(Received for publication, October 6, 1995; and in revised form, December 19, 1995)
From the
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 and b
by NADH or succinate
is inhibited when the particles are treated with both antimycin and
myxothiazol. Each inhibitor alone allows reduced b
and b
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
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
is also inhibited by any one of these reagents. These inhibitions
are also incomplete, and reduced b
is oxidized
through the leaks allowed by these inhibitors at least 10 times faster
than reduced b
. Heme b
can be
reduced in SMP via cytochrome c
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
as well. Reverse electron transfer to b
and b
is inhibited
partially by myxothiazol, much more by antimycin. Ascorbate + TMPD
also reduce b
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
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.
Ubiquinol-cytochrome c oxidoreductase (complex III, bc 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
,
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
, 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
(or cytochrome f in chloroplasts). In mitochondria, the
bis-heme cytochrome b is largely membrane-intercalated and
contains a low potential heme b
(E
-90 mV) near the outside surface of the inner
membrane and a high potential heme b
(E
+40 mV) at the center of the
membrane about 20 Å away from heme b
(5, 6, 11) . The
binuclear iron-sulfur protein (often referred to as the Rieske
iron-sulfur protein and here abbreviated as ISP) (
)has a
high E
(+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
has an E
=
+230 mV(12) . ISP and cytochrome c
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
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
and b
) and a high potential center (ISP and cytochrome c
).
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 to ISP and then to
cytochromes c
and c; (ii) a second
electron transfer from the one-electron oxidized quinol
(Q
) to b
(oxidation of
QH
at this site (Q
) involves the
translocation of 2H
outward); (iii) electron transfer
from b
to b
; (iv) electron
transfer from b
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
. The net result in two cycles is transfer of two
electrons from QH
out to cytochrome c and
translocation of 4H
across the membrane. Two sets of
inhibitors are known, which are classified as Q
and Q
site
inhibitors(5, 16, 17) . Myxothiazol (and
other inhibitors containing a
-methoxyacrylate moiety),
stigmatellin, mucidin, and undecylhydroxydioxobenzothiazole are
considered inhibitors of QH
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 Q
site
inhibitor (e.g. myxothiazol) are added together, then the
reduction of b
and b
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
-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
and yield Q
, which is the electron
donor to b
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
to the
high potential center, two protons are translocated from QH
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
and heme b
is b
.
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 undergoing oxidation 10 times faster than reduced b
(20) . These results are difficult to
reconcile with the Q cycle, because antimycin appears to inhibit the
oxidation of both b
and b
,
and the faster oxidation of b
than b
requires that the path of electrons from these
hemes through the leak in the antimycin block to be either separate or b
b
acceptor,
with the first step being considerably slower than the second. Neither
possibility agrees antimycin with the path being b
b
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.
It is well known that an I-side or an O-side
inhibitor blocks the oxidation, but not the substrate-induced
reduction, of b and b
.
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
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
and b
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
S
O
. In agreement with previous
results(3, 5, 30) , it is clear from Fig. 2that there appear to be two pathways for reduction of b
and b
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
and b
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 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
S
O
. The assay
temperature was 30 °C in this and subsequent
experiments.
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 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 and b
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). (
)The reoxidation of b through the leak in the
stigmatellin block showed two distinct phases, with the decay of
reduced b
beginning to plateau before the
reoxidation of b
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
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
undergoing reoxidation at
least 10 times faster than b
.
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 was
faster than that shown in Fig. 3A, which is
understandable because of the strong inhibitory effect of rotenone on
complex I.
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.
In view of these considerations, it was of interest to see
whether b could be reduced via c 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
0.025 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
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, as in Fig. 5, and both b
and b
after
addition of Na
S
O
(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
to Q-depleted yeast complex III promotes antimycin
binding. Because of the presence in Q-extracted SMP of
0.025 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
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, 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.
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 and b
via
cytochrome c
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
/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
-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
/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
/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
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
/ISP to Q then to b be sensitive to
either a Q
site or a Q
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 undergoing reoxidation much
faster than b
. These results indicate that the
faster reoxidation of b
cannot take place via the
slower reoxidation of b
. Therefore, the two hemes
would have to undergo reoxidation by separate paths (e.g.
b
via the Q
site and b
via the Q
site, see Fig. 1) or the reoxidation
path would have to be b
b
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
oxidation via the Q
site be
inhibitable by antimycin? By contrast, the path b
b
acceptor would agree with
the finding that reverse electron transfer from c
/ISP to b can be inhibited by either
antimycin or myxothiazol. It could be argued that reduced b
could still be oxidized at a faster rate via
reduced b
because of the more negative potential
of b
. Then, after oxidation of b
, reduced b
would decay at a
slow rate. In such a case, the oxidation rate of b
would have to be a measure of the leak allowed by antimycin or
myxothiazol. The b
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
through the leaks
allowed by these inhibitors should occur at the same rate, which it is
not. The reoxidation rate of b
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
is oxidized via b
and that antimycin and myxothiazol exert their
strongest, but different, inhibitory effects on electron transfer from b
to b
.
The above
considerations point to two problems with the Q cycle scheme. One is
that Q is placed between the b/b
and the ISP/c
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
site and by the O-side
inhibitors at the Q
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 and b
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
undergoing
oxidation much faster than reduced b
. 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
/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
to b
. 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 to b
,
next from b
to ISP/c
, and
least from QH
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
-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. If QH
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
. 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
is very likely oxidized via b
. One may, therefore, ask whether this single b oxidation path involves Q or leads directly to
ISP/c
.
(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 and b (see also (48) and (49) ). The fact that in Q-depleted bovine SMP
reduction of b
by ascorbate + TMPD is
inhibited by antimycin is also consistent with the data of Fig. 3regarding the antimycin sensitivity of the reoxidation of b
and b
and agrees with the
possibility that the electron path from ascorbate + TMPD to b
is c
/ISP
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
) is sufficient to give the result shown in Fig. 6A. However, the fact that the rate and extent of b
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
, not cytochrome b.
The results presented here are summarized in Fig. 7. This minimal scheme assumes that QH 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
and I
) bind to
cytochrome b. Neither inhibitor alone inhibits the oxidation
of QH
, but the presence of antimycin results in
destabilization of
Q
(32, 33, 34, 35) .
The oxidation of QH
results in outward translocation of
2H
. The scheme does not specify the destinations of
the first and the second electrons from QH
, i.e. as regards which heme, b
or b
, is reduced by which electron. Heme b
is oxidized via b
, with
either an I-side or an O-side inhibitor (I
or
I
) severely inhibiting electron transfer from b
to b
and at least 10 times
less severely from b
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
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
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
/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
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
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 and I
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.