Fig. 3, D and E, where the NADH concentration was the same but the myxothiazol concentration was 1 µM in D and 2 µM in E. It is seen that the duration of the b hemes maintained reduced at steady state was longer in E because of the greater inhibition of NADH oxidation by 2 µM myxothiazol at the level of complex I. However, in both experiments D and E, the b hemes underwent a biphasic reoxidation when the concentration of NADH decreased and the rate of electron transfer from complex I to complex III fell below the reoxidation rates of the b hemes. A double exponential analysis of the reoxidation kinetics of b in Fig. 3D is shown in Fig. 4. This is qualitatively similar to the analysis published previously for the reoxidation kinetics of the b hemes of complex III through the leak in the antimycin block(20) . Thus, it appears that regardless of whether the inhibitor is antimycin (an I-side inhibitor) or myxothiazol (an O-side inhibitor), the b hemes reoxidize through the leak in the block by either of these inhibitors in a biphasic manner, with b(L) undergoing reoxidation at least 10 times faster than b(H).


Figure 4: Double exponential analysis of the kinetics of reoxidation of NADH-reduced b hemes in the presence of myxothiazol. Myxothiazol and NADH were added at 1 and 50 µM, respectively. The rapid phase constitutes 50% of the total absorbance and has a rate constant of 0.046 s. The slow phase constitutes 50% of the total absorbance and has a rate constant of 0.0033 s.



It is perhaps important to add that, unlike the combined effects of an I-side and an O-side inhibitor on the reduction of the b hemes, the addition of both types of inhibitors did not cause greater inhibition of the reoxidation of these hemes. This is shown in Fig. 3F, where SMP were treated sequentially with antimycin and NADH and monitored at 563 - 575 nm. After reduction of the b hemes as shown, myxothiazol was added followed by rotenone to inhibit complex I. The addition of rotenone was necessary, because the combination of antimycin and myxothiazol results in a very slow rate of electron transfer to the b hemes (see Fig. 2A), not allowing NADH to become rapidly exhausted. Therefore, electron transfer from complex I to complex III had to be inhibited by rotenone to curtail the otherwise prolonged steady-state phase and demonstrate the reoxidation pattern of the b hemes. We have, however, checked the effect of rotenone added during the steady-state reduction phase in experiments similar to those of Fig. 3, A and D. In either case, the b hemes underwent a biphasic reoxidation, and the only difference was that in the former the reoxidation of b(L) was faster than that shown in Fig. 3A, which is understandable because of the strong inhibitory effect of rotenone on complex I.

Effect of Inhibitors on the Reduction of b(H) and b(L) via Cytochromes c and c(1) and the Rieske Iron-Sulfur Protein

It is well known that ascorbate plus TMPD feed electrons to the respiratory chain of SMP at the level of cytochromes c + c(1) and possibly the Rieske iron-sulfur protein. As seen in Fig. 5A, when added to bovine SMP, ascorbate + TMPD also partially reduced b(H), and when ATP was added to energize SMP, a further reduction of b took place. This further absorbance increase at 563 - 575 nm was largely due to the reduction of b(L) (data not shown). As seen in traces B and C of Fig. 5, myxothiazol or antimycin blocked the reduction of the b hemes by ascorbate + TMPD. These blocks were also incomplete (especially that of myxothiazol), and the combined addition of myxothiazol and antimycin did not cause greater inhibition (data not shown). (^3)Somewhat similar results have been reported by Miki et al.(31) . They showed that in proteoliposomes of bovine complex III, loaded with KCl and treated with ascorbate, a K diffusion potential induced by addition of valinomycin resulted in an antimycin and myxothiazol-sensitive reduction of cytochrome b. As seen in Fig. 5B, the addition of ATP to the system inhibited by myxothiazol caused an increase in absorbance at 563 - 575 nm, which was about half as much as the ATP-induced absorbance increase in the absence of myxothiazol (Fig. 5A). This partial ATP-induced absorbance increase was also due to the reduction of b(L) through the leak in the myxothiazol block. However, in Fig. 5C, where the inhibitor was antimycin, there was a decrease in absorbance at 563 - 575 nm upon ATP addition, which was reversed after uncoupling by FCCP. A possible interpretation of this result is that upon energization of the system by ATP, the leak through the antimycin block, but not through the myxothiazol block, allows reverse electron transfer from the b hemes to the Q pool and complexes I and II, thereby resulting in oxidation of the partially reduced b(H). This possibility agrees with the fact that the combined addition of myxothiazol (or MOA-stilbene) and antimycin prevented both the ATP-induced extra reduction and the oxidation of b (data not shown). This point aside, however, the significant message of Fig. 5is that reverse electron transfer from cytochromes c + c(1) and ISP to b(H) and b(L) can be inhibited by either myxothiazol or antimycin, which is in complete agreement with the data shown in Fig. 3, namely that reoxidation of b(H) and b(L) is also inhibited by either antimycin or myxothiazol.


