Crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformational switch of the Rieske iron–sulfur protein subunit

Cytochrome bc1 complexes (cyt bc1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiquinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction requires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron–sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron–sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-cycle mechanism of cyt bc1 function.

The cytochrome bc 1 complex (cyt bc 1 or bc 1 ), 2 also known as mitochondrial complex III, forms the mid-section of the cellu-lar respiratory chain (1). It is also an essential component of the photosynthetic apparatus of purple bacteria (2). A major function of cyt bc 1 is the pumping of protons across the membrane, which is coupled to electron transfer (ET) from its substrate, ubiquinol, to cyt c (cyt c 2 in bacteria). Depending on the organism, protein compositions of cyt bc 1 vary from 11 subunits in humans (Homo sapiens, Hsbc 1 ) (3) and cows (Bos taurus, Btbc 1 ) (4) to three or four subunits in photosynthetic bacteria such as Rhodobacter capsulatus (R. capsulatus, Rcbc 1 ) (5) or Rhodobacter sphaeroides (R. sphaeroides, Rsbc 1 ) (6). Common to all cyt bc 1 complexes are three catalytic subunits that contain prosthetic groups: cyt b, containing hemes b L and b H ; cyt c 1 , containing a c-type heme; and the Rieske iron-sulfur protein (ISP), containing an Fe 2 S 2 cluster.
The mechanism by which the cyt bc 1 carries out its ET-coupled proton translocation is known as the Q-cycle mechanism ( Fig. 1) (7). This mechanism requires a quinol oxidation site (Q o or Q P site) near the electrochemically-positive side of the membrane and a quinone reduction site (Q i or Q N site) near the negative side of the membrane. The presence of the two sites has been verified by crystal structures in the presence of various Q P and Q N site inhibitors and by mutational studies (8 -11). Essential to the Q-cycle mechanism is the bifurcated ET at the Q P site, in which the two electrons liberated from a ubiquinol substrate follow two different paths: one electron enters the high-potential chain (ISP, cyt c 1 , and cyt c) and the other follows the low-potential chain (b L , b H , and ubiquinone/ubisemiquinone). Despite the peculiarity of this proposed bifurcated ET, the Q-cycle mechanism is able to explain various experimental observations, including the H ϩ /e ratio, and the oxidant-induced cyt b reduction (12,13). Numerous hypotheses have been proposed to provide chemical explanations for the bifurcated ET (14).
The crystal structures of mitochondrial cyt bc 1 reveal several important features. 1) In the native enzyme, the extrinsic domain of the ISP (ISP-ED), a single electron carrier mediating ET from the membrane-embedded cyt b to cyt c 1 in the aqueous phase, exhibits a high degree of mobility and appears in distinctly different positions in different crystal forms (4,10,(15)(16)(17). 2

