(Received for publication, February 7, 1996; and in revised form, March 8, 1996)
From the
Purified cytochrome b-c complexes from beef
heart mitochondria and Rhodobacter sphaeroides were
reconstituted into potassium-loaded asolectin liposomes for studies of
the energy-dependent electron transfer reactions within the complexes.
Both complexes in a ubiquinone-sufficient state exhibit
antimycin-sensitive reduction of cytochromes b (both low and
high potential ones) upon induction of a diffusion potential by
valinomycin in the presence of ascorbate. Addition of N,N,N`,N`-tetramethyl-p-phenylenediamine (TMPD) to
the ascorbate-reduced potassium-loaded asolectin proteoliposomes
resulted in reduction of cytochrome b
. Upon
addition of valinomycin, the induced diffusion potential caused a
partial reoxidation of cytochrome b
and partial
reduction of cytochrome b
in beef heart
cytochrome b-c
complex in the presence of
antimycin and/or myxothiazol. Surprisingly, when ubiquinone-depleted
beef heart cytochrome b-c
complex liposomes were
treated under the same conditions, no cytochrome b
reduction was observed but only the oxidation of cytochrome b
, and the oxidation was not oxygen-dependent.
We explain this effect by b
, iron-sulfur protein
short-circuiting under these conditions, assuming that both antimycin
and myxothiazol markedly affect subunit b conformation. The
electrochemical midpoint potential of heme b
appears to be significantly higher than that of heme b
in the presence of myxothiazol, which cannot
be accounted for only by the potential-driven electron transfer between
these two hemes plus the shift in chemical midpoint potentials caused
by myxothiazol. A model for energy coupling consistent with structural
findings by Ohnishi et al. (Ohnishi, T., Schagger, H.,
Meinhardt, S. W., LoBrutto, R., Link, T. A., and von Jagow, G.(1989) J. Biol. Chem. 264, 735-744) is presented. This model is
a compromise between pure ``redox-loop'' and pure
``proton-pump'' mechanisms. Reoxidation of high potential
heme b is observed in an antimycin- or antimycin plus
myxothiazol-inhibited, ascorbate plus TMPD-prereduced R.
sphaeroides b-c
complex, upon membrane potential
development, suggesting that a similar electron transfer mechanism is
also operating in the bacterial complex.
Ubiquinol-cytochrome c oxidoreductase (b-c complex or complex III) accomplishes
vectorial proton translocation across the mitochondrial inner membrane
coupled to electron flow from ubiquinol to cytochrome c. To
date, experimental data on electron-transfer mechanisms are more or
less consistent with Mitchell's Q(
)-cycle
hypothesis(1, 2, 3, 4) . However,
the details of membrane energization resulting from charge separation
in the b-c
complex reactions remain unclear.
Electron transfer between the low and high potential cytochromes b (b and b
respectively) contributes about 40% to the total electrogenic
span of mitochondrial complex III(5, 6, 7) .
The remaining 60% of the membrane potential is believed to be
associated with vectorial proton
translocation(1, 6, 8, 9) . This 60%
portion of the total electrogenicity is closely related to the
structural arrangement of b-c
across the membrane.
On the basis of electrochemical and structural
studies(5, 6, 7, 8, 9, 10, 11, 12, 13) ,
different authors place the b
heme near the
outer (P) side of the mitochondrial membrane and b
heme in the middle(5, 7, 11) , or place b
in the middle of the membrane with b
on the inner (N)
side(6, 8, 9, 12) , or place the
hemes at approximately equal distance from the outer and inner
sides(13) , respectively.
Important data on electron
pathways and energy transduction has been obtained with the help of the
specific inhibitors myxothiazol and
antimycin(2, 3, 4, 5, 6, 7) .
Although it is generally believed that the binding sites of these two
inhibitors have essentially different locations (antimycin blocks
electron transfer between high potential b and quinone and
myxothiazol inhibits quinol oxidation by ISP and low potential b(2, 3, 4) ), the specific action of
the inhibitors remains somewhat ambiguous. To reconcile contradictory
data on the transmembrane arrangement of the b-c complex, Konstantinov (9) proposed that antimycin also
arrests redox-linked proton uptake and release in center o by
affecting low potential b and changing its E
/pH dependence (where E
indicates midpoint redox
potential)(14) . Furthermore, some spectroscopic and
potentiometric studies suggest the proximity of myxothiazol to both
center o and i(15, 16, 17) .
