Human Disease-related Mutations in Cytochrome b Studied in Yeast*

Several mutations in the mitochondrially encoded cytochrome b have been reported in patients. To characterize their effect, we introduced six “human” mutations, namely G33S, S152P, G252D, Y279C, G291D, and Δ252-259 in the highly similar yeast cytochrome b. G252D showed wild type behavior in standard conditions. However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation. The mutations G33S, S152P, G291D, and Δ252-259 were clearly pathogenic. They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc1 complex, as observed by immunodetection. Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc1 complex. Y279C caused a significant decrease of the bc1 function and perturbed the quinol binding. The EPR spectra showed an altered signal, indicative of a lower occupancy of the Qo site. The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Qo site properties. Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.

teins with large, hydrophilic domains, and cytochrome b, a predominantly hydrophobic protein consisting of eight transmembrane helices that contains two b-type hemes (b l and b h ) and forms the two quinol binding sites: Q o (site of quinol oxidation) and Q i (site of quinol reduction), located on opposite sides of the membrane. The catalytic mechanism of the bc 1 complex is essentially described by Mitchell's Q-cycle model. A quinol molecule binds at the Q o -site, is deprotonated, and transfers one electron through the "high potential" electron transfer chain consisting of the [2Fe-2S] cluster of the ISP and the c-type heme of cytochrome c 1 to the soluble acceptor, cytochrome c. Following a bifurcated pathway, a second electron is transferred across the membrane by the "low potential" pathway formed from hemes b l and b h and delivered to quinone bound at the Q i site, forming a stable semiquinone.
A number of mutations in the human cytochrome b have been linked with diseases. Nonsense or frameshift mutations that result in truncated cytochrome b almost invariably abolish complex assembly. The precise effect of the missense mutations is more difficult to predict, and their characterization is often hampered by the limited amount of tissue available. In this work, we use yeast mutants as models to characterize the deleterious effect of six mutations reported in patients, namely G33S, S152P, G252D, Y279C, G291D, and a short in-frame deletion of eight residues, ⌬252-259 (yeast numbering) (Fig. 1). Two other mutations of residue Tyr-279, Y279A and -W, were also analyzed. Yeast (Saccharomyces cerevisiae) and human cytochrome b share a very high degree of sequence similarity (55%), allowing the study of disease-related mutations in a convenient model system. G33S has been found in a patient with exercise intolerance (1). Gly-33 is located within transmembrane helix A, a hydrophobic environment at the Q i site and close to heme b h . S152P has also been reported in a patient with exercise intolerance (2). Ser-152 is located in a loop connecting helices cd1 and cd2 at the entry of the Q o binding pocket. The mutation G252D and the in-frame deletion of eight amino acids (residues 252-259) have been detected in patients suffering from cardiomyopathy (3) and exercise intolerance (1), respectively. These residues are located in the E-ef loop on the P side of the membrane. The mutation Y279C has been found in a patient with severe exercise intolerance and multisystem disorder (4). It has been also reported in atovaquone-resistant isolates of the parasite Plasmodium yoeli (5). Tyr-279 is in close proximity to the highly conserved "PEWY" motif region at the N-terminal region of the ef helix and 3.5 Å from residue 181, a ligand of the ISP [2Fe-2S] cluster. The mutation G291D is also associated with exercise intolerance (6). Gly-291 is located in transmembrane helix F1, in close vicinity of the Q o site. These human mutations were introduced into yeast cytochrome b, and their effects on the assembly and activity of the mutant bc 1 complexes were studied. The mutations G33S, S152P, ⌬252-259, and G291D were highly deleterious. Y279C had a less severe effect on the respiratory function. G252D has no effect in standard conditions. Respiratory competent clones were selected from mutants S152P and G291D. Compensatory mutations were identified in cytochrome b and in the hinge region of the ISP.
Generation of the Mutant Strains-The plasmid pBM5 carrying the wild type intronless sequence of the CYTB gene has been constructed by blunt end cloning of a PCR product of CYTB into the pCRscript vector (Stratagene). The mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. After verification of the sequence, the plasmids carrying the mutated genes were used for biolistic transformation. The mitochondrial transformation by microprojectile bombardment was adapted from Ref. 7 and described in Refs. 8 and 9.
