Biochimica et Biophysica Acta 1817 (2012) 2103–2109
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbabio
Oxygen dependent electron transfer in the cytochrome bc1 complex
Fei Zhou, Ying Yin, Ting Su, Linda Yu, Chang-An Yu ⁎
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA
a r t i c l e
i n f o
Article history:
Received 23 March 2012
Received in revised form 16 July 2012
Accepted 13 August 2012
Available online 19 August 2012
Keywords:
Cytochrome
Ubiquinone
Oxygen
Superoxide ion
Electron transfer
Bioenergetics
a b s t r a c t
The effect of molecular oxygen on the electron transfer activity of the cytochrome bc1 complex was
investigated by determining the activity of the complex under the aerobic and anaerobic conditions.
Molecular oxygen increases the activity of Rhodobacter sphaeroides bc1 complex up to 82%, depending on the
intactness of the complex. Since oxygen enhances the reduction rate of heme bL, but shows no effect on the
reduction rate of heme bH, the effect of oxygen in the electron transfer sequence of the cytochrome bc1
complex is at the step of heme bL reduction during bifurcated oxidation of ubiquinol.
© 2012 Published by Elsevier B.V.
1. Introduction
The cytochrome bc1 complex (bc1) is an essential energy transduction electron transfer complex in mitochondria and many aerobic and
photosynthetic bacteria [1–4]. This complex catalyzes the electron
transfer from ubiquinol (QH2) to cytochrome c (or c2) with concomitant
generation of a proton gradient and membrane potential for ATP
synthesis by the ATP synthase complex. All the bc1 complexes contain
three redox prosthetic group bearing subunits (cytochrome b, cytochrome c1, and Rieske iron–sulfur protein) with varying numbers
(ranging from 0 to 8) of non-redox prosthetic group bearing
(supernumerary) subunits. The cytochrome b subunit contains two
b-type hemes (bL and bH); the cytochrome c1 subunit carries a c-type
heme (c1); and Rieske iron–sulfur protein subunit possesses a high
potential [2Fe–2S] cluster (ISC). These three redox prosthetic group
bearing subunits are essential for catalysis, and their sequences are
highly conserved in all species [2]. The supernumerary subunits are not
involved in catalysis and probably have structural and regulatory
functions [5,6]. The bc1 complex from a photosynthetic bacterium
Rhodobacter sphaeroides has only one supernumerary subunit [7,8],
Abbreviations: ISP, iron–sulfur protein; ISC, iron–sulfur cluster; cyt., cytochrome;
CMC, critical micelle concentration; DM, n-dodecyl-ß-D-maltopyranoside; DOC, deoxycholate; LDAO, N,N-dimethyldodecylamine N-oxide; MCLA, 2-methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazol[1, 2-α]pyrazin-3-one, hydrochloride; O2•−, superoxide
anion; OG, n-octyl-D-gluocopyranoside; Q0C10BrH2, 2,3-dimethoxy-5-methyl-6-(10bromodecyl)-1,4-benzoquinol; SC, sodium cholate; SDS-PAGE, sodium doceylsulfate
polyacrylamide gel electrophoresis
⁎ Corresponding author. Tel./fax: + 1 405 744 6612.
E-mail address: [email protected] (C.-A. Yu).
0005-2728/$ – see front matter © 2012 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.bbabio.2012.08.004
subunit IV. This bacterial complex has been used as a model system for
the study of the mitochondrial complex.
Studies of bc1 have been extensive and much of structural and
functional information have been obtained [9–17]. The “protonmotive Q cycle” [4,18,19] is the most favored mechanism for electron and
proton transfers in bc1. The key step of the Q-cycle mechanism is the
bifurcation of electrons from QH2 at the QP site by ISC and heme bL.
