From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037
Received for publication, February 14, 2001, and in revised form, March 20, 2001
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ABSTRACT |
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Two sets of studies have been reported on the
electron transfer pathway of complex III in bovine heart
submitochondrial particles (SMP). 1) In the presence of myxothiazol,
MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated
with ascorbate and KCN to reduce the high potential components
(iron-sulfur protein (ISP) and cytochrome c1)
of complex III, addition of succinate reduced heme
bH followed by a slow and partial reduction of
heme bL. Similar results were obtained when SMP
were treated only with KCN or NaN3, reagents that inhibit
cytochrome oxidase, not complex III. The average initial rate of
bH reduction under these conditions was about
25-30% of the rate of b reduction by succinate in
antimycin-treated SMP, where both bH and
bL were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the
effect of the redox state of ISP/c1 on
cytochrome b reduction by succinate. 2) Reverse electron
transfer from ISP reduced with ascorbate plus phenazine methosulfate to
cytochrome b was studied in SMP, ubiquinone (Q)-depleted
SMP containing Quinol:cytochrome c (c2,
plastocyanin) oxidoreductases are energy-transducing enzyme complexes
found in the respiratory chains of all aerobic organisms as well as in
the electron transfer systems of chloroplasts and photosynthetic
bacteria (1-4). They differ in subunit composition and nature of their
quinol electron donor and their electron acceptor, but they are all
dimeric, contain a bis-heme cytochrome b, a unique binuclear
iron-sulfur protein and cytochrome c1 (or
f in chloroplasts), and pump protons with a H+/e
stoichiometry of 2. The x-ray crystal structure of the mitochondrial enzyme (complex III, bc1 complex) from cow,
chicken, and rabbit has been solved to about 3.0-Å resolution (5-7).
These structures show a linear sequence of electron carriers as
follows: Q Our own results (13-15), which could not be reconciled with the
Q-cycle hypothesis, are summarized below. All the experiments were done
with SMP to avoid the use of detergents, and the electron donors used
in forward electron transfer experiments were the respiratory chain
substrates, NADH and succinate.
1) Electron transfer from bH to
bL, and from bL to ISP,
is inhibited by either antimycin or myxothiazol (or HQNO, MOA-stilbene, or stigmatellin). These inhibitions are incomplete. The
bH and bL reoxidation
rates through the leak in the antimycin block were, respectively, 0.012 and 0.116 s 2) Consistent with the above results, it was shown that reverse
electron transfer from ISP/c1 (reduced with
ascorbate + TMPD) to b was inhibited not only by myxothiazol
(or stigmatellin or MOA-stilbene) but also by antimycin (15). The
Q-cycle hypothesis proposes that antimycin inhibits electron transfer
from bH to Q, for which there has been no direct
and unequivocal experimental support.
3) Similar results as in point 2 above were obtained with Q-extracted
SMP, in which the concentration of Q had been reduced 200-fold from
12.5 to This paper reports on the reduction in SMP of cytochrome b
by succinate in the absence and the presence of various complex III
inhibitors. It also reports on the features of reverse electron transfer from ascorbate + PMS via ISP to b in SMP,
Q-extracted SMP, and Q-replenished SMP. The results are in full
agreement with our previous findings and conclusions (13-15).
Chemicals--
Antimycin, sodium succinate, PMS, and FCCP were
obtained from Sigma; myxothiazol from Roche Molecular
Biochemicals; and stigmatellin and ascorbic acid from Fluka.
MOA-stilbene was a generous gift of Dr. G. von Jagow,
Universitatsklinikum, Frankfurt, Germany. The sources of other
chemicals were as indicated elsewhere (13-15).
Preparations of Bovine SMP, Q-depleted SMP, and Q-replenished
SMP--
SMP were prepared from bovine heart mitochondria in the
presence of 1.5 mM ATP (18) and Q-depleted SMP as described
previously (13). Ubiquinone-replenished SMP were prepared as follows.
