Hydrogen Bonding to the Substrate Is Not Required for Rieske Iron-Sulfur Protein Docking to the Quinol Oxidation Site of Complex III^*

Differential Inhibition of Mitochondrial and Bacterial cyt bc₁ Complexes by Famoxadone

Previously, we reported that binding of famoxadone to Btbc₁ induces docking of the ISP to the Q_P site (21). Unlike other Q_P site inhibitors such as stigmatellin and 3-Undecyl-2-hydroxydioxobenzothiazol, famoxadone does not make a direct H-bond with the ISP-ED because the shortest distance between His¹⁴¹ of the ISP and the phenylamino moiety of famoxadone is 6.1 Å (Fig. 3A). However, whether the fixation of the ISP-ED by famoxadone binding can be interpreted as part of the Q-cycle mechanism remains unclear. Thus, to understand this phenomenon, one would have to investigate the effect of famoxadone on cyt bc₁ of other organisms, which are evolutionarily remote from mammals or yeast, such as bacteria. Doubts that the capture of the ISP-ED induced by famoxadone binding is indeed part of a conserved mechanism were initially cast by the observation that the experimentally determined IC₅₀ values of 1.4 and 418 nm of isolated cyt bc₁ from photosynthetic bacterium Rhodobacter sphaeroides (Rsbc₁) and mitochondrial cyt bc₁ (Btbc₁), respectively, differ by a factor of nearly 300 (Fig. 4A). Further differences were noticed in the shapes of the inhibition curves for Rsbc₁ and Btbc₁, suggesting that Rsbc₁ might exhibit a different set of binding interactions with famoxadone.

Paradoxically, the difference in IC₅₀ values is not supported by the apparent conservation in the Q_P site, because all residues in contact with famoxadone in Btbc₁ are conserved, except for two residues: Phe²⁷⁶ and Ala²⁷⁷ (Pro³⁰⁰ and Phe³⁰¹ in Rsbc_1, Fig. 5). The closest distances between these two Btbc₁ residues and bound famoxadone are 4.53 and 3.72 Å, respectively (Fig. 3A). Judging by the alignment between Btbc₁ (PDB code 1L0L) and Rsbc₁ (PDB code 2FYN) structures, it seems unlikely that these two amino acid substitutions would result in a significant change in the binding of famoxadone, because modeling exercises indicate residue Phe³⁰¹ in Rsbc₁ appears to be in a good position to replace the function of Phe²⁷⁶ in Btbc₁.

Distinct Conformation of Famoxadone as the Q_P Site Occupant of Rsbc₁

To answer questions about the binding mode of the inhibitor famoxadone and its local and global effect, we determined the crystal structure of Rsbc₁ in complex with famoxadone (Fig. 1A, Table 1). There are four copies of cyt bc₁ in the Rsbc₁ crystal per asymmetric unit, each binding one molecule of famoxadone. Even though no non-crystallographic symmetry (NCS) restraints were applied to the inhibitor during refinement, the four famoxadone molecules superpose within 0.198 Å root mean square (RMS) deviation, allowing our analysis to focus on just one molecule.

The structure of the Rsbc₁·famoxadone complex reveals that famoxadone binds in the same location as in Btbc₁ (Fig. 3B). Like Btbc₁, the chiral environment of the Q_P site of Rsbc₁ selects the S-(−)-isomer of famoxadone for binding. Most importantly for mechanistic implications, the ISP-ED is found docking in the Q_P position, as evidenced by the strong peak of the anomalous difference Fourier density at the location of the 2Fe-2S cluster of ISP. At the same time, residue His¹⁵² (ligand to the 2Fe-2S cluster in Rsbc₁ and equivalent of His¹⁴¹ of Btbc₁) has a distance of 5.7 Å to the phenylamino group of famoxadone. This result confirms that recruitment of the ISP to the Q_P site does not depend on the H-bond formation between the ISP-ED and the inhibitor.

