Ubiquinol:Cytochrome c Oxidoreductase
EFFECTS OF INHIBITORS ON REVERSE ELECTRON TRANSFER FROM THE IRON-SULFUR PROTEIN TO CYTOCHROME b*

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta -methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0.06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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The mitochondrial ubiquinol:cytochrome c oxidoreductase (complex III, bc1 complex) is a homodimer of monomer molecular mass of ~ 240 kDa (1-4). Each monomer is composed of 11 subunits, of which 3 are redox proteins. These three proteins are a bis-heme cytochrome b containing a low potential heme bL (Em = ~-30 mV) and a high potential heme bH (Em = ~+90 mV),1 a [2Fe-2S] iron-sulfur protein (ISP, Em = +280 mV)2 with an EPR signal centered at g = 1.90, and a cytochrome c1 (Em = + 230 mV) (1, 7). In the mitochondrial respiratory chain, complex III transfers electrons from the ubiquinone pool to cytochrome c in a reaction that is coupled to outward proton translocation with a stoichiometry of H+/e = 2. When properly isolated, purified bovine heart complex III catalyzes the reduction of cytochrome c by ubiquinol-2 at a rate of 2500-3000 s-1 at 38 °C (8, 9).

Recently, x-ray crystallographic data have been published at 2.9 Å resolution by Yu and co-workers (2, 10-12) for about 80% of the bovine enzyme, at 3.0 Å resolution by Zhang et al. (3) for the chicken (as well as cow and rabbit) complex III, and at a more refined 3.0 Å resolution by Iwata et al. (4) for the bovine enzyme. In bovine complex III, cytochrome b is largely membrane-intercalated, with heme bL near the cytoplasmic surface of the molecule and 21 Å away from heme bH toward the matrix side. The iron-sulfur (FeS) cluster of ISP is 27 Å away from heme bL and 31 Å away from the c1 heme (2). With the chicken and the bovine complex III, Zhang et al. (3) noted that in the presence of the inhibitor stigmatellin the FeS cluster of ISP was located closer to bL than in the native crystals and 31 Å away from the c1 heme. They propose that the extramembranous domain of ISP can assume two conformations, a proximal conformation with the FeS cluster near bL (or the QO site of the Q cycle) and a distal conformation with the FeS cluster ~20 Å closer to the c1 heme. In a more recent report, Kim et al. (12) agree with the results and conclusions of Zhang et al. (3) regarding the effect of stigmatellin on the conformation of ISP. They further indicate that 5-undecyl-6-hydroxy-4,7-dioxobenzothiazol, but not myxothiazol, MOA-stilbene, and antimycin, also fixes the conformation of ISP near bL.

We have shown recently in energized submitochondrial particles (SMP) that reverse electron transfer from ISP/c1 to cytochrome b is inhibited more by antimycin, which binds near bH, than by myxothiazol, which binds near bL (13). Antimycin also inhibited reverse electron transfer from ISP/c1 to b in Q-extracted SMP, which contained <= 0.06 mol Q/mol cytochrome b or c1 (200-fold less than the unextracted SMP) and was incapable of oxidizing NADH or succinate by molecular oxygen (13). We have also shown that when SMP were treated with antimycin, KCN, and ascorbate plus TMPD to reduce the high potential centers of complex III, subsequent addition of NADH or succinate resulted in rapid and complete reduction of bH, and only the reduction of bL became slow and partial when ISP/c1 were prereduced (14). These and other results reported in Refs. 13 and 14 are not compatible with the Q cycle hypothesis, but they are fully consistent with the x-ray diffraction data of the three different groups mentioned, especially because these data do not show the presence of any Q at the "QO site" of the Q cycle.