Two mechanisms, the “sequential” [20–22] and “concerted” [23–26],
have been proposed for bifurcated QH2 oxidation. In the “sequential
oxidation” mechanism, the first electron of QH2 is transferred to ISC,
then to heme c1, and the resulting ubisemiquinone is used to reduce
heme bL then bH. In the “concerted oxidation” mechanism, the two
electrons from QH2 are transferred to ISC and heme bL simultaneously. The absence of detectable ubisemiquinone radical at the Qp site
(SQp) [27,28] favors the concerted oxidation mechanism and
questions the validity of the sequential oxidation mechanism, even
though several plausible explanations for the absence of Q-radical
have been offered, such as antiferromagnetic coupling between SQp
and ISC [20] or SQp being fast oxidized by heme bL [22]. It should be
noted that the detection of SQp under abnormal conditions has been
reported [29,30], but it has not been confirmed by other investigators.
The absence of SQp in a mutant bc1 lacking heme bL further indicates
that SQp is not involved in the bifurcated QH2 oxidation [28].
Furthermore, there is no evidence, so far, showing that ISC receives
an electron from QH2 before the other redox components [26].
Taking advantage of the unique EPR signatures of ISC [31], hemes
bL, bH, c1 [32,33], and ubisemiquinone radical [34,35], coupled with
the use of an ultra-fast microfluidic mixer and the freeze-quenching
device [36], the study of pre-steady state kinetics demonstrated that
the reduction of ISC and heme bL in beef bc1 by QH2 was reported [26]
2104
F. Zhou et al. / Biochimica et Biophysica Acta 1817 (2012) 2103–2109
to be similar with a t1/2 of 250 μs. These results are consistent with the
concerted oxidation mechanism.
In addition to its main functions of electron transfer and proton
translocation, the bc1 complex also produces O2•− [37,38]. The reaction
mechanism of O2• − generation by bc1 remains elusive. Production of
O2• − during electron transfer through bc1 is thought to have resulted
from the leakage of electrons from their normal pathways to react with
molecular oxygen. Under normal catalytic conditions, only a very small
amount (b2%) of electrons leak from bc1 to form O2• − [39,40]. This O2•−
generating activity increases when electron transfer is blocked by
antimycin, or when the electron transport chain becomes over reduced.
Electron leakage (or O2• − production) has been speculated to occur at
SQp [41,42] or reduced cyt bL [37,43], depending on the mechanism by
which bifurcation of ubiquinol proceeds in the Q-cycle model. Since the
existence of SQp is questionable and the mutant complex lacking heme
bL produces as much O2•− as the wild-type complex in the presence of
antimycin [28], neither SQp nor reduced heme bL can be the electron
source for O2•− formation. A new direct mechanism was proposed [44].
That is in a hydrophobic environment and in the absence of bL, O2 acts as
a low potential electron acceptor to participate in bifurcated oxidation
of QH2 with a high potential acceptor, ISP, to produce protonated
superoxide (O2·H) which diffuses to a hydrophilic environment (intermembrane space or matrix) and becomes deprotonated to produce
O2• −. In the presence of heme bL, O2 may bind to a site between heme bL
and QH2 to accept an electron (and H+) from QH2 to form O2H, which
then serves as electron donor for heme bL. In other words, O2 may
participate in bifurcated oxidation of QH2 by mediating electron
transfer from QH2 to bL. Alternatively, O2 can participate in bifurcated
oxidation of QH2 with ISC to generate O2H, which is then oxidized by bL
effectively. In either scenario, oxygen would affect the pre-steady state
reduction rate of cytochrome bL by ubiquinol and thus would enhance
the electron transfer activity of the bc1 complex. Herein we report the
effect of molecular oxygen on the electron transfer activity of bc1 and
the identification of heme bL reduction as the step in the electron
transfer sequence of bc1 that is affected by oxygen.
chamber. Q0C10 Br is a better substrate for QCR than Q0C10 and it has
been well documented [47]. The activity was measured by the
reduction of cytochrome c in a Shimadzu UV 2101 PC spectrophotometer at room temperature following the increase of A550. A millimolar
extinction coefficient of 18.5 was used to calculate the reduction of
cytochrome c. The pH and ionic strength in the assay mixture were
adjusted, if necessary, before measuring the activity.
