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INTRODUCTION |
Although the protonmotive Q cycle mechanism of the cytochrome
bc1 complex is generally well understood (1-3),
the redox behavior of cytochrome b during pre-steady state
reduction of the bc1 complex is not fully
understood. Cytochrome b reduction is triphasic, consisting
of a rapid partial reduction phase, a partial reoxidation phase, and a
slow rereduction phase. This behavior is puzzling, because the
reoxidation phase occurs while reduced substrate is still available,
and continued reduction of cytochrome b would be expected.
Previous examinations of the pre-steady state reduction kinetics of the
bc1 complex were limited to single wavelength
kinetics, and the spectral data, when collected, extended over time
ranges that were long relative to the half-times of the reactions
(4-9). The substrates used in these studies, succinate, duroquinol,
trimethylquinol, and ubiquinol, have relatively high redox potentials
and reduce only a small percentage of cytochrome b. This is
of concern because the high redox potential may predispose these
substrates to oxidize cytochrome b and thus introduce
artifacts into the pre-steady state kinetics in the absence of a low
potential reductant.
Several explanations for the triphasic reduction have been put forth.
One proposal is that ubiquinone formed at center P is not in rapid
equilibration with the quinone pool and oxidizes cytochrome
b at center N (5, 10). Crystal structures of the mitochondrial cytochrome bc1 complexes show a
pear-shaped and dimeric integral membrane protein that extends ~80 Å into the matrix and ~30 Å into the intermembrane space (11, 12).
There are two large cavities within the bc1
dimer that link center P of one monomer to center N of the second
monomer. The presence of these cavities may allow ubiquinol or
ubiquinone to exchange between these two sites without having to
diffuse into the membrane (11). It has also been suggested that the
decreased absorbance at the cytochrome b wavelength is not
true oxidation but rather spectral overlap of cytochrome
c1 that gives the appearance of cytochrome
b oxidation (8).
We have examined the pre-steady state reduction kinetics of the
Saccharomyces cerevisiae bc1 complex
by menaquinol using rapid scanning stopped flow spectroscopy.
Menaquinol (Em7 = 
74 mV; Ref. 13) reduces a
larger percentage of cytochrome b than does ubiquinol
(Em7 = +90 mV; Ref. 14) and rapidly reduces
cytochrome b through center P or center N (15). Rapid
scanning stopped flow spectroscopy allows the simultaneous monitoring
of the time course of reduction of cytochrome b and
c1 in a single reaction and the examination of
absorbance spectra at any time point during the reaction.
Our results show that triphasic reduction results from a bona
fide reoxidation and rereduction of cytochrome b and
that endogenous ubiquinone is responsible for triphasic reduction. We
propose that ubiquinone at center N within the oxidized
bc1 complex is responsible for the partial
reduction and reoxidation phases of the triphasic reduction by rapidly
oxidizing cytochrome b that has been reduced through center
P. Equilibration of the first electron from quinol oxidation through
center P between cytochrome bH and ubiquinone at
center N causes the partial reduction phase. When a second electron
from center P reduces ubisemiquinone to ubiquinol there is a partial
reoxidation of cytochrome b. After both the iron-sulfur
protein and cytochrome c1 are reduced,
menaquinol cannot be oxidized at center P, and menaquinol rereduces
cytochrome b through center N. The duration of the
reoxidation or lag phase is dependent upon the amount of ubiquinone
available to oxidize cytochrome b at center N after it has
been reduced by menaquinol. After the ubiquinone pool has been reduced
by a transhydrogenase reaction at center N, cytochrome b
remains reduced, and this causes the rereduction phase.
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EXPERIMENTAL PROCEDURES |
Materials--
Dodecyl maltoside was obtained from Roche
Molecular Biochemicals. DEAE-Biogel A was obtained from Bio-Rad.
Antimycin, diisopropyl fluorophosphate,
PMSF,1
2,3-dimethoxy-5-methyl-6-decyl benzoquinone ("decyl CoQ") and menaquinone were purchased from Sigma. Stigmatellin was purchased from
Fluka Biochemika. Yeast extract and peptone were from Difco. The yeast
coq2 mutant was obtained from Dr. Catherine Clarke (UCLA). The 2,3-dimethoxy-5,6-dimethyl benzoquinone was obtained from
Dr. Chang-an Yu (University of Oklahoma).
Preparation of Menaquinol--
A 100 mM stock
solution of menaquinone was prepared in ethanol. From this stock a 2 mM solution of menaquinone was prepared by dilution into 50 mM potassium phosphate, pH 6.0, + 250 mM
sucrose, 0.2 mM EDTA, 1 mM NaN3,
0.1% bovine serum albumin. Because menaquinone is insoluble in aqueous
buffers it precipitated from the solution. The menaquinone was reduced
with a 2-fold molar excess of sodium borohydride. As the menaquinone
was reduced it became soluble in the aqueous buffer and was mixed until
it was completely solubilized and no more bubbles were released. A
fresh menaquinol solution was prepared prior to each kinetic
experiment, kept under anaerobic conditions, and diluted into degassed
buffer immediately prior to its use. Control experiments established
that the reduction of the cytochrome bc1 complex
is caused by menaquinol and not by any residual sodium borohydride.