) In a large number of structures, ISP-ED is clustered
This work was supported by the Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1 and S2 and Table 1. The atomic coordinates and structure factors (codes 6NHH, 6NIN,and 6NHG) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 To whom correspondence should be addressed. E-mail: xiad@mail.nih.gov. 2 The abbreviations used are: bc 1   cro ARTICLE at the b-site: an ISP-binding site on the surface of the cyt b subunit near the Q P site. The distance between the Fe 2 S 2 of the ISP and the cyt c 1 heme is ϳ31 Å, which is too great for efficient ET to the high-potential chain (18). 3) In a small number of structures, the ISP-ED is found close to cyt c 1 (c 1 -site) and somewhere between the c 1 -site and the b-site (intermediate positions) (16,17). 4) The conformation of the ISP-ED can be modulated by two classes of cyt bc 1 inhibitors targeting the Q P site: one class is the P f -type, which fixes the ISP-ED at the b-site, and the other class is the P m -type, which mobilizes the ISP-ED (10,15,19). It was thus hypothesized that the ISP-ED functions as a controlled, swiveling single electron carrier that bridges the distance from ubiquinol to cyt c 1 in the aqueous phase, accomplishing the bifurcated ET at the Q P site (15,20).
The ISP-ED is roughly cone-shaped and at its tapered end (the tip) is an Fe 2 S 2 cluster coordinated by two internal cysteines and two surface-accessible histidine residues. This structural feature enables the ISP-ED, once oxidized, to dip into the volcanic crater-shaped binding site on the cyt b subunit (b-site), gaining access to the Q P site. Although the ISP-ED is bound at the b-site, it extracts one electron as well as one proton from ubiquinol, producing transiently a ubisemiquinol free radical at the Q P site and a reduced ISP-ED. To force a bifurcated ET to occur, the reduced ISP-ED is postulated to stay bound at the Q P site until the remaining electron associated with the ubisemiquinol enters the low-potential chain. Subsequently, with the removal of the reaction product ubiquinone into the membrane phase, the reduced ISP-ED is released and delivers the electron to cyt c 1 , while the proton enters the aqueous phase (15).
The requirement of a mobile ISP-ED for the function of cyt bc 1 was elegantly demonstrated in bacterial cyt bc 1 systems by introducing mutations that altered the mechanical flexibility of residues in the "neck" region of ISP (21,22) and by engineering a disulfide bridge between cyt b and ISP-ED (23). Foreshortening the neck region or substituting flexible residues with rigid ones (Ala 3 Pro and Gly 3 Val) in the ISP inactivated the entire complex without affecting the assembly or complex integration into the membrane (24). Likewise, using a mutant cyt bc 1 that carries a K70C mutation in the ISP subunit and an A185C mutation in the cyt b subunit (ISP K70C Cytb A185C Rsbc 1 ), Xiao et al. (23) showed that in an oxidizing environment that forms and maintains the disulfide bridge the enzyme's function was abolished but could be restored by reduction.
It was demonstrated that the ISP-ED of bacterial cyt bc 1 can be immobilized at the b-site by P f -type inhibitors such as stigmatellin or famoxadone (6,25). Despite the fact that a large number of mechanistic studies of the cyt bc 1 complex were conducted using photosynthetic bacteria such as R. sphaeroides and R. capsulatus for their ease of molecular manipulability and the demonstration that their enzyme's function requires the ISP-ED to be conformationally flexible, this very transition from a fixed to a mobile conformation has never been structurally demonstrated for any bacterial cyt bc 1 complex. Furthermore, the sequence of events for the ISP-ED to transition from a fixed state in the b-site to a mobile state and subsequently to approach the c 1 -site has not been clearly defined. Although the functional significance for the existence of the b-site is wellestablished, that for the c 1 -site has been controversial. The question is whether a fixed c 1 -site is truly a requirement for the function of cyt bc 1 . In this work, we demonstrate the existence of an ISP-ED conformation switch in bacterial cyt bc 1 by solving the crystal structure of cyt bc 1 in complex with azoxystrobin (Rsbc 1 /azo). We further show that the ISP-ED adopts multiple positions when it is in the mobile state. This was accomplished by enhancing the anomalous signal of iron atoms by collecting data from a crystal of bovine mitochondrial cyt bc 1 in complex with azoxystrobin (Btbc 1 /azo) at an incident wavelength of 1.739 Å near the Fe absorption edge. Our data corroborate with the highly transient nature of the c 1 -state when ISP-ED is mobile (26).

Structure of Rsbc 1 arrested in the b-state reveals the correct position of the engineered disulfide bond
We previously introduced a double-cysteine mutation in Rsbc 1 to show that the flexibility of the ISP-ED is important for the cyt bc 1 function (23). The mutation sites were K70C in the ISP subunit and A185C in the cyt b subunit, resulting in a mutant designated ISP K70C cytb A185C Rsbc 1 . To verify the correct formation of the disulfide bond, we crystallized ISP K70C cytb A185C Rsbc 1 in the presence of stigmatellin, a known P f -type inhibitor that arrests the ISP-ED at the b-site. The structure ( Fig. 2A) of ISP K70C cytb A185C Rsbc 1 /stg was determined with three independent dimers per crystallographic asymmetric unit (Table 1). In the structure, the ISP-ED is trapped in the b-site, and the disulfide bridge between residues Cys-70 of the ISP and Cys-185 of cyt b is correctly formed (Fig.  2B). All three dimeric cyt bc 1 molecules, with a total of six disulfide bridges, are superimposable. Importantly, the structure Figure 1. Q-cycle mechanism The cyt bc 1 complex can be seen as made of a high-potential chain (ISP, cyt c 1 , and cyt c) located in the intermembrane space (IMS) of mitochondria and a low-potential chain (hemes b L and b H ) embedded in the inner membrane (IM). There are two reaction sites: quinone reduction (Q N ) and quinol oxidation (Q P ) sites. At the Q P site, the two electrons from a ubiquinol (QH 2 ) diverge: the first one taking the high-potential chain and the second one going into the low-potential chain, reducing ubiquinone/ubisemiquinone (Q/SQ . ) at the Q N site. The Q and QH 2 at the Q N and Q P sites are in free exchange with those present in the membrane-bound quinone/quinol pool shown inside the ellipse in the dashed line.

Binary conformation switch of ISP in bacterial cyt bc 1
proves that the residues chosen for mutation to cysteine are properly aligned when the ISP-ED is at the b-site and the disulfide bridges are formed under normal oxidizing conditions. The engineered inter-subunit disulfide bond changes the position of the ISP-ED domain only slightly from its position in the WT Rsbc 1 /stg complex published earlier (Fig. S1) (6).