To 52 mg of dry Q-depleted SMP were added 0.5 ml of spectrograde n-pentane and 1.0 µmol of Q10 in ~ 0.1 ml of n-pentane. The mixture was homogenized in a glass
homogenizer on ice, transferred to a test tube, and centrifuged for
1-2 min at top speed in a tabletop clinical centrifuge. The
supernatant was removed, and the pellet was dispersed in 0.5 ml of
n-pentane and centrifuged as before. The pentane-washed
pellet was dried under vacuum and suspended by homogenization in 0.5 ml
of a buffer containing 0.25 M sucrose and 10 mM
Tris acetate, pH 7.5. The readdition of Q10 in this manner
restored Assays--
Reduction of cytochrome b was monitored
at 563 minus 575 nm in a buffer containing 0.25 M sucrose,
5 mM MgCl2, and 50 mM Tris-HCl, pH
8.0. Other conditions are given in the figure and table legends. Absorbance changes were monitored by an SLM DW-2000 dual wavelength spectrophotometer. The data shown were collected and stored in a
computer on line to the spectrophotometer. Rapid mixing of SMP (plus
additives) with succinate for the data of Fig. 1 and Table I was
achieved using the stopped-flow attachment (MilliFlow) of Spectronic
Instruments. The light path of the observation chamber was 10 mm, and
the dead time, as specified, by MilliFlow was 2 ms. However, as stated
below, the overall system did not allow a reliable monitoring of
absorbance changes due to reduction of the chromophores under study
below 0.1 s.
Effect of Inhibitors on Reduction of Cytochrome b by
Succinate--
Fig. 1 shows
spectrophotometric traces of the progress of cytochrome b
reduction upon rapid mixing of succinate with SMP pretreated with
antimycin (trace A), myxothiazol (trace B), and
KCN (trace C). The extent of b reduction 0.2 s after mixing as estimated from these and similar experiments are
given in Table I. The 0.2-s absorbance
increases may represent the initial rates of cytochrome
b reduction, but the limitations of the spectrophotometer did not allow the initial rates up to 0.1 s to be reliably
recorded, and we prefer to report the results without any assumptions
and extrapolations. The data are averages of three to six experiments, using three separate preparations of SMP. To avoid complications, the
SMP were used as prepared without treatments for further activation of
succinate dehydrogenase. The NADH oxidase activity of SMP is several
times higher than that of their succinoxidase activity, but NADH could
not be used in these experiments, because complex III inhibitors like
myxothiazol and MOA-stilbene also inhibited complex I, albeit to a
smaller extent. These limitations aside, the data of Table I contain
the following significant features.
(i) It is seen that when SMP were treated with myxothiazol or
MOA-stilbene, or with antimycin plus ascorbate and KCN (where complete
prereduction of c + c1 was achieved),
succinate was still able to reduce b but to a smaller extent
than when they were treated with antimycin alone. As will be seen
below, antimycin, unlike the other inhibitors used in Table I, affords
complete reduction of both hemes bH and
bL.
(ii) It is seen that in the presence of KCN or NaN3, which
inhibit cytochrome oxidase, not complex III, the 0.2-s extent of cytochrome b reduction was somewhat less than, or roughly in
the same range as, the extent of b reduction in the presence
of myxothiazol or MOA-stilbene. The first entry in Table I also shows
that the steady-state extent of b reduction by our coupled
SMP in the absence of any inhibitor was in the same range as those
mentioned above and that addition of the uncoupler FCCP nearly
completely abolished this steady-state level of b reduction.
The results with KCN (or NaN3) as the inhibitor are
particularly interesting, because the pattern of b reduction
in the presence of KCN (or NaN3) is essentially the same as
that in the presence of the complex III inhibitors that bind between
bL and ISP. These results are shown in Fig.
