Q O Site Deficiency Can Be Compensated by Extragenic Mutations in the Hinge Region of the Iron-Sulfur Protein in the bc 1 Complex of Saccharomyces cerevisiae

The mitochondrial bc 1 complex catalyzes the oxida- tion of ubiquinol and the reduction of cytochrome (cyt) c . The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd 1 amphipathic helix in the quinol-oxidizing (Q O ) site. The resulting mutant was respiration-defi- cient and was affected in the quinol binding and electron transfer rates at the Q O site. An intragenic suppres- sor mutation was selected (A144F (cid:1) F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 Å from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc 1 complex. They were located about 20 Å from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c 1 during enzyme turnover. Our results suggested that the compensatory closest

The membrane-bound mitochondrial bc 1 complex (b 6 f complex in chloroplasts and cyanobacteria) is a key component of the respiratory and photosynthetic electron transfer chains (see Refs. 1-5 for reviews). It catalyzes the transfer of electrons from ubiquinol to cytochrome c and couples this electron transfer to the vectorial translocation of protons across the inner mitochondrial membrane. It contributes to the proton-motive force, which is subsequently used by the ATP synthase to produce ATP. All bc complexes, from bacteria to higher eukaryotes, contain three subunits forming the catalytic core of the enzyme and carrying four redox centers as follows: the iron-sulfur protein (ISP) 1 with a [2Fe-2S] cluster; the monohe-mic cyt c 1 ; the dihemic mitochondrially encoded cytochrome b with a low potential b L heme (E m7 around Ϫ50 mV, where E m indicates equilibrium redox midpoint potential) located near the positive side of the membrane; and a high potential b H heme (E m7 around ϩ90 mV) located on the negative side of the membrane. In eukaryotic cells, in addition to these three conserved subunits, as many as eight additional subunits are found whose functions are poorly understood (5). The bc 1 complex catalytic activity is best described by the modified Q cycle (6 -9). Electrons are delivered into a bifurcated pathway at the Q O site. A first electron is transferred from quinol to the ISP (in the so-called high potential electron transfer pathway to cyt c 1 and the soluble cyt c), resulting in the formation of an unstable semiquinone which then transfers a second electron to hemes b L and b H (in the so-called low potential pathway) across the membrane. At the Q I site, on the negative side of the membrane, quinone is reduced to semiquinone by heme b H . Two molecules of ubiquinol at the Q O site are thus required to reduce a quinone to quinol at the Q I site. Although several models have been proposed to account for the bifurcated electron transfer at the Q O site (10 -15), the mechanism at the molecular level is not completely understood. Several threedimensional structures of eukaryotic bc 1 complexes have been obtained in the presence or absence of different inhibitors (16 -19), and more recently, the yeast bc 1 complex structure was obtained at 2.3 Å, including bound water molecules (20). These different structures as well as biochemical and spectroscopic data (21)(22)(23)(24)(25)(26)(27)(28)(29) suggest that the extrinsic carboxyl-terminal domain of the ISP carrying the [2Fe-2S] cluster moves between a position close to cyt b (proximal conformation or "b" state) and a position close to cyt c 1 (distal conformation or "c 1 " state), thus solving the apparent incompatibility between distances and rate of electron transfer between the redox centers (16 -19). This movement is made possible by the presence of a flexible "hinge" region (sequence 85 TADVLAMAK 93 in the yeast Saccharomyces cerevisiae) located after the transmembrane domain of the ISP. However, recent data (30,31) obtained with the cyt b 6 f complex indicate that the ISP linker domain is insensitive to changes that increase or decrease its length or flexibility.
Cyt b is a hydrophobic integral membrane protein with eight transmembrane helices connected via extramembranous loops * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed. Tel. and is a central component for quinol oxidation and inhibitor binding. Numerous biochemical studies and crystallographic data showed the important role of the cd amphipathic helix of cyt b in the catalytic mechanism at the Q O site. Studies of mutations in the region Trp-142 3 Thr-145 in the cd 1 helix in different organisms have shown the importance of this region for the electron transfer at the Q O site and for the quinol and inhibitor binding. Examination of this region shows that all the residues are very well conserved. Among them, only Ala-144 had not been mutagenized previously. In this study, we replaced the alanine at position 144 by a phenylalanine in yeast cyt b. The mutation caused a respiration-deficient phenotype and drastically decreased the bc 1 activity because of quinolbinding deficiency. Analysis of suppressors obtained from this non-functional mutant showed that an intragenic suppressor mutation in the Q O domain of cyt b (short distance interaction) and several extragenic suppressor mutations in the flexible hinge region of the iron-sulfur protein (long range interactions) could restore the activity. The detailed characterization of the suppressors provide genetic and biochemical information on the close interactions between the quinol-oxidizing Q O site in cyt b and the ISP via the hinge region.