ISP-ED is in the mobile state as revealed by the structure of Rsbc 1 inhibited by azoxystrobin
To demonstrate the ISP-ED conformation switch in bacterial cyt bc 1 , we used a P m -type inhibitor, namely azoxystrobin, in a crystallization experiment with Rsbc 1 (Rsbc 1 /azo), as binding of azoxystrobin in Btbc 1 promotes the mobile state of the ISP-ED. This experiment was expected to be challenging, due to the enhanced mobility of the ISP-ED and with it the loss of a rigid hydrophilic surface that could contribute to crystal contacts. With just cyt c 1 remaining in a rigid conformation, the chances of crystallizing Rsbc 1 /azo were considered small. Nev-ertheless, crystallization conditions were established after extensive screening, and the resulting crystals of Rsbc 1 /azo diffracted X-rays to 3 Å resolution (Table 1). Consistent with the structures reported previously (6), crystal contacts are formed between neighboring ef1 helices through pairs of -stacked tryptophan residues (Trp-313) flanked by molecules that appear to be lipids.
The structure of Rsbc 1 /azo has at its core a cyt b homodimer with a total of 16 membrane-spanning helices (Fig. 3A) that are flanked by pairs of C-terminal helices of cyt c 1 and N-terminal helices of the ISP. The large functional head domains of both cyt c 1 and the ISP extend into the periplasm. Unlike the static head domain of cyt c 1 , the ISP-ED is mobile and disordered in both halves of the cyt bc 1 dimer, with no clearly defined electron density to model the polypeptide chain (Fig. 3B). In fact, both ISP-EDs are so disordered that the Fe 2 S 2 cluster can only be located with the help of its anomalous signal, which is obtained from the anomalous difference Fourier map using the coefficients F(h,k,l) -F(-h,-k,-l) with a phase of model Ϫ90°. This calculation produced several peaks Ͼ3 near the subunits cyt b and cyt c 1 in addition to the expected peaks for the b L , b H , and c 1 heme iron atoms (Table 2). However, in each case their occupancies are low compared with those of the heme groups b L , b H , and c 1 . The highest nonheme iron peak is 6 above average and is 19.2 Å from the c 1 heme iron. We assigned this peak to the Fe 2 S 2 cluster of the ISP-ED of the second monomer (model B). A model for this ISP-ED was constructed but had a low occupancy of 0.25. It should be noted that this ISP-ED conformation is only one of many conformations that exist (see below). Indeed, structure refinement of Rsbc 1 /azo with the ISP-ED included produced no better refinement statistics than without it. Furthermore, the highest Fe 2 S 2 anomalous peak of model B is slightly offset (ϳ2 Å) from that of model A ( Table 2). Taken together, the binding of azoxystrobin allows the ISP-ED to be in a mobile state with no specific positional preference. This degree of mobility is also observed in mitochondrial cyt bc 1 in the structures Btbc 1 /azo (PDB code 1SQB) and Ggbc 1 /azo (PDB code 3L71). The latter is a homodimer with one ISP-ED (chain E) disordered and the other (chain R) ordered. It occupies the c 1 Ј position, far from cyt b. It should be mentioned that the disorder of the ISP-ED is not an indication of the absence of the ISP subunit as a whole, because the N-terminal helix of the ISP is clearly visible in the electron density from residues 13 to 52 of the neck region (Fig. 3B).

Binding of azoxystrobin to the Q P site of Rsbc 1
In each of the cyt b subunits, a single azoxystrobin molecule ( Fig. 4A) is firmly bound at the Q P site, displacing ubiquinol and thus preventing any direct reduction of the Fe 2 S 2 cluster of the ISP. The toxophore of azoxystrobin is a planar E-␤-methoxy methyl acrylate group forming a hydrogen bond (2.79 Å) through the ester's carbonyl group with the backbone amide of Glu-295 (Fig. 4B), which is part of the highly conserved PEWY motif. At the same time, the side chain of Glu-295 is rotated toward the aqueous phase, compared with its inward conformation when P f -type inhibitors such as UHDBT or stigmatellin are bound (19). The flat toxophore (3-methoxy methylacrylate) is positioned between Phe-144 and Tyr-147. The attached Heme groups are depicted as ball-and-stick models with carbon atoms in black, nitrogen in blue, and oxygen in red. The Fe 2 S 2 cluster of ISP is shown as balls with iron atoms in orange and sulfur in green. B, electron density in the vicinity of the disulfide bridge (magenta) between ISP (shown in yellow) and cyt b (shown in green). The continuous density around this bridge (K170C of ISP and A185C of cyt b) shows that it formed correctly to arrest ISP-ED in the b-state.