As in the Btbc₁·famoxadone structure, the inhibitor bound to Rsbc₁ is capable of forming an H-bond between atom O6 and the backbone amide nitrogen atom of Glu²⁹⁵ over a distance of 2.69 Å and between atom N1 and the conserved water molecule (W1) (Fig. 3B). Unexpectedly, the terminal phenoxy group adopts a position essentially opposite to that found in Btbc₁·famoxadone, which is quantifiable by a change in torsion angle (C12-C11-O14-C15) from −116° to +145° (Fig. 6A). However, this difference in positions of the phenoxy group is consistent with the observation that the residues lining the entrance to the Q_P pocket are quite variable between cyt b subunits of different organisms. The adaptation of the terminal phenoxy group to a different environment appears to be based on both exclusion and attraction. The exclusion is caused by the substitution of Ala²⁷⁷ of Btbc₁ with Phe³⁰¹ in Rsbc₁ (Fig. 5), which would not allow the phenoxy group to be in the same position as in Btbc₁. Being flexible to swing around, the aromatic phenoxy group of famoxadone finds stabilization by aromatic attractions from Phe¹⁶⁶, Phe³³⁷, and Phe¹⁴⁴ (Figs. 3B and 5A), which correspond to Ile¹⁵⁰, Ala²⁹⁵, and Phe¹²⁸ in the bovine sequence.

Famoxadone has been noted to prefer aromatic environments (21), which is demonstrated most convincingly by the 13-fold increase in IC₅₀ value for the F129L mutation in yeast (30). Thus, the combined effect of the naturally occurring aromatic residues Phe¹⁶⁶ and Phe³³⁷ in the cyt b subunit of Rsbc₁ (as opposed to the respective residues Leu¹⁵⁰ and Ala²⁹⁵ in Btbc₁) might cause the stabilization of famoxadone in the present conformation and explain the difference in IC₅₀ values compared with Btbc₁ (Fig. 4A).

Mapping the interactions between Q_P site residues and bound famoxadone, it became immediately apparent that Rsbc₁ uses an overlapping but non-identical set of residues from Btbc₁ for famoxadone binding (Fig. 5). Most notably, Rsbc₁ uses Phe¹⁶⁶, Met³³⁶, and Phe³³⁷ to engage famoxadone, whereas the equivalent residues Leu¹⁵⁰, Leu²⁹⁴, and Ala²⁹⁵ in Btbc₁ remain uninvolved with the inhibitor. Conversely, not all interactions observed in Btbc₁ are also found in the Rsbc₁·famoxadone complex, as seen in residues Phe⁹¹, Tyr⁹⁵, Phe²⁷⁶, Ala²⁷⁷, and Ile²⁹⁸ in Btbc₁ (Phe¹⁰⁵, Tyr¹⁰⁹, Pro³⁰⁰, Phe³⁰¹, and Ile³⁴⁰ in Rsbc₁) (Fig. 5). Thus, these structural observations qualitatively explain the differences in IC₅₀ values for famoxadone binding to Btbc₁ and Rsbc₁, which could not be predicted based on sequence alignment.

Oxazolidinedione-type Inhibitors Induce Docking of the ISP-ED to the Q_P Site without Direct H-bonding to the ISP and Inhibitor-bound Btbc₁ Structures Reveal a Conserved Core-binding Motif

Superposition of the cyt b subunits of Rsbc₁ and Btbc₁ complexed with famoxadone shows a good structural alignment of the oxazolidinedione core of famoxadone and its two directly connected aromatic rings (Fig. 6A), which raises the question as to whether the difference in binding affinity of famoxadone between Rsbc₁ and Btbc₁ is entirely due to the difference in interaction with the terminal phenoxy group. To evaluate the amount of binding energy contributed by various portions of famoxadone other than the phenoxyl group, we measured the binding of two additional oxazolidindione-type compounds, 5-methyl-5-(4,6-difluorophenyl)-3-phenylamino-2,4-oxazolidinedione (jg144) and 5-methyl-2-(methylsulfanyl)-5-phenyl-3-(phenylamino)-3,5-dihydro-4H-imidazol-4-one(fenamidone), to both Btbc₁ and Rsbc₁ (Fig. 4, B and C). The chemical structures of jg144 and fenamidone closely resemble that of famoxadone except that they do not have the conformationally flexible terminal phenoxy group (Fig. 2, B and C). Although jg144 replaces the phenoxy group and the hydrogen atom H9 of famoxadone each with fluorine, fenamidone features a substituted imidazole ring in place of the oxazolidinedione ring.

The IC₅₀ values for jg144 inhibition were determined to be 1.1 and 1.2 nm, respectively, for Rsbc₁ and Btbc₁ (Fig. 4B). Similarly, the IC₅₀ values for fenamidone were 3.5 and 8.3 nm, respectively, for Rsbc₁ and Btbc₁ (Fig. 4C). More importantly, the profiles of the jg144 and fenamidone inhibition curves are very similar regardless of whether they bind to Rsbc₁ or Btbc₁, indicating that similar interactions are employed for their binding.