The ubiquinone analogue, 2,3-dimethoxy-5,6-dimethyl benzoquinone, was
dissolved directly in 50 mM potassium phosphate, pH 6.0, 250 mM sucrose, 0.2 mM EDTA, 1 mM
NaN3, 0.1% bovine serum albumin and reduced with
borohydride. This quinol was used as substrate for the experiments in
Fig. 4
Purification of Cytochrome bc1 Complex--
Two
pounds of Red Star baker's yeast were washed once with distilled water
and once with disruption buffer (100 mM Tris, 250 mM sorbitol, 5 mM MgCl2, 150 mM potassium acetate, 1 mM dithiothreitol, pH
8.0). The washed yeast cells were resuspended by adding 40 ml of
disruption buffer and frozen by slowing pouring the suspension as a
thin stream into liquid nitrogen. The frozen yeast were blended in
liquid nitrogen in a stainless steel Waring blender for a total of 5 min at 1-min intervals. Additional liquid nitrogen was periodically added to prevent the thawing of the cells.
The lysed cell powder was thawed under warm water with the addition of
1 mM diisopropyl fluorophosphate and 1 mM PMSF.
The cell debris was sedimented at 3000 × g for 10 min,
and the pellet was washed once in disruption buffer and sedimented at
3000 × g for 10 min. The supernatants were combined,
and the mitochondrial membranes were sedimented at 20,000 × g for 30 min. The mitochondrial membranes were washed twice
in 50 mM Tris acetate, 0.4 M mannitol, 2 mM EDTA, pH 8.0, containing 1 mM diisopropyl
fluorophosphate and once in 150 mM potassium acetate, 50 mM Tris acetate, 2 mM EDTA, pH 8.0. Mitochondrial membranes were stored in 150 mM potassium acetate, 50 mM Tris acetate, 2 mM EDTA, 50%
glycerol, pH 8.0, at 20 °C. Membrane protein concentrations were
determined by a modified Lowry method (16)
To purify the bc1 complex mitochondrial
membranes were suspended at 10 mg/ml in 50 mM Tris-HCl, 1 mM MgSO4, 1 mM PMSF, pH 8.0, and
0.8 g of dodecyl maltoside/g of membrane protein was added and
slowly stirred for 45 min at 4 °C. The membrane extract was
clarified by centrifugation at 100,000 × g for 90 min.
After the addition of 100 mM NaCl and stirring for 60 min,
the extract was loaded onto a 1.5 × 20 cm DEAE-Biogel A
chromatography column equilibrated with 50 mM Tris-HCl, 1 mM MgSO4, 1 mM PMSF, 100 mM NaCl, pH 8.0. After loading, the column was washed with
two column volumes of the same buffer and eluted with six column
volumes of a linear gradient of 100-400 mM NaCl in 50 mM Tris-HCl, 1 mM MgSO4, 1 mM PMSF, pH 8.0. The bc1 complex
eluted at approximately 280 mM NaCl. The combined
bc1 fractions were concentrated to ~50 µM cytochrome bc1 complex (17)
using Amicon Centriprep 30 tubes.
The yeast mutant coq2 was grown in 80 liters of YPD and
was harvested by centrifugation. The cytochrome
bc1 complex from coq2 was isolated
as described above.
Kinetic Measurements--
Kinetic measurements were performed at
room temperature by rapid scanning stopped flow spectroscopy, using an
OLIS Rapid Scanning Monochromator (On-Line Instrument Systems, Inc.,
Bogart, GA) equipped with a 1200 lines/mm grating blazed at 500 nm.
This produced a spectrum of 75 nm width, centered at 555 nm, with a
resolution of 0.4 nm. The dead time of the instrument was ~2 ms, and
the end of this period was chosen as time 0, after which data were collected at 1000 scans/s.
Reactions were started by mixing 2 µM
bc1 complex in 50 mM potassium
phosphate, pH 6.0, containing 250 mM sucrose, 0.2 mM EDTA, 1 mM NaN3, and 1.0 mg/ml
bovine serum albumin against an equal volume of buffer containing
menaquinol. An oxidized spectrum was obtained by mixing the oxidized
bc1 complex against buffer and averaging the
data set to a single scan. For each experiment three data sets were
averaged, and the oxidized spectrum was subtracted from each scan. From
the three-dimensional data set, which is comprised of wavelength,
absorbance, and time, we examined the time course of cytochrome
b and c1 reduction at 563.3 and 554.6 nm, respectively.
The rates of ubiquinone reduction by menaquinol were measured in an
Aminco DW2A spectrophotometer. The instrument was in split beam mode,
and equal concentrations of decyl ubiquinone were in each cuvette.
Menaquinol was added to one cuvette, and the rate of ubiquinone
reduction was monitored by the decrease in absorbance at 281 nm. This
wavelength was chosen because it is an isosbestic point for
menaquinol/menaquinone. These experiments were performed using the same
buffer used in the stopped flow experiments, with the addition of
0.05% dodecylmaltoside to keep decyl ubiquinone in solution.
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RESULTS |
Kinetics of Reduction of the Cytochrome bc1 Complex by
Menaquinol--
Menaquinol is a preferable substrate for reduction of
the cytochrome bc1 complex because the
oxidation-reduction potential (Em7 = 74 mV;
Ref. 13) is low enough to reduce all of cytochrome bH and a portion of cytochrome
bL, in addition to the Rieske iron-sulfur cluster and cytochrome c1. We previously
established that menaquinol rapidly reduces the
bc1 complex through the catalytic centers P and
N and that menaquinol reduction via center P and N is not dependent
upon endogenous ubiquinone (15). By using menaquinol to reduce the
bc1 complex and monitoring the reaction with
rapid scanning stopped flow spectroscopy, it is possible to examine the
time course of cytochrome b and c1
reduction in a single reaction and to obtain time resolved optical
spectra at 1-ms intervals during the reaction.
