Effect of Famoxadone on Photoinduced Electron Transfer between
the Iron-Sulfur Center and Cytochrome c1 in the
Cytochrome bc1 Complex*
Kunhong
Xiao
,
Gregory
Engstrom§,
Sany
Rajagukguk§,
Chang-An
Yu
,
Linda
Yu
,
Bill
Durham§, and
Francis
Millett§¶
From the
Department of Biochemistry and Molecular
Biology, Oklahoma State University, Stillwater, Oklahoma 74078 and the
§ Department of Chemistry and Biochemistry, University of
Arkansas, Fayetteville, Arkansas 72701
Received for publication, November 14, 2002, and in revised form, January 6, 2003
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ABSTRACT |
Famoxadone is a new cytochrome
bc1 Qo site inhibitor that
immobilizes the iron-sulfur protein (ISP) in the b
conformation. The effects of famoxadone on electron transfer between
the iron-sulfur center (2Fe-2S) and cyt c1 were
studied using a ruthenium dimer to photoinitiate the reaction. The rate
constant for electron transfer in the forward direction from 2Fe-2S to
cyt c1 was found to be 16,000 s
1
in bovine cyt bc1. Binding famoxadone decreased
this rate constant to 1,480 s
1, consistent with a
decrease in mobility of the ISP. Reverse electron transfer from cyt
c1 to 2Fe-2S was found to be biphasic in bovine cyt bc1 with rate constants of 90,000 and 7,300 s
1. In the presence of famoxadone, reverse electron
transfer was monophasic with a rate constant of 1,420 s
1.
It appears that the rate constants for the release of the oxidized and
reduced ISP from the b conformation are the same in the
presence of famoxadone. The effects of famoxadone binding on electron
transfer were also studied in a series of Rhodobacter
sphaeroides cyt bc1 mutants involving
residues at the interface between the Rieske protein and cyt
c1 and/or cyt b.
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INTRODUCTION |
The cytochrome (cyt)1
bc1 complex (ubiquinol:cytochrome c
oxidoreductase) is an integral membrane protein in the electron
transport chains of mitochondria and many respiratory and
photosynthetic prokaryotes (1). The complex translocates four protons
to the positive side of the membrane per two electrons transferred from ubiquinol to cyt c in a Q-cycle mechanism (2). A
key-bifurcated reaction occurs at the Qo site in which the
first electron is transferred from ubiquinol to the Rieske iron-sulfur
center (2Fe-2S) and then to cyt c1 and cyt
c (1-3). The second electron is transferred from
semiquinone in the Qo site to cyt bL
and then to cyt bH and ubiquinone in the
Qi site. Qo site inhibitors can be divided into three classes. Class Ia inhibitors such as myxothiazol and
(E)-
-methoxyacrylate-stilbene alter the heme spectrum of
cyt bL, class Ib inhibitors such as UHDBT alter
the EPR spectrum of 2Fe-2S, and class Ic inhibitors such as
stigmatellin alter both (1-3). X-ray crystallographic studies have
revealed that these different classes of inhibitors occupy different
subsites in the Qo pocket and have different effects on the
mobility of the extramembrane domain of the Rieske iron-sulfur protein
(ISP) (4-8). Stigmatellin binding increases the midpoint redox
potential (Em) of 2Fe-2S by ~200-250 mV (9) and
immobilizes the ISP in the b conformation with the His-161
ligand on reduced 2Fe-2S in hydrogen-bonding contact with stigmatellin
in the Qo site (4-8). In contrast,
(E)-
-methoxyacrylate-stilbene binding to the
Qo site completely eliminates the anomalous scattering signal for 2Fe-2S close to cyt bL, indicating
the release of the ISP from the b conformation to a mobile
state (7). These studies led to the proposal that the ISP functions as
a mobile shuttle as it transfers an electron from Q-H2 in
the Qo site to cyt c1 (4-8).
Experimental support for the mobile shuttle mechanism has been obtained
from cross-linking and mutational studies that immobilize the ISP or
alter the conformation of the neck region (10-19).
Famoxadone and azoxystrobin are new Qo site inhibitors that
have significantly different effects on cyt bc1
than other Qo site inhibitors (20-22). X-ray
crystallographic studies revealed that famoxadone binding in the
Qo site is stabilized by a network of interactions among
the three aromatic groups on famoxadone and aromatic residues in the
binding pocket (Fig. 1, top) (23). Famoxadone binding leads
to extensive conformational changes on the surface of cyt b
and triggers a long range conformational change in the ISP from the
mobile state to a state with 2Fe-2S proximal to cyt b (23).