Figure 5: Reduction of the b hemes in SMP by ascorbate plus TMPD and the effect of energization. The experimental conditions were the same as in Fig. 2, except that FCCP was absent and 10 mM KCN was present. SMP concentration was 1.28 mg/ml. Inhibitor concentration was 4 µM myxothiazol (Myxo, trace B) or 1 µM antimycin A (Ant A, trace C). Where indicated, 10 mM ascorbate, 0.2 mM TMPD, 2 mM ATP, or 2 µM FCCP was added.



Reduction of b(H) by Ascorbate+TMPD in Ubiquinone-extracted Bovine SMP and in Ubiquinone-deficient Yeast SMP

According to the Q cycle, there are two separate Q binding sites and two species of Q, one produced at the Q(o) site upon one-electron oxidation of QH(2) by ISP and the other produced at the Q(i) site upon reduction of Q by b(H). The latter is supported by the finding that there is in SMP a stable, antimycin-sensitive Q (32, 33, 34, 35) . However, direct evidence for the existence of the former is weak(36) , and reduction of isolated ISP at neutral pH by QH(2) has been shown to be 10^4-10^5 times slower than the rate of electron transfer through complex III (37) . The inability of QH(2) to act as a substrate for ISP agrees with the findings of Yu and co-workers (38, 39, 40, 41) that, in Q-depleted bovine and yeast systems, azido derivatives of Q, which can act as complex III substrates, label upon photoactivation cytochrome b and a small molecular weight subunit, but not ISP. It has been shown in R. capsulatus chromatophore membranes that removal of Q and its redox state affect the EPR line-shape of ISP at g(x)(42) . These results are consistent with the Q cycle scheme in which Q/QH(2) interact directly with ISP at the Q(o) site. Another possibility is that the conformation of cytochrome b is altered by Q/QH(2) binding,and this conformation change is communicated to ISP affecting its EPR spectrum.

In view of these considerations, it was of interest to see whether b could be reduced via c(1) and ISP in the absence of Q. For this purpose, two sets of experiments were conducted, one with Q-extracted bovine SMP and the other with Q-deficient yeast SMP. Extraction of Q from bovine SMP was performed according to Ernster et al.(43) . Treatment of bovine SMP with polar solvents or with nonpolar solvents in the presence of water completely and irreversibly destroys the rotenone-sensitive NADH-Q reductase activity of complex I(44) . The procedure of Ernster et al. ((43) , see also (45) ) extracts Q with n-pentane from lyophilized mitochondria, as a result of which complex I activity is lowered but not completely destroyed. We applied this procedure to lyophilized SMP, with eight cycles of extraction for more extensive removal of Q. Analysis of the Q content of bovine SMP and the Q-extracted particles showed that, per mg of protein, SMP contained 5 nmol of Q and the Q-extracted preparation used in the experiments described below leq0.025 nmol of Q. This means that the Q content of SMP was lowered by 200-fold in the extracted particles, and its molar ratio relative to cytochrome b or c(1) was diminished from 12.5 to 0.06. Extraction of Q resulted in 97% and >99% loss, respectively, of antimycin-sensitive succinate-cytochrome c and NADH-cytochrome c reductase activities, and reconstitution of the extracted particles with added Q restored these activities by 86 and 27%, respectively. The Q-reconstituted particles were uncoupled. Otherwise, their inhibitor-response properties were the same as those described above for unextracted SMP.