We determined and refined the structure for the complex Btbc₁·fenamidone at 2.65 Å resolutions (Table 1) and reanalyzed the structure of Btbc₁·jg144 (PDB code 2FYU) at 2.25 Å resolution. In both cases, their respective ISP-EDs are docked at the Q_P site, as indicated by the high anomalous peak heights of the 2Fe-2S clusters (Table 2). The arrest of the ISP-ED is a characteristic trait of P_f-type inhibitors (13), and here the observed shortest distances between His¹⁴¹ of the ISP and the bound inhibitors are 6.34 and 6.74 Å, respectively, for jg144 and fenamidone (Fig. 3, C and D, Table 2). Thus, no direct H-bond is formed between the ISP and the inhibitors.

The structures of both Btbc₁·jg144 and Btbc₁·fenamidone showed no significant differences in side chain rotamers from Btbc₁·famoxadone (Fig. 6B). This in turn suggests that the terminal phenoxy group of famoxadone is capable of sampling the environment around the entrance of the Q_P site without perturbing any side chains in the Q_p site. The consistency of interactions and the measurements showing little difference in IC₅₀ values between Btbc₁ and Rsbc₁ for jg144 and fenamidone firmly establish the structural basis for the three-ring unit as the toxophore of the oxazolidinone-type inhibitors (29).

Mechanistic Implications Concerning the Bifurcated Electron Transfer at the Q_P Site

Quinol oxidation at the Q_P site requires strict, consecutive bifurcated ET steps that are subject to a high degree of control, an idea that has received support over the years by a substantial amount of experimental evidence and features a bimodal ISP-ED conformation switch at its core (14, 15). Although most structural data on this switch were first obtained from Btbc₁, structural information of several avian mitochondrial cyt bc₁ (Ggbc₁) complexes recently made public in the Protein Data Bank (PDB codes 3L74 and 3L73) is in perfect agreement with the former. However, the conformational switch of the ISP-ED in bacterial bc₁ has been difficult to observe crystallographically. This is because all structures of bacterial cyt bc₁ complexes obtained so far were crystallized with bound stigmatellin, and it has been difficult to crystallize them in a different P_f-type inhibitor such as famoxadone. Conceivably, crystallizing bacterial cyt bc₁ bound with a P_m-type inhibitor would be even more difficult. Prior to this work, complexes of bacterial bc₁ were crystallized only in the presence of the P_f–type inhibitor stigmatellin with the ISP-ED immobilized at the Q_P position (4, 12, 17). In this work, we show for the first time that another P_f-type inhibitor, namely famoxadone, is able to fix the conformation of ISP-ED without providing a direct H-bond to the ISP subunit, which allows successful crystallization of Rsbc₁.

Quinol oxidation at the Q_P site is initiated with the recruitment to and/or stabilization and proper alignment of the ISP-ED at the Q_P site, which was proposed to take place via a direct H-bond between one of the histidine ligands to the 2Fe-2S cluster of ISP (His¹⁴¹ in Btbc₁ and His¹⁵² in Rsbc₁) and the hydroxyl group of its substrate ubiquinol. This proposal was based on the observation that in high resolution crystal structures, polar atoms from the enzyme's ISP-ED and the inhibitor (specifically stigmatellin and 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole (11, 19)) appeared within H-bond distance. Contrary to this view, structural studies with Btbc₁ demonstrated that famoxadone is capable of immobilizing the ISP-ED at the Q_P site in the absence of a direct H-bond to the ISP (13, 21). The question becomes whether the presence or absence of the H-bond can support the required bimodal conformation switch of the ISP-ED.

The bifurcated ET at the Q_P site begins with the binding of substrate QH₂, which also triggers the docking of ISP-ED. An H-bond forms between a hydroxyl group of QH₂ and a histidine of the ISP-ED. After deprotonation, the QH₂ transfers its first electron to the 2Fe-2S cluster of the ISP, which is an energetically favorable process. The second electron of QH₂, however, has to travel to the Q_N site via the hemes, b_L and b_H, eventually reducing a ubiquinone (Q) or ubisemiquinone (Q^•) molecule (Fig. 1B). It is important to emphasize (i) that to avoid short circuit reactions, the 2Fe-2S cluster cannot be allowed to leave the Q_P site and return oxidized until the product has been completely removed and/or replaced by QH₂ and (ii) that although the ISP-ED remains at the Q_P site, the H-bond should always exist between the reduced ISP and the product ubiquinone (Q) after the completion of the two-electron transfer reaction (Fig. 7). In other words, the H-bond between the substrate and the ISP cannot control the conformation switch of the ISP-ED.