In contrast to stigmatellin, famoxadone increases the
Em of 2Fe-2S by only 26 mV and
immobilizes both the oxidized and reduced
ISP in the b conformation.2 This is
consistent with the finding that famoxadone is more deeply buried in
the Qo site than stigmatellin and that it does not form a
hydrogen bond with the His-161 ligand on reduced 2Fe-2S (5, 8, 23).
Azoxystrobin also immobilizes the ISP in the b conformation but has only a minor effect on the Em of 2Fe-2S,
decreasing it by 24 mV.2
In this paper, the effects of famoxadone and azoxystrobin on electron
transfer between the Rieske iron-sulfur center and cyt c1 are studied using the binuclear ruthenium
complex, Ru2D, to rapidly photooxidize or photoreduce cyt
c1 (10). Binding famoxadone to the
Qo site of bovine cyt bc1 decreased
the rate constant for electron transfer from 2Fe-2S to cyt
c1 from 16,000 s
1 to 1,480 s
1, consistent with a decrease in the mobility of ISP.
Famoxadone binding also decreased the rate constant for
reverse electron transfer from cyt
c1 to 2Fe-2S to 1,420 s
1,
indicating that the rate constants for the release of oxidized and
reduced ISP from the b conformation are the same in the
presence of famoxadone. Famoxadone binding was also found to decrease
the rate of electron transfer from 2Fe-2S to cyt
c1 in R. sphaeroides cyt
bc1 as well as mutants involving residues at the
interface between the Rieske protein and cyt c1
and/or cyt b.
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EXPERIMENTAL PROCEDURES |
Materials--
Ru2D was prepared by a modification
of the method of Downard et al. (24). Bovine cyt
bc1 was purified as described by Yu et
al. (25). Wild-type and mutant R. sphaeroides cyt
bc1 were prepared as described by Tian et
al. (11). Paraquat, succinate, TMPD, p-benzoquinone,
and antimycin A were obtained from Sigma; stigmatellin was purchased
from Fluka; and [Co(NH3)5Cl]2+
was synthesized (26). N-Dodecyl-
-D-maltoside
was obtained from Anatrace. Succinate cytochrome c reductase
(SCR) was purified as described previously (27).
Generation and Expression of R. sphaeroides Cyt bc1
Mutants--
Mutations were constructed by site-directed mutagenesis
using the Altered Sites system from Promega. The single-stranded
pSELNB3503 (28) was used as the template for mutagenesis, and
oligonucleotides used were as follows: K70C (ISP),
5'-GTCAAGTTCCTCGGCTGCCCGATCTTCATCCGCCGCCGCACCGAGGCCGACATCG-3'; D143C (ISP), 5'-GGCGGCGTGTCGGGTTGCTTCGGGGGCTGGTTCT-3';
F148C (ISP), 5'-GGGGGCTGGTGCTGCCCCT-3'; P150C (ISP),
5'-TTCGGGGGCTGGTTCTGCTGCTGCCACGGATCGCACTA-3'; G153C (ISP),
5'-TGGTTCTGCCCCTGCCACTGCTCGCACTACGACAGTGCC-3'; S154A (ISP),
5'-CCCTGCCACGGAGCGCACTACGACAGT-3'; Y156W (ISP),
5'-CACGGATCGCACTGGGACAGTGCCGGCCGTA-3'; K164C (ISP),
5'-AACGTGAACTGCGGCTTCATGCT
ACAGTGCCGGCCGTATCCGGTGCGGCCCCGCGCCCGAGAACC-3'; P166C (ISP),
5'-CGTATCCGGAAGGGCTGCGCGCCCGAGAACCTG-3'; V64C (cytb), 5'-GTCACCGGCATCTGCCTTGCGATGCAT-3'; G89C (cytb),
5'-AACGTGAACTGCGGCTTCATGCT-3'; M92C(cytb),
5'-AACGGCGGCTTCTGCCTGCGCTACCTGCATGC-3'; A185C (cytb), 5'-GCTGCTCGGCGGCCCGTGCGTGGACAATGCCA-3'; Y302C (cytb),
5'-CTGCCCTTCTGCGCGATCCTGCGCGC-3'; I326C (cytb),
5'-CATCAGCTTCGGCATCTGCGACGCCAAGTTCTTCGGCGTGCTCGCGATGT-3'; K70C(ISP)/A185C(cytb),
5'-GTCAAGTTCCTCGGCTGCCCGATCTTCATCCGCCGCCGCACCGAGGC CGACATCG-3'/5'-GCTGCTCGGCGGCCCGTGCGTGGACAATGCCA-3';
P33C(ISP)/G89C(cytb), 5'-GGGGCCGCCGTCTGGTGCCTGATCAACCAAATG-3'/5'-AACGTGAACTGCGGCTTCATGCT-3'; and N36C(ISP)/G89C(cytb),
5'-TGGCCGCTGATCTGCCAAATGAATCCGTC- 3'/5'-AACGTGAACTGCGGCTTCATGCT-3'.