Binary conformation switch of ISP in bacterial cyt bc 1
o-phenyloxy ring is nearly orthogonal to the acrylate moiety due to steric constraints (Fig. 4B), but it is also ideally positioned to wedge between Pro-294 of the PEWY motif and Gly-158 of the cd1 helix. The pyrimidine ring forms an aromaticaromatic (Ar-Ar) pair with Phe-298 and prevents Ile-162 of the cd1 helix from returning to a fully relaxed position. The o-cyano-phenoxy group is surrounded by the hydrophobic residue Met-140 and the aromatic groups of Phe-337 and Phe-301 at ideal distances for Ar-Ar interactions.

Multiple positions of the ISP-ED are also observed in the structure of mitochondrial cyt bc 1 in complex with azoxystrobin
The fact that iron atoms can appear as significant peaks in anomalous difference Fourier maps and thus mark the positions of the Fe 2 S 2 clusters in Rsbc 1 /azo inspired us to re-visit the structure of mitochondrial cyt bc 1 with the P m -type inhibitor azoxystrobin bound. To study this, we grew crystals of Btbc 1 in the presence of azoxystrobin (Btbc 1 /azo) and collected a diffraction data set using an incident wavelength of ϭ 1.739 Å near the iron K absorption edge (FeK: 1.743 Å) ( Table 1). Using a Btbc 1 crystal is particularly advantageous over other alternatives because of its stability, very high space group symmetry of I4 1 22 (highly redundant data), and non involvement of the ISP-ED in crystal contacts. The crystal structure not only shows enhanced anomalous signals for the element iron (Table 2), but indeed it dramatically improves the signal for all sulfur atoms in cofactors, standard cysteine and methionine residues, as well as those of phosphorus atoms in lipids (Fig. 5A). Although our re-investigation of the structure of Btbc 1 /azo does not substantially differ from our previously published structure (PDB code 1SQB) or its avian counterpart (PDB code 3L71) (both were collected at 1.0 Å incident radiation), it does, however, allow better visualization of the ISP-ED in its mobile state ( Table 2).
Although the ISP-EDs are disordered, the presence of four Ͼ3.5 peaks for the Fe 2 S 2 clusters indicate that, despite the absence of obvious restraints (domain-domain interactions, crystal contacts), the ISP-ED still finds local minima between cyt b and cyt c 1 (Fig. 5A).

ISP-ED conformation switch is characterized by the change from a fixed to a mobile state and induced by different types of Q P site inhibitors
The ISP-ED conformation switch was first suggested in a structural study of mitochondrial cyt bc 1 , in which different inhibitors gave rise to dramatically different peak heights in anomalous difference Fourier maps for the Fe 2 S 2 cluster at its cyt b-binding site (10). This phenomenon was subsequently used as a basis for classification of cyt bc 1 inhibitors that target the Q P site (19); those that give rise to strong peaks for the Fe 2 S 2 cluster are called P f -type inhibitors, and those that lead to no or weak anomalous peaks are termed P m -type inhibitors. However, this switch between conformations is often misinterpreted as evidence that the ISP-ED moves from the fixed b-site to a similarly rigid c 1 -site (27)(28)(29), and thus it deserves further clarification.
The accumulation of a large number of homologous structures of cyt bc 1 from different species, including bacteria, yeast, birds, and mammals, that were crystallized in different space groups and under various conditions allowed comparisons aimed at distinguishing lattice-induced stabilization of the ISP-ED from natural, low-affinity sites. However, this comparison is often biased toward ISP-ED models built by crystallographers, who naturally select the most prominent conformation and consequently ignore all the others. To overcome this deficiency, in this work we performed structure superposition