Under conditions of continuous turnover, where the catalytic reaction
is zero order with respect to ubiquinol and cytochrome c,
the turnover number of the yeast bc1 complex
approaches 200 s
1 (17). From this catalytic activity one
can estimate that the half-time for the transit of a single electron
through the enzyme from ubiquinol to cytochrome c would
occur within approximately 5 ms. It is clearly not possible to monitor
pre-steady state reduction of the bc1 complex
under conditions where the reaction is zero order with respect to
menaquinol, because much of the reaction would occur within the 2-ms
mixing time of the instrument. However, by lowering the concentration
of menaquinol, the pre-steady state reduction can be monitored on a ms
time scale under conditions where the reduction is first order with
respect to menaquinol.
The traces in Fig. 1 show the time course
of reduction of cytochrome b and c1 when 1 µM bc1 complex is reduced with 6 µM menaquinol. Cytochrome c1
reduction was monophasic and with this low concentration of menaquinol
occurred at 4.6 ± 0.2 s1. In contrast, cytochrome
b reduction was triphasic and consisted of a rapid partial
reduction phase, a partial reoxidation phase, and slow rereduction
phase. During the partial reduction phase ~30% of the cytochrome
b was reduced and reached its maximum value at 70 ms. During
the reoxidation phase ~20% of the cytochrome b remained
reduced, and the minimum value was reached at 280 ms. The rereduction
phase was monophasic and occurred at a rate of 1.9 ± 0.4 s1.
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Fig. 1.
Pre-steady state reduction of the cytochrome
bc1 complex by menaquinol in the absence
of inhibitors. The traces show the time course of the
reduction of 1 µM cytochrome bc1
complex by 6 µM menaquinol. The traces labeled cyt
b and cyt c1 correspond to the absorbance
changes at 563.3 and 554.6 nm, respectively. The inset shows
the reduced minus oxidized spectra of the bc1
complex at 70 ms, 280 ms, and 2 s during the reaction. Each
spectrum is an average of 15 individual spectra collected at 1-ms
intervals over the 15-ms intervals indicated by the horizontal
bars labeled A, B, and C.
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Time resolved spectra averaged across 15-ms intervals before (at 70 ms), during (at 280 ms), and after (at 2 s) the triphasic reduction confirm that the apparent reoxidation phase was caused by the
net oxidation of cytochrome b and not by spectral overlap of
cytochrome c1. From the absorption spectra
A and C in the inset of Fig. 1, one
can calculate that the expected absorbance for cytochrome b
at 563.3 nm in spectrum B would be 0.031-0.043, absent any
reoxidation of the cytochrome b. Instead, in the time
interval between 70 and 280 ms, the absorbance at 563.3 nm decreased to 0.012 in spectrum B. This decrease cannot be accounted for
by spectral overlap from cytochrome c1, because
this would require a >0.02 decrement in the c1 spectrum at
563.3 nm, which is greater than the absorbance increase (~0.016)
because of c1 reduction at the 554.6 nm
absorbance maximum of reduced c1. Furthermore, any small decrease in absorbance at 563.3 nm because of
c1 reduction must also be included in spectra
A and C.
We conclude that our prior interpretation of the triphasic reduction is
correct (6). That is, the reaction consists of an initial partial
reduction of cytochrome b through center P, followed by and
possibly partially coinciding with reoxidation through center N. When
the iron-sulfur protein and cytochrome c1 become
reduced, further reduction of cytochrome b linked to reduction of the high potential acceptors at center P is no longer possible. At this point in the reaction, corresponding to ~400 ms in
Fig. 1, rereduction of cytochrome b resumes through center N
at a slower rate.
At high concentrations of menaquinol (e.g. 200 µM), the rate of b reduction does not appear
to be triphasic. We thus examined the effects of increasing the
menaquinol concentration on the time course of cytochrome b
and c1 reduction as shown in Fig. 2. As the menaquinol concentration
increased, the rate of cytochrome c1 reduction
increased and remained monophasic, and at 100 µM menaquinol the rate was 28 ± 1 s1. There was a
hyperbolic relationship between menaquinol concentration and the rate
of cytochrome c1 reduction, and a double
reciprocal plot produced a Vmax for
c1 reduction of 40 ± 4 s1
(data not shown). When the menaquinol concentration is no longer rate-limiting for c1 reduction, electron
transfer from menaquinol to the iron-sulfur becomes limiting (18).
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Fig. 2.
Effect of increasing menaquinol concentration
on the pre-steady state reduction of the cytochrome
bc1 complex. The traces
show the time course of reduction of cytochromes (cyt)
b and c1 when 1 µM
cytochrome bc1 complex is reduced by 12, 25, 50, or 100 µM menaquinol.
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For cytochrome b, increasing the menaquinol concentration
increased the rate of the partial reduction phase, such that at 100 µM menaquinol this phase occurred during the 2-ms mixing
time (Fig. 2). As the concentration of menaquinol increased, there was
a gradual elimination of the reoxidation phase and an increase in the
rate of the rereduction phase. At 100 µM menaquinol, the reoxidation phase was absent, and the trace consisted of a very rapid
partial reduction, a brief plateau, and a slower reduction phase, at a
rate of 21 ± 2 s1. The tracings in Fig. 2
demonstrate that the apparent lack of triphasic reduction at high
concentrations of menaquinol results from increasingly fast reduction
and rereduction phases as the menaquinol concentration is increased,
such that the three phases of the reaction coalesce into an apparently
biphasic reduction.