A plate-mating procedure (28) was used to mobilize the pRKDfbcFmBmCHQ
plasmid in Escherichia coli S17-1 cells into R. sphaeroides BC17 cells as described previously (12). Growth of
E. coli cells and plasmid-bearing R. sphaeroides
cells were carried out as described previously (12). The identity of
the mutations was confirmed by DNA sequencing before and after
photosynthetic or semi-aerobic growth of the cells as described
previously (12). Mutant cytochrome bc1 was
purified as described by Xiao et al. (12).
Determination of Enzyme Activity and Redox Potential of the
2Fe-2S Cluster in Mutant Cyt bc1--
The cyt
bc1 activity was determined in an assay mixture
containing 100 mM Na+/K+ phosphate
buffer, pH 7.4, 300 µM EDTA, 100 µM cyt
c, and 25 µM 2,3-dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinol
(Q0C10BrH2) at 23 °C using the
method described by Xiao et al. (12). The redox potentials
of 2Fe-2S in cyt bc1 mutants were determined as
described previously (12). The reduction of cyt
c1 was followed by measuring the increase of the
-absorption (553-545 nm) in a Shimadzu UV2101 PC spectrophotometer.
The reduction of 2Fe-2S was followed by measuring the negative CD peak
at 500 nm of partially reduced complex minus fully oxidized complex in
a JASCO J-715 spectropolarimeter (29-31). The same samples were used
for the absorption and CD measurements. The redox potentials of 2Fe-2S were calculated from the redox states of heme c1
and 2Fe-2S at pH 8.0 using 280 mV for the midpoint redox potential of
heme c1 (32).
Flash Photolysis Experiments--
Transient absorbance
measurements were carried out by flash photolysis of 300-µl solutions
contained in a 1-cm glass semi-microcuvette. The excitation light flash
was provided by a Phase R model DL1400 flash lamp-pumped dye laser
using coumarin LD 490 to produce a 480-nm light flash of <0.5-µs
duration. The detection system has been described by Heacock et
al. (33). Samples typically contained 5 µM cyt
bc1 in a buffer with 0.01% dodecylmaltoside. In
photoreduction experiments, 10 mM aniline and 1 mM 3CP were used as sacrificial donors, and catalytic
concentrations of horse cyt c and bovine cyt oxidase (20 nM) were present to maintain cyt bc1
in the oxidized state. In photooxidation experiments, paraquat or
[Co(NH3)5Cl]2+ was used as
sacrificial acceptors, and
Q0C10BrH2 was used to reduce cyt
bc1. To regenerate reduced quinol throughout the
flash experiments, 1 mM succinate and 50 nM SCR
were included. Redox mediators included p-benzoquinone
(
m = +280 mV) and TMPD (
m = +275 mV) used in
concentrations of 10 and 2 µM, respectively. The
experiments were carried out aerobically to rapidly reoxidize the
highly absorbing reduced paraquat.
 |
RESULTS AND DISCUSSION |
Effects of Famoxadone and Azoxystrobin on Electron Transfer between
2Fe-2S and Cyt c1 in Bovine Cyt bc1--
An
important goal toward understanding the mechanism of electron transfer
in cyt bc1 is to determine what factors control the conformation of the ISP in each state of the complex, the dynamics
of the changes between the different conformations, and the rate of
electron transfer in each of the conformations. X-ray crystallographic
studies are providing valuable information on the conformations of the
ISP including the b conformation (Fig. 1, top), the
c1 conformation (Fig. 1, bottom), and
intermediate and mobile conformations (4-8). However, it has been
difficult to determine the kinetics of electron transfer from 2Fe-2S to cyt c1 (34, 35) as well as the dynamics of ISP
conformational changes. The development of the ruthenium photoreduction
method provides an opportunity to measure electron transfer between
2Fe-2S and cyt c1 in both the forward and
reverse directions and thus provides kinetic information on two
different initial redox states of cyt bc1 (10).