Binary conformation switch of ISP in bacterial cyt bc 1
using not only the positions of Fe 2 S 2 from different models but also anomalous difference density maps, if available, resulting in a distribution of Fe 2 S 2 locations in 3D space ( Fig. 5B and Table 3). From the distribution, we observed the following. 1) When the Q P site is occupied by the P f -type inhibitors (stigmatelin, UHDBT, etc.), there is one strong Fe 2 S 2 anomalous peak, located at the bottom of the ISP-docking site. This position is frequently referred to as the b-site. 2) When the Q P site is not occupied by substrates or inhibitors, Fe 2 S 2 can appear in three different positions, and its corresponding anomalous peaks are weak. The most frequent position is near the b-site and is displaced on average from it by 1.5 Å (Nat site, Fig. 5B). A second position, represented by one model (PDB code 1BE3), has the Fe 2 S 2 near the heme c 1 , a position referred to as the c 1 -site. The same position was found to overlap with weak anomalous peaks from P m -type inhibitor-bound cyt bc 1 crystals. A third position, also represented by only one model (PDB code 1BGY), is 7 Å from the second site and is referred to as the intermediate site (Int site, Fig. 5B). Again, this site also overlaps with anomalous peaks from apo-and P m -type inhibitor-bound cyt bc 1 crystals. 3) When the Q P site is bound with P m -type inhibitors (azoxystrobin, MOAS, etc.), the Fe 2 S 2 positions are further displaced from the apo-site, spanning the distance between the "Int" site and the "Nat" site, as exemplified by the 3.8 Å shift from the apo-site for the azoxystrobin-bound structure (Fig. 5B). We term this site the bЈ-site. It has a rms deviation from its mean position of 7.3 Å. There are three additional sites marked also by weak anomalous peaks. One overlaps with the c 1 -site. Another was previously observed in a number of avian cyt bc 1 structures inhibited by P m -type inhibitors (PDB code 6NHH, 3L70, etc.), referred to as the c 1 Ј-site. The third Fe 2 S 2 position has not previously been reported and is designated the bЉ-site.
Based on the distribution of the anomalous peaks and following published conventions, we propose the following. 1) ISP-ED is at the b-site (therefore, the cyt bc 1 is in the b-state) when it associates tightly with the cyt b subunit, permitting the first electron and proton transfer from the substrate to the ISP. The b-site is approximated by the binding of P f -type inhibitors. At this position, the ISP-ED features a very large anomalous peak, and the rms deviation for the Fe 2 S 2 position is 0.72 Å. 2) In the absence of substrate at the Q P site, the ISP-ED switches to a mobile conformation, which is facilitated by the binding of P m -type inhibitors at the Q P site. The Fe 2 S 2 cluster can appear in either the bЈ-site, bЉ-site, c 1 -site, or c 1 Ј-site, indicating that the positions of the ISP-ED are not well-determined. Thus, we further define the conformation switch of the ISP-ED as the change from a fixed state at the b-site to a mobile state, detached from the b-site (Fig. 5B) (15). In the fixed state, the ISP-ED docks firmly at the b-site with its Fe 2 S 2 cluster penetrating into and being part of the Q P site, where the substrate ubiquinol and inhibitors bind. By contrast, the ISP-ED is in the mobile state that requires the ISP-ED to be detached by at least 1.5 Å from the b-site, which is triggered by the displacement of the cd1/cd2 helices. It becomes apparent that the liberated ISP-ED is very mobile but may still favor distinct positions on its path to the cyt c 1 for the electron transfer. Interestingly, the c 1 Ј-, bЈ-, and the bЉ-sites are located along the path of an arc that may define the preferred trajectory for the movement of the ISP-ED (Fig. 5B).

ISP-ED conformation switch is critical to the Q-cycle mechanism
The key question surrounding the bifurcated ET at the Q P site is how the second electron of ubiquinol is prevented from following the first electron, which enters the high-potential chain consisting of ISP (E m, 7 ϭ ϩ285 mV for Rsbc 1 ), cyt c 1 (E m, 7 ϭ ϩ295 mV), and soluble cyt c 2 (E m, 7 ϭ ϩ295 mV). The Azoxystrobin is shown as a CPK model and lipid molecules as ball-and-stick models. The two cyt c 1 subunits with covalently linked c-type hemes are shown in blue. The bound Sr 2ϩ ions (silver spheres) stem from a crystallization additive. Both yellow ribbon models of the iron-sulfur protein are highly disordered and are shown in a position that places their Fe 2 S 2 clusters in or near a position with detectable anomalous signal from their iron atoms. The parallel horizontal lines delineate the boundary of the membrane bilayer. B, electron density for the Rsbc 1 /azo dimer with cyt b in red, cyt c 1 in blue, and ISP in yellow. Although the N-terminal helix of ISP has a good electron density, the head domain is largely disordered.