MATERIALS AND METHODS
Media, Growth Conditions, in Vivo Phosphorylation Efficiency, and Preparation of Mitochondria-Growth conditions have been described previously (32). Growth yield, phosphorylation efficiency, and growth rate were determined as outlined in Refs. 33 and 34. Preparation of mitochondria was performed according to the method of Guerin et al. (35) with slight modifications (32).
Generation of the Site-directed Mutants-The plasmid pYGT19, which carries the HpaII-BglII yeast mitochondrial DNA fragment containing the WT intronless sequence of the cyt b gene, has been cloned into the AccI-BamHI site of pUC13 and was kindly given by Dr. J. Lazowska (CNRS, Gif sur Yvette, France). The plasmid pRG415.RIP containing the RIP gene (36) was kindly given by Prof. B. Trumpower (Dartmouth Medical School, Hanover, NH). The mutagenesis was performed using QuickChange Site-directed Mutagenesis kit (Stratagene) according to the manufacturer's recommendations. After verification of the sequence, the plasmids carrying the mutated cyt b or RIP gene were used for transformation. The generation of the cyt b mutant was performed by biolistic transformation as described previously (37). For the generation of the RIP mutants, the plasmids carrying mutated versions of the RIP gene were used to transform a strain, JPJ1/B78, which combined a deletion of the genomic RIP gene with the mutant A144F cyt b. The strain derived from JPJ1, kindly given by Prof. B. Trumpower (36), was constructed by cytoduction (genetic method used both with yeast and mammalian cells to transfer the mitochondrial genome from a donor strain or cell line into the 0 derivative of a recipient strain).
Yeast Strains for Suppressor and Biochemical Analysis-The diploid strain B78 (Table I), generated by biolistic transformation, carries the mitochondrial cyt b mutation A144F. BM2 is an isogenic WT strain. Respiration-competent clones (i.e. suppressor clones) were selected on respiratory medium using the diploid strain B78. These clones were then sporulated. Respiration-competent haploid clones were analyzed as described previously (38) to determine the heredity of the suppressor mutations. After identification of the suppressor mutations, four pairs of diploid strains (R3, R8, R20, and R26) were constructed by crossing spores that harbored the same mutations in the RIP gene, in combination with the mutant (A144F) or the WT cyt b (Table I). Biochemical analyses were performed using the diploid strains homozygous for WT or mutant RIP and harboring the WT or mutant cyt b.
Activities of the Whole Respiratory Chain and of Its Various Segments-Activities have been monitored as described previously (39,40). The I 50 value for inhibitors is defined as the concentration required to decrease the DBH 2 -cyt c reductase activity by 50% and is expressed in moles of inhibitor per mol of cyt b for each strain. The relative inhibitor titer I 50 r is the ratio between the I 50 in a mutated strain and the I 50 obtained with the WT strain and indicates the resistance of a strain to the inhibitor in comparison with the WT strain (41). Extinction coefficients of 6.22, 16, 18, and 24 mM Ϫ1 cm Ϫ1 were used for calculation of NADH, DB, cyt c ϩ c 1 , and cyt b, respectively.
Spectral Analysis-Mitochondrial membranes were suspended in MR3 buffer, and optical absorption spectra were obtained at 25°C as described previously (32). Redox Titrations-Optical redox titrations were carried out at 25°C as described by Dutton (42), with a flow of argon gas, using a dual wavelength DW2000 SLM-Aminco spectrophotometer. Reductive and oxidative titrations were conducted with sodium dithionite and potassium ferricyanide, respectively. Mitochondrial membranes were suspended in 50 mM MOPS buffer, pH 7. The following redox mediators were used: potassium ferricyanide ( Presteady-state Cytochrome Reduction Kinetics by the Center O and Center I Pathways-Cyt b and cyt cc 1 reduction kinetics were monitored with a dual wavelength Aminco DW2A spectrophotometer equipped with a rapidly stirred reaction cuvette, as described previously (40).
EPR Spectroscopy-EPR spectra were recorded at liquid helium temperatures on a Bruker ESP 300E X-band spectrometer equipped with an Oxford Instruments liquid helium cryostat and temperature control system. Mitochondrial membranes were suspended in 0.65 M sorbitol, 10 mM Tris-HCl, 2 mM EDTA, 0.1% bovine serum albumin, pH 6.5.
Molecular Representation-Molecular representation was carried out by using Swiss-Pdb Viewer software (43). The x-ray coordinates of the yeast mitochondrial bc 1 complex in the presence of the inhibitor stigmatellin (20) (accession number 1EZV in the Brookhaven Protein Data Bank) were used to visualize the regions of cyt b and ISP described in this study and to calculate the closest distances between selected atoms.

Generation of the Respiration-deficient Mutant, Selection and
Genetic Characterization of the Suppressors-The yeast mutant harboring the site-directed mutation A144F in the mitochondrially encoded cyt b was generated by the biolistic method. The conserved residue Ala-144 is located in the cyt b cd 1 helix in the Q O site (Fig. 1). The replacement of alanine by the bulky phenylalanine is likely to perturb the Q O -binding pocket and alter the bc 1 activity. The mutant strain was indeed unable to grow on respiratory medium ( Table I).