Moreover, it is becoming clear that the measured rates of electron
transfer are probably rate-limited by conformational changes in the ISP
(36). The binuclear complex Ru2D contains the
2,2':4',4":2",2
-quaterpyridine ligand, which bridges
the two ruthenium atoms (Fig.
2A, inset) (24).
Ru2D has a charge of +4, which allows it to bind with high
affinity to the negatively charged domain on cyt
c1 (10). The electrochemical properties of
Ru2D are similar to those of the widely used ruthenium
tris-bipyridine complex (Table I).
The metal-to-ligand excited state of Ru2D with a lifetime
of 0.5 µs is both a strong oxidant and a strong reductant and can
rapidly oxidize or reduce cyt c1 in the presence of appropriate sacrificial electron acceptors or donors (10).

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Fig. 1.
Top, x-ray crystal structure of
bovine cyt bc1 in the presence of famoxadone
(23). Famoxadone is colored purple, the cyt
c1 and cyt bL hemes are
red, and the 2Fe-2S center is represented by a
Corey-Pauling-Koltun model. The ISP is blue, and cyt
b is gray. Residues 252-268 in the ef
loop, which change conformation as a result of famoxadone binding are
colored orange. Residues 163-171 in the neck-contacting
domain are colored yellow. The residues in the ISP and cyt
b that were mutated are shown as sticks.
Bottom, x-ray crystal structure of bovine cyt
bc1 P6522 crystals
(c1 conformation) (6). ISP is blue,
cyt c1 is yellow, and the residues
that were mutated are shown as sticks. R. sphaeroides numbering is used.
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Fig. 2.
Electron transfer from 2Fe-2S to cyt
c1 in bovine cyt
bc1 following photooxidation of cyt
c1. A, a sample containing
5 µM bovine cyt bc1, 20 µM Ru2D, and 10 mM paraquat in 20 mM Tris-Cl, pH 8.0, at 24 °C was treated with 10 µM Q0C10BrH2, 1 mM succinate, and 50 nM SCR to reduce 2Fe-2S
and cyt c1. The sample was subjected to a 480-nm
laser flash to photooxidize cyt c1, and electron
transfer from 2Fe-2S to cyt c1 was followed at 552 nm.
The smooth curve is a biphasic fit with rate constants of
16,000 ± 3,000 s 1 and 250 ± 50 s 1 and relative amplitudes of 66 and 34%.
Inset, Ru2D with ruthenium atoms represented by
Corey-Pauling-Koltun models and nitrogens by small balls. B,
famoxadone (30 µM) was added to the sample in
A and subjected to a laser flash to photooxidize cyt
c1. Note the change in time scale. The
monophasic transient has a rate constant of 1,480 ± 250 s 1. C, stigmatellin (45 µM) was
added to the sample in B and subjected to flash photolysis.
D, the sample in A was treated with 50 µM azoxystrobin and subjected to flash photolysis.
The monophasic transient has a rate constant of 3,400 ± 600 s 1.
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To study electron transfer in the forward direction, the ruthenium
dimer Ru2D was used to rapidly photooxidize cyt
c1 in bovine cyt bc1 with
cyt c1 and 2Fe-2S was initially reduced (Fig.
2A). The excited state of Ru2D oxidizes cyt
c1 within 1 µs according to Scheme
1. Only one of the two ruthenium centers
in Ru2D is photoexcited in this experiment, and this one is
represented in Scheme 1. The sacrificial electron acceptor paraquat was
present in the solution to oxidize Ru(II*) and/or Ru(I). The rapid
photooxidation of cyt c1 shown by the initial
decrease in 552-nm absorbance was followed by biphasic reduction of cyt
c1 with rate constants of k1 = 16,000 ± 3,000 s
1 and
k2 = 250 ± 50 s
1 (Fig.