2. The spectra of Fig. 2 show in the range 540-580 nm the reduction of SMP chromophores by
Na2S2O4 minus the reduction of
those chromophores by succinate in SMP treated with antimycin
(trace A), MOA-stilbene (trace B), stigmatellin (trace C), and KCN (trace D). As seen in
trace A (antimycin), the chromophores not reduced by
succinate and subsequently reduced by
Na2S2O4, were c + c1. In traces B (MOA-stilbene) and
C (stigmatellin) the chromophores not reduced by succinate
were c + c1 as expected, but also
bL with an Role of Ubiquinone in the Redox Reactions of Complex III--
In
Fig. 3, traces A and
C show the reduction of cytochrome b,
respectively, in SMP and Q-extracted SMP by reverse electron transfer
from ISP, which was reduced by ascorbate + PMS. As mentioned above, the
SMP and the Q-extracted SMP used, contained per mole of cytochrome
b or c1, respectively, 12.5 and
Returning to traces A and C of Fig. 3, it is seen
that there was essentially no difference in the rate and the extent of
bH reduction via ISP between the SMP containing
12.5 mol of Q/mol of complex III and the Q-extracted SMP containing
less than 0.06 mol of Q/mol of complex III. Traces B and
D of Fig. 3 show that pretreatment of the particles with
antimycin inhibited the rate and the extent of
bH reduction by reverse electron transfer from ISP, fully consistent with our previous finding that in forward electron transfer antimycin inhibits electron transfer from
bH to bL, and much less
effectively from bL to ISP (13). It is also seen
that antimycin inhibited the Q-extracted SMP (trace D) less
than the normal SMP, which is consistent with the finding of Tsai and
Palmer (17) that addition of Q to Q-extracted yeast complex III
increased the affinity of the enzyme for antimycin. In Fig. 3,
traces A and C show that addition of antimycin
after bH reduction had little or no effect. This
will be discussed in connection with Fig.
4 below.
Fig. 4 shows the reduction of bH by
ISP reduced by ascorbate + PMS in SMP (trace A),
Q-extracted SMP (trace B), and Q-extracted SMP replenished
with Q10 (trace C). Before addition of ascorbate + PMS, the particles were treated with 3 µM stigmatellin.
By comparison with traces A and C of Fig. 3, it
is seen that treatment of the particles with stigmatellin decreased the
rate and enhanced the extent of bH reduction. As
pointed out elsewhere, the increase in the extent of b
reduction in the presence of stigmatellin is surprising, because it has
been reported that in isolated complex III stigmatellin increases the
Em of ISP by 250 mV (24, 25). This increased
reduction of bH in the presence of stigmatellin does not happen when the inhibitor is myxothiazol, MOA-stilbene, or
ethoxyformic anhydride, which ethoxyformylates a histidine residue of
ISP (15, 26, 27).3 However,
the important point is that the slower and the greater extent of
bH reduction in the presence of stigmatellin
clearly show the absence of any difference in the progress of
bH reduction between SMP (trace A),
Q-extracted SMP (trace B), and Q-replenished SMP
(trace C).
A second point of interest in Fig. 4 is what happened when antimycin
was added to the reaction mixtures after the reduction of
bH had reached near completion. It is seen in
trace A that addition of antimycin resulted in near complete
oxidation of bH. This antimycin-induced
reoxidation of bH happened only to a small extent in trace B with Q-extracted SMP, but was restored to
a considerable extent with the Q-replenished SMP (trace C).
The Q-cycle hypothesis specifies that antimycin inhibits electron transfer from bH to Q (1-4). There has been no
experimental proof of this assumption. However, the results of Fig. 4
clearly demonstrate that antimycin does not inhibit electron transfer
from bH to Q. Rather it promotes the oxidation
of bH by Q, possibly because antimycin lowers
the Em of bH by
20-40 mV (23). The reason that in Fig. 3 addition of
antimycin did not cause bH reoxidation is that
under those conditions bH was kept reduced via
the leak in the antimycin block. In Fig. 4, the combination of
stigmatellin and antimycin caused a severe inhibition of
bH reduction. This combined with the lowering of
the Em of bH by antimycin resulted in its oxidation by the Q pool.
Conditions Affecting Cytochrome b Reduction--
An examination of
Table I shows essentially three sets of data on the effect of
inhibitors on cytochrome b reduction by succinate. (i) In
SMP treated with antimycin alone, both hemes bH
and bL were reduced with a combined
The results with antimycin, myxothiazol, or MOA-stilbene as the sole
complex III inhibitor, or with antimycin + myxothiazol, can be
rationalized in terms of the Q-cycle, but the results with KCN or
NaN3, or with antimycin added to SMP containing prereduced ISP/c1, cannot be. If the Q-cycle mechanism were
operative in the latter condition, one would expect (a)
reduced ISP/c1 to disallow the single-electron
oxidation of QH2 at the QP site of the Q-cycle and the formation of Q
The results with KCN and NaN3 are even more at variance
with expectations from the Q-cycle hypothesis. Here there were no complex III inhibitors, and there were several high potential electron
sinks (SMP contain twice as much cytochrome oxidase as complex III) to
afford multiple single-electron oxidations of QH2 at the
QP site, thereby producing multiple Q Role of Ubiquinone in Complex III--
Figs. 3 and 4 show clearly
that the reduction of cytochrome b by reverse electron
transfer from reduced ISP is unaffected by extraction of Q from SMP to
the extent that its concentration in SMP is lowered from 12.5 to
As mentioned above, the Q-cycle hypothesis states that antimycin
inhibits electron transfer from bH to Q at the
QN site, for which there has been no direct experimental
proof. That this assumption is also incorrect is clear from the results
of Fig. 4, which show that addition of antimycin promotes the oxidation
of bH by Q. The reason for the antimycin effect
is that it lowers the Em of
bH by 20-40 mV (23) and, in combination with
stigmatellin, inhibits further bH reduction by
reverse electron transfer via ISP. As a result reduced
bH is oxidized by Q, which binds near this heme.