The stringent respiratory growth defect provided an easy handle to select suppressors. Respiration-competent clones were selected on respiratory medium. The heredity of the suppressor mutations was determined. One mitochondrial and several nuclear suppressors were found. The cyt b gene of the suppressor clones was sequenced, which confirmed the presence of the A144F mutation. As expected, a second mutation was observed in the cyt b gene of the mitochondrial suppressor clone. The secondary mutation, F179L, is located in the Q O region, 4 Å from the primary mutation (Fig. 1). It is likely that the replacement of phenylalanine by the smaller and hydrophobic residue leucine reduces the steric hindrance induced by A144F and restores the function in the Q O region.
The nuclear suppressors were identified by directly sequencing the most probable candidate gene, which was the nuclearly encoded gene RIP, coding for the ISP. Among the different subunits of the bc 1 complex, the ISP is located the closest to the Q O region and Ala-144. Its hydrophilic carboxyl-terminal domain is known to move during catalysis between a position close to cyt b and a position close to cyt c 1 (17)(18)(19)28). Four mutations were observed as follows: T85A (found in four independent clones), A90G, A92D, and A92T. The mutations were all located in the hinge or tether region of the ISP (Fig. 1).
In order to confirm the suppressor effect of the mutations in RIP, these changes were introduced by site-directed mutagenesis in the plasmid-borne RIP gene (see "Materials and Methods"). The plasmids carrying the mutated RIP were then used to transform a strain that carries both a deletion of genomic RIP and the cyt b mutation A144F (JPJ1/B78). The transformation of this strain with the plasmids carrying the mutated RIP gene (T85A, A90G, A92D, or A92T) restored the respiratory growth competence, whereas the transformation with the WT version of RIP did not restore the respiratory growth. This demonstrated that the changes in the tether region compensated for the deleterious effect of the mutation A144F in the Q O domain.
A detailed analysis of the effect of A144F mutation and of the suppressor mutations was then performed. The strains used for the analysis were diploid strains homozygous for the nuclear suppressor (RIP, ripT85A, A90G, A92D, or A92T) and carry the WT or the mutant A144F cyt b (Table I). They were constructed as described under "Materials and Methods." Growth Characteristics-Whereas the mutant A144F was unable to grow on respiratory substrates (ethanol and glycerol) and exhibited a lower growth yield (25%) on fermentable substrates like galactose, the respiration-competent suppressors

TABLE I Genetic background and growth characteristics of the strains used in this study
The strains were constructed as described under ''Materials and Methods.'' Growth on respiratory medium (2% glycerol, 2% EtOH) was checked on plates. The doubling time (growth rate) was measured in liquid media. The phosphorylation efficiency (mol of ATP synthesized/mol of substrate consumed) was calculated as described previously (33) and is referred as growth yield. NA, non-applicable; ND, not determined. Functional Interaction between Cyt b and Iron-Sulfur Protein grew on respiratory substrates with a lower growth rate than the WT (5-6 h versus 3 h and 40 min) but with the same growth yield, showing no uncoupling. Note that the strains harboring the isolated ISP mutations (ISP T85A, A90G, A92D, and A92T) without the original cyt b mutation A144F exhibited a growth rate and yield on respiratory and fermentable substrates similar to the WT (Table I).
Cytochrome bc 1 Content-A144F showed a decrease of about 30% in the cyt b content and about 50% in the ISP (measured by the presence of the EPR-detectable [2Fe-2S] cluster) indicating that the assembly of its bc 1 complex was perturbed (Table II). However, this decrease alone could not account for the respiratory growth deficiency. Some of the suppressor strains exhibited a slight decrease of 25-35% in the content of the [2Fe-2S] cluster (A144FϩISP T85A, A144FϩISP A92D), whereas the strains with the ISP mutations alone showed a WT level of cyt b and ISP. No significant differences were observed for the cyt c ϩ c 1 and aa 3 content in the strains tested.
Redox Potentiometry-In order to test whether the redox potentials of the hemes were affected, potentiometric redox titrations were carried out with the A144F mutant, its intragenic suppressor A144FϩF179L, and the WT. No major changes of the midpoint potential were observed both for the hemes b L and b H of the mutant A144F (Ϫ65 and ϩ75 mV, respectively) and of the revertant A144FϩF179L (Ϫ70 and ϩ85 mV, respectively) when compared with the WT (Ϫ55 and ϩ95 mV, respectively) ( Table II). As expected, no significantly different midpoint potentials were obtained for the cyt c ϩ c 1 (between ϩ270 and ϩ285 mV). Therefore the respiratory deficiency of A144F could not be attributed to a modification of the physicochemical properties of the b hemes of cyt b. This was in agreement with the rather long distances of about 12 and 23 Å between Ala-144 and heme b L and b H , respectively (Fig. 1).