Most of the
electrochemical data for the cytochrome b-c complex were obtained from submitochondrial particles,
mitochondria or chromatophores. For studying electron transfer within
the complex, several advantages can be gained by the use of the
purified cytochrome b-c
complex, reconstituted in
asolectin liposomes. The influence of other components of the electron
transfer chain is eliminated, and the effect of quinone can be studied
using quinone-depleted and -replenished b-c
complexes. Recently Miki et al.(13) performed
an elegant study of reverse electron transport in purified cytochrome b-c
complexes reconstituted into potassium-loaded
vesicles. Potential induced reduction of b
,
without apparent b
reduction, was observed in
the presence of ascorbate. When high potential b
is prereduced by an ascorbate and TMPD mixture, an induced
membrane potential causes low potential b
heme
reduction at the expense of high potential b
heme. The important role of quinone in b
reduction was shown, as was the inhibitory effect of antimycin
and myxothiazol on reverse electron transport(13) . However, a
detailed study of the effects of inhibitors on energy-linked electron
transfer in the mitochondrial cytochrome b-c
complex, in the presence and absence of quinone, was not
performed(13) . We have examined potential induced reduction of
cytochromes b in intact and Q-depleted preparations of beef
heart b-c
complex at saturating concentrations of
antimycin and myxothiazol. Additionally, we describe the energy-linked
cytochrome b reduction in the Rhodobacter sphaeroides cytochrome b-c
complex reconstituted into
liposomes.
Asolectin (crude soybean phospholipids) was obtained from Associate Concentrates (Woodside, Long Island, NY) and partially purified according to Sone et al.(18) . Horse heart cytochrome c, valinomycin, nigericin, MOPS, and sodium ascorbate were purchased from Sigma. N,N,N`,N`-Tetramethyl-p-phenylenediamine (TMPD) and safranine O were purchased from Aldrich. Myxothiazol was a product of Boehringer Mannheim (Mannheim, Germany). Antimycin A was a product of U. S. Biochemical Corp., and 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one hydrochloride (MCLA) was a gift from Dr. Anraku (University of Tokyo).
The mitochondrial cytochrome b-c complex was
purified as described earlier(19) . The R. sphaeroides cytochrome b-c
complex was prepared by a
method involving Triton X-100 solubilization followed by DEAE-Bio-Gel A
and DEAE-Sepharose 6B column chromatography in the presence of 0.01%
dodecyl maltoside. This method (17) was developed by adaptation
of those of Yu and Yu (20) and Ljungdahl et
al.(21) . Quinone-depleted mitochondrial cytochrome b-c
complex was prepared by repeated ammonium
sulfate precipitation, removing 90% of the phospholipids and quinone,
and subsequent asolectin replenishment(22) . The specific
activity of mitochondrial b-c
complex at room
temperature was better than 10 µmol of cytochrome c reduced per min/nmol of b heme. Whereas the activity of
quinone- and phospholipid-depleted preparations was less than 10% that
of the intact mitochondrial b-c
complex, the
activity of the phospholipid-replenished preparation was better than
90%. The activity of the bacterial b-c
complex was
more than 2 µmol of cytochrome c reduced per min/nmol of b heme. Activity assay mixtures contained 50 mM sodium/potassium phosphate buffer, pH 7.0, 0.3 mM EDTA,
50 µmol of cytochrome c and 25 µmol of
Q
C
H
.
Reconstitution of
potassium-loaded proteoliposomes was performed by the cholate dialysis
method(13, 23, 24) . To prepare mitochondrial b-c complex proteoliposomes, acetone-washed
phospholipids at 40 mg/ml were sonicated at 0 °C until clear, in
buffer containing 10 mM MOPS, pH 7.4, 100 mM KCl, and
1.65% sodium cholate. Intact or quinone-depleted b-c
complex was added to give a protein/asolectin (w/w) ratio of
1:40. The phospholipid/protein mixture was incubated for 30 min at 0
°C and dialyzed at 4 °C against 10 mM MOPS buffer
containing 100 mM KCl for 36 h with 2 buffer changes. To
remove external potassium, proteoliposomes were dialyzed against 10
mM MOPS buffer, pH 7.4, containing 100 mM NaCl, for
10 h with 4 buffer changes. The procedure was modified for the
bacterial b-c
complex to provide better stability
and reconstitution. Phospholipids were sonicated in buffer containing
10 mM MOPS, pH 7.9, containing 300 mM KCl and 1.65%
sodium cholate. The protein/asolectin (w/w) ratio in the final mixture
was 1:45-1:50. Sodium cholate was removed by dialysis against 10
mM MOPS buffer, pH 7.9, containing 300 mM KCl for 24
h with 3 buffer changes. Replacement of external potassium with sodium
was done by dialyzing the proteoliposomes against 10 mM MOPS
buffer, pH 7.9, containing 300 mM NaCl, for 10 h with 4 buffer
changes.
The oxidation control ratio, defined as the ratio of the
specific activities in the presence and absence of valinomycin plus
nigericin (23) , was more than 10 for proteoliposomes with
mitochondrial b-c complexes and about 2 for
reconstituted bacterial b-c
complexes. In the
presence of ionophores, reconstituted beef heart b-c
complex exhibited a specific activity close to that of dispersed
complex. Stimulation of reconstituted bacterial b-c
complex by valinomycin and nigericin gives a specific activity of
about 0.9 µmol of cytochrome c reduced per min/nmol of b heme, less than 50% of the dispersed complex. Attempts to
use other detergents to improve the oxidation control of the bacterial b-c
complex were not successful. 0.5 or 1% sodium
cholate or 0.5-1.5% dodecyl maltoside or
decanoyl-N-methylglucamide did not improve the specific
activity or the oxidation control ratio.