Binary conformation switch of ISP in bacterial cyt bc 1
second electron actually enters the low-potential chain that is formed by two b-type heme groups (b L , E m, 7 ϭ Ϫ90 mV; b H , E m, 7 ϭ ϩ50 mV) (30 -32). The distance of ϳ31 Å between Fe 2 S 2 and cyt c 1 heme iron, as seen in crystal structures of cyt bc 1 bound with established P f -type inhibitors, precludes an efficient ET between the two redox groups (Fig. 6A and Table  S1). Thus, it was proposed that the ET between Fe 2 S 2 and cyt c 1 is achieved by the oscillatory motion of the iron-sulfur protein's extrinsic domain (ISP-ED). At the end point of this motion, the Fe 2 S 2 and cyt c 1 are brought close enough for efficient ET (Table S1). Indeed, the ISP-ED has been modeled in crystal structures as close as 15-21 Å from the Fecenter of the c-type heme group (Fig. 6A) (17). Furthermore, the flexibility of the ISP-ED was shown to be essential for the function of cyt bc 1 in an elegant biochemical study (22). However, such a model is at odds with the kinetic measurements because the ET rate between ISP and cyt c 1 was experimentally measured at 60,000 s Ϫ1 for Rsbc 1 (Sadoski et al. (26)), whereas the enzymatic turnover rate of Rsbc 1 is only 83 s Ϫ1 (2.5 mol of cyt c/min/nmol of cyt b) (33). In other words, it is not a lack of flexibility of the ISP-ED that prevents the second electron from entering the high-potential chain. Furthermore, it is unable to explain the observation that cyt b reduction precedes that of cyt c 1 in pre-steadystate quinol oxidation experiments (34,35).
The ISP-ED conformation switch offers a satisfactory mechanism for bifurcated ET at the Q P site (Esser et al. (15)) (Fig. 6B), utilizing the architecture of the enzyme that has the ability to control the docking and undocking of the ISP-ED from the cyt b subunit. Thus, although the single-electron carrier ISP-ED remains immobilized at the b-site, the electron it carries cannot be delivered to the cyt c 1 and neither can it accept a second electron; therefore, the high-potential chain is discon-nected. The initial one-electron oxidation creates an unstable ubisemiquinone radical with a very-low redox potential (E m, 7 ϭ Ϫ60 mV) (36), leaving the low-potential chain (b L , b H , and Q/Q ⅐ ) as the only choice for the second electron.
We previously showed that the conformation switch of the ISP-ED is achieved by modulating its affinity to cyt b through substrate-induced movements of the cd1/cd2 helices. Fixation of the ISP-ED at the b-site is due to the increased affinity of the ISP-ED to the b-site in the presence of P f -type inhibitors as a result of cd1 helix movement, which alters the ISP-ED-binding site. By contrast, the P m -type inhibitors move the cd1 helix in the opposite direction, reducing the affinity for the ISP-ED (15).

Implications concerning development of novel inhibitors that target cyt bc 1
Azoxystrobin is an effective cyt bc 1 inhibitor of both Btbc 1 and Rsbc 1 . As a classical Q P -site inhibitor of the P m -type, the measured IC 50 values for azoxystrobin in Rsbc 1 and Btbc 1 are 1.34 and 1.03 nM, respectively (Fig. S2), which is corroborated by the nearly identical binding mode of the inhibitor revealed by comparison of the structures of Btbc 1 and Rsbc 1 in complex with azoxystrobin (Fig. 3B). High-resolution structures of the complexes afford us a detailed analysis for the observed resistance against azoxystrobin in certain mutant strains of the yeast. One frequently observed mutation (G143A) in cyt b impedes binding and thus renders pathogens resistant to azoxystrobin (Fig. 3C), which is caused by the smaller gap between the highly conserved residues Pro-294 and Gly-158 (the residues Pro-271 and Gly-143 in yeast), into which the aromatic ring of azoxystrobin intercalates. Although the G143A mutation is often the sole reason for azoxystrobin resistance in a The crystal of Btbc 1 /azo has a space group symmetry of I4 1 22 with one monomer per crystallographic asymmetric unit. The crystal of Rsbc 1 /azo has a space group symmetry of P2 1 with a dimer in the asymmetric unit. b All anomalous difference Fourier maps were calculated using the data in the resolution range between 25 and 5 Å. Figure 4. Chemical structure of azoxystrobin and its binding environment in Rsbc 1 . A, chemical structure of azoxystrobin. B, stereographic diagram of the bound azoxystrobin in stick model in the Q P pocket overlaid with the blue difference electron density cage for azoxystrobin. The electron density was generated with phenix.polder and contoured at 5. Residues lining the Q P pocket are shown as a stick model. The hydrogen bond between azoxystrobin and the main-chain amide N atom of Glu-295 is shown as the dotted line in red. C, superposition of the Q P site between the Btbc 1 /azo and Rsbc 1 /azo structures. The poses of azoxystrobin molecules bound to the Q P sites of Rsbc 1 (yellow sticks) or Btbc 1 (cyan sticks) are nearly identical.