2A). The rate constant k1 has been
assigned to electron transfer from reduced 2Fe-2S to photooxidized cyt
c1, whereas k2 is
correlated with the oxidant-induced reduction of cyt
bH (Scheme 2)
(10). The rate constant k2 thus represents
electron transfer of the first electron from Q-H2 to 2Fe-2S
and cyt c1 followed rapidly by the transfer of
the second electron from the semiquinone to cyt
bL and cyt bH (10). The
experimental rate constant k1 is much smaller
than the theoretical value predicted for electron transfer between
2Fe-2S and cyt c1 in the crystallographic c1 state, 1 × 106-2 × 107 s
1 (5, 10). Therefore, it appears that
the measured rate constant for electron transfer is "gated" by
changes in the conformation of the ISP (10). This interpretation is
consistent with previous studies showing that k1
has a large temperature dependence with an activation energy of 59 kJ/mol (10). X-ray diffraction studies have indicated that the ISP is
largely in the b conformation when both cyt
c1 and 2Fe-2S are reduced (37). Therefore, the
rate constant of 16,000 s
1 could represent the rate of
the conformational change from the b state of the ISP to the
c1 state where rapid electron transfer can occur.
The addition of 30 µM famoxadone to bovine cyt
bc1 led to a single phase of reduction of
photooxidized cyt c1 with a rate constant of
1,480 ± 250 s
1, and no reduction of cyt
bH was observed at 562 nm (Fig. 2B). The rate constant was independent of the concentration of famoxadone over the range of 10-100 µM, consistent with a large
binding constant. These results indicate that famoxadone binds strongly
to the Qo site and decreases the rate constant for electron
transfer from 2Fe-2S to cyt c1 from 16,000 to
1,480 s
1. It appears that famoxadone binding
significantly decreases the rate constant for the conformational change
from the b state to the c1 state. The
amplitude of the 552-nm transient for cyt c1 reduction in the presence of famoxadone is 31% of the amplitude of the
initial photooxidation, approximately the same as in the absence of
famoxadone (28%). Because 2Fe-2S and cyt c1
have the same redox potentials at pH 8.0, the electron transfer from
2Fe-2S to cyt c1 would reach equilibrium with
cyt c1 50% reduced and the amplitude of the cyt
c1 reduction would be expected to be 50% of the
initial photooxidation. It is quite possible that the ISP is lost from
a fraction of cyt bc1 molecules during
purification, accounting for the smaller amplitude of the observed cyt
c1 reduction. The similar reduction amplitudes
in the presence and absence of famoxadone are consistent with the
finding that famoxadone binding has only a small effect on the redox
potential of 2Fe-2S, increasing it by 26 mV. The addition of
stigmatellin completely eliminated the reduction of cyt
c1, indicating that stigmatellin displaced famoxadone from the Qo site and locked reduced ISP strongly
in the b conformation (Fig. 2C). This indicates
that stigmatellin has a much higher affinity for the Qo
site than famoxadone. The addition of 30 µM azoxystrobin
to bovine cytochrome bc1 resulted in a single
phase of cyt c1 reduction with a rate constant
of 3,400 ± 600 s
1 (Fig. 2D). This result
is consistent with x-ray crystallographic studies showing that
azoxystrobin binding immobilizes the ISP in the b
conformation.2 The addition of 30 µM
stigmatellin to bovine cyt bc1 treated with 30 µM azoxystrobin led to the elimination of cyt
c1 reduction, indicating that stigmatellin
displaced azoxystrobin from the Qo site.
To study reverse electron transfer from cyt c1
to 2Fe-2S, Ru2D was used to photoreduce cyt
c1 in oxidized bovine cyt
bc1 according to Scheme
3 (Fig. 3). Electron transfer from
excited state Ru(II*) to cyt c1 was complete in
1 µs, the lifetime of the excited state of Ru2D.