Respiratory Rates and Activities of the Mitochondrial Complexes-As shown in Table III, A144F exhibited less than 15% of the WT succinate and NADH oxidase activities, which was not enough to support respiratory growth. In the suppressors, these activities increased to 20 -60% of WT values. The strains with isolated ISP mutations (T85A, A90G, and A92D) exhibited WT level activities. Similar results were obtained for the succinate and NADH-cyt c reductase activities. In most of the mutants, the percentage of NADH oxidase and NADH-cyt c reductase activities compared with WT was smaller than the percentage of succinate oxidase and succinate-cyt c reductase activities (Table III). Complex II has a lower activity (0.18 mol of Q reduced min Ϫ1 ⅐mg Ϫ1 ) than NADH-Q reductase (1.8 mol of NADH oxidized min Ϫ1 ⅐mg Ϫ1 ) and is the limiting step be-tween succinate and O 2 . Therefore, a decreased activity of complex III affects to a greater extent the NADH oxidase and NADH-cyt c reductase activities than the succinate oxidase and succinate cyt c reductase activities (see Refs. 39, 46, and 47 for a theoretical analysis). In A144F, the maximum activity of complex II was only 40%, whereas it remained at the WT level in the suppressor A144FϩF179L. This decreased activity at the complex II level was observed previously in other cyt b mutants, suggesting a direct interaction between complexes II and III and/or a down-regulation of the expression of complex II in respiratory complex III-deficient mutants (39,48,49).
As expected, the bc 1 complex turnover was severely decreased in A144F (8%). Its activity ranged from 16 to 28% in the suppressors. In the strains harboring the ISP mutations alone (T85A, A90G, and A92T), the maximum complex III activity was significantly higher than in the WT (115-141%, Table III), and no direct correlation was observed between the increased complex III and complex IV activities (Table III).
Kinetics of cyt b and cyt c 1 Reduction-In order to investigate which step was modified in the overall steady-state electron transfer from ubiquinol to cyt c in the mutant and suppressor strains, we measured the kinetics of reduction of both cyt b and cyt c 1 (Fig. 2). According to the Q cycle mechanism (6 -9), cyt b can be reduced either by the center O pathway (in the presence of the center I inhibitor antimycin, Fig. 2B) or by the thermodynamically unfavorable center I pathway (in the presence of the center O inhibitor myxothiazol, Fig. 2C). The kinetics provide a global value for the rate of arrival and binding of the substrate to its site and the rate of the various electron transfer steps occurring between this binding site and cyt b or c 1 . The measurement of these reduction kinetics, however, is limited by the lag time of the apparatus, which is about 50 ms. The traces presented in Fig. 2 followed pseudo first-order kinetics. Apparent rate constants were calculated from these kinetics. The rates of cyt c ϩ c 1 reduction by the high potential pathway (involving the ISP and cyt c 1 ) in A144F was decreased to about 10% of the WT rates ( Fig. 2A). In the suppressors, the rates were about 30 -55% of the WT, whereas the strains harboring the isolated ISP mutations A92T and A90G exhibited WT rates ( Fig. 2A). The cyt b reduction kinetic through center O was decreased to about 25% in A144F (Fig. 2B). It was less affected in the suppressors (40 -80% of WT) and similar to the WT in the strains with isolated ISP mutations. The cyt b reduction kinetic by the center I pathway was not affected in A144F and in the suppressor A144FϩF179L (Fig. 2C).
Catalytic Efficiency and Inhibitor Binding at the Q O Site-The bc 1 complex steady-state activity can be described as a TABLE II Cytochrome and ISP contents and redox midpoint potentials of the b-and c-type cytochromes of the bc 1 complex Cytochrome content is expressed as a percentage of the WT content. For the WT strain, the contents in dithionite-reduced cytochrome c ϩ c 1 , b, and a ϩ a 3 were 0.59, 0.45, and 0.15 nmol/mg protein, respectively. Values are expressed as averages based on several experiments. ͓2Fe-2S͔ cluster content is expressed as a percentage of the WT content and normalized for cyt b concentration. It was determined by the amplitude of the EPR g y signal in the presence of stigmatellin. The E m7 values were obtained by fitting the amplitude of the absorption ␣ band of the corresponding cytochrome during potentiometric titration (42). ping-pong mechanism in which ubiquinol and cyt c interact with two independent sites. In order to determine the apparent second-order rate constant (k min ) that characterizes the binding of the substrate to its site (50, 51), we measured steadystate DBH 2 cyt c reductase activities by varying the concentration of the reducing substrate DBH 2 while maintaining the other substrate, cyt c, at saturating levels. The constant k min (Table IV) can be calculated from the ratio of maximal turnover (V max /[bc 1 ]) to apparent K m (it should be borne in mind that K m is a complex parameter in this mechanism). For A144F, k min was 4 times lower than for the WT (3.8 ϫ 10 6 versus 14.5 ϫ 10 6 M Ϫ1 s Ϫ1 ), which indicates that the interaction of ubiquinol (DBH 2 ) with the bc 1 complex at the Q O site was affected. It is restored to a near WT level in the suppressor A144FϩF179L. The binding site for ubiquinol is thought to overlap with the binding sites of inhibitors such as myxothiazol and stigmatellin, within a buried hydrophobic region of cyt b (52). Therefore, we measured the I 50 inhibitor titers for myxothiazol

. Kinetics of reduction of the bc 1 complex via the center O and center I pathways.