The most unusual aspect of these results is that cytochrome
b goes partially reoxidized under conditions where the
menaquinol pool remains highly reduced. For example, at 50 ms during
the triphasic reduction with 12 µM menaquinol (Fig. 2),
~0.3 µM cytochrome b has been reduced, and
>90% of the menaquinol remains in the reduced form. Contrary to what
would be expected, during the ensuing 100 ms cytochrome b
undergoes partial reoxidation while the calculated concentration of
menaquinol is >10 µM, and the potential of the menaquinol pool at pH 6 is < 60 mV. As discussed below, the
lack of equilibration of cytochrome b with the menaquinol
pool suggests that triphasic cytochrome b reduction is
caused by the presence of ubiquinone at center N that oxidizes
cytochrome b and that this ubiquinone does not rapidly
equilibrate with the menaquinol pool.
Kinetics of Reduction of the Cytochrome bc1 Complex by
Menaquinol in the Absence of Ubiquinone--
To determine whether
ubiquinone is responsible for triphasic cytochrome b
reduction, we isolated the cytochrome bc1
complex from a yeast mutant (coq2) that lacks ubiquinone because of
the deletion of a gene for an enzyme in the ubiquinone biosynthetic pathway (19). The coq2 mutant is unable to respire but can grow on
fermentable carbon sources. Using this mutant to obtain bc1 complex lacking ubiquinone avoids any damage
to the enzyme that might result from extraction of ubiquinone with
organic solvents and eliminates the possibility that any residual
ubiquinone remains in the bc1 complex.
Having previously shown that menaquinol can reduce the b and
c1 cytochromes through center N and center P in
the bc1 complex isolated from the coq2 mutant
(15), we examined the time course of the reduction of cytochrome
b and c1 over a range of menaquinol concentrations as shown in Fig. 3. The
most obvious difference in the pre-steady state reduction of the
bc1 complex from the mutant lacking endogenous
ubiquinone is that reduction of cytochrome b is not
triphasic but rather a rapid monophasic reaction. A similar lack of
triphasic reduction was reported in two previous studies (20, 21). With
6 µM menaquinol the rate of cytochrome b
reduction was monophasic and occurred at 9.1 ± 1.9 s1. The rate increased linearly with menaquinol
concentration such that at 50 µM menaquinol the rate was
~90 s1, with a large portion of the reduction occurring
during the 2-ms mixing time. These results are similar to what is seen
in the presence of antimycin (15), which blocks electron transfer
through center N.
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Fig. 3.
Absence of triphasic cytochrome b
reduction in cytochrome bc1 complex
lacking endogenous ubiquinone. The traces show the time
course of reduction of cytochromes (cyt) b and
c1 when 1 µM cytochrome
bc1 complex isolated from the coq2 yeast
mutant is reduced by 6, 12, 25, or 50 µM
menaquinol.
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In addition, at each of the menaquinol concentrations tested, the rate
of cytochrome c1 reduction was about four times
slower than observed with the bc1 complex from
wild type yeast. Using 6 µM menaquinol, the rate of
cytochrome c1 reduction occurred at 0.9 s1 and increased with menaquinol concentration such that
at 50 µM menaquinol the rate was 4.0 s1.
With the bc1 complex from the wild type yeast,
the corresponding rates were 3.3 and 18 s1. An
explanation for the decreased rate of c1
reduction is discussed below.
The lack of triphasic reduction in the coq2
bc1 complex is not dependent on the low
potential of the menaquinol substrate, because the same effect is
observed with ubiquinol. As shown in Fig.
4, reduction of cytochrome b
by 2,3-dimethoxy-5,6-dimethyl benzoquinol, a ubiquinol analogue, in the
bc1 complex from the wild type yeast is
triphasic but is monophasic in the coq2 bc1 complex. The only apparent difference in the reduction of b
in the coq2 bc1 complex by
ubiquinol versus menaquinol is that the transient
over-reduction and equilibration by reoxidation at the end of the
reaction is more pronounced with ubiquinol (Fig. 4, bottom
panel) than with menaquinol (Fig. 3).
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Fig. 4.
Pre-steady state reduction of the cytochrome
bc1 complex by ubiquinol in the presence
or absence of endogenous ubiquinone. The top panel
shows the time course of cytochrome b and
c1 reduction when 1 µM native
cytochrome bc1 complex is reduced by 150 µM 2,3-dimethoxy-5,6-dimethyl benzoquinol, a ubiquinol
analogue. The bottom panel shows the time course of
cytochrome b and c1 reduction when 1 µM ubiquinone-deficient cytochrome
bc1 complex is reduced by 150 µM
ubiquinol analogue.
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Restoration of Triphasic Cytochrome b Reduction in the
bc1 Complex from the coq2 Mutant by Ubiquinone--
To
confirm that ubiquinone is responsible for triphasic cytochrome
b reduction, we added back ubiquinone to cytochrome
bc1 complex isolated from the coq2
mutant. Adding back various amounts of ubiquinone to the
ubiquinone-deficient cytochrome bc1 complex alters the reduction by menaquinol as shown in Fig.