Fig. 6shows the results of b reduction by ascorbate + TMPD in Q-extracted (traces A and B) and Q-reconstituted (traces C and D) SMP. All preparations were treated with KCN and ascorbate, and then 200 µM TMPD was added where shown by vertical arrows, and the reduction of b was monitored at 563 - 575 nm. Where indicated, 2 µM antimycin (4-fold molar excess relative to cytochrome b) was also added before addition of ascorbate and TMPD. It is seen in trace A that addition of TMPD resulted in b reduction in the Q-depleted SMP and in trace C that Q reconstitution had little or no effect on the rate and the extent of b reduction. The species of b heme reduced by ascorbate + TMPD was b(H), as in Fig. 5, and both b(H) and b(L) after addition of Na(2)S(2)O(4) (data not shown). As seen in trace B (Fig. 6), antimycin partially inhibited b reduction by ascorbate + TMPD in the Q-extracted SMP. This inhibition was somewhat greater in the Q-reconstituted system (Fig. 6, trace D), which agrees with the finding of Tsai and Palmer (46) that addition of Q(6) to Q-depleted yeast complex III promotes antimycin binding. Because of the presence in Q-extracted SMP of leq0.025 nmol of Q/mg of protein, additional experiments were done with SMP prepared from mitochondria isolated from a Q-deficient yeast mutant(25, 47) . Before use, SMP from the wild-type and Q-deficient yeast were also assayed for their Q content, but none was detected in the mutant SMP. As seen in traces E and F of Fig. 6, ascorbate + TMPD were able to reduce cytochrome b in Q-deficient yeast SMP, and pretreatment of the particles with antimycin showed little or no inhibition. These results agree with the reports of others on Q-depleted bovine and yeast complex III(48, 49) . They found that Q extraction did not impair the oxidation of cytochrome b via ISP/c(1) by ferricyanide. Although not shown, experiments similar to those of Fig. 2, Fig. 3, Fig. 5, and Fig. 6were also performed with SMP from wild-type yeast. In all respects, the results were qualitatively the same as those shown for bovine SMP, except that the yeast SMP were uncoupled and exhibited no increased b reduction upon ATP addition in experiments like those of Fig. 5.


Figure 6: Reduction of the b hemes in Q-extracted bovine SMP and Q-deficient yeast SMP. Traces A-D, Q-extracted bovine SMP (A and B) or Q-reconstituted bovine SMP (C and D) were used at 1.27 mg/ml. The reaction medium was the same as in Fig. 2. Antimycin A was added at 2 µM. Traces E and F, SMP prepared from the mitochondria of the Q-deficient yeast mutant were used at 2.0 mg/ml. In E and F, the assay medium, at pH 6.3, contained 0.6 M sorbitol, 10 mM potassium phosphate, 2 mM EDTA, 0.1 mM MgCl(2), and 20 mM KCl(27) . Antimycin A was added at 10 µM. In all experiments 10 mM KCN and 10 mM ascorbate were present, and then 200 µM TMPD was added where indicated by an arrow.



One other point should be mentioned. Like Ernster et al. (43) before us, we found in antimycin-treated, Q-extracted bovine SMP that succinate addition results in a slow reduction of cytochrome b, which could be considered due to the presence in these particles of a residual amount of Q. However, similar results have been reported by Clejan and Beattie (25) with Q-deficient yeast mitochondria and have been found by ourselves with Q-deficient yeast SMP, except that in our experiments the extent of succinate-induced b reduction was less. It is possible that in the absence of Q the low potential b of complex II can slowly reduce the b of complex III.


DISCUSSION

In the studies reported above, there are three sets of data that are difficult to reconcile with the Q cycle mechanism of electron transfer and the sites of action of the I-side and the O-side inhibitors as specified in Fig. 1.