Spectral measurements were carried out on Shimadzu UV-2101PC and SLM-AMINCO DW-2000 spectrophotometers. The presence of a potassium-diffusion potential was confirmed by the safranine method (13, 25) . Five µl of proteoliposomes were added to 1 ml of medium, containing 5 µM safranine, 10 mM MOPS, pH 7.4, and 100 mM NaCl. The reaction was initiated by adding 0.3 µg of valinomycin. To collapse the gradient 1 µg of nigericin was added. The absorbance was followed at 525 nm.
To prepare samples for the recording of potential induced cytochrome spectra, 100 µl of proteoliposomes were diluted four times in 0.5-ml cuvette with the corresponding NaCl-containing dialysis buffer and, if not otherwise stated, reduced for 5 min with 10 mM ascorbate or for 30 min with 10 mM ascorbate plus 100 µM TMPD. Inhibitors and uncouplers solutions were made in ethanol. Unless otherwise stated, the antimycin- and myxothiazol-to-cytochrome b ratios were 4:1 and 8:1, respectively. Transmembrane diffusion potentials were induced by adding 0.3 µg of valinomycin to 0.4 ml of proteoliposomes. Spectra were taken every 10 s in the 540-580-nm region. The time necessary to record one spectrum was 6 s; the slit width was 2 nm; and the sampling interval varied from 0.05 to 0.2 nm. Deconvolution of the spectra was performed using extinction coefficients from West et al.(7) . The ethanol concentration in samples was kept at less than 1%.
In the absence of ion exchange, both sides of the proteoliposome membrane have approximately equal electric potential. This was tested by safranine, an optical probe for the membrane potential (13, 25) (see ``Experimental Procedures''). Valinomycin acts selectively as a potassium cation uniportor. Rapid diffusion of cations through the membrane (along the concentration gradient) creates an excess of positive charges on the outer side of the membrane and an excess of negative charges on the inner side of the membrane. A valinomycin-induced electrical potential (diffusion potential), negative on the inside, shifts the equilibrium in those reactions in which charges are moved across the membrane. The diffusion potential caused reduction of cytochrome b in vesicles pretreated with ascorbate (Fig. 1). The direct cytochrome b reduction by ascorbate in the absence of induced diffusion potential was negligible, less than 5% of cytochrome b was reduced after a 30-min incubation of vesicles with 10 mM ascorbate. In agreement with Miki et al.(13) , nigericin or excess valinomycin collapsed the diffusion potential and reversed the cytochrome b reduction.
Figure 1:
Energy-dependent
reduction of cytochrome b in reconstituted mitochondrial b-c complex. 0.3 µg of valinomycin
was added to 0.4 ml of proteoliposomes in 10 mM MOPS/NaOH, pH
7.4, 100 mM NaCl, containing 1.23 µM cytochrome b and 5 mM ascorbate, at room temperature; curves
a-d represent difference spectra between valinomycin added
and valinomycin-free ascorbate-reduced proteoliposomes. a, 1
min after valinomycin addition; b, 3 min after valinomycin
addition; c, after reduction is completed (6 min after
valinomycin addition); d, after reduction by solid sodium
dithionate. The curves were vertically shifted to avoid overlapping.
The upper inset is an enlargement of curve a in the lower panel.
Contrary to the report of Miki et al.(13) , we observed reduction of both hemes b and b
. At the beginning
of the reaction, reduction of b
was more
obvious, the absorption peak being at 563 nm, with a characteristic
shoulder at 558 nm. As reduction proceeds, the peak position shifts to
a shorter wavelength, indicating an increasing contribution of reduced b
heme. After potential induced b reduction was complete (about 40-45% of total
dithionite-reducible cytochrome b), the peak maximum was 562.3
nm. Deconvolution of this final absorption using extinction
coefficients given by West et al.(7) gives a
composition of 75% b
and 25% b
. Reduction of both cytochromes b is
completely inhibited by antimycin, as observed by Miki et
al.(13) . This suggests that the electron is input from
center i. The observed ``transient'' character of
low potential b heme reduction is apparently caused by the
gradual loss of the diffusion potential.
The presence of low
concentrations (up to 2 Q/cytochrome b) of exogenous quinone
(QC
) enhances cytochrome b reduction
to 60-65% of the dithionite-reducible cytochrome b (Fig. 2). The reduction rate is significantly greater than
in the absence of exogenous Q. At Q/cytochrome b ratios
greater than 1, transient energy-dependent cytochrome c
partial reoxidation is clearly seen ( Fig. 3a),
and cytochrome b
reduction is more obvious (Fig. 3). Cytochrome b reduction, as well as transient
cytochrome c
reoxidation, is totally abolished by
the addition of antimycin (not shown). Cytochrome c
oxidation is an indirect indication for the quinone being reduced
at center o. The purified cytochrome b-c
complex contains less than 1 mol of quinone per mol of cytochrome c
, most of the quinone molecules may be located at
center i (or associated with cytochrome b
) and
little in center o. Added exogenous Q will bind to center o (or to cytochrome b
) and this Q may be able to
accept the first electron from cytochrome b
via b
and the second electron from ISP-cytochrome c
upon induction of diffusion potential.