5. Over the range of ubiquinone added,
the kinetics of cytochrome c1 reduction was
monophasic. With 1 µM ubiquinone added, the rate of
cytochrome c1 reduction occurred at ~3.3
s1 and decreased only slightly to 2.5 s1
with 8 µM ubiquinone. It was surprising that adding
ubiquinone did not significantly slow the rate of cytochrome
c1 reduction, because it could conceivably act
as a competitive oxidant of menaquinol. If reduction of the ubiquinone
pool was a prerequisite to reduction of the complex through center P,
one would expect a much larger decrease in the rate of cytochrome
c1 reduction as the ubiquinone content
increased. This also suggests that in the oxidized
bc1 complex ubiquinone does not interfere with
menaquinol access to center P.
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Fig. 5.
Restoration of triphasic cytochrome
b reduction by addition of ubiquinone to the
cytochrome bc1 complex isolated from a
mutant lacking ubiquinone. The traces show the time
course of reduction of cytochromes (cyt) b and
c1 when 1 µM
bc1 complex isolated from the coq2 yeast
mutant is reconstituted with 1, 2, 4, or 8 µM decyl
ubiquinone and then reduced by 50 µM menaquinol.
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The addition of ubiquinone to the ubiquinone-deficient cytochrome
bc1 complex restored triphasic cytochrome
b reduction (Fig. 5). With 1 µM ubiquinone
added, the time course of cytochrome b reduction consisted
of a rapid partial reduction phase, a small lag phase, and a slower
reduction phase. The rapid reduction phase comprised ~35% of the
total absorbance change and was complete within 50 ms. As the
ubiquinone concentration increased, the reduction of cytochrome
b clearly became triphasic and consisted of a rapid partial
reduction phase, a lag phase, and a slow reduction phase. Also, the
portion of the cytochrome b reduced during the rapid reduction phase decreased, and the lag phase became longer. With 8 µM ubiquinone added, only 25% of the cytochrome
b was reduced during the rapid partial reduction phase that
was complete within 50 ms. The lag phase extended for over 500 ms and
was followed by the slow reduction phase.
It has been previously shown that the reduction of the Q pool occurs in
parallel with the rereduction phase of triphasic reduction (22). Thus,
the ubiquinone pool is unlikely to be immediately reduced upon the
addition of menaquinol. As we increased the ubiquinone concentration we
apparently slowed the reduction of the quinone pool, which apparently
must occur for cytochrome b to remain reduced. Our results
show that the rapid reduction of cytochrome b through center
P is not affected by exogenous ubiquinone, although the equilibration
of bH with the quinone pool appears to be
shifted, because less cytochrome b is reduced during the
initial phase of the triphasic reduction as the ubiquinone
concentration is increased.
Fig. 6 shows the effects of increasing
menaquinol concentration on the time course of cytochrome b
and c1 reduction when 2 µM
ubiquinone was added to 1 µM ubiquinone-deficient
cytochrome bc1 complex. Over the range of
menaquinol concentrations tested, the reduction of cytochrome
c1 was monophasic and increased from 1.6 s1 at 6 µM menaquinol to 8.2 s1 at 50 µM menaquinol. Because there was a
linear relationship between menaquinol and the rate of cytochrome
c1 reduction, the reaction is limited by
menaquinol concentration, and not by the rate of electron transfer from
the iron-sulfur protein to cytochrome c1.
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Fig. 6.
Effect of increasing menaquinol concentration
on the time course of cytochrome bc1
complex reduction when ubiquinone is added to isolated cytochrome
bc1 complex from a yeast mutant lacking
ubiquinone. The traces show the reduction of
cytochromes (cyt) b and c1
when 1 µM cytochrome bc1 complex
isolated from the coq2 yeast mutant is reconstituted with 2 µM decyl ubiquinone and then reduced by 6, 12, 25, or 50 µM menaquinol.
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For cytochrome b, the reduction was clearly triphasic at the
lower menaquinol concentrations and became increasingly biphasic as the
menaquinol concentration increased. The proportion of the cytochrome
b reduced during the fast reduction phase remained constant
at ~25%, and this change occurred during the 2-ms dead time at the
higher menaquinol concentrations. The lag between the two phases
decreased as the menaquinol concentration increased, consistent with
the more rapid reduction of the quinone pool by menaquinol.
Effect of Exogenous Ubiquinone on Triphasic Cytochrome b Reduction
in Wild Type Complex--
We also examined the effects of increasing
the ubiquinone concentration on the reduction of cytochrome
b in cytochrome bc1 complex
containing endogenous ubiquinone. The bc1
complex from the wild type yeast contains approximately one ubiquinone
per enzyme monomer, as determined by extraction of the purified enzyme. Fig. 7 shows the effects of exogenous
ubiquinone on the time course of cytochrome b and
c1 reduction by 25 µM menaquinol.
In the absence of exogenous ubiquinone, cytochrome
c1 reduction was monophasic and occurred at 11 s1. As the ubiquinone concentration was increased, the
kinetics of cytochrome c1 reduction remained
monophasic, and with 8 µM added ubiquinone the rate
decreased to 6 s1. Because increasing the total
ubiquinone concentration by 8-fold only decreased the rate of
cytochrome c1 reduction by one-half, these
results suggest that menaquinol reacts directly with center P and not
via the ubiquinone, but that excess ubiquinone competes weakly with
menaquinol for center P.