As pointed out by Zhang et al. (3), the possible movement of the extramembranous domain of ISP between bL and c1 has important mechanistic implications, because in its proximal position the FeS cluster of ISP would be 31 Å away from the c1 heme, a distance incompatible with rapid electron transfer. However, whether in the absence of stigmatellin or 5-undecyl-6-hydroxy-4,7-dioxobenzothiazol the extramembranous domain of ISP can move so close to bL is not known. Nor is it known whether in the uninhibited enzyme the redox states of cytochrome b, ISP and c1 affect the movement of the extramembranous domain of ISP between its proposed proximal and distal positions. This paper examines features of the bis-heme cytochrome b reduction in SMP in the absence and the presence of complex III inhibitors when it is reduced by the respiratory chain substrates, NADH and succinate, and when it is reduced by reverse electron transfer from ISP/c1. The results confirm our previous data that Q is not an obligatory electron carrier between b and ISP and suggest that when ISP and c1 are both reduced, reduced ISP inhibits the rapid and complete reduction of bL by respiratory substrates.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Chemicals-- Antimycin A, EFA, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were obtained from Sigma. Myxothiazol was from Boehringer Mannheim. Stigmatellin, ascorbic acid, and TMPD were from Fluka. NADH was from CalBiochem. ATP was from Amersham Pharmacia Biotech. Potassium ferricyanide was from Fisher Chemicals. MOA-stilbene was a generous gift of Dr. G. von Jagow (Universitatsklinikum, Frankfurt, Germany). The sources of other chemicals were as indicated elsewhere (13-16).

Preparation of Bovine SMP-- SMP were prepared from bovine heart mitochondria in the presence of 1.5 mM ATP during sonication as described previously (16). Extraction of Q from SMP and determination of the Q content of SMP and Q-extracted SMP were the same as previously reported (13). Protein concentration was determined by the method of Lowry et al. (17).

Treatment of SMP with EFA-- Treatment of SMP with EFA and the activity measurements were done essentially as described before (18). Briefly, SMP at 10 mg/ml were incubated with 6.9 mM EFA at 0 °C for 30-40 min in a buffer containing 0.25 M sucrose and 10 mM Tris-HCl, pH 7.5. The succinate-cytochrome c reductase activity of SMP after EFA treatment was 5-10% of that of the untreated SMP. To prevent the inactivation of succinate dehydrogenase by EFA, either 10 mM malonate or 15 mM fumarate was added during incubation of SMP with EFA (18).

Assays-- Reduction of cytochrome b was monitored spectrophotometrically at 563 minus 575 nm in a buffer containing 0.25 M sucrose, 5 mM MgCl2, and 50 mM Tris-HCl, pH 8.0. SMP concentration was 1.1-1.14 mg/ml. Results were essentially the same at 565 minus 575 nm. Reduction of cytochromes c/c1 was monitored at 550 minus 540 nm. At this wavelength pair, contribution from the reduction of the b hemes was negligible. KCN, ascorbic acid (neutralized with NaOH), and TMPD were added at 10, 1.0, and 0.1 mM, respectively. Inhibitors of complex III were added from an ethanolic solution at the final concentrations indicated: antimycin A (2 µM), myxothiazol (4 µM), stigmatellin (4 µM), and MOA-stilbene (4 µM). 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. When the spectra of the cytochromes were recorded, addition of dithionite to SMP gave rise to a small increase in absorbance below 556 nm, which was not related to the absorbances of the cytochromes. Spectra recorded in the presence of dithionite were corrected for this absorbance increase. Assay temperature was 30 °C throughout.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Complete Reduction of bH and Incomplete Reduction of bL-- As stated above, the x-ray diffraction data of complex III crystals from three different groups (2-4, 12) do not show any Q located near bL and ISP (i.e. the crucial QO site of the Q cycle). Kim et al. (12) state that in their native complex III crystals the QO binding pocket is empty and that when these crystals were soaked with either Q-6 or Q-10 difference density maps had their highest peaks at the antimycin-binding site, i.e. near bH. Moreover, Rich and co-workers (19) have concluded from their recent studies that there is no detectable ubisemiquinone at the QO site, thus making the proposed chemistry of bifurcated QH2 oxidation at the QO site of the Q cycle hypothesis even more complicated. These recent data agree with our previous results (13, 14) as well as with those of Palmer and co-workers (20, 21) that Q is not an obligatory electron carrier between cytochrome b and ISP/c1. In the Q cycle terminology, the complex III inhibitors binding near bH (e.g. antimycin, funiculosin, and 2-nonyl-4-hydroxyquinoline-N-oxide) are referred to as Qi site inhibitors, and those binding near bL (e.g. myxothiazol, MOA-stilbene, mucidin, and stigmatellin) as QO site inhibitors. However, in view of the absence of evidence for a Q binding site near bL and ISP, we shall refer, as before (13), to these two sets of inhibitors, respectively, as inside or N-side inhibitors, and outside or P-side inhibitors.