Figure 2:
Energy-dependent reduction of cytochrome b in potassium-loaded reconstituted proteoliposomes in the
presence and absence of exogenous quinone. The traces illustrate the time course of absorption at the 562-575
wavelength pair after valinomycin addition in the absence added
QC
(a) and presence of 0.87
µM (b) and 3.85 µM Q
C
(c). The cytochrome b concentration was 1.09 µM. Other conditions were as
in Fig. 1.
Figure 3:
Energy-dependent absorption spectra of
reconstituted proteoliposomes in the presence of exogenous quinone. The
reaction mixture contained 1.33 µM cytochrome b,
15 mM ascorbate, and 2 µM QC
. Other conditions were as in Fig. 1. Difference spectra between the current system state and
the initial ascorbate reduced state were taken after 0.5 min (a), 2 min (b), and after reduction is completed
(after 3 min) (c).
At
higher Q/cytochrome b ratios (more than 2), a decrease of the
potential induced cytochrome b reduction was observed (Fig. 4). Miki et al.(13) , who first reported
the effect, ascribed it to direct reoxidation of reduced cytochrome b by oxidized quinone in center i. We
think that this effect is more likely caused by a limited rate of
electron transfer from ascorbate to cytochrome c
or ISP. As the excess exogenous Q in center o may accept
more electrons from cytochromes b upon induction of diffusion
potential, it acts as an electron sink and thus decreases the extent of
cytochrome b
reduction. Since to complete the Q
reduction at the Qo site it is necessary to receive an electron from
ISP/cytochrome c
, and reduction of ISP/cytochrome c
by ascorbate is not rapid enough, a transient
oxidation of cytochrome c
is expected. This
factor, together with the limited lifetime of the diffusion potential,
causes the apparent inverse dependence of potential induced cytochrome b reducibility upon quinone concentration at high Q/cytochrome b ratios.
Figure 4:
Effect of exogenous quinone concentration
on the potential induced cytochrome b reduction. , in
the presence of 5 mM ascorbate;
, 10 mM ascorbate;
, 20 mM ascorbate. Total cytochrome b (100%) was 1.16 µM, and 1-µl aliquots of
Q
C
were added in ethanol to give indicated
quinone/cytochrome b ratio. Other conditions were as in Fig. 1.
Unfortunately the transient nature of cytochrome c reoxidation makes correlation of the increase of c
reoxidation with the decrease of cytochrome b reduction difficult. To further investigate the reason for
diminished cytochrome b reduction in the presence of an excess
of quinone, we studied the dependence of cytochrome b reduction on the quinone/cytochrome b ratio, at different
ascorbate concentrations. The results are shown in Fig. 4. At
high quinone concentrations, cytochrome b reduction was
proportional to the ascorbate concentration. At high ascorbate
concentrations, maximal cytochrome b reduction persists to
higher quinone/cytochrome b ratios, supporting our explanation
of this phenomenon.
Ascorbate is able to reduce b(27) in the presence of TMPD, and
addition of valinomycin to this system resulted in a reduction of b
as described by Miki et
al.(13) , without the reoxidation of b
. Addition of antimycin to ascorbate
plus TMPD-reduced and reconstituted b-c
complex
liposomes caused a slow partial reoxidation of prereduced b
and the well-known red shift of the reduced b
maximum (15) . The rate of the
reoxidation is increased when an excess of exogenous quinone was added
to the system. In the absence of exogenous quinone, the reoxidation is
slow enough to allow identification of the potential dependent
reaction. Fig. 5illustrates difference spectra recorded
immediately after the addition of valinomycin (a) and after
the subsequent addition of nigericin (b). The
nigericin-treated sample serves as a control of potential dependent
reactions. Contrary to Gopher and Gutman (5) and Konstantinov et al.(6) , who independently reported partial
oxidation of prereduced b
with no reduction of b
, we observed partial reoxidation of high
potential heme b
together with partial reduction
of low potential heme b
. The studies of Gopher
and Gutman (5) and of Konstantinov et al.(6) were performed on submitochondrial particles (SMP)
poised with succinate/fumarate. Therefore, we compared potential
dependent reactions of antimycin-inhibited proteoliposomes in the
presence and absence of a 10 mM fumarate/1 mM succinate poising medium containing a catalytic amount of
succinate-ubiquinone reductase. We found that the energy-dependent
partial reoxidation of high potential heme b
together with partial reduction of low potential heme b
in the reconstituted proteoliposomes were not
changed upon addition of 10 mM fumarate/1 mM succinate to the medium. Deconvolution of difference spectra using
the extinction coefficients from West et al.(7) gives a b
oxidized/b
reduced ratio about 1.15
(average of three experiments).