Binary conformation switch of ISP in bacterial cyt bc 1
fungal pathogens (with cross-resistance to numerous other strobilurin-derived pesticides), a performance penalty for the cyt bc 1 activity is incurred, as the binding of ubiquinol is also negatively affected.
The binding pose of azoxystrobin in Rsbc 1 compares well with those found in the structures of mitochondrial cyt bc 1 (Btbc 1 , PDB code 1SQB) (Fig. 4C) and (Ggbc 1 , PDB code 3L71). In the first crystal structure (PDB code 1SQB) with bound azoxystrobin, the methyl acrylate ester was assigned the s-cis conformation because of ambiguity in the electron density. Although the hydrogen bond to the Glu-295 amide exists even in the s-cis conformation, the energetically more favorable s-trans conformation was found in the higher-resolution structure of Ggbc 1 (PDB code 3L71) and was confirmed in our current Btbc 1 /azo structure. In Rsbc 1 , the bound azoxystrobin molecule adopts an overall conformation that displays a very small rms deviation from that in mitochondrial Btbc 1 after careful superposition of the cyt b subunits. How- Figure 5. ISP-ED mobility of Btbc 1 /azo revealed by anomalous difference Fourier. A, peaks from anomalous difference Fourier using X-ray diffraction data collected near the iron-absorption edge. The enhanced anomalous peaks in Btbc 1 /azo (magenta) contoured at 3.5 reveal four prominent sites for Fe 2 S 2 -labeled bЈ-, bЉ-, c 1 Ј-, and c 1 -sites, respectively. The stronger signal of the bЈ-site led to a model of ISP-ED with high-atomic displacement factors. The outline of the ISP-ED at the c 1 Ј-position, which was not modeled in the crystal structure, is indicated by dotted lines. Similarly, the weaker sites bЉ and c 1 Ј were not modeled. B, alignment of cyt bc 1 structures based on superpositions of the cyt b subunits to a single monomer (PDB code 6NHG and Btbc 1 /azo, this work). The cyt b and cyt c 1 subunits superimpose well and are rendered here as the green and blue molecular surfaces, respectively. The positions of the ISP subunit are represented by positions of the Fe 2 S 2 cluster (from available experimental coordinates in PDB in stick models) and by anomalous difference Fourier peaks, contoured at 3.5-3.8 values in red (PDB code 1SQX, Btbc 1 /stg), white (PDB code 2FYU, Btbc 1 /jg144), dark cyan (PDB code 1NTZ, Btbc 1 /native), yellow (PDB code 1SQQ, Btbc 1 /MOAS), magenta (6NHG, Btbc 1 /azo), and blue (PDB code 6NHH, Rsbc 1 /azo). PDB identifiers correspond to the published position of the ISP-ED. The arc in black represents a possible trajectory for ISP movement. The inset features a magnified view of the ISP-docking site showing the b-site represented by the Fe 2 S 2 position from the structure of cyt bc 1 bound with stigmatellin, the bЈ-site from structures of cyt bc 1 in the apo or P m -type inhibitor-bound state.

Protein purification of Rsbc 1 and Mtbc 1
The purification procedure for Rsbc 1 , published earlier (37), was used with minor changes to obtain protein suitable for crys- Figure 6. Controlled ISP-ED motion switch and the bifurcated ET mechanism. A, structure of the dimeric Rsbc 1 /azo represented by the prosthetic groups b L , b H , c 1 , and Fe 2 S 2 in stick models. The bound azoxystrobin molecules are shown as ball models. The ISP-ED subunit was mobile and is shown as a molecular surface in the two extreme conformations: one is at the b-site (gray) and another is near the c 1 Ј-site (orange). Distances between pairs of prosthetic groups are also indicated. B, Q-cycle mechanism is modified to incorporate the conformation switch of ISP-ED, which allows the enforcement of the bifurcated ET at the Q P site.

Binary conformation switch of ISP in bacterial cyt bc 1
tallization. Briefly, chromatophore membranes were prepared from cells harboring the plasmid pRKDfbcFBC H , which codes for a WT Rsbc 1 lacking subunit IV ( ⌬-IV Rsbc 1 ), by disrupting cells with a French press followed by differential centrifugations. To purify the His 6 -tagged Rsbc 1 , the chromatophore suspensions were adjusted to a cyt b concentration of 25 M with 50 mM Tris-HCl (pH 8.0 at 4°C), containing 20% glycerol, 1 mM MgSO 4 and 1 mM phenylmethylsulfonyl fluoride. Dodecyl-Dmaltopyranoside (DDM) solution (10% w/v) was added dropwise to a final detergent-to-protein ratio of 0.57 (mg/mg). After centrifugation, the supernatant was loaded onto a nickelnitrilotriacetic acid column, which was washed with 6 column volumes of buffer A (50 mM Tris-HCl, pH 8.0, at 4°C, 200 mM NaCl, 0.01% DDM), 6 column volumes of buffer A in the presence of 5 mM histidine and 4 column volumes of buffer B (50 mM Tris-HCl, pH 8.0, at 4°C, 200 mM NaCl, 0.5% ␤-octyl glucoside (␤-OG)) in the presence of 5 mM histidine. The desired protein fractions were eluted with buffer B containing 200 mM histidine and concentrated with Centriprep-50 to a final concentration of 300 M.
The Btbc 1 was prepared starting from highly-purified succinate-cyt c reductase, as reported previously (38). The cyt bc 1 particles were solubilized by deoxycholate, and contaminants were removed by a 15-step ammonium acetate fractionation. The purified cyt bc 1 complex was recovered in the oxidized state from the precipitates formed between 18.5 and 33.5% ammonium acetate saturation. The final product was dissolved in a buffer containing 50 mM Tris-HCl, pH 7.8, and 0.66 M sucrose. The protein stock solution with a protein concentration of 30 mg/ml was stored at Ϫ80°C. The concentrations of cyt b and c 1 were determined spectroscopically using millimolar extinction coefficients of 28.5 and 17.5 mM Ϫ1 cm Ϫ1 for cyt b and c 1 , respectively.