A, reduction kinetics of cyt c ϩ c 1 via the center O pathway. A mixture of NADH (2 mM) and KCN (2 mM) was used to initiate the cyt reduction (marked by an arrow). Reaction was followed in the dual mode at the wavelength pair 551/540 nm. B, reduction kinetics of cyt b (562/575 nm) via the center O pathway (in the presence of the center I inhibitor antimycin). NADH (2 mM) was used to initiate the reduction. C, reduction kinetics of cyt b via the center I pathway (in the presence of the center O inhibitor myxothiazol). The same final amount of substrate-reducible cyt c ϩ c 1 (A) or cyt b (B and C) was present in the WT and mutant strains, although some mutated strains exhibit a slower reduction not completed in this time scale. and stigmatellin in A144F and its intragenic suppressor A144FϩF179L. The relative inhibitor titer values (I 50 r ϭ I 50 mutant/I 50 WT) showed that the interaction of the Q O inhibitors was not drastically affected despite the fact that the interaction with quinol at the Q O site was modified. Only a slight increase in sensitivity toward myxothiazol (I 50 r ϭ 0.6) and stigmatellin (I 50 r ϭ 0.8 -0.9) was observed (Table IV). Q O Site Occupancy Examined by EPR Spectroscopy-The Q O site occupancy was monitored by using the EPR signal characteristic of the interaction between the ISP [2Fe-2S] cluster and the quinone/quinol bound at the Q O site. It is well known that a g x signal centered at g ϭ 1.80 is observed when the Q pool is fully oxidized (E h around 200 mV) and that this signal shifts to a lower g x value of around 1.77 when the Q pool is fully reduced (E h around 0 mV) (10,53,54). When the Q O site is empty, because of either mutation or extraction of quinone, the value of the g x signal shifts to a lower value of around 1.76 (10). A value of g x ϭ 1.78 is observed when the Q O site is occupied by stigmatellin (55).
As expected, the WT sample reduced with ascorbate showed an EPR spectrum with a g y signal centered at g ϭ 1.893 and a g x trough at g ϭ 1.80, characteristic of a Q O site fully occupied with oxidized quinone (Fig. 3A). In A144F, the ISP content was about 50% of the WT, as judged by the amplitude of the g y signal ( Fig. 3A; note the different scale in the mutant and the WT). Table II presents the quantification of the [2Fe-2S] cluster in the different strains. In A144F, the g x trough was less pronounced and shifted to a lower value of g x ϭ 1.775, indicating a partially empty Q O site (Fig. 3A). The g y signal was upfield shifted, possibly indicating a modified interaction between the ISP cluster and cyt b. In the intragenic suppressor A144FϩF179L, the g x trough was centered at the classical g ϭ 1.80 value but exhibited a shallow trough compared with that of the WT. This shallow trough centered at g x ϭ 1.80 is also observed in the extragenic suppressors, A144FϩISP T85A (Fig.  3A), A144FϩISP A90G, and A144FϩISP A92D, T (results not shown). This means that both intragenic and extragenic suppressors recovered quinone occupancy in the Q O site but not to the WT level. Note that the strains with the ISP mutations alone, exemplified by ISP T85A (Fig. 3A), exhibited a deep g x trough centered at g ϭ 1.80 like the WT.
When the WT sample was reduced with dithionite ( Fig. 3B) and the Q pool was fully reduced, the g x signal was shifted from g x ϭ 1.80 (Q pool oxidized, Fig. 3A) to a value of g x ϭ 1.78, thus showing the response of the EPR g x signal of the [2Fe-2S] cluster to the redox state of the Q pool. In A144F, the g x signal was almost the same when the Q pool was oxidized or reduced (1.775 versus 1.78). This was likely because of the low quinone occupancy in the Q O site. The result was in agreement with the low catalytic efficiency for quinol (k min ) observed in the mutant (Table IV). By contrast, the intra-and extragenic suppressors and the strains with isolated ISP mutations responded to the redox state of the Q pool like the WT.