As mentioned above, we have shown that addition of NADH or succinate to SMP pretreated with antimycin, KCN, and ascorbate (± TMPD) results in the rapid and complete reduction of bH. Only the reduction of bL became slow and incomplete under these conditions (14). These results do not agree with the Q cycle hypothesis, which requires that blocking of the Qi site by antimycin and prereduction of ISP/c1 by ascorbate should inhibit the reduction of both bH and bL by NADH or succinate. Treatment of SMP with myxothiazol, MOA-stilbene, or stigmatellin in the presence or the absence of KCN and ascorbate (± TMPD) resulted in a similar pattern of substrate reduction of bH and bL (14). Heme bH was rapidly reduced, whereas bL was only partially reduced. Furthermore, when SMP were pretreated with myxothiazol, KCN, and ascorbate and then succinate was added to reduce cytochrome b, subsequent addition of K3Fe (CN)6 to oxidize ISP/c1 also resulted in the rapid oxidation of the partially reduced bL, but bH remained in rapid electronic communication with the reduced Q pool and could not be oxidized by ferricyanide (14).

Fig. 1A shows an example of the partial reduction of cytochrome b when NADH was added to SMP treated with MOA-stilbene. However, as seen in Fig. 1B, no complex III inhibitor is needed to obtain a partial b reduction. In this experiment, the SMP were treated only with KCN, and no complex III inhibitor was added, but the pattern of b heme reduction upon addition of NADH was essentially the same as in Fig. 1A and the experiments described above. Cytochrome b reduction was partial (Fig. 1B), and what was not reduced was a portion of bL (Fig. 1C). In this experiment, the partial reduction of bL is referable to the fact that addition of NADH results first in the reduction of the high potential electron carriers on the oxygen side of cytochrome b, and this condition inhibits the complete reduction of bL. Thus, there are essentially two conditions in SMP that inhibit the complete reduction of bL by NADH or succinate: (i) prereduction of ISP/c1 and (ii) presence of a P-side complex III inhibitor. Neither condition inhibits the rapid and complete reduction of bH. As stated above, prereduction of ISP/c1 in the presence of an N-side inhibitor (e.g. antimycin) results in a very slow and partial reduction of bL by NADH or succinate (14). This also applies to prereduction of ISP/c1 in the presence of a P-side inhibitor, in the sense that the partial reduction of bL by NADH or succinate also becomes slower. However, the fact that both b hemes are rapidly and completely reduced by either succinate or NADH in antimycin-treated SMP (13) indicates that the partial reduction of bL under conditions (i) and (ii) is not an inherent feature of the system. Nor can we relegate the complete reduction of bL in the presence of antimycin and its incomplete reduction in the presence of a P-side inhibitor, respectively, to favorable and unfavorable Em changes effected by these inhibitors. According to Howell and Robertson (7), antimycin lowers the Em of bL by 20 mV, myxothiazol raises it by 10-30 mV, and stigmatellin causes no detectable change. The reason for the incomplete reduction of bL under conditions (i) and (ii) must therefore rest in the changes effected on complex III by the P-side inhibitors or by reduced ISP/c1.


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Fig. 1.   Incomplete reduction by NADH of the cytochrome b of complex III in SMP when the particles were pretreated with MOA-stilbene (A) or KCN (B). In A and B the reduction of cytochrome b was monitored at 563 minus 575 nm as described under "Experimental Procedures." The concentration of SMP here and in the experiments of Figs. 2-4 was 1.1-1.14 mg/ml. C, trace 1 shows the reduction of bH + bL at 563 nm and c + c1 at 553 nm after addition of NADH to KCN-treated SMP in B. Trace 2 is the absorbance difference between Na2S2O4-reduced minus NADH-reduced SMP in B, showing the spectrum of the remainder of bL with the major alpha  peak at 566 nm and shoulder due to the second alpha  peak at ~558 nm. MOA, MOA-stilbene.