In this work, we present evidence from the famoxadone-bound Rsbc₁ structure and two Btbc₁ structures with bound oxazolidinedione-type inhibitors, jg144 and fenamidone, showing that docking of the ISP-ED to the Q_P site, both in mitochondrial and bacterial cyt bc₁, does not require a direct H-bond between the ISP and the inhibitor. This lends strong support for the hypothesis that electron bifurcation at the Q_P site is achieved through a controlled bimodal conformational change of the ISP-ED (14, 15).

Crystal Contacts Do Not Significantly Influence the Conformation of ISP-ED

The head domain of ISP in Rsbc₁ is involved in crystal-packing contacts, whereas that of Btbc₁ is not. This observation provides a good opportunity to qualitatively assess how much influence crystal contacts have on the conformation of the intrinsically moving domain. For the purpose of an unbiased comparison, we also determined the structure of the doubly inhibited (stigmatellin and antimycin) ternary complex of Rsbc₁ (Table 1). Antimycin is a Q_N site inhibitor and is not known to influence ISP-ED conformation; thus, its presence is of no consequence for this analysis. Stigmatellin inhibited Rsbc₁ has been structurally characterized before (4). However, the newly determined structure that we are using as a reference was obtained from the same batch of protein under the same conditions, and importantly, the crystals features the same space group P1, the same number of molecules per asymmetric unit (two dimers) and has the same cell dimensions and crystal packing. To quantify how much the ISP-ED moves in response to a change in the Q_P site occupant, we superposed only the Cα atoms of the cyt b subunits of the different complexes (stigmatellin versus famoxadone) and calculated the respective RMS deviations of the head domain of the ISP, cyt b, and cyt c₁ subunits (Table 3). Although the Cα traces of cyt b superimpose very well (<0.4 Å), the traces of the ISP-ED no longer coincide: they display RMS deviations as large as 2.55 Å (139 Cα) and 2.86 Å (124 Cα), respectively, for Rsbc₁ and Btbc₁. This is significant, because the corresponding deviations of the head domain of cyt c₁ (a domain that is spatially close to ISP-ED and located in the same aqueous phase) amounted to an RMS deviation of only 0.60 Å (Rsbc₁) and 0.93 Å (Btbc₁), respectively. It is worth noting that the RMS deviations of ISP-ED in Rsbc₁ and Btbc₁ are nearly the same despite the fact that the ISP-ED of the Rsbc₁ is clearly involved in crystal contacts, unlike the ISP-ED in Btbc₁, which has none. The similarities in RMS deviations between Rsbc₁ and Btbc₁ suggest that the crystal contacts in Rsbc₁ play only a secondary role in determining the final conformation of the ISP-ED once ISP-ED is docked at the Q_P site.

The angular displacement of the ISP-EDs between Btbc₁ and Rsbc₁ when bound with different inhibitors can also be visualized using vectors (Fig. 8A), which is calculated based on superposition of cyt b subunits only between the structures with bound antimycin and famoxadone. The red vectors in the ISP trace (yellow trace) of the stigmatellin-inhibited Rsbc₁ represent the connection to the Cα atoms of the famoxadone-inhibited complex. The blue vectors represent the equivalent repositioning of the Cα atoms of the ISP-ED in Btbc₁. This vector diagram illustrates qualitatively that the amount of change (vector length) is approximately the same in both sets. However, the direction in which the ISP-ED is displaced in Rsbc₁, versus Btbc₁, is characterized by an angle of 58° (represented by half-transparent triangles). It is conceivable that, as a consequence of packing forces in Rsbc₁ crystals, the ISP-ED undergoes an angular adjustment but importantly maintains the proper distance from the Q_P site of cyt b.

Implications Concerning the Design and Use of Broad Spectrum Antifungal Agents

Despite considerable pressure that must exist to maintain the intricate function of cyt b and its active sites, several residues, individually or in groups, may have changed in the course of evolution. These changes are seen as the root cause of intrinsic drug resistance by target site mutations. By determining the structure of Rsbc₁ in complex with famoxadone and comparing it with that of Btbc₁, we uncovered the structural basis for the effects of sequence polymorphisms on the binding of inhibitors to cyt bc₁ of different organisms, which were manifested by varied efficacies of inhibitors against cyt bc₁ complexes. This structural observation is consistent with measurements of IC₅₀ values, indicating that famoxadone inhibits Rsbc₁ more strongly than Btbc₁ (Fig. 4A); it agrees also with the binding energy of −12.0 kcal/mol and −12.8 kcal/mol, respectively, estimated by molecular docking experiments for mitochondrial and bacterial cyt bc₁ complexes. The conformation of famoxadone in the binding pocket shows sensitivity to the environment and is characterized by a tendency to optimize Ar-Ar interactions (21).