That the electron acceptor from reduced bH is Q
is supported by the data of traces B and C of
Fig. 4, which show that this antimycin-promoted
bH oxidation happens only to a very small extent
in Q-extracted SMP (trace B), but to a considerable extent
when Q10 is added to the Q-extracted SMP.
In summary, the results presented here are in complete agreement with
our previous reports and conclusions as depicted in Fig.
5, which shows a linear path of electron
transfer from QH2
Finally, the electron transfer pathway of Fig. 5 agrees with recent
rapid kinetic measurements of cytochromes b and
c1 reduction. Using purified bovine complex III
and a photoreleasable decylubiquinol as substrate, Chan and co-workers
(37) have shown that cytochrome b is reduced with a rate
constant of 270 s0.06 mol of Q/mol of complex III, and Q-replenished
SMP. The results showed that Q was not required for electron transfer
from ISP to b, a reaction that was inhibited by antimycin
(also by myxothiazol or MOA-stilbene as reported elsewhere). It was
also shown that antimycin did not inhibit electron transfer from
b (bH) to Q, in clear contrast to
the assumption of the Q-cycle hypothesis regarding the site of
antimycin inhibition.
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bH
bL
ISP
c1.1
The bis-heme cytochrome b is the only transmembranous
redox protein of complex III, with heme bL
located near the cytoplasmic surface of the enzyme, and heme
bH located 21 Å away from
bL toward the matrix side. ISP and
c1 are largely extramembranous on the
cytoplasmic side, where the extramembranous domain of ISP containing a
[2Fe-2S] cluster oscillates between bL and
c1 to facilitate rapid electron transfer.
Ubiquinone (Q) binds near bH. In the Q-cycle
hypothesis, first proposed by Mitchell in 1975 (8-10), two separate
Q-binding sites are conceived, a Q reduction site (Qi or
QN site) near bH and a
QH2 oxidation site (QO or QP site)
between bL and ISP. Intensive research in the
intervening years has not produced any direct evidence for the presence
of Q or QH2 between bL and ISP. The x-ray crystal structures of the complex III of three different species
as reported by three different groups also have failed to locate Q at
this site, even when excess Q6 or Q10 was
diffused into bovine complex III crystals
(11).2 However, kinetic
results and EPR spectra of ISP in Q-extracted and Q-replenished
preparations have been interpreted in favor of the presence of one or
two molecules of Q (with 10-fold different binding affinities)
functioning between bL and ISP (1-4, 12).
1, and through the leak in the
myxothiazol block were, respectively, 0.0033 and 0.046 s
1, indicating that myxothiazol is the
stronger inhibitor and that electron transfer from
bH to bL is the more
sensitive step to either inhibitor (13). Because in these experiments
reduced bL was oxidized through the leak by
these inhibitors 10-12 times faster than reduced
bH, the results suggested that forward electron transfer in complex III proceeds from bH to
bL (13). There is, however, no experimental
support for the Q-cycle view that forward electron transfer proceeds
from bL to bH.
0.06 mol of Q/mol of cytochrome b or
c1, and the rate of forward electron transfer
from succinate and NADH to cytochrome c had been reduced by
97% and >99%, respectively (13, 15). These results, (i.e.
antimycin-sensitive reverse electron transfer from
ISP/c1 to b in the Q-extracted SMP),
which suggested noninvolvement of Q as an obligatory electron carrier between ISP and b, predated the inability of the three
different laboratories to find Q between ISP and b in the
x-ray crystal structures of several different preparations of complex
III from three different species (5-7, 11). The contention of the
proponents of the Q-cycle hypothesis is that antimycin, which binds in
complex III crystals near bH, and stigmatellin,
myxothiazol, and MOA-stilbene, which bind in complex III near
bL and ISP, are all structural analogues of Q
(1-4, 16). There is, however, no clear experimental support for this
contention, because none of these inhibitors have been shown to compete
with Q for binding to complex III, whereas there is published evidence
that the presence of Q favors antimycin binding (17).