(i) The finding that the reduction of b(H) and b(L) via cytochrome c(1) and ISP by ascorbate + TMPD is inhibited by antimycin (see also (31) ). The Q cycle as depicted in Fig. 1shows two paths of reverse electron transfer from c(1)/ISP to cytochrome b: one path inhibitable by myxothiazol, the other by antimycin. Therefore, the fact that reduction of b by ascorbate + TMPD is partially inhibited by myxothiazol (or MOA-stilbene) is not surprising and is consistent with the fact that myxothiazol alone can inhibit b oxidation essentially as much as myxothiazol plus antimycin (Fig. 3). One can also argue that the reason myxothiazol alone can inhibit reverse electron transfer to b is that the path via the Q(i)-site involves coupled proton translocation in reverse from the cytosolic to the matrix side, which in a nonenergized system is energetically uphill and prohibited. However, it is clear that as one attempts, on the basis of these arguments, to justify that inhibition by myxothiazol alone of reverse electron transfer from c(1)/ISP to b is not inconsistent with the Q-cycle, one is compounding by the same arguments the problem of rationalizing the fact that this reverse electron transfer to b is also inhibitable by antimycin alone. In other words, why should electrons coming via c(1)/ISP follow the antimycin-sensitive path, which involves the energetically disfavored reverse cytosolic-to-matrix proton translocation (in the absence of membrane energization by ATP) when the myxothiazol-sensitive path for reaching b via the Q(o) site is open? To put it more simply, if, according to the Q cycle, ubiquinone is both the reductant and the oxidant of b, why should electron transfer from c(1)/ISP to Q then to b be sensitive to either a Q(i) site or a Q(o) site inhibitor alone when electron transfer from NADH or succinate to Q then to b is not (Fig. 2, E and F)?

(ii) The fact that the reoxidation of substrate-reduced b in SMP through the leak in either an I-side or an O-side inhibitor is biphasic, with b(L) undergoing reoxidation much faster than b(H). These results indicate that the faster reoxidation of b(L) cannot take place via the slower reoxidation of b(H). Therefore, the two hemes would have to undergo reoxidation by separate paths (e.g. b(H) via the Q(i) site and b(L) via the Q(o) site, see Fig. 1) or the reoxidation path would have to be b(H) b(L) acceptor, with the first step being inhibitable more by either an I-side or an O-side inhibitor than the second step. Clearly, the assumption of separate pathways would present essentially the same problems as discussed in the first point, namely, why should b(L) oxidation via the Q(o) site be inhibitable by antimycin? By contrast, the path b(H) b(L) acceptor would agree with the finding that reverse electron transfer from c(1)/ISP to b can be inhibited by either antimycin or myxothiazol. It could be argued that reduced b(L) could still be oxidized at a faster rate via reduced b(H) because of the more negative potential of b(L). Then, after oxidation of b(L), reduced b(H) would decay at a slow rate. In such a case, the oxidation rate of b(L) would have to be a measure of the leak allowed by antimycin or myxothiazol. The b(L) reoxidation rates as determined previously in the presence of antimycin (20) and here in the presence of myxothiazol are, respectively, 0.116 and 0.046 s. However, if this mechanism were correct, then the much slower decay of reduced b(H) through the leaks allowed by these inhibitors should occur at the same rate, which it is not. The reoxidation rate of b(H) through the antimycin leak is 0.012 s(20) and through the myxothiazol leak is 0.0033 s, nearly 4-fold slower (see the legend to Fig. 4). These data agree, however, with our interpretation that b(H) is oxidized via b(L) and that antimycin and myxothiazol exert their strongest, but different, inhibitory effects on electron transfer from b(H) to b(L).

The above considerations point to two problems with the Q cycle scheme. One is that Q is placed between the b(H)/b(L) and the ISP/c(1) centers and is assigned the dual role of the reductant as well as the oxidant of b. This point will be discussed under the third section. The second is the sites assigned for inhibition of specific electron transfer steps by the I-side inhibitors at the Q(i) site and by the O-side inhibitors at the Q(o) site. This point is discussed below.

The results shown here indicate that the I-side and the O-side reagents inhibit the following reactions. (i) The first is reduction of b(H) and b(L) by respiratory substrates. To achieve this inhibition, both an I-side and an O-side inhibitor must be added, suggesting the presence of an I-side-sensitive as well as an O-side-sensitive path for b reduction. Neither path appears to be completely inhibitable by the available reagents (Fig. 2). (ii) The second is oxidation of the b hemes. Inhibition of the oxidation of the b hemes can be achieved by either an I-side or an O-side inhibitor. This block by either type of inhibitor is also incomplete and allows a slow biphasic oxidation of the b hemes, with reduced b(L) undergoing oxidation much faster than reduced b(H). Addition of both an I-side and an O-side inhibitor does not cause greater inhibition of the reoxidation of these hemes. Consistent with these results, reverse electron transfer from c(1)/ISP to b can also be inhibited (again incompletely) by either an I-side or an O-side inhibitor. (iii) The third is electron transfer from b(H) to b(L). This reaction is inhibited (also incompletely) by either an I-side or an O-side inhibitor.