Crystallization of Rsbc 1 in complex with azoxystrobin and the double-cysteine mutant (ISP K70C cytb A185C Rsbc 1 ) inhibited by stigmatellin
The ⌬-IV Rsbc 1 variant at a concentration of 57 mg/ml was incubated with 3.5 mM azoxystrobin in the presence of 3.5 mM fresh sodium ascorbate for 24 h at 4°C. The solution was diluted with a buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 200 mM histidine, 0.5% ␤-octyl glucoside, 10% glycerol, 5 mM NaN 3 and 1.25 mM sodium ascorbate. This produced a 14.25 mM solution of the ⌬-IV Rsbc 1 /azo complex, which was screened for optimal crystal growth in four different concentrations of sucrose monocaprate (SMC) (0.12, 0.18, 0.24, or 0.30%) and at different concentrations of PEG 400 (5, 6, 7 or 8%). Prior to the addition of the PEG 400, each drop was augmented with 10 mM Sr(NO 3 ) 2 . The best crystals grew in standard 24-well, sitting drop plates at 16°C in the presence of 7% PEG 400 and 0.12% SMC over a reservoir of 100 mM Tris-HCl, pH 7.0, 384 mM NaCl, 16% PEG 400, 12.8% glycerol, 5 mM NaN 3 , and 9 mM tris(carboxymethyl) phosphine.

Crystallization of Btbc 1 in complex with azoxystrobin
A solution of the purified Btbc 1 was adjusted to a final concentration of 20 mg/ml in a buffer containing 50 mM MOPS at pH 7.2, 20 mM ammonium acetate, 20% (w/v) glycerol, and 0.16% sucrose monocaprate. The inhibitor azoxystrobin (ChemService) was dissolved in DMSO to a concentration of 100 mM and mixed with native Btbc 1 in a molar ratio of 5:1. The inhibitor/Btbc 1 solution was incubated at 4°C overnight before it was used for crystallization. This solution was set up for crystallization as described previously (Xia et al. (4)). Crystals of cyt bc1 with bound azoxystrobin appeared within 3-4 weeks and were cryo-protected at a glycerol concentration of 30 -40% (w/v).

X-ray diffraction data collection, structure determination, and refinement
Rsbc 1 crystals were flash-frozen in liquid propane without additional cryoprotectant. A crystal of ISP K70C cytb A185C Rsbc 1 / stg diffracted X-rays to about 3.6 Å Bragg spacing at beamline 22 ID (Ser-CAT) of the Advanced Photon Source at the Argonne National Laboratory (APS, ANL) and displayed the symmetry of space group C2. Structure solution proceeded by rigid-body refinement of the isomorphous high-resolution trimer of Rsbc 1 /stg (PDB code 2QJY). The refinement protocol was carried out initially in the absence of a disulfide bridge, resulting in maps with clear evidence for electron density between the residues forming an inter-subunit Cys-Cys bridge. The structure of the mutant was refined and rebuilt in Phenix (phenix-online.org) and COOT (Crystallographic Object Oriented Toolkit Software), respectively.
For the Rsbc 1 /azo crystal, a complete data set to 3.0 Å Bragg spacing was collected. The crystal belonged to the space group P2 1 and contained one Rsbc 1 dimer per asymmetric unit. The structure was solved by molecular replacement (Molrep, CCP4) using a trimmed dimer from a high-resolution structure (PDB code 2QJY). While having very poor densities, the mobile head domains of ISP (residues 52 to 187) were positioned in the near-"c 1 " positions based on the anomalous signal for the Fe 2 S 2 cluster. The low occupancies of the mobile ISP extrinsic domains were fixed at 0.25 and grouped b-factor refinement was applied.
Diffraction images of a Btbc 1 /azo crystal were collected at beamline 5 ID (DND-CAT, APS, ANL) specifically using an X-ray wavelength of ϭ 1.739 Å at the K absorption edge of iron. The crystal diffracted initially well beyond the edge of the detector, limiting the extent of the recordable data to 2.8 Å Bragg spacing. The highly-redundant data set has strong anomalous data stemming from iron with contributions from sulfur and phosphorus atoms.

Binary conformation switch of ISP in bacterial cyt bc 1
Author contributions-L. E. and D. X. conceptualization; L. E., F. Z., and D. X. data curation; L. E., F. Z., and D. X. formal analysis; L. E. and D. X. validation; L. E. and D. X. investigation; L. E. and D. X. visualization; L. E., F. Z., C.-A. Y., and D. X. methodology; L. E. writing-original draft; L. E. and D. X. writing-review and editing; C.-A. Y. and D. X. resources; D. X. supervision; D. X. funding acquisition; D. X. project administration.