Because of the absence of an apo Rsbc₁ structure, the only comparisons that can be made are between complexes of Rsbc₁ with famoxadone and stigmatellin (Fig. 8B) and the equivalent pair of Btbc₁ complexes (Fig. 8C). The comparison made it immediately clear that the accommodation of famoxadone in the Q_P pocket requires adjustments of torsion angles in both the inhibitors and cyt b residues. Direct steric interactions force Phe²⁹⁸ (Phe²⁷⁴ in Btbc₁) to adopt two distinct conformations depending on the binding of either stigmatellin or famoxadone in both Rsbc₁ and Btbc₁. Similarly pronounced are changes in the positions of Glu²⁹⁵ (Glu²⁷¹ in Btbc₁) in response to the inhibitors. In the Rsbc₁·famoxadone structure, Glu²⁹⁵ returns to the solvent channel or to the “native” position as observed in Btbc₁ structures because of the loss of a hydrogen bond to the inhibitor (8).

From the Rsbc₁ structure, Phe¹⁶⁶, Met³³⁶, and Phe³³⁷ (Leu¹⁵⁰, Leu²⁹⁴, and Ala²⁹⁵ in Btbc₁) appear to play a major role in repositioning the terminal phenoxy moiety of famoxadone. Although this is convincing based on qualitative energy considerations, the situation seems subtler in the recently published Ggbc₁ structure with famoxadone (35), showing the same moiety in an orientation nearly identical to the one found in Rsbc₁ but in contrast to that in Btbc₁. Despite the higher sequence identity between Ggbc₁ and Btbc₁ (Fig. 5), residue Phe¹⁵¹ in the avian Gg cyt b is also a phenylalanine, as in Rsbc₁ (Rsbc₁ Phe¹⁶⁶). However, residue Phe³³⁷ of Rs cyt b coincides with the non-aromatic Ala²⁹⁶ in Gg cyt b, implying that a single change in the amino acid composition (Leu¹⁵⁰Phe) at the entrance to the Q_P pocket is sufficient to stabilize an alternate conformation of famoxadone to the one that was first found in Btbc₁. This observation underscores the importance of Ar-Ar interaction in inhibitor binding.

The fact that famoxadone is able to adapt to the different microenvironments of the target sites of a wide range of organisms suggests two important principles for designs of broad spectrum inhibitors: one is the flexibility to adopt different conformations as needed, and the other is the ability to fully engage in Ar-Ar interactions. A single pair of Ar-Ar interactions could provide as much as 1.3 kcal/mol of free energy (36), which can be exploited either for the enhancement of specificity or for the evasion of resistance and is also consistent with previous observations that the phenoxyphenyl group of famoxadone is most tolerant to variations (29).

Expression of Δ-sub IV Rsbc₁

The purification procedure for Rsbc₁, published earlier (37), was used with minor changes to obtain protein for crystallization. Briefly, chromatophore membranes were prepared from cells harboring the plasmid (pRKDfbcFBC_H) coding for Δ-sub IV wild type Rsbc₁ protein, by disrupting cells with a French press followed by differential centrifugations. To purify His₆-tagged Rsbc₁, the chromatophore suspensions were adjusted to a cyt b concentration of 25 μm with 50 mm Tris-Cl (pH 8.0 at 4 °C), containing 20% glycerol, 1 mm MgSO₄, and 1 mm PMSF. Dodecyl-d-maltopyranoside (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 on a nickel-nitrilotriacetic 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% β-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 μm.

Crystallization of Btbc₁ in Complex with Inhibitors

The final concentration of purified Btbc₁ complex was adjusted to 20 mg/ml in a solution containing 50 mm MOPS buffer at pH 7.2, 20 mm ammonium acetate, 20% (w/v) glycerol, and 0.16% sucrose monocaprate. This solution was further incubated with a 5× molar excess amount of the desired inhibitor and set up for crystallization as described in previous publications (8, 21). Crystals were treated with a cryoprotectant in 5% increments until the final glycerol concentration reached 42% and then cryo-cooled in liquid propane.