Added to the above is the fact that compounds such as antimycin and myxothiazol are stoichiometric inhibitors. They bind to the cytochrome b molecule at different sites (4) and cause maximal inhibition at 1 mol of inhibitor/mol of complex III monomer. Therefore, to reconcile their pleiotropic inhibitory effect with their stoichiometric binding, it may be concluded that they cause inhibition by altering the conformation of the cytochrome b molecule, not by blocking a single specific electron transfer step. The data reported above suggest that this inhibitor-induced conformation change of cytochrome b restricts electron transfer most from b(H) to b(L), next from b(L) to ISP/c(1), and least from QH(2) to the b hemes. This proposed inhibitor-induced conformation change of the cytochrome b molecule as the basis of the pleiotropic inhibitory effect of stoichiometric inhibitors agrees with the effect of inhibitors on the EPR spectra of b and b in R. sphaeroides complex III (50) as well as with an early finding that at 1 mol/mol of complex III monomer, antimycin inhibits the resolution of complex III into a particulate b-rich and a soluble c(1)-rich fraction by 6 M guanidine HCl(51) .

Another point of interest is that the reductant of complex III is the two-electron donor QH(2). If QH(2) were to donate its two electrons by separate paths to b, then one could rationalize the need for an I-side and an O-side inhibitor to block b reduction by QH(2). One could also rationalize the fact that an EPR-detectable ubisemiquinone is produced in this process and its appearance in a stable form is inhibited by antimycin (5, 32, 33, 34, 35) . To this extent, these considerations are consistent with the Q cycle. However, in disagreement with the Q-cycle, our data suggest that there appears to be only a single reoxidation path for the b hemes, which is inhibitable by either antimycin or myxothiazol, and furthermore that b(H) is very likely oxidized via b(L). One may, therefore, ask whether this single b oxidation path involves Q or leads directly to ISP/c(1).

(iii) Reduction by ascorbate+TMPD of b in Q-depleted bovine SMP and in Q-deficient yeast SMP. The results presented in Fig. 6are consistent with the possibility that Q is not an obligatory electron carrier between ISP/c(1) and b (see also (48) and (49) ). The fact that in Q-depleted bovine SMP reduction of b(H) by ascorbate + TMPD is inhibited by antimycin is also consistent with the data of Fig. 3regarding the antimycin sensitivity of the reoxidation of b(L) and b(H) and agrees with the possibility that the electron path from ascorbate + TMPD to b(H) is c(1)/ISP

b(L)

b(H), with the slashes denoting inhibition by antimycin. The reason that reduced b(L) does not accumulate here is the same as in the experiments of Fig. 5, i.e. this low potential heme requires a membrane potential to become reduced by ascorbate + TMPD.

It could be argued, on the other hand, that the residual amount of Q present in the extracted bovine SMP (0.06 mol of Q/mol of cytochrome b or c(1)) is sufficient to give the result shown in Fig. 6A. However, the fact that the rate and extent of b(H) reduction by ascorbate + TMPD are nearly identical in trace A (Q-depleted bovine SMP) and trace C (Q-reconstituted bovine SMP, see also trace A of Fig. 5for unextracted bovine SMP) mitigates this argument. The data on Q-deficient yeast SMP do not agree with such a view either. Another concern could be that ascorbate + TMPD might feed electrons directly to cytochrome b and somehow antimycin interferes with this nonenzymatic reaction. In this regard, the following points may be added. (i) We have repeated all the experiments involving ascorbate + TMPD with ascorbate alone as the reductant and the only difference was that the presence of TMPD increased the rate of b reduction. (ii) It has been shown by Davidson et al.(52) in chromatophore membranes of R. capsulatus mutants lacking ISP that ascorbate (+phenazine methosulfate) reduces only cytochrome c(1), not cytochrome b.