After addition of stigmatellin, the ISP [2Fe-2S] cluster is fixed in a position close to cyt b. His-161 of ISP is H-bonded to the carbonyl group of the stigmatellin polar head (17,19,20). A144F was still able to bind stigmatellin as shown by the g x trough centered at g x ϭ 1.775 as in the WT (Fig. 3C). In the intragenic suppressor A144FϩF179L, the stigmatellin g x signal was present but shifted to higher field at g x ϭ 1.763. This shift is also observed for the extragenic suppressors A144FϩISP T85A and for the single mutant ISP T85A (g x Ϸ1.77), although to a lesser extent. The EPR data showed that the inhibitor could bind in the mutated strains, which was in agreement with the inhibitor titration experiments (Table IV). The shift of the stigmatellin-induced g x signal observed in suppressors may indicate either a modification of the positioning of stigmatellin in the Q O pocket, without alteration of its affinity, or a slight modification of the ISP position in contact with cyt b in the presence of stigmatellin because of the secondary mutations. 1 Complex Function-Several mutations in the Q O site of bc 1 /b 6 f complexes have been described previously. They affect the electron transfer rates, the quinol binding, and/or the affinity for the Q O site inhibitors myxothiazol, stigmatellin, or DBMIB (see Ref. 56 for a review). Some of these mutations are localized in the amphipathic helix cd 1 , which is critical for the quinol and inhibitor binding, and for the docking of ISP on cyt b during catalysis. For example, mutations of Trp-142, located 3.5 Å from Cys-163 of the ISP cluster region, cause myxothiazol hypersensitivity or resistance and impair the reduction of the high potential pathway (48), most likely by an incorrect docking of the ISP on cyt b. In the bacterial bc 1 complex, Gly-143 is involved in myxothiazol resistance and in quinol binding (57,58). In the b 6 f complex, a mutation at the same position leads to resistance to stigmatellin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (59). In fungal plant pathogens, mutation G143A was reported to cause resistance to fungicides (60). Thr-145 is located 8 Å from stigmatellin and 3.7 Å from Leu-162 of the ISP cluster domain. Mutations of Thr-145 impair the quinol oxidation, shift upfield the g x signal of the ISP, and cause myxothiazol hypersensitivity (61). 2 No mutations of Ala-144, on this cd 1 helix, have been described previously.

Role of the Conserved cyt b Ala-144 in the bc
We introduced the mutation A144F by the biolistic method. The resulting mutant was unable to grow on respiratory substrates. It exhibited very low respiration rates with NADH or succinate as substrates (less than 15% of the WT rates), and its bc 1 complex turnover was drastically decreased by more than 90% (Table III). The analysis of the bc 1 complex assembly in A144F showed a decrease of 30% in cyt b content and of about 50% in EPR-detectable [2Fe-2S] cluster content (Table II). 2 A. S. Saribas, M. Tokito, and F. Daldal, personal communication.

TABLE IV
Catalytic properties of the bc 1 complex with the substrate DBH 2 (V m , K m , and k min ) and resistance to inhibitors myxothiazol and stigmatellin V m represents the maximum DBH 2 cyt c reductase activity (mol⅐cyt c reduced/ s Ϫ1 /mol⅐cyt b) measured at 550/540 nm with a dual wavelength spectrophotometer. K m for DBH 2 was deduced from the titration of the cyt c reductase activity with increasing amounts of the substrate DBH 2 and plotted with a Lineweaver-Burk representation (not shown). k min (V m /K m ) represents the enzyme catalytic efficiency for the substrate DBH 2 . The inhibitor titers I 50 (concentration of inhibitor required to reduce the DBH 2 cyt c reductase activity by 50%) were 0.86 mol⅐myxothiazol/mol⅐cyt b and 0.26 mol⅐stigmatellin/mol⅐cyt b in the WT strain. The relative inhibitor titer (I 50 r ) is the ratio of I 50 obtained with the mutants relative to that in the WT.

Functional Interaction between Cyt b and Iron-Sulfur Protein
However, this could not entirely explain the very low steadystate activity of the bc 1 complex. Redox potentiometry data indicated that there was no significant change in the midpoint potentials of the two b hemes. This was to be expected because Ala-144 is located far from the two hemes. The electron transfer steps at the Q O site, both the reduction kinetics of cyt b and of the high potential pathway (involving ISP and cyt c 1 ), were severely decreased, whereas the electron transfer at the Q I site was not affected (Fig. 2). Interaction of quinol/quinone with the Q O site was found to be modified in the mutant because the catalytic efficiency with decylubiquinol, as measured by the k min , was four times lower in the mutant than in the WT (Table  IV). This was confirmed by EPR spectroscopy. The typical [2Fe-2S] g x signal, which was centered at g ϭ 1.80 when the Q pool was oxidized in the WT, was shifted upfield in the mutant (g ϭ 1.775, Fig. 3A). This was similar to signals observed in bc 1 complexes partially depleted of Q (10). This upfield shift can be rationalized in the three-dimensional structure of the bc 1 complex. When the quinone/quinol site occupancy is low or nil, the equilibrium of ISP position would be displaced from a position closer to cyt b (b state) to a more distal position to the Q O site, hence shifting the EPR g x signal toward a higher field. In addition, the g y signal was upfield-shifted in the A144F mutant; this may reflect a change in the H-bonding network of the [2Fe-2S] cluster resulting from a modified interaction with cyt b.