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Fig. 7.
Effect of exogenous ubiquinone on the time
course of the reduction of the cytochrome
bc1 complex. The traces
show the reduction of cytochromes (cyt) b and
c1 when 1 µM cytochrome
bc1 complex containing endogenous ubiquinone is
mixed with 2, 4, or 8 µM decyl ubiquinone and then
reduced by 25 µM menaquinol.
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In contrast to cytochrome c1, the addition of
ubiquinone had a much more dramatic effect upon the kinetics of
cytochrome b reduction. As the ubiquinone concentration was
increased cytochrome b reduction remained triphasic and
became progressively more obvious. The percentage of the cytochrome
b reduced during the rapid partial reduction phase decreased
from 45% in the absence of exogenous ubiquinone to 30% in the
presence of 8 µM. This suggests that additional
ubiquinone alters the distribution of an electron between bH and ubiquinone at center N. In addition, the
duration of the reoxidation phase dramatically increased with
increasing ubiquinone concentrations. In the absence of additional
ubiquinone the lag phase was only ~25 ms, but with 8 µM
exogenous ubiquinone the lag extended for over 200 ms. These results
are consistent with the interpretation that the ubiquinone pool must be
reduced for the rereduction phase to proceed. If ubiquinone is
available, cytochrome b that has been reduced through center
P or N will be continually oxidized at center N. As the ubiquinone pool
becomes reduced, less is available to oxidize cytochrome b,
thus allowing cytochrome b to remain reduced.
Effect of Exogenous Ubiquinone on Cytochrome b Reduction through
Center N in Wild Type and Ubiquinone-deficient Cytochrome
bc1 Complex--
To confirm that the rereduction phase
requires the reduction of the ubiquinone pool, we examined the effects
of exogenous ubiquinone on the time course of cytochrome b
reduction through center N. In these experiments stigmatellin was
included to block reduction through center P. The top left
panel in Fig. 8 shows the effect of
exogenous ubiquinone on the time course of cytochrome b
reduction in wild type cytochrome bc1 complex
through center N. In the absence of exogenous ubiquinone, cytochrome
b reduction was biphasic with 80% reduced at 8.7 s1 and 20% reduced at 1.1 s1. When 2 µM ubiquinone was added the rates decreased to 70%
reduced at 5.0 s1 and 30% reduced at 0.4 s1.
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Fig. 8.
Effect of exogenous ubiquinone on the time
course of cytochrome b reduction in native and
ubiquinone-deficient cytochrome bc1
complex through center N or center P. The top left
panel shows cytochrome b reduction when 1 µM native cytochrome bc1 complex
is mixed with 2 µM decyl ubiquinone and then reduced by
25 µM menaquinol in the presence of stigmatellin. The
top right panel shows cytochrome b reduction when
1 µM ubiquinone-deficient cytochrome
bc1 complex is mixed with 2 µM
decyl ubiquinone and then reduced by 25 µM menaquinol in
the presence of stigmatellin. The bottom left panel shows
cytochrome b and c1 reduction when 1 µM ubiquinone-deficient cytochrome
bc1 complex is reduced by 25 µM
menaquinol in the presence of antimycin. The bottom right
panel shows cytochrome b and c1
reduction when 1 µM ubiquinone-deficient
bc1 complex mixed with 2 µM decyl
ubiquinone is reduced by 25 µM menaquinol in the presence
of antimycin.
|
|
The top right panel in Fig. 8 shows a similar experiment
with bc1 complex from the yeast mutant lacking
endogenous ubiquinone. Without the addition of ubiquinone cytochrome
b reduction was biphasic with 66% reduced at 17 s1 and 33% reduced at 1.2 s1. With 2 µM ubiquinone the fast rate decreased to 50% reduced at
10 s1 and 50% reduced at 0.7 s1. Thus,
b reduction by menaquinol through center N is approximately twice as fast in the absence of ubiquinone and addition of two equivalents of ubiquinone to the ubiquinone-deficient complex reduced
the rate to approximately that seen with the complex containing endogenous ubiquinone.
Effect of Exogenous Ubiquinone on Cytochrome b and c1
Reduction through Center P in Ubiquinone-deficient Cytochrome
bc1 Complex--
To confirm that exogenous ubiquinone had
little effect upon the reactions at center P, we examined the time
course of cytochrome b and c1
reduction in the presence of antimycin in the absence or presence of
exogenous ubiquinone. The bottom left panel in Fig. 8 shows
the time course of cytochrome b and
c1 reduction in the ubiquinone-deficient
cytochrome bc1 complex. The results show that
cytochrome b was biphasic with 50% occurring at 22 s1 and 50% occurring at 5.0 s1. Cytochrome
c1 reduction was monophasic and occurred at a
rate similar to the slow rate of cytochrome b reduction at
4.3 s1.
The bottom right panel in Fig. 8 shows the effects of
exogenous ubiquinone on the kinetics of cytochrome b and
c1 reduction within the cytochrome
bc1 complex from the mutant lacking endogenous ubiquinone. Again, cytochrome b reduction was biphasic and
occurred with rates of 21 s1 and 2.7 s1.
Cytochrome c1 reduction was monophasic and
occurred at 2.6 s1. Thus, two equivalents of ubiquinone
had no effect upon the fast phase of b reduction through
center P.