The results presented here are summarized in Fig. 7. This minimal scheme assumes that QH(2) binds to cytochrome b and is oxidized in two single-electron steps, which are conformationally blocked when the two types of inhibitors (designated in the scheme as I(1) and I(2)) bind to cytochrome b. Neither inhibitor alone inhibits the oxidation of QH(2), but the presence of antimycin results in destabilization of Q(32, 33, 34, 35) . The oxidation of QH(2) results in outward translocation of 2H. The scheme does not specify the destinations of the first and the second electrons from QH(2), i.e. as regards which heme, b(L) or b(H), is reduced by which electron. Heme b(H) is oxidized via b(L), with either an I-side or an O-side inhibitor (I(1) or I(2)) severely inhibiting electron transfer from b(H) to b(L) and at least 10 times less severely from b(L) to ISP. The oxidation of reduced cytochrome b results in the translocation by the cytochrome b molecule of 2H outward. This agrees with the finding of Beattie (53) that modification by dicyclohexylcarbodiimide of cytochrome b in yeast complex III results in greater inhibition of proton translocation than electron transfer. Finally, electrons go from ISP to c(1) and therefrom out to c. It is important to emphasize that Fig. 7is intended only to summarize the results shown here in graphic form, and the arrows for proton translocation have been dashed to emphasize lack of experimental support at this time. In addition, it should be added that Fig. 7does not attempt to explain two important features of complex III. One feature is the phenomenon of oxidant-induced transient extra reduction of cytochrome b(18, 19) . This phenomenon is easily explained by the Q cycle, as discussed above. However, there is no experimental proof that QH(2) is the direct electron donor to ISP and not to b. On the contrary, the data of Fig. 6suggest that Q is not required for electron transfer between c(1)/ISP and b (see also (48) and (49) ). The other feature is that in an antimycin-treated system, the b hemes cannot be reduced by QH(2) in the absence of ISP(30) . The Q cycle hypothesis accounts for this feature as well, but in view of the data of Fig. 5and 6, it is now imperative to demonstrate unequivocally that QH(2) is the obligatory electron donor of ISP or to seek a different explanation for the latter feature.


Figure 7: Scheme summarizing the experimental data of this report. I(1) and I(2) indicate I-side and O-side inhibitors, without specifying which is I-side inhibitor and which O-side. Dashed arrows are hypothetical proton-translocation steps. Asc, ascorbate; M and C, the matrix and the cytosolic sides, respectively, of the mitochondrial inner membrane.




FOOTNOTES

*
This work was supported by United States Public Health Service Grant DK08126. This is publication 9020-MEM from The Scripps Research Institute, La Jolla, CA. 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.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: ISP, iron-sulfur protein of complex III; EPR, electron paramagnetic resonance; Q and QH(2), oxidized and reduced ubiquinone, respectively; b(H) and b(L), the high and low potential hemes, respectively, of the bis-heme cytochrome b of complex III; c(1), cytochrome c(1); HQNO, 2-n-heptyl-4-hydroxyquinoline-N-oxide; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; TMPD, N,N,N`,N`-tetramethyl-p-phenylenediamine; MOA-stilbene, (E)-methyl-3-methoxy-2-(4`-trans-stilbenyl)acrylate; SMP, submitochondrial particles.

(^2)
At concentrations geq60 µM, HQNO caused rapid oxidation of both b(L) and b(H). This effect could not be prevented by pretreatment of SMP with antimycin.

(^3)
HQNO and stigmatellin could not be used as inhibitors of reverse electron transfer in the presence of ascorbate + TMPD. In the presence of HQNO, the otherwise slow reduction of b(H) by ascorbate (no TMPD) was greatly enhanced. Stigmatellin had a similar effect. Also, in a reaction mixture containing cytochrome c and buffer, addition of stigmatellin promoted the reduction of cytochrome c by ascorbate.


ACKNOWLEDGEMENTS

We thank Dr. D. S. Beattie, West Virginia University, Morgantown, WV, for the gift of wild-type and ubiquinone-deficient mutant strains of S. cerevisiae and C. Munoz for the preparation of bovine heart mitochondria.


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