2.3. Fast kinetic measurements
To determine the rate of electron transfer between quinol and
heme b or between quinol and heme c1, bc1 was mixed with an equal
volume of Q0C10BrH2, at room temperature, in an Applied Photophysics stopped-flow reaction analyzer SX.18MV (Leatherhead,
United Kingdom). The concentration of bc1 was 10 μM (based on
cytochrome b) in 100 mM phosphate buffer, pH 8.0, at 4 °C, containing 1 mM EDTA, 1 mM NaN3 and 0.01% DM. The concentration of
Q0C10BrH2 was 200 μM in 0.5 mM phosphate buffer, pH 6.0, containing 1 mM EDTA, 1 mM NaN3, and 0.01%DM. Reductions of cytochrome
b and cytochrome c1 in the wild-type were monitored by the increase
of absorption difference of A561–580 nm and A552–540 nm, respectively,
with a photodiode array scan between 600 and 500 nm. Reductions of
heme bL in the H111N and of heme bH in the H198N mutant complexes
were determined for the increase in A565–580 and A561–580, respectively.
When an inhibitor was used, bc1 was treated with a 5-fold molar excess
of the inhibitor over heme c1, for 15 min at 4 °C, prior to the experiment.
Because the concentration of Q0C10BrH2 used was 20 times higher
than that of bc1, the reactions between bc1 and quinol were treated
as pseudo first-order reactions. To determine electron transfer rates
under anaerobic conditions, the enzyme and substrate solutions
were deoxygenated before introducing to the stopped-flow reaction
analyzer.
2.4. Determination of superoxide generation
Cytochrome c (horse heart, type III) and superoxide dismutase
were purchased from Sigma. 2-Methyl-6‐(4-methoxyphenyl)-3, 7dihydroimidazol [1, 2-α] pyrazin-3-one, hydrochloride (MCLA) was
obtained from Molecular Probes, Inc. N-Dodecyl-D-maltopyranoside
(DM) and N-octyl-D-glucopyranoside (OG) were from Anatrace. 2, 3Dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinol(Q0C10BrH2)
was prepared as previously reported [45]. All other chemicals were of
the highest purity commercially available.
Superoxide produced from the oxidation of ubiquinol by bc1 was
determined by measuring the chemiluminescence of MCLA-O• −
adduct. Equal volumes of solutions A and B [48] were mixed in an
Applied Photophysics stopped-flow reaction analyzer SX.18MV, by
leaving the excitation light off and registering light emission [49].
Reactions were carried at 23 °C. Solution A contains 50 mM
phosphate buffer, pH 7.4, 1 mM KCN, 1 mM NaN3, 1 mM EDTA, 0.1%
bovine serum albumin, 0.01% DM and 5.0 μM (based on cytochrome
c1). Solution B contains 50 μM Q0C10BrH2 and 4 μM MCLA in the same
buffer. Concentrated stock solution of Q0C10BrH2 was made in 95%
ethanol containing 1 mM HCl. The final concentration of ethanol in
solution B is less than 1%. Once the reaction started, the produced
chemiluminescence, in voltage, was consecutively monitored for 5 s.
2.2. Enzyme preparations and assays
3. Results and discussion
The wild-type and mutant cytochrome bc1 complexes from
R. sphaeroides were prepared and assayed according to the methods
developed in our laboratory [46]. To determine the electron transfer
activity in bc1 under anaerobic conditions, a Thunberg cuvette was used.