Figure 5: Energy-dependent intracytochrome b electron transfer in antimycin- and/or myxothiazol-inhibited proteoliposomes. Proteoliposomes containing 1.12 µM cytochrome b were prereduced with 10 mM ascorbate, 0.1 mM TMPD for 20 min. Then the appropriate inhibitor was added, and the resulting spectrum taken as a base line. Spectra were recorded immediately after potential development. a, antimycin-inhibited proteoliposomes, antimycin/cytochrome b ratio = 4; b, nigericin was added to valinomycin-treated sample a spectra; c, myxothiazol-inhibited proteoliposomes, myxothiazol/cytochrome b ratio = 8; d, combination of myxothiazol and antimycin inhibition.
For comparison, we examined the
effect of myxothiazol plus antimycin, on ascorbate plus TMPD-prereduced b-c complexes. Antimycin and myxothiazol block
electron input and output to both centers i and o,
and according to West et al.(7, 26) ,
development of a diffusion potential causes electron transfer from b
to b
(Fig. 5d).
In this reaction b
undergoes partial reoxidation
and b
undergoes partial reduction. The average
of the b
oxidized/b
reduced ratio was 1.1 (three experiments), which is close to that
in the presence of merely antimycin. Apparently, no or very slow b
center o electron transfer
occurs in antimycin-inhibited proteoliposomes.
In the
succinate/fumarate poised SMP, Konstantinov et al.(6) observed an energy-linked reduction of cytochrome b via center i, induced by the addition
of ATP, with no further reduction of b
. Later
West (26) showed that membrane potential, created by aeration
of a suspension of mitochondria in the presence of ascorbate and
hexammineruthenium chloride, forces electrons from the Q pool through
center i into b
with no change in the
redox state of b
in mitochondria equilibrated
with succinate/fumarate in the presence of myxothiazol. Under such
conditions, high potential cytochrome b
is
significantly reduced while electron input from center o is
blocked.
In the myxothiazol-inhibited and ascorbate plus
TMPD-reduced proteoliposomes, the high potential cytochrome b was reduced. In this system an induced
diffusion potential caused a reduction of b
at
the expense of heme b
. The ratio b
-oxidized/b
-reduced was
1.0 (Fig. 5c). If we assume that the partial
reoxidation of b
resulted from the lack of an
electron donating system (succinate/fumarate/succinate-ubiquinone
reductase) in the reconstituted liposomes, the data obtained from the
myxothiazol-inhibited proteoliposomes are consistent with the data
obtained with SMP and mitochondria(6, 26) .
In the
absence of TMPD, the b was not reduced, and
addition of valinomycin to myxothiazol-inhibited proteoliposomes caused
a AA-sensitive reduction of b
, while b
remained oxidized (Fig. 6). Under
similar conditions, reduction of cytochrome b was observed by
Miki et al.(13) , but these authors did not analyze
the species of cytochromes b reduced and erroneously assumed
the reduced cytochrome to be b
. This low
potential heme b
reduction was readily abolished
by adding nigericin. An excess of exogenous quinone, which serves as
``redox buffer,'' increases the level of cytochrome b
reduction to 20-25% of the cytochrome
with no observable b
reduction. Hence,
myxothiazol inhibits the potential dependent b
reduction but not the potential dependent b
reduction.
Figure 6:
Difference spectrum between energized and
nonenergized myxothiazol-inhibited proteoliposomes. Proteoliposomes
containing 1.12 µM cytochrome b and indicated
amount of quinone were prereduced with 10 mM ascorbate for 10
min. Then myxothiazol was added, and the resulting spectrum taken as a
base line. Spectra were recorded immediately after potential
development. Myxothiazol concentration was 7 µM. a, in the absence of QC
; b,
in the presence of 2.93 µM Q
C
.
Ascorbate plus TMPD reduced the cytochrome b in quinone-depleted liposomes much like in the
intact liposomes. Potential development in Q-depleted cytochrome bc
liposomes showed potential dependent electron
movement, from high to low potential hemes (Fig. 7, a and b). The reduction pattern, in the absence of quinone,
is similar to that of the antimycin- and myxothiazol-treated
Q-sufficient complex. The average b
-oxidized/b
-reduced
ratio is 1.1. According to the Q-cycle
model(1, 2, 3, 4) , since quinone
depletion of the b-c
complex blocks electron
transfer in both centers i and o, no additional
influence of antimycin and myxothiazol on Q-depleted preparation should
be expected.
Figure 7: Energy-dependent intracytochrome b electron transfer in antimycin- and/or myxothiazol-inhibited quinone-deficient proteoliposomes. Proteoliposomes containing 1.05 µM cytochrome b were prereduced with 10 mM ascorbate, 0.1 mM TMPD for 20 min. Then the appropriate inhibitor was added and the resulting spectrum taken as a base line. Spectra were recorded immediately after potential development. a, no inhibitor added; b, nigericin added to spectrum a; c, antimycin added; d, myxothiazol added; e, combined effect of antimycin and myxothiazol.