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85% of the succinoxidase activity of the SMP. Protein
concentration was determined by the method of Lowry et al.
(19).
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Fig. 1.
Effects of inhibitors on the reduction of
cytochrome b by succinate. SMP were treated with
antimycin (trace A), myxothiazol (trace B), KCN
(trace C), or antimycin and myxothiazol (trace
D), then mixed with sodium succinate in a stopped-flow attachment
of the SLM DW-2000 dual wavelength spectrophotometer. The progress of
cytochrome b reduction was monitored at 563 minus 575 nm.
For other details, see Table I and "Results"
Summary of the effects of inhibitors on the reduction by succinate of
cytochrome b in SMP
1% by volume. The SMP
concentration of cytochrome b, as estimated from the
absorbance difference at 563 minus 575 nm of
Na2S2O4 reduced minus oxidized samples, using
an
= 20 mM
1 b heme (20), was
0.40-0.43 nmol/mg of protein.
A0.2 s, absorbance
increase at 563 minus 575 nm due to cytochrome b reduction
0.2 s after rapid mixing of succinate with SMP ± inhibitors
(see Fig. 1).
peak at ~566 nm. In trace
D (KCN, which inhibits cytochrome oxidase) succinate had reduced a
major portion of c + c1. However, as
in traces B and C, heme bL
was not reduced by succinate but by the subsequence addition of
Na2S2O4. As was shown elsewhere,
the same pattern of b reduction was obtained when succinate
was added to SMP pretreated with antimycin plus ascorbate and KCN (14).
In this and in conditions of traces B, C, and
D of Fig. 2, bH was reduced by
succinate as shown in Fig. 1 and Table I, and the reduction of
bH was followed by a much slower and partial
reduction of bL (14, 15). Indeed, in
traces B and C of Fig. 1, the reduction of
bH was largely completed in <0.2 s., and the
subsequent slow absorbance increase was due mainly to the partial
reduction of bL. What these biphasic
b reductions have in common is the absence of
oxidized ISP/c1 in electronic communication with bL. MOA-stilbene,
myxothiazol, and stigmatellin inhibit electronic communication between
bL and ISP; KCN does not inhibit complex III,
but by inhibiting cytochrome oxidase allows
ISP/c1 to become reduced (the 0.2-s extent of
c + c1 reduction by succinate in the
presence of KCN was about the same as that shown for b
reduction in Table I for the experiment in the presence of
NaN3). Treatment of SMP with antimycin alone allows both
b hemes to become reduced by succinate, but prereduction of
ISP/c1 in antimycin-treated SMP by ascorbate
plus KCN inhibits rapid bL reduction, allowing
only bH to be initially reduced (Table I).
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Fig. 2.
Difference spectra of chromophores reduced by
Na2S2O4 minus those reduced by
succinate in SMP treated with inhibitors. SMP at 1.08 mg/ml were
treated with 2 µM antimycin (A), 4 µM MOA-stilbene (B), 4 µM
stigmatellin (C), or 10 mM KCN (D).
Then sodium succinate at a final concentration of 6 mM was
added, and the absorbance of the sample was recorded in the wavelength
range shown at a scan rate of 1.0 nm/s. The data were
stored in a computer on line to the spectrophotometer. Then a few
grains of Na2S2O4 were thoroughly
mixed into each reaction mixture and the absorbance of the mixture
recorded as before. The traces shown are the difference
absorbencies of the Na2S2O4 reduced
minus the succinate reduced spectra. Assay temperature was 30 °C
here and in the experiments of Figs. 3 and 4.
0.06 mol of Q, and as compared with SMP the NADH- and
succinate-cytochrome c reductase activities of the
Q-extracted SMP, had been reduced by 99 and 97%, respectively (13). It
should also be mentioned here that Daldal and co-workers (21) have
shown that when added to chromatophore membranes of a Rhodobacter
capsulatus mutant lacking ISP, ascorbate + PMS reduced cytochrome
c1, but not cytochrome b.