Reverse Electron Transfer from ISP/c1 to b-- In SMP, cytochrome b can be reduced by reverse electron transfer via reduced ISP/c1 when the particles are treated with KCN and ascorbate plus TMPD (Fig. 2A). The reduction of cytochrome b is slow and partial, in keeping with the thermodynamically uphill nature of the process, and the electrons accumulate in the higher potential heme bH. As was shown previously, this reaction is strongly inhibited by antimycin and unaffected when the Q content of SMP was reduced 200-fold by extraction to <= 0.06 mol Q/mol cytochrome b or c1 (13). The rate of reverse electron transfer to b is also inhibited by myxothiazol and MOA-stilbene (81% inhibition, Fig. 2B) but to a much smaller extent than by antimycin (94% inhibition, Fig. 2C).3 We had shown earlier that in SMP complex III is inhibited by incubation of the particles with EFA and that the inhibition is completely reversed by subsequent treatment of SMP with hydroxylamine, indicating that EFA ethoxyformylates the imidazole nitrogen(s) of one or more essential histidyl residues (18). The inhibition appeared to block electron transfer between b and c1, and subsequent studies with complex III and isolated ISP showed that EFA modifies ISP and alters its EPR spectrum (22). Stigmatellin also alters the EPR line shape of ISP in complex III. However, pretreatment of complex III with EFA completely prevented the effect of stigmatellin on the EPR signal of ISP, and pretreatment with stigmatellin largely prevented the effect of EFA, suggesting overlapping sites of ISP modification by the two inhibitors (22). As seen in Fig. 2D, treatment of SMP with EFA inhibits (by ~70%) the initial rate of electron transfer from ascorbate/TMPD to b, further confirming that this reaction involves ISP as an intermediate electron carrier.


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Fig. 2.   Effects of MOA-stilbene (B), antimycin A (C), and EFA (D) on the reduction of cytochrome b by reverse electron transfer via ISP/c1 using ascorbate + TMPD as electron donors in KCN-treated SMP. The trace in A is the control experiment in the absence of a complex III inhibitor. Assay conditions, concentrations of inhibitors and conditions for treatment of SMP with EFA are given under "Experimental Procedures." The reduction of cytochrome b was monitored at 563 minus 575 nm. MOA, MOA-stilbene; Ant A, antimycin A; Asc, ascorbate.

As pointed out elsewhere (13), the stronger inhibition by antimycin than by myxothiazol (and MOA-stilbene, Fig. 2B) of reverse electron transfer from ISP/c1 to b does not agree with the Q cycle scheme. This is because in order for this reaction to be antimycin-sensitive in the Q cycle, the electrons from ISP would have to go to Q at the QO site. Then the reduced Q would have to become protonated from the P-side, move to the Qi site, reduce bH via the antimycin inhibition site, and deposit protons to the N-side. Clearly, this reverse proton translocation from the P-side to the N-side cannot take place in an unenergized system, and we have shown that the reverse electron transfer reactions shown in Fig. 2 and elsewhere (13) are unaffected in the presence of the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

Recent x-ray crystallographic data of complex III have shown that, unlike myxothiazol and MOA-stilbene, stigmatellin fixes the extramembranous domain of ISP near bL (3, 12). Also in our reverse electron transfer experiments, stigmatellin showed a different effect than myxothiazol and MOA-stilbene. As seen in Fig. 3A, treatment of SMP with stigmatellin slowed down the rate of b reduction by reverse electron transfer but considerably increased its extent (compare with Fig. 2A). As in the case of Fig. 2, the reduced b in the presence of stigmatellin was also bH (see below). It has been reported by Ohnishi, von Jagow, and co-workers (23, 24) that stigmatellin raises the Em of ISP in cytochrome reductase preparations by 250 mV. Because in our reverse electron transfer experiments, the extent of b reduction was much greater in the presence than in the absence of stigmatellin, we were concerned that stigmatellin may be promoting b reduction by a mechanism not involving ISP and an antimycin-sensitive pathway. However, as seen in Fig. 3 (B and C), b reduction by reverse electron transfer was inhibited when EFA- or antimycin-treated SMP were subsequently treated with stigmatellin, KCN, ascorbate, and TMPD. These results suggest that in the presence of stigmatellin the path of electrons from ISP/c1 to b remains unaltered. Therefore, the greater extent of b reduction in the presence of stigmatellin must somehow be a consequence of the increased proximity of the FeS cluster of ISP to bL, even though the reported 250 mV increased Em of ISP in the presence of stigmatellin is difficult to reconcile with the results. It should be pointed out, however, that these Em measurements were made in detergent-treated and fractionated preparations (23, 24). Whether the Em of ISP in intact SMP treated with stigmatellin is the same or different remains to be determined.