However, in the presence of antimycin, ascorbate plus
TMPD-reduced cytochrome b underwent partial reoxidation upon
potential development in the Q-depleted preparation (Fig. 7c). Difference spectra between
valinomycin-treated and valinomycin-free samples show a negative
absorption peak at 562.5 nm, with no b reduction. The oxidation of high potential b heme
occurred rapidly, within the time required to obtain a spectrum.
Addition of nigericin restored the initial cytochrome b spectrum. Surprisingly, myxothiazol had a similar effect on
energized, quinone-depleted vesicles. In the presence of myxothiazol,
oxidation of b
, with no apparent effect on b
, was observed with a negative absorption peak
at 561.5 (Fig. 7d). The combined effect of myxothiazol
and antimycin was the same as that of antimycin alone (Fig. 7e). A possible explanation is that, at least in
Q-depleted preparations, there exists an electron pathway, sensitive to
antimycin and myxothiazol, that is not compatible with the Q-cycle
model. However, it is not known whether or not this hypothetical
pathway only appears in the presence of antibiotic under the altered
phospholipid environment.
Using MCLA, an indicator of
superoxide(28) , did not result in a change in luminescence
upon adding valinomycin to the sample containing either Q-depleted or
Q-sufficient proteoliposomes that were prereduced by ascorbate plus
TMPD and antimycin-inhibited (data not shown). Preliminary flushing of
ascorbate plus TMPD-reduced, Q-depleted proteoliposomes with argon did
not change their response to potential development in the presence of
antimycin or myxothiazol. Thus generation of
O in the system can be excluded and
oxygen cannot be considered as possible electron acceptor.
The
remaining quinone in Q-depleted preparations is not likely to account
for the observed phenomenon since it is not likely to be moving rapidly
between Q-depleted and Q-sufficient complexes. The b-c complex is roughly a cylinder, one-third part of which is buried
in the phospholipid bilayer(11) . If we neglect differences in
density between the complex and the membrane, the average distance
between neighboring complexes is approximately 11 complex diameters,
much greater than the distance between hypothetical centers o and i. Besides, in reconstituted mixtures of intact and
quinone-deficient b-c
complexes, cytochrome b reducibility is linearly proportional to the intact complex
percentage (data not shown).
Figure 8:
Energy-dependent reactions in
reconstituted bacterial b-c complex. 0.3
µg of valinomycin was added to 0.4 ml of potassium-loaded
proteoliposomes (0.95 µM cytochrome b) prereduced
by 10 mM ascorbate (a and b) or 10 mM ascorbate plus 0.1 mM TMPD (c and d) in
10 mM MOPS/NaOH, pH 8.0, 300 mM NaCl, at room
temperature. After the potential induced reaction is completed, a
difference spectrum is recorded with the valinomycin-free sample as a
reference; a, diffusion potential reduced cytochrome b; b, difference spectrum between potential reduced
and ascorbate + TMPD-reduced proteoliposomes; c,
potential induced reoxidation of b
in
antimycin-inhibited reconstituted bacterial b-c
; d, potential induced reoxidation of b
in antimycin plus myxothiazol-inhibited, reconstituted bacterial b-c
.
In
the presence of myxothiazol and antimycin, or antimycin alone, b in bacterial cytochrome b-c
proteoliposomes was reduced by ascorbate plus TMPD as in the case
of the mitochondrial complex. The prereduced cytochrome b was
oxidized upon potential development (Fig. 8, c and d). Although no measurement of heme b
reduction was carried out, because of high turbidity, the
reduction of cytochrome b
was expected. The
electron transfer sequence within the bacterial complex appears to be
identical to that of the mitochondrial system.
In the lack of detailed direct structural data on the spatial
organization of the b-c complex, the study of the
potential dependent reactions plays an important role for clarification
of not only energy transduction mechanisms, but also of the closely
related problem of the geometric arrangement of the complex across the
membrane. According to Gopher and Gutman (5) and Konstantinov et al.(6) , energy-linked oxidation of prereduced b
in antimycin-inhibited submitochondrial
particles occurs through center o, while b
is in equilibrium with the succinate/fumarate pair. This is
consistent with a P side location for b
. On the
other hand, based on the facts that high potential heme reduction in
center i is energy-independent (6, 26) and
that high potential heme b was reduced by membrane-impermeable
redox agents from the N side(8, 12) , Konstantinov (9) places b
electrically on the inner
side and b
in the middle of the membrane.
According to his model, antimycin A, an inhibitor of electron transfer
between Qi (center i) and high potential b
, also inhibits proton extrusion in center o. This can explain the energy-independent redox state of heme b
in antimycin-inhibited
SMP(5, 6) .
Some structural data on the arrangement
of the b hemes became available after the study of the
spin-spin interactions of paramagnetic probes with redox active sites
of reconstituted b-c complex(11) . The
data obtained by Ohnishi et al. (11) exclude the
location of the low potential b
heme in the
middle of the membrane and the location of the high potential b
heme close to the matrix side surface.