Purified bacterial bc1 was diluted with 50 mM TrisCl buffer, pH 8.0,
containing 200 mM NaCl, and 0.01% DM to a final cytochrome b
concentration of 1 μM. An appropriate amount of diluted bc1 solution
was added to the assay mixture (1.35 mL) containing 100 mM
phosphate buffer, pH 7.5, 100 μM cytochrome c, and 1 mM EDTA in
the main chamber of a Thunberg cuvette. Aliquots of a stock solution of
Q0C10BrH2 in 95% ethanol containing 1 mM HCl was added to 150 μL of
aqueous solution in the sidearm of the Thunberg cuvette to a final
concentration of 25 μM. To achieve anaerobic conditions, the Thunberg
cuvette was subjected to a repeated vaccum and flashing with argon for
3 min before Q0C10BrH2 in the side arm was mixed with bc1 in the main
3.1. Comparison of the electron transfer activities of bc1 in the presence
and absence of oxygen
2. Experimental procedures
2.1. Materials
It has been demonstrated that the electron transfer from ubiquinol
to cytochrome c catalyzed by bc1 is accompanied with the generation of
minute superoxide [37–43]. Therefore, it would be expected that bc1
has a higher electron transfer activity under the anaerobic condition
than that under aerobic condition, since no electron leaking would
occur in the absence of oxygen. Also, the deoxygenated process, which
often involves repeated evacuating and flushing with argon, would
cause an increase of the concentrations of substrate and enzyme in the
assay system and thus would enhance the apparent activity. Recently,
we have made a systematic comparison of the electron transfer
activities of bc1 in the presence and absence of oxygen under carefully
controlled conditions. All the factors that might affect the activity
F. Zhou et al. / Biochimica et Biophysica Acta 1817 (2012) 2103–2109
Table 1
Summary of electron transfer activities of various cytochrome bc1 complexes in the
presence and absence of oxygen.
Preparations
a
Rsbc1
Rsbc1 + IV
RsΔIV
c1-14Gly-IV-6His
Specific activity, μmol c reduced/min/
nmol b
Aerobic
Anaerobic
3.0 ± 0.3
4.2 ± 0.5
0.6 ± 0.1
3.8 ± 0.5
2.0 ± 0.3
2.3 ± 0.4
0.5 ± 0.1
2.1 ± 0.4
a
Rsbc1, wild-type, 4 subunit complex; IV, recombinant, purified subunit IV;
Rsbc1 + IV, wild-type complex added IV; RsΔIV, Rsbc1 lacking subunit IV; c1-14Gly-IV6His, Rsbc1 with subunit IV fused to Cytc1 C-terminal through 14 glycine residues. Each
data point represents an average of eight experiment results.
affinity, which renders the deoxygenating protocol used in the present
study insufficient.
Since the subunit IV fused R. sphaeroides bc1 has a large extent of
oxygen effect on its electron transfer activity, this bacterial complex
was used throughout this entire study.
3.2. Effect of oxygen concentration in the assay mixture on electron
transfer activity of bc1
As shown in Fig. 1 (the curve with circles), when the electron transfer
activity of a given concentration of bc1 was measured using assay
mixtures containing various concentrations of oxygen, the activity
increased as the oxygen concentration in the assay mixture increased.
The oxygen concentration in the air saturated assay mixture containing
50 mM phosphate, pH 7.4, was assumed to be 250 μM, and that in the deoxygenated assay mixture was assumed to be 0 μM. The oxygen
concentrations in between these two concentrations were obtained by
mixing various portions of air-saturated and deoxygenated assay
mixtures in a specially designed non-air-space mixing cuvette. The
electron transfer activity of bc1 increased as the oxygen concentration in
the assay mixture increased, showing a hyperbolic titration curve. The
concentration of oxygen in the assay mixture for 50% activation was at
100 μM.
When the superoxide generations by bc1 were examined under
increasing oxygen concentrations, a more complicated curve (parabolic) was obtained (see Fig. 1, the curve with crosses). Little
superoxide is generated by bc1 at a low oxygen concentration.
When the oxygen concentration in the assay mixtures was higher
than 160 μM, the superoxide production was proportional to the
concentration of oxygen in the assay mixture.
3.3. Comparison of ionic strength effect, pH profile, and activation
energies between activities of bc1 assayed under aerobic and anaerobic
conditions
Since molecular oxygen can activate the electron transfer activity
of bc1, it is important to see whether or not the oxygen-activated and
-nonactivated electron transfer activities in bc1 has the same
200
Relative oxygen
dependent activity (%)
measurements, including the temperature, enzyme concentration, and
substrate quinol reduction state used, were eliminated.