Kinetics of Ubiquinone Reduction by Menaquinol in the Absence or
Presence of the Cytochrome bc1 Complex--
To clarify the
role of endogenous ubiquinone on reduction of the enzyme by menaquinol,
we measured the rate of ubiquinone reduction by menaquinol in the
absence or presence of ubiquinone-deficient cytochrome
bc1 complex. In the absence of enzyme, the
reduction of 20 µM ubiquinone by 100 µM
menaquinol occurred with a half-time of 10.0 s (data not shown).
This rate is so slow that menaquinol is unlikely to reduce the
cytochrome bc1 complex via endogenous ubiquinone
but rather reduces the enzyme directly.
Cytochrome bc1 complex catalyzed the
transhydrogenation reduction of ubiquinone by menaquinol. With 0.05 µM enzyme the half-time for reduction of 20 µM ubiquinone by 100 µM menaquinol
decreased to 7.5 s, and with 0.25 µM enzyme the
half-time decreased to 3.2 s. Extrapolation of the rates to the
concentration of enzyme used in our pre-steady state reduction
experiments resulted in a half-time of 1.2 s. This rate is fast
enough to account for the rereduction phase observed during triphasic
reduction and for the extension of the reoxidation phase resulting from
the addition of exogenous ubiquinone. This activity was measured in the
presence of stigmatellin and was blocked by the presence of antimycin,
confirming that this transhydrogenase reaction is catalyzed at center N
(21).
| |
DISCUSSION |
We re-investigated the triphasic reduction of cytochrome
b to determine whether the apparent triphasic reduction
results from a true reoxidation of the b (6) or is an
artifact resulting from declining absorption in the
c1 spectrum and overlap at the wavelength (563 nm) typically used to monitor b reduction (7, 8). We also
wanted to know whether triphasic reduction is uniquely dependent on the
relatively high reduction potential of the substrates typically
employed to elicit this reaction, and finally, we sought to clarify the
role, if any, of endogenous ubiquinone in the triphasic reaction.
Our results clearly establish that in the absence of inhibitors
pre-steady state reduction of cytochrome b is a triphasic reaction, even when the potential of the substrate is significantly lower than that of ubiquinol and the bH heme.
Time resolved optical spectra during the triphasic reduction
demonstrate that cytochrome b undergoes partial reduction,
partial reoxidation, and then rereduction. The reaction pattern changes
from triphasic to biphasic and eventually to apparently monophasic as
the concentration of menaquinol is increased and the reaction
approaches first order.
Our results also show that endogenous ubiquinone is responsible for the
triphasic reduction of cytochrome b. In the absence of
endogenous ubiquinone we no longer observed the triphasic reduction of
cytochrome b, but upon ubiquinone addition triphasic
reduction was restored. These results agree with previous studies, in
which extraction of ubiquinone from the cytochrome
bc1 complex eliminated triphasic reduction (20),
and triphasic reduction was restored upon ubiquinone addition (21). Our
finding that the rates of the three phases depend on the ubiquinone
content of the bc1 complex explains why in some
instances the reaction was reportedly biphasic (20).
We have previously shown that menaquinol can rapidly reduce cytochrome
b through center N in the absence of endogenous ubiquinone when center P is blocked (15). Endogenous ubiquinone or ubiquinone added to the ubiquinone-deficient bc1 complex
apparently inhibits reduction of cytochrome b through center
N. If stigmatellin is present to block reduction of b
through center P, the rate of b reduction through center N
is slower in bc1 complex in which ubiquinone is
present than in the ubiquinone-deficient complex. Addition of one
equivalent of ubiquinone to the ubiquinone-deficient bc1 complex slowed the reduction through center
N in the presence of stigmatellin but had no effect on the fast phase
of b reduction through center P, measured in the presence of
antimycin. When antimycin is present ubiquinone would be blocked from
binding to center N but could bind to center P. Although we saw no
effect on the fast phase of b reduction, there was a 2-fold
decrease in the slow phase of b reduction and the rate of
cytochrome c1 reduction. This may result from
ubiquinone oxidizing the iron-sulfur protein after it has been reduced
by menaquinol, thus slowing the apparent rate of cytochrome
c1 reduction.
The partial reoxidation of cytochrome b that occurs under
conditions where the potential of the substrate is low enough to reduce
cytochrome b requires that an oxidant for cytochrome
b must be electronically isolated from the ubiquinol or
menaquinol pool. To allow for this it was proposed that ubiquinone
formed at center P moves to center N and creates a locally high
ubiquinone concentration in proximity to bH that
is not in rapid equilibrium with the ubiquinol pool (5). The
possibility of exchange of ubiquinone between center P and N is
supported by the crystal structure, which shows a channel that may
connect center P of one monomer with center N of the other monomer
(11). However, in our experiments pre-existent ubiquinone residing at
center N in the wild type complex would obviate the necessity of any such movement.
We propose that within the oxidized bc1 complex
ubiquinone occupying center N prevents the rapid reduction of
cytochrome b through center N and oxidizes cytochrome
b that has been reduced via center P. This occupancy creates
the partial reduction and reoxidation phases. As long as ubiquinone is
available cytochrome b will remain oxidized, and as the
ubiquinone pool becomes reduced the rereduction phase proceeds. The
transhydrogenase catalyzed equilibration of ubiquinone with menaquinol
at center N is slow relative to bH oxidation by
ubiquinone, and this rate difference allows cytochrome
bH to be oxidized and then slowly reduced during the rereduction phase as it equilibrates with the menaquinol pool.