Crystallization of Famoxadone-inhibited Rsbc₁

A solution of Δ-sub IV Rsbc₁ (57 mg/ml) in a buffer containing 0.5% β-OG was treated overnight with a 3-fold molar excess of famoxadone (Chem. Service Inc.). The solution was diluted 4-fold with buffer consisting of 50 mm Tris-HCl, pH 8.0, 0.3% β-OG, 200 mm histidine, 150 mm NaCl, 10 mm sodium ascorbate, and 10% glycerol. The resulting solution of 14 mg/ml Rsbc₁ in 0.35% β-OG was augmented with sucrose monocaprate (0.06%, ∼0.5 critical micelle concentration) and 10 mm strontium nitrate. PEG 400, added to a final concentration of 7%, served as the precipitant. The solution was allowed to stand overnight at 4 °C. Any precipitate was centrifuged off, and 5 μl of the supernatant was used in sitting drop vapor diffusion crystallization trays over 1 ml of 100 mm Tris-HCl, pH 8.0, 20% glycerol, 600 mm NaCl, 26% PEG 400, and 5 mm NaN₃. After several weeks, small red crystals appeared which froze cleanly in liquid propane without requiring additional cryoprotectants.

Diffraction Data Collection, Structure Determination, and Refinement

Crystals of the tetragonal Btbc₁·fenamidone and the triclinic famoxadone-inhibited Rsbc₁ were stable when cryo-cooled to 100 K, permitting several hours of data collection at the SER-CAT Beamline (ID22) of the Advanced Photon Source, Argonne National Lab. Raw diffraction frames were processed with the program package HKL2000 (38). A diffraction data set for the Btbc₁ crystal was phased with coordinates of the 11-subunit apo structure of Btbc₁ (PDB code 1NTM) (23). The program REFMAC (39) was employed for initial rigid body refinement followed by iterative maximum likelihood and TLS (Translation, Libration, and Screw tensor) refinement. In between REFMAC runs, sigma A (40) weighted 2F_o − F_c and F_o − F_c Fourier maps were calculated and used to identify and build bound ligand and solvent molecules manually with the program Coot (41). The data set for the Rsbc₁ was phased with molecular replacement using an edited version of the Rsbc₁ dimer (PDB code 2QJY) in MolRep (CCP4) (42), producing a solution with two dimers/unit cell. Care was taken in the subsequent refinement to allow the head domain of the ISP to adjust to a position possibly different from that in stigmatellin-inhibited complexes. Rigid body refinement, simulated annealing, positional, and atomic displacement (ADP, TLS) refinement were carried out with phenix.refine 1.8–1512 (43). 4-fold NCS restraints were applied throughout the refinement. However, no NCS restraints were imposed on the four famoxadone molecules during refinement, yet they are superposed within 0.2 Å RMS deviation (average from mean structure), allowing our analysis to focus on just one molecule. Manual adjustments of the coordinates were carried out in O (44) and Coot 0.6. As the last step, famoxadone was fitted into the difference density. Final refinement statistics are given in Table 2.

Measurement of IC₅₀ Values for bc₁ Inhibitors

The activities of Mtbc₁ and Rsbc₁ were assayed following the reduction of substrate cyt c. The bc₁ preparation was diluted to a final concentration of 0.1 and 1 μm, respectively, for Btbc₁ and Rsbc₁ in a buffer containing 50 mm Tris-HCl, pH 8.0, 0.01% β-DDM, and 200 mm NaCl. To a 2-ml assay mixture containing 100 mm phosphate buffer, pH 7.4, 0.3 mm EDTA, and 80 μm cyt c, Q₀C₁₀BrH₂ was added to a final concentration of 25 μm, and the solution was split evenly into two cuvettes. To one cuvette, 5 μl of diluted bc₁ solution was added, and we immediately began recording the cyt c reduction at 550-nm wavelengths for 100 s in a two-beam Shimadzu UV-2250 PC spectrophotometer at room temperature. The amount of cyt c reduced at a given period of time was calculated using a millimolar extinction coefficient of 18.5 mm⁻¹ cm⁻¹. To measure the effect of bc₁ inhibitors, bc₁ was preincubated with various concentrations of inhibitors for 5 min on ice prior to the measurement of bc₁ activity. The IC₅₀ value for each inhibitor was calculated by a least squares procedure fitting the equation (Y = A_min + (A_max −A_min)/(1 + 10^{(X-logIC₅₀)})) implemented in a commercial package Prism, where A_max and A_min are maximal and minimal activities, respectively.