Table 1 summarizes the electron transfer activities, expressed as
μmol cytochrome c reduced per min per nmol cytochrome b, of various
bc1 preparations assayed under aerobic and anaerobic conditions. To
our surprise, all the purified R. sphaeroides bc1 (wild-type and mutants)
tested had higher activity when assayed under aerobic condition, as
compared to that assayed under anaerobic condition. Molecular oxygen
increases the specific electron transfer activity of bacterial bc1 up to 82%,
depending on the structural intactness of the complex. The complex
that has a better intact structure shows a larger extent of increase in
activity by molecular oxygen. The wild-type complex (as prepared),
which is somewhat deficient in subunit IV, shows a 33% increase in
activity by molecular oxygen. When the wild type complex is
replenished with the purified, recombinant subunit IV, the electron
transfer activity increases and the activity enhancement by oxygen also
increases. The subunit IV added wild type complex (IV + wild-type)
increases electron transfer activity by 82% in the presence of oxygen. In
line with these results, the subunit IV lacking complex (RsΔIV) has a
less than 15% activity increase by oxygen. The subunit IV fused
mutant (c1-14Gly-IV-6His) complex [46], which was prepared by in line
fusing the N-terminus of subunit IV to the C-terminus of cytochrome c1
with a 14-glycine linker between the two fusing subunits, and a
6-histidine tag at the C-terminus of subunit IV, shows an activity
increase of 80% by molecular oxygen. These results suggest that the
subunit IV of the R. sphaeroides complex plays an important role in
oxygen enhanced electron transfer activity.
To be sure that the observed oxygen effect is real and not due to
artifacts introduced during sample preparations, the activity was
assayed by introducing the oxygenated and de-oxygenated bc1
solutions to the deoxygenated and oxygenated assay mixtures,
respectively, with the exact identical manner. When oxygenated bc1
was introduced to the de-oxygenated assay mixture, the activity was
quickly decreased to the same level as that of the deoxygenated bc1
when it was added to the anaerobic assay mixture. On the other hand,
when the deoxygenated bc1 was added to the oxygenated assay
mixture, the activity is almost the same as that of the bc1 without
being subjected to the deoxygenated process, suggesting that oxygen
is quickly re-associated to the bc1 complex. These results confirm that
the oxygen effect on the activity observed is real and not due to the
artifacts introduced during sample manipulation.
It should be mentioned that the beef mitochondrial complex, which
is known to have a more stable structure and higher electron transfer
activity than the subunit IV-added or subunit IV-fused bacterial
complex, showed 22% of the electron transfer activity increase (from
18 to 22 μmol cytochrome c reduced per nmol cytochrome b per min)
by oxygen. This result seems contradictory to the conclusion drawn
from the study of the R. sphaeroides complexes in which the extent of
the electron transfer activity increase by molecular oxygen is correlated
to the structural intactness of the complex. One possible explanation is
that the mitochondrial enzyme might have a much higher oxygen
2105
150
100
50
0
0
50
100
150
200
250
Oxygen ( M)
Fig. 1. Oxygen dependent electron transfer and superoxide generation activities of the
c1-14Gly-IV-6His Rsbc1 complex. The curve with circles (–○–○–) represents the net
increase of cytochrome bc1 activity and the curve with cross (–x–x–) is the superoxide
generating activity from the oxidation of ubiquinol by Rsbc1 under various oxygen
concentrations. Reactions and measurements were performed as described in
“Experimental procedures”. Air-saturated (250 μM) and deoxygenated (0 μM) solutions were proportionally mixed to give a desired oxygen concentration before starting
reactions. Each data point represents an average of four experiment results. The
electron transfer activity under anaerobic condition is used as 100%. The 100%
superoxide generation activity is obtained under aerobic condition, which is equal to
0.25 V chemillumination.
F. Zhou et al. / Biochimica et Biophysica Acta 1817 (2012) 2103–2109
properties or derived from the same origin. To achieve this end, the
activities of bc1 obtained under aerobic and anaerobic assay
conditions were examined and compared for the ionic strength
effects, pH profiles, and activation energies. When the bc1 activity was
assayed under the various ionic strength conditions, both activities,
aerobically and anaerobically, decreased in the same manner as the
concentration of the phosphate buffer in the assay mixture increased.