The direct reduction of ubiquinone by menaquinol is slow relative to
the transhydrogenase reaction at center N. This allows ubiquinone to
oxidize cytochrome bH during triphasic reduction that has been reduced through center P and to oxidize
bH that has been reduced by menaquinol through
center N. Thus, the addition of endogenous ubiquinone extends the
reoxidation phase and slows the rereduction phase by reoxidizing
cytochrome b faster than it can be reduced through either
center P or N.
In the ubiquinone-deficient complex, menaquinone is apparently unable
to rapidly reoxidize cytochrome b through center N, because
either menaquinone formed at center P is unable to move within the
enzyme to center N for steric reasons or, if it moves, it cannot
oxidize bH in the presence of excess menaquinol,
because it is a poor oxidant (Em7 = 74 mV).
This results in the lack of triphasic reduction. This limitation only
manifests under conditions of the pre-steady state experiments. The
cytochrome c reductase activity of the ubiquinone-deficient
complex was 100 s1 with menaquinol as substrate, compared
with 70 s1 with ubiquinol as substrate, indicating that
reoxidation of bH by menaquinone is not
thermodynamically limited under conditions of catalytic turnover. This
is consistent with the oxidation of substrate at center P by the
iron-sulfur protein being the rate-limiting step within the catalytic
cycle (18) and not the oxidation of cytochrome
bH by either ubiquinone or menaquinone.
When ubiquinol was used as a substrate we only observed triphasic
reduction when endogenous ubiquinone was present. This shows that
endogenous ubiquinone occupying center N is responsible for the
oxidation of cytochrome b during triphasic reduction. This does not exclude the possibility that ubiquinone formed at center P can
move directly to center N. However, the water-soluble ubiquinone analogue used as substrate for reduction of the quinone-deficient complex would not be expected to cycle effectively from one center to
the other within the hydrophobic interior of the enzyme.
In the ubiquinone-deficient complex cytochrome b can be
rapidly reduced under pre-steady state conditions through both center N
and center P, and triphasic reduction is not observed. In those complexes in which reduction occurs through center N, the reduction is
uncoupled from c1 reduction. Consequently, the
extent to which cytochrome b is reduced through center N can
be estimated by the shortfall in c1 reduction in
the ubiquinone-deficient complex compared with the wild type complex.
The extent of c1 reduction is only slightly less
in the absence of endogenous ubiquinone (Fig. 3 versus Fig.
2), indicating that most of the complexes are reduced through the
thermodynamically preferred center P pathway.
The decreased rate of c1 reduction seen in the
ubiquinone-deficient complex (Fig. 3 versus Fig. 2) is
comparable with the decreased rate of c1
reduction that is observed in the presence of antimycin. This results
from the fact that reduction of the high potential centers of the
bc1 complex, the iron-sulfur protein and
cytochrome c1, is dependent upon the
availability of oxidized low potential centers, which in turn is
affected by the redox equilibration between bH
and ubiquinone. This aspect of the Q cycle mechanism is discussed in
more detail elsewhere.2
In our mechanism, within the native oxidized complex, ubiquinone would
occupy center N and be capable of rapidly oxidizing cytochrome
b reduced through center P. Thus, when menaquinol is oxidized at center P one electron reduces the iron-sulfur protein, and
the second electron reduces cytochrome b. Because ubiquinone occupies center N, a single electron in cytochrome b will
equilibrate between bH and Q, forming the
species (bH·Q). The
equilibration of the first electron between bH
and Q causes the partial reduction of cytochrome b. When a
second menaquinol molecule is oxidized at center P, both the
iron-sulfur protein and cytochrome c1 become
reduced, which prevents any subsequent reactions at center P. A second
electron enters cytochrome b and reduces
(bH·Q) to
bHQH2, which causes the partial
reoxidation phase.
Equilibration of the first electron introduced through center P between
bH and Q at center N is consistent with an
electronically coupled complex between bH and
Q
(23, 24) and with their relative midpoint potentials. At room
temperature, the midpoint potential of bH in
yeast was reported to be +60 mV, and the potentials for the two
half-reactions converting ubiquinone to ubiquinol at center N were
calculated to be 110 mV (Q/Q) and 200 mV
(Q/QH2), respectively (25). Assuming
Em7 for the Q/QH2 couple to be +90 mV (14) in the absence of any preferential binding, these potentials reflect approximately 100 times tighter binding of Q than
QH2 at center N. It would be expected that a single
electron would equilibrate between bH and the Q,
but when a second electron enters cytochrome b via center P
cytochrome bH would remain oxidized and
QH2 would be formed and displaced from center N by Q. The semiquinone at center N cannot rapidly exchange with the ubiquinol pool, which accounts for the existence of a stable semiquinone at
center N (26).
Our model is consistent with the nonequilibrium experiments where the
formation of a semiquinone at center N paralleled the rapid reduction
phase of cytochrome b (4). The semiquinone concentration
decreased in parallel with the reoxidation phase and increased in
parallel with the rereduction phase. In the presence of antimycin or
upon ubiquinone extraction, the kinetics of cytochrome b
reduction was biphasic, and no semiquinone was formed (20). These
results agree with our interpretation that ubiquinone at center N, and
the stabilization of the semiquinone creates the first phase of
triphasic cytochrome b reduction.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Biochemistry, Dartmouth Medical School, 7200 Vail, Hanover, NH
03755. Tel.: 603-650-1621; Fax: 603-650-1389.
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