The sacrificial electron donors aniline
and 3CP were present in the solution to reduce Ru(III). The
photoreduction of cyt c1 was followed by
biphasic oxidation with rate constants of 90,000 ± 15,000 s
1 and 7,300 ± 1,200 s
1 and relative
amplitudes of 57 and 43%, respectively (Fig. 3A). The
difference in kinetics compared with forward electron transfer is
apparently the result of the initial redox states of the enzyme. X-ray
diffraction studies have indicated that a smaller fraction of ISP is in
the b conformation in the fully oxidized complex than in the
complex with cyt c1 and that 2Fe-2S is reduced
(37). It is reasonable to assign the fast phase to the mobile
conformation of the ISP and the slow phase to the conformation
initially in the b state (10). With this assumption, the
fast phase would be gated by fluctuations in conformation between the
mobile state and the c1 state, whereas the slow
phase would be gated by the conformational change from the b
state to the mobile state. The addition of famoxadone resulted in a
single phase of cyt c1 oxidation with a rate
constant of 1,420 ± 200 s
1 (Fig. 3B).
The amplitude of the single phase in the presence of famoxadone was
nearly the same as the sum of the two phases in the absence of
inhibitor. This result is consistent with the finding that ISP is
nearly all in the b conformation in the presence of
famoxadone (23). The rate constant kd for the change in conformation from the b state to the mobile state must
therefore be much smaller than the rate constant kf
from the mobile state to the b state in the presence of
famoxadone. The rate constant kd is 1420 s
1 in the presence of famoxadone, whereas the rate
constant kf could be 90,000 s
1 or even
higher. It is interesting that the rate constants for forward and
reverse electron transfer are the same in the presence of famoxadone.
This indicates that the rate constant kd does not
depend on the redox state of 2Fe-2S, consistent with the finding that
famoxadone binding changes the redox potential of 2Fe-2S by only 26 mV.
The temperature dependence of kd gives activation
parameters of
H
= 19.1 ± 1.8 kJ/mol
and
S
=
121 ± 6 J/mol·K for the conformational change from the
b state to the mobile state in the presence of famoxadone
(Fig. 4).

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Fig. 3.
Reverse electron transfer from cyt
c1 to 2Fe-2S in bovine cyt
bc1 following photoreduction of cyt
c1. A, a sample containing
5 µM oxidized bovine cyt bc1 and
25 µM Ru2D in 5 mM sodium
phosphate, pH 7.0, 10 mM aniline, 1 mM 3CP, and
0.01% lauryl maltoside at 24 °C was excited with a 480-nm laser
flash to photoreduce cyt c1. Electron transfer
from cyt c1 to 2Fe-2S was followed at 552 nm.
Catalytic concentrations (50 nM) of cyt c and
cyt oxidase were present in the solution to reoxidize cyt
bc1 between flashes. The smooth curve
is a biphasic fit with rate constants of 90,000 ± 15,000 s 1 and 7,300 ± 1,200 s 1 and relative
amplitudes of 57 and 43%. B, famoxadone (30 µM) was added to the solution in A and
subjected to flash photolysis. The monophasic transient has a rate
constant of 1,420 ± 200 s 1.
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Fig. 4.
Temperature dependence of rate constant for
electron transfer from cyt c1 to 2Fe-2S in
bovine cyt bc1 inhibited with
famoxadone. The 552-nm transients were recorded under the
same conditions as in Fig. 3B. The solid line is
the best fit to the Eyring equation: ln(k/T) =  H /RT + S /R + ln(h/kB) with
H = 19.1 ± 1.8 kJ/mol and
S = 121 ± 6 J/mol·K.
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The present studies indicate that famoxadone binding significantly
decreases the rate constant for the release of the ISP from the
b conformation in cyt bc1. X-ray
crystallography studies have revealed that famoxadone binding leads to
significant conformational changes in three domains on the surface of
cyt b: residues 163-171 that contact the neck region of the
ISP; residues 262-268 in the ef loop that are part of the
ISP-docking crater; and residues 252-256 in the middle of the
ef loop that connects the Qo site to the other
two surface domains (Fig. 1, top) (23). The ef loop may play a role in relaying famoxadone-induced conformational changes in the Qo pocket to the ISP crater and the neck
contact domain to decrease the rate of release of the ISP. The linkage between the conformation of the Qo site and the dynamics of
the ISP movement could be a key to how the enzyme promotes the transfer of the first electron from Q-H2 to 2Fe-2S but inhibits the
transfer of the second electron from semiquinone to 2Fe-2S. The
potential role of the ef loop in regulating the dynamics of
ISP domain movement during the catalytic cycle is particularly intriguing.