In the presence of oxygen, the specific activity of bc1, defined as μmol
cytochrome c reduced per min per nmol b, decreased from 5.8 to 0.2
when the phosphate buffer concentration increased from 25 mM to
500 mM. In the absence of oxygen, the specific activity of bc1
decreased from 2.1 to 0.2 when the phosphate buffer concentration
increased from 25 mM to 500 mM. These results indicate that the
oxygen-dependent and -independent electron transfer activities in
bc1 have a similar ionic strength effect.
The activities obtained both aerobically and anaerobically exhibit
a similar pH profile. They both increased as the pH values in the assay
mixture increased from 6 to 8.5 and then decreased drastically. Since
the bc1 activity obtained under aerobic condition has a similar ionic
strength effect and pH profile as those of the activity obtained under
anaerobic conditions, these two activities are very likely to derive
from the same reaction. In other words, both oxygen-dependent and
oxygen-independent activities in bc1 share the same rate limiting
step in their reaction sequences.
To further establish that the oxygen-dependent and -independent
electron transfer activities in bc1 share the same origin (rate limiting
step), the activation energies of these two reactions were examined.
Fig. 2 shows the Arrhenius plots for electron transfer from QH2 to
cytochrome c catalyzed by bc1 in the presence and absence of oxygen.
No significant differences in the activation energies were obtained
from these two plots; these are 14.8 kJ/mol and 14.2 kJ/mol for the
reactions catalyzed by bc1 under aerobic and anaerobic conditions,
respectively. This result further supports the idea that both oxygendependent and -independent reactions are involved in the same rate
limiting step.
know which step is regulated by oxygen. Toward this end, we
determine the pre-steady state rates of reduction of hemes bL and bH
in bc1 by QH2, under aerobic and anaerobic conditions, using a
stopped-flow apparatus. Fig. 3A shows time tracings of the reduction
of heme bH in the c1-14Gly-IV-6His Rsbc1 complex, by QH2, under
aerobic and anaerobic conditions. The reduction rate of heme bH was
determined by measuring the increase in the difference absorption
between 561 and 580 nm. The rate of heme bH reduction was found to
be higher in the presence of oxygen than that in the absence of
oxygen. Since heme bL once being reduced by QH2 is rapidly oxidized
by heme bH, the observed difference in rates of heme bH reduction in
the presence and absence of oxygen could result from the difference
in rates of heme bL reduction between these two conditions. The rates
of heme bL reduction in the IV-fused bc1 complex under aerobic and
anaerobic conditions were obtained by following the increase in
absorption difference between 565 nm and 580 nm (see Fig. 3B). The
reduction rate of heme bL is much higher under the aerobic condition
than that under the anaerobic condition, even though these
measurements may be somewhat compromised with the spectral
overlapping between hemes bL and bH.
To further confirm that the oxygen is participated at the heme bL
reduction step, the rates of heme bH reduction in the stigmatellintreated (Fig. 4A) and in the antimycin-treated (Fig. 4B) complexes
were measured under aerobic and anaerobic conditions. These
0.020
A
k1= 79
A561-580
2106
0.010
k1=38
3.4. Effect of oxygen on the pre-steady state reduction rate of cytochrome
bL in bc1 complex by ubiquinol
Since there are multiple steps involved in the electron transfer
from ubiquinol to cytochrome c catalyzed by bc1, it is important to
0.0
0
0.02
0.04
0.06
0.08
0.1
0.08
0.1
Time (s)
1.4
B
1.2
k1 = 97
0.008
14.8 kJ/mole
0.8
A565-580
In(activity)
1.0
0.6
0.004
k1= 43
14.2 kJ/mole
0.4
0.2
0.0
3.30
3.35
3.40
1/T,
3.45
3.50
3.55
103K
Fig. 2. Arrhenius plots for the electron transfer activities of subunit IV fused Rsbc1
complex. The ubiquinol-cytochrome c oxidoreductase activities were assayed under
aerobic (―) and anaerobic (- - -) conditions as described in “Experimental
procedures.” The natural logarithms of activities were plots against the reciprocal of
absolute temperature in Arrhenius plots. The slopes of the least squares fitting straight
lines fitted by Kaleidagraph were used to estimate their activation energies. Each data
point represents an average of eight experiment results.