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Fig. 3.   Effects of EFA and antimycin A on the reduction of cytochrome b by reverse electron transfer via ISP/c1 in SMP treated with stigmatellin. A, reduction of b in SMP treated with stigmatellin, KCN, ascorbate and TMPD. B and C, effects of SMP treatment with EFA (B) and antimycin (C) on the promotion by stigmatellin of b reduction by reverse electron transfer. The experimental conditions were the same as in Fig. 2 and as described under "Experimental Procedures." Stig, stigmatellin; Asc, ascorbate; Ant A, antimycin A.

As seen in Fig. 4A, when b was reduced by addition to SMP of KCN, ascorbate, and TMPD, subsequent addition of antimycin resulted in partial reoxidation of the reduced b. The extent of reoxidation upon antimycin addition was greater when EFA-treated SMP was used (Fig. 4B), suggesting a balance between the rates of b reduction by reverse electron transfer and its reoxidation as a consequence of antimycin addition. Fig. 4C shows that the b heme (bH, see Fig. 4E) reduced by reverse electron transfer in the presence of stigmatellin was also reoxidized upon addition of antimycin. These results suggested that the recipient of electrons from bH is ubiquinone. Antimycin would inhibit electron flow from ISP/c1 to bH (via bL; Fig. 5A) and also would lower the Em of bH by 20-40 mV (7), resulting in the oxidation of bH by Q. This conclusion proved correct, because when the experiment of Fig. 4C was repeated with Q-extracted SMP, the reduction of bH via ISP/c1 was unaltered, but subsequent addition of antimycin caused only a small degree of reoxidation (Fig. 4D). In Fig. 4E, trace 1 shows the absorption peaks of the hemes reduced (bH heme, lambda max at 562 nm; c + c1 hemes, lambda max at 553 nm) in the experiment of Fig. 4C after addition to SMP of KCN, stigmatellin, ascorbate, and TMPD, and trace 2 shows the absorption of the heme (bH, lambda max at 562 nm) oxidized after addition of antimycin where indicated to the SMP of trace 1. The experiments of Figs. 2-4 were repeated with ascorbate plus phenazine methosulfate instead of TMPD, and the results were essentially the same. Davidson et al. (25) have shown that in Rhodobacter capsulatus mutants lacking ISP ascorbate + phenazine methosulfate reduce the c-type cytochromes but not cytochrome b.


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Fig. 4.   Reoxidation by antimycin of the cytochrome b (bH) reduced by reverse electron transfer via ISP/c1. A, control SMP. B, EFA-treated SMP. C, stigmatellin-treated SMP. D, Q-extracted SMP treated with stigmatellin. E, trace 1 shows the reduction of bH (lambda max at 562 nm) and c + c1 (lambda max at 553 nm) after the addition of ascorbate plus TMPD to SMP treated with stigmatellin and KCN (C). Trace 2 shows the difference in absorbance before minus after addition of antimycin A in C. EFA treatment and Q extraction of SMP were done as described under "Experimental Procedures." Other conditions were the same as in Fig. 3. Asc, ascorbate; Ant A, antimycin A; Stig, stigmatellin.


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Fig. 5.   A, proposed pathways of electrons and protons in complex III. This scheme is essentially the same as that in Ref. 13, except that here ISP (i.e. the extramembranous domain thereof) is shown to oscillate (heavy curved arrows) between bL and c1, in agreement with Refs. 3, 4, and 12. For simplicity, the suggestion of cross-electron transfer between complex III monomers at the level of ISP/c1 (3, 12) has not been included. B, depiction of the suggestion that when ISP and c1 are both prereduced by ascorbate (Asc) and TMPD, reduced ISP is repulsed by reduced c1, moves near bL, and interferes with its reduction via the Q pool. C, depiction of the paths of inhibition (wavy lines) by N-side (N) and P-side (P) inhibitors (see Ref. 13). The scheme also shows the suggestion that the combination of the two inhibitors (N+P) alters the conformation of cytochrome b and the binding site of Q/QH2 (wavy cytochrome b border), thereby inhibiting the off rate of ubiquinone. Large, hollow N and P refer, respectively, to the negative (matrix) and positive (cytosolic) sides of the mitochondrial inner membrane.