Effects of Famoxadone on Electron Transfer between 2Fe-2S and Cyt
c1 in R. sphaeroides Cyt bc1--
Electron
transfer in the forward direction was studied using Ru2D to
photooxidize cyt c1 in R. sphaeroides
cyt bc1 according to Scheme 1 in the presence of
the sacrificial electron acceptor [Co(NH3)5Cl]2+. The reduction of
photooxidized cyt c1 was biphasic with rate constants of k1 = 80,000 ± 15,000 s
1 and k2 = 1,500 ± 300 s
1 (Fig. 5A).
k1 is attributed to electron transfer from
2Fe-2S to cyt c1, whereas
k2 is due to subsequent electron transfer from Q-H2 to 2Fe-2S and cyt c1 followed
by electron transfer from the semiquinone to cyt
bL and cyt bH (Scheme 2)
(10). Previous studies of the effects of temperature, pH, and redox
potential demonstrated that k1 is not
rate-limited by true electron transfer in the c1 state but rather is gated by conformational changes from the
b state and the mobile state to the active
c1 state (36). According to this analysis, the
population of the c1 state is small but the rate
constant for electron transfer from 2Fe-2S to cyt
c1 in the c1 state is
much larger than the observed value of k1, 80,000 s
1. The addition of famoxadone leads to monophasic
reduction of cyt c1 with a rate constant of
4,800 ± 800 s
1 (Fig. 5B). There is no
reduction of cyt bH in the presence of famoxadone, consistent with displacement of Q-H2 from the
Qo site. The most reasonable explanation of
these results is that famoxadone binding stabilizes the b
state of the ISP and that the observed rate constant
k1 is gated by the rate constant
kd for the release of the ISP from the b
state to the mobile state and the c1 state. The
rate constant kf for the conformational change from
the mobile state to the b state is expected to be much
larger than kd in the presence of famoxadone and could be 80,000 s
1 or larger. Electron transfer was also
studied in the reverse direction using Ru2D to photoreduce
cyt c1 in the oxidized complex according to
Scheme 3 in the presence of the sacrificial electron donors aniline and
3CP. Oxidation of photoreduced cyt c1 was
biphasic with rate constants of 84,000 ± 15,000 s and 4,800 ± 800 s
1 and relative amplitudes of 60 and 40%,
respectively. The addition of famoxadone led to monophasic oxidation of
cyt c1 with a rate constant of 6,800 ± 1,200 s
1. It appears that the Rieske protein is largely
in the b conformation in the presence of famoxadone and the
rate of release to the c1 conformation is
decreased. The fact that the rate constants for forward and reverse
electron transfer are nearly the same in the presence of famoxadone
indicates that the rate constant kd does not depend
on the redox state of 2Fe-2S.

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Fig. 5.
Electron transfer from 2Fe-2S to cyt
c1 in R. sphaeroides
cyt bc1 following photooxidation of
cyt c1. A, a solution
containing 5 µM R. sphaeroides cyt
bc1 and 20 µM Ru2D in
20 mM sodium borate, pH 9.0, and 5 mM
[Co(NH3)5Cl]2+ was treated with
Q0C10BrH2 to reduce cyt
bc1 and excited with a 480-nm laser flash. The
smooth curve is a biphasic fit with rate
constants of 80,000 ± 15,000 s 1 and 1,500 ± 300 s 1. B, famoxadone (30 µM)
was added to the sample in A and subjected to flash
photolysis. The monophasic transient has a rate constant of 6,800 ± 1,200 s 1.
|
|
Effect of Mutations in the Iron-Sulfur Protein and cyt
b on Electron Transfer in Cytochrome bc1--
Mutations in
R. sphaeroides cyt bc1 were generated
by site-directed mutagenesis to characterize the interaction of the ISP with cyt c1 and with cyt b (Fig. 1).
Flash photolysis studies were carried out to determine the effects of
these mutations on the rate constants k1 for
electron transfer from 2Fe-2S to cyt c1 and
k2 for electron transfer from Q-H2
to 2Fe-2S as described above (Table II).