Nevertheless, the low potential b heme is somewhat buried
within the b-c
complex, the effective distance
from the heme to the outside surface being approximately 1.6 nm.
Recently Miki et al.(13) studied the energy-linked
reduction of cytochrome b in the reconstituted
potassium-loaded proteoliposome system. They proposed an alternative
model of potential-linked reversed electron transfer in the beef heart
cytochrome b-c complex reconstituted into
potassium-loaded phospholipid vesicles, which can be described in terms
of the redox-loop mechanism. The conclusions made by these authors
imply that the high and low potential hemes are at approximately equal
distances from N and P sides of the membrane, respectively. However, no
study of potential dependent reactions in antimycin- and/or
myxothiazol-inhibited reconstituted proteoliposomes was performed to
confirm this model.
Our results described in this paper support a proton motive mechanism that is a compromise between two extremes, a pure redox-loop mechanism and a pure proton-pump mechanism. The possibility of this compromise was stressed before(1, 6, 9, 29) , and the influence of antimycin on the electrogenic reactions within the frame of the corresponding model was discussed(9, 12) .
We assume that both antimycin and myxothiazol not only inhibit the
electron transfer in the centers i and o, but also
change the conformation of the cytochrome b subunit comprising
both high potential and low potential b hemes, thus affecting
redox-linked electrogenic proton movement. The ability of antimycin to
modify the E/pH dependence of cytochrome b and to affect the electrogenic proton extrusion from center o has been already discussed(9, 12, 14) .
We obtained some insight concerning the effect of myxothiazol on the
properties of cytochrome b when EPR spectra of inhibited and
uninhibited R. sphaeroides cytochrome b-c
complexes were studied. Both b
and b
EPR signals (g = 3.49 and g =
3.76 respectively) were shifted upfield in the presence of myxothiazol,
suggesting that the inhibitor strongly affects both b cytochromes(17) .
The evidence for a conformation
change caused by antimycin and/or myxothiazol is also provided by our
observation of energy-dependent b oxidation in
the presence of myxothiazol and/or antimycin in Q-depleted b-c
complexes. According to the Q-cycle model (2, 3, 4) , the first electron of quinol is
passed to iron-sulfur protein (ISP) and then passed to cytochrome c
. The resulting semiubiquinone is able to reduce
the low potential cytochrome b. The low potential cytochrome b transfers the electron to high potential cytochrome b, which reduces quinone in center i. Low potential
cytochrome b is not able to directly exchange electrons with
ISP. If it did, the electron would not recycle through the high
potential cytochrome b but would transfer from b
to the iron-sulfur protein which has a more
positive midpoint potential. According to this model, in the absence of
quinone there should be no communication between the cytochromes b and other parts of the electron transfer chain. The only possible
electron movement is between transmembranous b hemes.
Contrary to the Q-cycle model, we observed potential-linked b reoxidation, with no change in the b
redox state, in Q-depleted antimycin- and/or
myxothiazol-inhibited proteoliposomes. A possible explanation is b
-ISP short-circuiting. If the high potential
cytochrome b is rapidly equilibrating with an external redox
medium through b
and ISP, an electron will move
from reduced heme b
, along the electric
diffusion potential, to b
located near the P
side, which immediately passes the electron to an external acceptor.
The short-circuiting is a consequence of conformational changes caused
by the presence of inhibitors (antimycin and/or myxothiazol) and Q
deficiency, and it is observed only if both factors are present.
Conformational change affecting both cytochromes b, in
Q-depleted preparations, has been clearly demonstrated by EPR studies (30) .
The electron pathway and redox coupling to proton
translocation in the Q-sufficient b-c complex can
be illustrated as follows (Fig. 9). The model includes some of
the features proposed by Konstantinov and
co-workers(6, 9, 12) , and it is consistent
with the structural findings made by Ohnishi et
al.(11) . This model implies the existence of at least two
redox-linked ionizable
groups(1, 12, 14, 29) , one closer
to the outside and another closer to the inside, the pK values
of which depend on the redox states of both hemes b and,
possibly, on the redox states of quinone in centers i and o. We assume that myxothiazol and antimycin significantly
change the responses of these groups to the redox processes in the b-c
complex.
Figure 9:
A model for reverse electron transfer
coupled to potential induced proton translocation in reconstituted b-c complex. The energy-dependent steps
are shown by bold arrows; dashed lines represent
coupling of electron transfer to proton translocation steps in
uninhibited (A), antimycin-inhibited (B), and
myxothiazol-inhibited b-c
complex (C). Horizontal arrows represent nonelectrogenic electron and
proton transfers. See text for further
description.