In the mutant A144F, the interaction with the Q O inhibitors myxothiazol and stigmatellin was not drastically modified (Table IV and Fig. 3C) showing that position 144 is not directly involved in stigmatellin and myxothiazol binding. This suggested that the quinol and stigmatellin binding domains are not strictly identical and/or that the affinity of stigmatellin for bc 1 complex, which is very high (k d ϭ 10 Ϫ10 M) (62), was less affected by the mutation than the affinity of the substrate Note that all the spectra in one layer (A-C) are scaled to the same amplitude of the peak to trough g y signal and that the scale is different (two times) for the mutant A144F (due to a lower amount of the ISP in this strain). See Table II for the quantification of the ISP in the different strains analyzed. Spectra obtained in the presence of dithionite (B) exhibited a lower g y signal due to a negative contribution of the complex II iron-sulfur clusters. EPR conditions are as follows: temperature, 15 K; microwave frequency, 9.42 GHz; microwave power, 6.3 milliwatts; modulation frequency, 100 kHz; and modulation amplitude, 1.6 milliteslas. quinol, which is much lower (in the micromolar range). In the bc 1 complex structure, despite its close distance with stigmatellin (6 Å), the side chain of Ala-144 points toward the amphipathic helix cd 2 and the transmembrane helix D and not toward stigmatellin (Fig. 1). This may explain the weak effect of the mutation on the inhibitor binding. Sequence alignments of cyt b show that Ala-144 is well conserved in bc 1 complexes and is replaced by a valine in b 6 f complexes (63,64). The replacement of Ala-144 by the bulkier residue phenylalanine is likely to cause steric hindrance with Phe-179 and other amino acids surrounding position 144 as visualized by computer simulations. The presence of two opposing phenylalanine residues (A144F and Phe-179) is likely to result in a reorganization of the positioning of several amino acids in this region and possibly to a local displacement of the backbone of the cd 1 helix toward the quinol binding pocket, which could thus impede the quinol binding and electron transfer at the Q O site as observed in our study.
Second Site Suppressor Mutations in cyt b and in the Hinge Region of the Iron-Sulfur Protein-In order to obtain information on the functional interactions between the Q O site and other domains of the bc 1 complex, we selected respirationcompetent clones from the deficient mutant A144F, and we identified the suppressor mutations. Two types of suppressors were found, F179L in cyt b and several long distance suppressors in the ISP.
The strain harboring the cyt b mutations A144FϩF179L recovered the ability to grow on respiratory substrates, although with a longer doubling time than the WT (Table I).
Phe-179 and Ala-144 are separated by only 4 Å (Fig. 1). The steric hindrance caused by the bulky side chain of A144F could be compensated by the replacement of Phe-179 by the smaller hydrophobic residue leucine. Phe-179 is highly conserved among the cyt b sequences from bc 1 and b 6 f complexes, from bacteria to higher eukaryotes (see cyt b sequence alignments in Refs. 4 and 64). It is replaced by other hydrophobic residues, leucine in Mycobacter, isoleucine in Aeropyrum pernix, and methionine in Chlorobium limicola. It thus seems that a hydrophobic residue is required in this region. Interestingly, Aquifex aeolicus has phenylalanine in position 144, but the smaller residue valine is found in position 179 instead of the common phenylalanine residue.
This suppressor A144FϩF179L exhibited a WT level of cyt b and ISP, as judged by the optical and EPR signals. The strain recovered 28% of the bc 1 complex turnover (8% in the original mutant A144F, Table III), but it was still affected in the cyt b and cyt c ϩ c 1 presteady-state reduction kinetics by the center O pathway as shown in Fig. 2. The catalytic efficiency for quinol was close to WT, as judged by the k min value (Table IV). The quinone/quinol binding was also probed by EPR spectroscopy. The spectra showed that A144FϩF179L behaved more like the WT, with an EPR [2Fe-2S] g x signal centered at g ϭ 1.80 when the Q pool is oxidized. This indicated a better interaction with the substrate than in A144F. Interestingly, although the mutant A144F exhibited a binding of stigmatellin similar to the WT strain (Table IV), the suppressor A144FϩF179L exhibited an upfield shift of the EPR g x signal in the presence of stigmatellin (from g ϭ 1.775 in the WT to g ϭ 1.763 in the suppressor, Fig. 3C). This could be due to a modification of the positioning of stigmatellin in the Q O site without altering its affinity (Table IV) loop (17, 19, 20)) in the presence of this inhibitor. This latter proposal is supported by the analysis of Rhodobacter capsulatus mutants; mutations in the ISP clus-ter region at Leu-136 (Leu-162 in yeast) caused a drastic shift toward lower or higher g x values in the presence of stigmatellin, depending on the amino acid replacement (21). ISP Leu-162 (yeast numbering), which points outside the [2Fe-2S] cluster and is in direct contact with helix cd 1 of cyt b (at 2.8 and 3.9 Å from cyt b Asn-149 and Val-146, respectively), is very important for the docking of the ISP and the quinol oxidation. Mutations at Leu-162 likely perturb this docking, decreasing quinol binding and modifying the EPR signal in the presence of stigmatellin.