The effects of famoxadone on the kinetics of selected mutants was also
examined (Table III). The ISP mutant P166C is completely inactive in photoinduced electron transfer, suggesting that this mutation may have led to a critical alteration in
the conformation of the ISP. The P150C mutation near the 2Fe-2S center
dramatically decreases the rate constant k2 for
electron transfer from Q-H2 to 2Fe-2S down to only 4 ± 1 s
1 but does not significantly affect the rate
constant k1 for electron transfer from 2Fe-2S to
cyt c1. It appears that the structural change
caused by this mutation greatly affects the interaction of the ISP with
the cyt b peptide but does not affect the dynamics of the
interaction of the ISP with cyt c1. Famoxadone
binding decreased the rate constant k1 somewhat
more than for wild-type cyt bc1, indicating that
the ISP was held more tightly in the b conformation (Table
III). The K70C mutation on the ISP decreases k2
down to 250 ± 50 s
1 but does not affect
k1, indicating a significant effect on the interaction of ISP with the cyt b peptide but no effect on
the interaction with cyt c1. In contrast, the
D143C mutation decreases k1 down to 20,000 ± 4,000 s
1 but does not affect
k2. This finding suggests that the acidic residue Asp-143 on the surface of the ISP may be involved in the interaction with cyt c1. Surprisingly,
famoxadone binding only decreased k1 to
10,700 ± 2,000 s
1, indicating that this mutant is
not held as tightly in the b conformation as wild-type ISP.
The G153C mutation affects both rate constants, decreasing
k1 to 13,000 ± 2,600 s
1 and
k2 to 450 ± 80 s
1. This
residue close to the 2Fe-2S center appears to be important for the
interaction with both cyt b and cyt
c1. Famoxadone binding to the G153C mutant
decreased the rate of electron transfer from 2Fe-2S to cyt
c1 to 1,500 ± 300 s
1,
indicating that this mutant is held more tightly in the b
conformation than wild-type ISP. Among the cyt b mutations,
only Y302C affected the photoinduced kinetics, decreasing
k1 to 12,000 ± 2,000 s
1 but not greatly
affecting k2. It appears that the ISP is held more tightly in the b conformation in this mutant than in
wild-type enzyme. Earlier studies have shown that the rate constant
k1 for electron transfer from 2Fe-2S to cyt
c1 was not greatly affected by ISP mutations
S154A and Y156W, which decrease the redox potential of 2Fe-2S
significantly (36). These results provided evidence that
k1 is not rate-limited by true electron transfer
in the c1 state but is gated by conformational
changes from the b state and from the mobile state to the
c1 state. The effects of famoxadone binding on
k1 in the S154A and Y156W mutants were
comparable with the effect on wild-type cyt bc1.
Xiao et al. (12) have previously shown that the formation of
a disulfide cross-link between ISP and cyt b in the
K70C/A185C mutant led to a complete loss of steady-state activity,
providing experimental evidence for the mobile shuttle mechanism of the
ISP. The K70C/A185C mutant was totally inactive in photoinduced
electron transfer, providing further confirmation of the mobile
shuttle mechanism. Xiao et al. (38) previously prepared the
P33C/G89C and N36C/G89C mutants where each has a disulfide cross-link
between the tail region of the ISP and cyt b. These mutants
both have good steady-state enzyme activity, providing evidence for the
intertwined dimer structure of cyt bc1. The
photoinduced electron transfer kinetics is not greatly affected by
cross-linking in these mutants, providing further evidence that the
tail region of the ISP is not involved in the mobile shuttle
mechanism.
 |
FOOTNOTES |
*
This work was supported by National Institutes of
Health Grant GM20488 (to F. M. and B. D.), NCRR COBRE 1 P20 RR15569
(to F. M. and B. D.), and GM30721 (C.-A. Y.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
479-575-4999; Fax: 479-575-4049; E-mail: millett@comp.uark.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M211620200
2
Z. L. Zhang, D. Xiao, Q. Byron, C.-H. Tai, B. D. Jordan, L. Yu, and C.-A. Yu, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
cyt, cytochrome;
Qo, quinol reduction site o;
UHDBT, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole;
ISP, Rieske
iron-sulfur protein;
Em, midpoint redox potential;
2Fe-2S, Rieske iron-sulfur center;
Ru2D, [Ru(bpy)2]2(qpy)(PF6)4;
qpy, 2,2':4',4":2",2
-quaterpyridine;
Q0C10BrH2, 2,3-dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinol;
TMPD, tetramethylphenylenediamine;
SCR, succinate cytochrome c
reductase;
3CP, carboxyl-2,2,5,5-tetramethyl-1-pyrolidinyloxy free
radical;
QH2, ubiquinol.
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.