Furthermore, as was shown elsewhere with ascorbate + TMPD, the
b heme reduced by reverse electron transfer is
bH (Em
+90 mV), whose
Em is 120 mV more positive than that of
bL (Em
30 mV) (22, 23).
Here, we have used PMS instead of TMPD, because PMS affords a faster
reduction of ISP by ascorbate; as a result the extent and the rate of
bH reduction via ISP are somewhat greater.
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Fig. 3.
Reduction of cytochrome b by
reverse electron transfer from reduced ISP and the inhibition of this
process by antimycin in SMP (traces A and
B) and in Q-depleted SMP (traces C
and D). SMP and Q-depleted SMP in
sucrose-Tris buffer were treated with 10 mM KCN, 1.0 mM ascorbic acid (neutralized with NaOH), 2 µM antimycin where indicated, and 0.05 µM
PMS. Because the extraction of Q from SMP involved removal of sucrose
by washing of the particles, lyophilization, and eight extractions with
n-pentane, it was felt that instead of protein
concentration, the relative SMP concentrations here and in Fig. 4
should be stated as their Na2S2O4
reducible cytochrome b levels (see Table I). These
concentrations were 0.59 µM cytochrome b in
traces A and B and 0.50 µM
cytochrome b in traces C and D.
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Fig. 4.
Reduction of cytochrome b by
reverse electron transfer from reduced ISP in stigmatellin treated SMP
(trace A), Q-depleted SMP (trace B),
and Q-replenished SMP (trace C). Concentrations
of KCN, ascorbate, PMS, and antimycin were the same as in Fig. 3.
Stigmatellin concentration was 3 µM. Concentrations of
SMP, Q-depleted SMP, and Q-replenished SMP were in terms of cytochrome
b, respectively, 0.58 µM, 0.60 µM, and 0.76 µM.
DISCUSSION
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ABSTRACT
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A0.2 s of 28-30. (ii) In SMP treated with
antimycin plus myxothiazol (or antimycin plus MOA-stilbene), the
reduction of both b hemes by succinate was strongly
inhibited, in agreement with previous findings (1-4, 13). (iii) In SMP
treated with myxothiazol, MOA-stilbene, KCN, or NaN3, or in
ISP/c1 prereduced SMP treated with antimycin,
only heme bH was initially reduced with an
average
A0.2 s of 8 ± 1.5.
A0.2 s similar to when the inhibitor was myxothiazol.
A0.2 s at best in the same range as when the
SMP were treated with a QP site inhibitor such as
myxothiazol or MOA-stilbene. As stated above, the common feature among
the conditions that greatly inhibit bL
reduction, but allow bH reduction (apparently at
a rate lower than that of bH reduction by SMP
treated with only antimycin), is the absence of oxidized
ISP/c1 in electronic communication with
bL. Trumpower and co-workers (28, 29) have shown
recently that lowering the Em of ISP by mutation in
yeast decreases the initial rate of cytochrome b reduction by menaquinol in purified complex III. These results together with the
data of Table I suggest that the redox state of
bL, which would be affected by the
Em of ISP, influences the reduction of
bH by the nearby QH2. In other
words, for rapid reduction of bH by
QH2, bL must be maintained at a high
oxidation state, which ISP with an Em of +280 mV can
achieve. As the Em of ISP is lowered, the reduced
level of bL increases, and the rate of
bH reduction by QH2 drops. The same
result is obtained when ISP is prereduced, its electronic communication with bL is largely inhibited by myxothiazol (or
MOA-stilbene), or its oxidation is prevented by inhibition of
cytochrome oxidase with KCN or NaN3.
