Effect of Famoxadone on Photoinduced Electron Transfer between the Iron-Sulfur Center and Cytochrome c1 in the Cytochrome bc1 Complex*

Kunhong XiaoDagger , Gregory Engstrom§, Sany Rajagukguk§, Chang-An YuDagger , Linda YuDagger , Bill Durham§, and Francis Millett§

From the Dagger  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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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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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)-beta -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)-beta -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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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-beta -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 alpha -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 (epsilon m = +280 mV) and TMPD (epsilon 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.

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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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Table I
Spectral and electrochemical properties of ruthenium complexes

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.


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Scheme 1.  


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Scheme 2.  

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 Delta HDagger  = 19.1 ± 1.8 kJ/mol and Delta SDagger  = -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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Scheme 3.  


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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) = -Delta HDagger /RT + Delta SDagger /R + ln(h/kB) with Delta HDagger  = 19.1 ± 1.8 kJ/mol and Delta SDagger  = -121 ± 6 J/mol·K.

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.


                              
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Table II
Kinetic properties of R. sphaeroides cyt bc1 mutants


                              
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Table III
Effect of famoxadone on electron transfer from 2Fe-2S to cyt c1 in R. sphaeroides cyt bc1 mutants


    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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