0.000
0
0.02
0.04
0.06
Time (s)
Fig. 3. Time trace of cytochrome b reduction by Q0C10BrH2 in c1-14Gly-IV-6His Rsbc1
complex under aerobic (——) and anaerobic (――) conditions. Reactions and
measurements were performed as described under “Experimental procedures” and
monitored by photodiode array. (A) Reductions of cytochrome bH were determined
from the increase at A561–580. (B) Reductions of cytochrome bL were determined from
the increase at A565–580. Each curve is a representative measurement of eight
experiment results.
F. Zhou et al. / Biochimica et Biophysica Acta 1817 (2012) 2103–2109
experimental designs were based on the Q-cycle mechanism in which
heme bH can be reduced by QH2via two routes: one is from the
forward reduction via heme bL through bifurcated oxidation of QH2
with the iron–sulfur cluster at the Qp site, and the other is from the
reversed reduction via bound ubisemiquinone at the QN site.
Stigmatellin is a Qp site inhibitor which blocks the heme bH reduction
through the forward reaction, whereas antimycin is a QN site inhibitor
which blocks the heme bH reduction through the reversed reaction. In
the stigmatellin-inhibited complex, the heme bH reduction is
achieved through the reversed reaction and in the antimycininhibited complex, the heme bH reduction is achieved through the
forward reaction.
If oxygen is involved in the heme bL reduction step, one should see
a difference in the rates of heme bH reduction obtained under aerobic
and anaerobic conditions in the antimycin-treated complex and see
no difference in the rates of heme bH reduction obtained under these
two conditions in the stigmatellin-treated complex. These are indeed
the case as shown in panels B and A of Fig. 4, respectively. These
results further confirm that the oxygen participates at the site of the
reduction of heme bL.
One way to unambiguously confirm that molecular oxygen is
involved in the heme bL reduction step of bc1 catalyzed QH2
reduction, is to examine the oxygen effect on bc1 containing only
one type of heme b, either bL or bH. The availability of the H111N
mutant complex in which heme bH is knocked out by replacing a
histidine-111 ligand with asparagine [28] and the H198N mutant
complex in which the heme bL is knocked out by replacing an H-198
ligand with asparagine, enable us to undertake this approach. Fig. 5
shows the time tracings of the heme bL reduction in the H111N
mutant complex, by ubiquinol, under aerobic and anaerobic
k1 = 63
0.02
k1 = 63
A561-580
0.01
0
0
conditions. When this mutant complex was reduced with QH2, the
rate of heme bL reduction was faster in the presence of oxygen than
that in the absence of oxygen. Since there is no heme bH present in
this mutant complex, the oxygen effect observed must be on heme bL
only. On the other hand, when the H198N mutant complex was
reduced with QH2, the reduction kinetic of heme bH obtained in the
presence and absence of oxygen was the same (data not shown). This
is expected because in this mutant complex reduction of heme bH is
proceeded through the QN site. The presence of oxygen effect on the
rate of heme bL reduction in the H111N mutant complex and the
absence of such effect on the rate of heme bH reduction in the H198N
mutant complex further confirm that oxygen is involved in the heme
bL reduction step in bc1.
3.5. Effect of superoxide dismutase on the oxygen increased rate of heme
bL reduction in the H111N mutant complex
Since the rate of heme bL reduction in the H111N mutant complex
increased in the presence of oxygen and this mutant complex has
been reported to produce more superoxide than that of the wild-type
complex, it is of interest to see whether or not the oxygen enhanced
heme bL reduction by QH2 is due to the increased superoxide
production by this mutant complex. If the oxygen increased rate of
heme bL reduction is due to the increased superoxide production,
then an addition of superoxide dismutase to this mutant complex
should diminish the oxygen enhanced rate of heme bL reduction.