0.06 mol/mol of complex III. As stated above, Daldal and co-workers
(21) have shown that in chromatophore membranes from a R. capsulatus mutant lacking ISP, ascorbate + PMS (which were used in
our studies here as electron donors in the experiments of Figs. 3 and
4) reduced cytochrome c1, but not cytochrome
b. Furthermore, Fig. 3 shows that 2 µM antimycin inhibited reverse electron transfer from ISP to
bH, which agrees with our finding that antimycin
strongly inhibits electron transfer between bL
and bH (13, 30). The inhibition of this reaction
by antimycin, as well as by myxothiazol and ethoxyformic anhydride,
which modifies ISP (27), further supports the contention that in the
experiments of Figs. 3 and 4 heme bH is reduced
via bL by reverse electron transfer from reduced
ISP. The fact that Q is not required for this process agrees with the
inability of three independent groups to find Q between ISP and
bL in the x-ray diffraction patterns of complex
III from three different species (5-7). It has been stated, without
any experimental support, that after oxidation of QH2 at
the QP site of the Q-cycle the oxidized Q leaves this site
(1). This hypothetical explanation rationalizes the inability of
investigators to find Q between ISP and bL in
the x-ray diffraction patterns of complex III, even when Q6
or Q10 was diffused into the crystals (5-7, 11). However, the assumption that oxidized Q leaves the QP site is
totally inconsistent with the data of Figs. 3 and 4, and with the well
documented earlier results regarding ATP-driven reverse electron
transfer in mitochondria from the level of cytochrome c to
NAD (Ref. 31 and see also Ref. 32). Clearly, if Q were an obligatory
electron carrier between ISP and cytochrome b and absent in
the oxidized form at this location, such reverse electron transfer via
complexes III and I to NAD could not take place.
bH
bL
ISP
c1.
Electron transfer between bH and
bL is strongly inhibited by antimycin, which
binds to the N side of cytochrome b near heme
bH, and by myxothiazol, MOA-stilbene, and
stigmatellin, which bind to the P side of cytochrome b
between bL and ISP (13, 30). In agreement with
the lack of any direct and unequivocal literature evidence to the
contrary, our results show the absence of a requirement for Q for
electron transfer between ISP and cytochrome b. Obligatory
single electron transfer from QH2 on the N side to
bH results in the formation of Q
View larger version (15K):
[in a new window]
Fig. 5.
The electron transfer pathway of complex
III. Curved arrows indicate the oscillation of the
extramembranous domain of ISP between bL and
c1 (1, 6). Wavy lines show the
reactions inhibited by P side (myxothiazol, MOA-stilbene, stigmatellin)
and N side (antimycin, HQNO) inhibitors (13). Dashed arrows
are hypothetical proton translocation steps. Hollow N and
P indicate the negative (matrix) and the positive
(cytoplasmic) sides of complex III, respectively.
1, and cytochrome
c1 with a rate constant of 60 s
1, 4.5 times slower. In addition, using
purified yeast complex III and menaquinol as substrate, Trumpower and
co-workers (28, 29) have shown that cytochrome b is reduced
at an initial rate of 58 s
1, but cytochrome
c1 at a rate of 8.4 s
1, 7 times slower. Attempting to
rationalize their unexpected results in terms of the Q-cycle
hypothesis, Chan and co-workers (37) state that the much slower rate of
cytochrome c1 reduction relative to cytochrome
b reduction indicates the slow rate of electron transfer
from ISP to c1. However, Millett and co-workers
(38) have shown that in purified bovine complex III electron transfer from ISP to cytochrome c1 occurs with a rate
constant of 16,000 s
1.
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ACKNOWLEDGEMENTS |
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We thank Calixto Munoz for the preparation of bovine heart mitochondria and Thu Ho for expert technical assistance.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant DK08126. This is publication number 13697-MEM from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 858-784-8092;
Fax: 858-784-2054; E-mail: hatefi@scripps.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101446200
2 Kim et al. (11) stated that the QO pocket was empty in their complex III crystals. Nevertheless, difference density maps indicated that diffused Q6 or Q10 was localized only near bH.
3 Preliminary experiments have suggested that in SMP from Saccharomyces cerevisiae, the extent of cytochrome b reduction by ascorbate + PMS was also little affected by stigmatellin (A. Matsuno-Yagi and Y. Hatefi, unpublished data).
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ABBREVIATIONS |
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The abbreviations used are: Q and QH2, respectively, oxidized and reduced ubiquinone; Q10, ubiquinone containing 10 isoprenoid units at position 6 of the benzoquinone ring; bH and bL, respectively, the high potential and the low potential b hemes of complex III cytochrome b; ISP, iron-sulfur protein; c1, cytochrome c1; SMP, bovine-heart submitochondrial particles; HQNO, 2-n-heptyl-4-hydroxyquinoline-N-oxide; MOA-stilbene, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; PMS, phenazine methosulfate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; EPR, electron paramagnetic resonance; Asc, ascorbate.
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