From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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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), 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 = ~ 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 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.
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.
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.
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
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.
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,
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 -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.
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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).
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.
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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 peak at 566 nm and shoulder due to the second
peak at ~558 nm.
MOA, MOA-stilbene.
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.
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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.
max at 562 nm;
c + c1 hemes,
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,
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 ( max at 562 nm) and
c + c1 (
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.
c1]
[bL - ISP*
c1,
bL
ISP* - c1]
[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.
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ACKNOWLEDGEMENT |
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We thank C. Munoz for the preparation of bovine heart mitochondria.
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FOOTNOTES |
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* 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.
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).
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ABBREVIATIONS |
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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.
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REFERENCES |
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