Since the extent of oxygen effect observed on the reduction rate of
heme bL, in the H111N mutant complex is the same as that observed
in the superoxide dismutase added mutant complex, free superoxide
anion is not involved in the oxygen enhanced reduction of heme bL.
3.6. Oxygen plays a regulatory role on the bifurcated oxidation of
ubiquinol
A
0.03
2107
0.02
0.04
0.06
0.08
0.1
Aerobic organisms rely on oxygen for the generation of energy to
support cell growth and maintain cellular functions. Cellular energy is
produced by a coupling reaction between the electron transport chain
and ATP synthase complex. When oxygen is limited, the activity of
cytochrome c oxidase will be decreased and less supply of reduced
cytochrome c is needed. Consequently, less activity of bc1 complex is
needed to generate reduced cytochrome c and thus less supply of
reduced ubiquinol, generated from succinate or NADH by their
Time (s)
0.05
B
0.04
0.008
k1 = 35
A565-580
0.03
k1 = 24
0.02
0.004
k1= 20
k1 =14
0.01
0
0
0.02
0.04
0.06
0.08
0.1
Time (s)
Fig. 4. Time trace of cytochrome b reduction by Q0C10BrH2 in Rsbc1 in the presence of
inhibitor stigmatellin (A) and antimycin A (B) under aerobic (——) and anaerobic
(――) conditions. Reactions and measurements were performed as described under
“Experimental procedures” and monitored by photodiode array. Reductions of
cytochrome b were determined from the increase at A561–580. Each curve is a
representative measurement of eight experiment results.
0.000
0
0.02
0.04
0.06
0.08
0.1
Time (s)
Fig. 5. Time trace of cytochrome b reduction by Q0C10BrH2 in Rsbc1 mutant H111N
under aerobic (―) and anaerobic (――) conditions. Reactions and measurements were
performed as described under “Experimental procedures” and monitored by photodiode array except for the concentration of bc1 complex of 10 μM based on cytochrome
c1. Reductions of cytochrome bL were determined from the increase at A565–580. Each
curve is a representative measurement of eight experiment results.
2108
F. Zhou et al. / Biochimica et Biophysica Acta 1817 (2012) 2103–2109
Fig. 6. Schematic expressions of the bifurcated oxidation of QH2 by bc1 in the presence
and absence of oxygen. The size of the arrows indicates the relative electron transfer
rates.
respective dehygrogenases, complex II or complex I, will be
consumed. How does the molecular oxygen enhance the electron
transfer activity of bc1? As shown in Fig. 6, there are three possible
ways that heme bL can accept electron from QH2. (i) Heme bL can
accept electron from QH2 via protonated superoxide (O2·H) better
than it can accept electrons (+ protons) directly from QH2. In other
words, O2 acts as a mediator between QH2 and heme bL. Our
preliminary data indicates that one of the two detected xenon (O2)
binding sites (Xe2) is located between the QH2 binding site and heme
bL, supporting this mediator idea. (ii) Heme bL can accept an electron
from both QH2 and free O2·H which is generated from bifurcated
oxidation of QH2 by ISC and molecular oxygen directly, as described
in a recent report [44] which shows that an intact cytochrome b is not
necessary for generation of superoxide from QH2 by bc1 complex. (iii) In
the absence of molecular oxygen heme bL can only accept electron from
QH2 at a slower rate. Scenarios i and ii will result in the enhancement of
electron transfer by oxygen. Further exploration is needed before the
true mechanism of the oxygen enhancement of the bifurcated oxidation
of ubiquinol can be firmly established.
Acknowledgements
This work was supported by grants GM30721 from the National
Institutes of Health, and the Oklahoma Agricultural Experiment
Station (projects #1819 and #2372) from the Oklahoma State
University.
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