Fig. 9A shows the
events postulated to take place during the potential induced reverse
electron transfer in uninhibited proteoliposomes. The reduced quinone
in center i reduces heme b, and an
ionizable group in the vicinity of the heme accepts a proton from the
quinone molecule. Simultaneously, proton uptake from the P side by
another redox-linked ionizable group occurs, coupled to the heme b
reduction by quinol in center i. The
diffusion potential drives an electron from high potential heme b
to low potential heme b
. The low potential heme passes the electron
and the proton from the redox-linked ionizable group to the quinone
molecule, and proton release to the N side, coupled to the low
potential heme oxidation in center o by quinone, occurs. The second
electron and the second proton necessary to completely reduce Qo come from the P side through cytochrome c
-ISP
chain. The quinone reduced in center o freely diffuses to
center i. As a result of multiple turnovers of the
Q-b-b-Q loop with three electrogenic processes
involved, proton uptake from the P side, proton release to the N side,
and electron movement from heme b
to heme b
, the overall Q-pool and cytochrome b redox potential lowers, and the cytochrome b reduction
induced by the diffusion potential is observed. The electrogenic proton
uptake from P side is included on the picture because a marked distance
(1.6 nm) between the low potential heme and the outside surface was
reported(11) .
In the antimycin-inhibited proteoliposomes,
electron transfer from center i to b is
blocked, and proton uptake from the P side is inhibited. Apparently,
proton release to the N side is also inhibited by antimycin (Fig. 9B). Heme b
rapidly
exchanges electron with Q/QH
pair in center o. However, since significant components of the electrogenic
span are lost, the oxidation of low potential heme b
by Q to yield QH
does not occur to a measurable
extent due to the more negative midpoint redox potential of the
Q/QH
pair, and we observe reduction of low potential
heme together with partial oxidation of high potential heme b
after the diffusion potential development. In
SMP the Q/QH
pair is rapidly equilibrated with the
Q-pool by stoichiometric amounts of succinate-ubiquinone reductase,
which forms a supercomplex with the b-c
complex (31) . Since the electron transfer from b
to Q/QH
is not accompanied by proton
translocation, the redox potential of the heme b
in energized SMP is equal to that established by the
succinate/fumarate pair(5, 6) .
We observed that b remains oxidized while cytochrome b
is partially reduced after potential
development in the myxothiazol-inhibited ascorbate-reduced
proteoliposomes. Since the electron from donor (ISP) is blocked by the
inhibitor, the reducing power is apparently supplied by the Q/QH
pair. The excess exogenous quinone creates a ``redox
buffer,'' and the chemical potential of the Q/QH
pair
will remain essentially constant during the course of the reaction. In
the presence of exogenous quinone, 20-25% of cytochrome b
can be reduced with little or no b
reduction. A rough estimate of the apparent
electric potential difference between the two b hemes is 160
mV or more, with b
heme being more reducible
than b
heme in the presence of a diffusion
potential. On the other hand, other experiments with antimycin- or
myxothiazol- plus antimycin-inhibited, or quinone-depleted
preparations, show that the induced potential difference between the
two hemes is less than 100 mV, with b
heme being
less reducible than b
heme. The difference
cannot be accounted for by a myxothiazol influence on the b heme chemical midpoint potentials because both b cytochromes titrate in the presence of myxothiazol with E
values 15-30 mV more positive than in its
absence(16) .
Our explanation for the effect is illustrated
in Fig. 9C. In the myxothiazol-inhibited b-c complex, the electron transfer from Qi to high potential heme is electroneutral. The electron is passed
to the high potential heme together with a proton, which binds to a
redox-linked ionizable group in the vicinity of the b
heme. It is likely that myxothiazol affects a redox-linked
ionizable group, located in the vicinity of center o, and thus
no proton is taken up from the P side. Even if the proton uptake from P
side is not inhibited by myxothiazol, its contribution to the total
electrogenic span may be rather small, since the low potential heme is
close to the P side. On the next step, b
heme is
oxidized by b
heme. In the uninhibited b-c1 complex, the proton that came from the Qi remains with the b
heme and is released to
the N side only on the subsequent step, when b
heme is reoxidized by Qo (Fig. 9A). The
electron transfer from b
heme to b
heme is not electrogenic enough to reduce b
heme to a greater extent than b
heme (see experiments with antimycin- plus
myxothiazol-inhibited proteoliposomes and Q-depleted proteoliposomes).
Probably, due to the influence of myxothiazol, the proton bound near
the b
heme is released to the N side during the
electron transfer from high potential to low potential heme. This
proton movement from the deeply buried b
heme to
the N side accounts for more than 60 mV of apparent electric potential
difference between the b hemes and contributes to low
potential heme reduction with b
heme remaining
significantly oxidized.
The important aspect of this work was to
analyze the influence of antimycin and myxothiazol on the potential
dependent reactions in the b-c complex. The
results obtained show that these inhibitors not only block electron
conductivity between the Q-pool and cytochromes b, but they
affect the conformation of the cytochrome b subunit and,
possibly, the coupling of proton translocation to electron transfer in
the b-c
complex.
The observation of a potential
induced cytochrome b reduction, in reconstituted bacterial b-c complexes, is consistent with there being
significant structural similarity between mitochondrial and bacterial
ubiquinol-cytochrome c oxidoreductases. Further detailed study
of the bacterial b-c
response to membrane
potential in reconstituted proteoliposomes is in progress in our lab.