Extragenic suppressors of A144F were found in the hinge region of the ISP (A144FϩISP T85A, A90G, A92D, T). This ISP hinge region has been the target of an extensive site-directed mutagenesis to change the length and flexibility of this linker domain (22)(23)(24)(25)(26)(27)(28). A recent report on the Synechococcus cyt b 6 f complex showed that the complex activity was not sensitive to modification of the hinge region (30), and the recent threedimensional structure of the cyt b 6 f complex indicated that the amplitude of the ISP movement was small (65). In S. cerevisiae, the mutation ISP T85I caused temperature sensitivity, hypersensitivity to 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), resistance to myxothiazol, and a 30% decrease of the bc 1 complex activity (66). Mutations in Ala-90 (A90F, I) and Ala-92 (A92C, F, R) affected only slightly the bc 1 complex activity (26) despite the increased size of the replacing residue, which could reduce the flexibility of the linker domain. In R. capsulatus, a suppressor mutation was found in the cyt b ef loop in position 286 (Leu-263 in yeast), which compensated the bc 1 defect caused by the insertion of one alanine in the ISP hinge region (67). Recently, in R. capsulatus, Darrouzet and Daldal (68) reported the study of mutations of residue 288 in cyt b ef loop (Thr-265 in yeast), which was found to be important for the docking of the ISP during catalysis. The authors reported the selection of suppressor mutations in the ISP hinge region (positions Val-88 and Ala-90, yeast numbering). This demonstrated a functional interaction between the cyt b ef loop and the linker domain of the ISP.
In our study, carried out on the S. cerevisiae bc 1 complex, the extragenic suppressor mutations in the hinge region of the ISP, namely T85A, A90G, A92D, A92T, are located 18 -21 Å from the primary mutation cyt b A144F (Fig. 1). The suppressors partially restored the respiratory growth competence. The bc 1 activity ranged from 16 to 22% of the WT value, and the cyt b and cyt c ϩ c 1 presteady-state reduction kinetics by the center O pathway were still affected (Fig. 2). The EPR spectra showed that in the suppressor mutants (exemplified by A144FϩISP T85A, Fig. 3A), the quinol site occupancy was recovered but not to the WT level (shallower g x signal). The g x signal responded well to the redox state of the Q pool with a typical signal at g ϭ 1.78 when the Q pool was reduced (Fig. 3B). In the presence of stigmatellin, the characteristic g x signal was present but slightly shifted toward a higher magnetic field although to a lesser extent than in A144FϩF179L (see above). The isolated mutation ISP T85A also exhibited the same upfield shift of the g x signal in the presence of stigmatellin. This suggested that the modification of the interaction of the [2Fe-2S] cluster with stigmatellin was probably because of the substitution T85A in the ISP hinge region.
It is worth noting that the mutants with the ISP mutations alone (ISP T85A, A90G, A92T) exhibited a significantly better maximum DBH 2 cyt c reductase activity than the WT strain (Table III). These changes might give an optimized flexibility to the hinge region or allow a better orientation of the [2Fe-2S] cluster in the Q O site that would give a slightly more efficient maximum DBH 2 cyt c reductase activity.
Computer simulations showed that the replacing side chains in the ISP hinge region in mutants T85A, A90G, A92T, D could be sterically accommodated. However, rearrangements of the hydrogen bonds around these residues may occur. Inspection of stigmatellin-bound bc 1 complex structure showed that T85A could cause the loss of an H-bond to the side chain of ISP Asp-87. In ISP A92D, T, additional H-bonds to cyt b Gln-112, at 3.6 Å from Ala-92, can be expected. Additional H-bonds were proposed in the ISP A86L mutant (A86L is in interaction with the side chain of Leu-89 (69)). The movement of the ISP between cyt b and cyt c 1 requires the "unwinding" of the small ␣-helix in the tether region. Change in the H-bond network in this helical domain could change the flexibility of this hinge region and modify the positioning and/or the movement of the ISP, which could restore partially the Q O site catalysis in the suppressors.
In conclusion, besides the importance of the residue Ala-144 in the cyt b cd 1 helix for the quinol binding in the Q O site, our results revealed in the eukaryotic bc 1 complex, the functional interaction between the cyt b cd 1 helix in the Q O site and the ISP, mediated via the hinge region of the ISP. Similar interaction has been shown previously between the cyt b ef loop and ISP in bacterial bc 1 complex (67,68). The studies carried out both on bacterial and eukaryotic bc 1 complexes demonstrate the importance of the interaction of the ISP with the ef loop and the amphipathic cd 1 helix of cyt b, which are important, both structurally and functionally, for the formation of the Q O site during enzyme turnover. Our results also suggest that the compensatory mechanism of the extragenic suppressor mutations is most likely due to the repositioning of the ISP [2Fe-2S] cluster, caused by slight changes of the hinge region structure.
Thus, it appears that mutations in this hinge domain can finely tune the flexibility, the mobility, and the positioning of the ISP to allow the optimum quinol oxidation and movement of the ISP between cyt b and cyt c 1 during catalysis.