The data of Fig. 4 (C and D) confirm our previous results and conclusions that Q is not an obligatory electron carrier between cytochrome b and ISP (13). The fact that the x-ray crystallographic data of complex III from three different groups (2-4, 12) do not show the presence of Q near bL and ISP also agrees with our results. Another interesting point is suggested by the results of Fig. 4. It is well known that a combination of an N-side and a P-side inhibitor strongly inhibits the reduction of cytochrome b by NADH or succinate. However, as seen in Fig. 4C, the combination of stigmatellin and antimycin did not inhibit the rapid reoxidation of b. On the basis of our finding that the stoichiometric inhibitors antimycin, myxothiazol, MOA-stilbene, and stigmatellin appear each to inhibit three redox reactions of the bis-heme cytochrome b (Fig. 5C), we proposed that these inhibitors act by causing conformational changes in cytochrome b (13), a suggestion that agreed with the results of others (26, 27). We now suggest further that the conformation changes effected by the combination of an N-side and a P-side inhibitor retards the off-rate of Q bound to cytochrome b. In such a case, the reduction of b by NADH or succinate in SMP treated with, for example, antimycin and myxothiazol (or stigmatellin) would be inhibited, because the oxidized Q bound to b would not come off rapidly to be replaced by reduced Q. However, in the experiment of Fig. 4C, there would be sufficient oxidized Q bound to b to reoxidize the partially reduced bH.

As regards the inhibition of bL reduction when ISP/c1 are prereduced (Fig. 1B and Ref. 14), the following considerations may be of interest. As stated above, Zhang et al. (3) have suggested that the extramembranous domain of ISP moves during electron transfer between its proximal position near bL and its distal position near c1. These electron transfer steps may be graphically illustrated as follows, with the asterisked electron carriers indicating reduced species and the length of the lines between them indicating proximity: [bL* - ISP --- c1] right-arrow [bL - ISP* --- c1, left-right-arrows  bL --- ISP* - c1] right-arrow [bL --- ISP - c1*]. In other words, oxidized ISP would move near reduced bL, and reduced ISP would move near oxidized c1. However, by prereduction of ISP/c1 with ascorbate or as in the experiment of Fig. 1B where the oxidation of reduced ISP and c1 was prevented by inhibiting cytochrome oxidase with KCN, we create a situation that does not exist under normal electron transfer conditions. We propose that when both ISP and c1 are reduced, the reduced ISP is repulsed from the proximity of reduced c1 to reside near oxidized bL, i.e. [bL - ISP* --- c1*], and that the proximity of reduced ISP to bL inhibits the rapid and complete reduction of this heme. It is also possible that the P-side inhibitors binding near bL exert a similar effect on its reducibility. Our new results and conclusions agree with the tentative electron transfer scheme published earlier (13, 14). In Fig. 5, this scheme has been expanded to incorporate (i) the conclusion of Zhang et al. (Ref. 3; see also Ref. 12) regarding the movement of the extramembranous domain of ISP between bL and c1 for facilitated electron transfer; (ii) our proposal that when ISP and c1 are both reduced, reduced ISP is repulsed from the vicinity of reduced c1, moves near bL, and interferes with the rapid and complete reduction of bL via the Q pool; and (iii) our suggestion that the combination of an N-side and a P-side inhibitor alters the conformation of cytochrome b in such a manner that the off-rate of Q becomes inhibited.

    ACKNOWLEDGEMENT

We thank C. Munoz for the preparation of bovine heart mitochondria.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant DK-08126. This is publication number 11931-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.

1 In Refs. 5 and 6, the Em values of bH and bL are given as +40 mV and -90 mV, respectively.

3 The greater inhibition by antimycin of bH reduction by reverse electron transfer may be in part because antimycin lowers the Em of bH by 20-40 mV (7).

    ABBREVIATIONS

The abbreviations used are: ISP, iron-sulfur protein of complex III; SMP, bovine heart submitochondrial particles; b, cytochrome or heme b; c1, cytochrome or heme c1; Q, oxidized ubiquinone; QH2, reduced ubiquinone; EPR, electron paramagnetic resonance; MOA-stilbene, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; EFA, ethoxyformic anhydride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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