Isolation and Genetic Analysis of the Revertants-Diploid strains respiratory growth deficient generated by biolistic transformation were used to select revertants. The mutants were subcloned. Several subclones were grown in YPD, and then incubated on respiratory medium (YPG). Respiratory competent clones appeared after a 1-or 2-week incubation. Four to 15 independent revertants (each from different subclones) were then sporulated. Respiratory competent haploid clones were analyzed as described in Ref. 10 to determine the mitochondrial or nuclear heredity of the reversion mutation. For the mitochondrial reversion, the cytochrome b gene of the revertant was sequenced to identify the secondary change. The nuclear reversions were identified by directly sequencing the most probable candidate gene, which was the nuclearly encoded gene RIP, coding for the ISP. The cytochrome b gene FIG. 1. Location of the mutations in the bc 1 structure. The figure was prepared using the coordinates of the yeast enzyme (Protein Data Bank accession code 1KYO) with VMD (24,25). The cytochrome b polypeptide backbone is represented in orange, the ISP in cyan. Cytochrome b residues involved in disease-associated point mutations are shown in green, and the location of the ⌬252-259 deletion is indicated in white. The location of the compensatory mutations in the ISP and cytochrome b are shown in yellow and purple, respectively. Stigmatellin bound at the Q o site is shown in pink, and the position of residue Ala-144 in blue.
was also sequenced to confirm the presence of the primary mutation.
For characterization on the effect of nuclear reversions in ISP, strains combining the mutations in ISP with either the WT or mutant cytochrome b were constructed by cytoduction (11), by transferring the mitochondrial genome from a donor strain into the rho o derivative of the recipient strain harboring the ISP mutation.
Preparation of Decylubiquinol-10 mg of 2,3-dimethoxy-5-methyl-ndecyl-1,4-benzoquinone (decylubiquinone), an analogue of ubiquinone (Sigma), was dissolved in 400 l of nitrogen-saturated hexane. An equal volume of aqueous 1.15 M sodium dithionite was added, and the mixture was shaken vigorously until colorless. The upper, organic phase was collected, and the decylubiquinol recovered by evaporating off the hexane under nitrogen. The decylubiquinol was dissolved in 100 l of 96% EtOH (acidified with 10 mM HCl) and stored in aliquots at Ϫ80°C. Decylubiquinol concentration was determined spectrophotometrically from absolute spectra, using ⑀ 288 -320 ϭ 4.14 mM Ϫ1 cm Ϫ1 .
Preparation of Crude Mitochondrial Membranes and Measurement of Cytochrome c Reductase Activity-Wild type and mutant yeast strains were grown to stationary phase (48 h) in 200 ml of YPD cultures at 28°C. The cells (ϳ2 g wet weight per culture) were harvested by centrifugation at 4000 ϫ g for 10 min. Cell pellets were washed by resuspension in 40 ml of 50 mM potassium phosphate, 2 mM EDTA (pH 7.5) and centrifuged as before. The harvested cells were resuspended in 10 ml of 50 mM potassium phosphate, 2 mM EDTA (pH 7.5) supplemented with 0.2 mM phenylmethylsulfonyl fluoride and 0.05% (w/v) bovine serum albumin prior to disruption in a Retsch MM300 glass bead mill operating at 30 Hz for 10 min at 4°C. Membranes were separated from cell debris by centrifugation at 10,000 ϫ g for 20 min. The supernatant was centrifuged at 100,000 ϫ g for 90 min and the pelleted membranes were suspended in a minimal volume of 50 mM potassium phosphate (pH 7.5), 2 mM EDTA containing 10% (v/v) glycerol. Resuspended membranes (2 M bc 1 ) were stored in 100-l aliquots at Ϫ80°C.
Cytochrome c reductase activity measurements were assayed in 50 mM potassium phosphate (pH 7.5), 2 mM EDTA, 10 mM KCN, 0.025% (w/v) lauryl maltoside, and 30 M equine cytochrome c at room temperature. Membranes were diluted to 2.5 nM cytochrome bc 1 complex (determined from the dithionite reduced minus ferricyanide-oxidized difference spectra, using ⑀ 562-575 ϭ 28.5 mM Ϫ1 cm Ϫ1 (12). Cytochrome c reductase activity was initiated by the addition of decylubiquinol (5-100 M). Reduction of cytochrome c was monitored in a Cary 4000 spectrophotometer at 550 versus 542 nm. Initial rates (computer-fitted as zero-order kinetics) were measured as a function of decylubiquinol concentration, and V m and K m values were derived from Eadie-Hofstee (v versus v/[S]) plots. All rate measurements were performed in triplicate.
Spectroscopic Analysis of Cytochromes in Whole Cells-Spectra were generated by scanning of cell suspensions with a single beam spectrophotometer built in-house operating at room and cryogenic temperatures (77 K). The cells, grown on YPD plates for 48 h, were re-suspended at a concentration of around 200 mg (room temperature spectra) or around 400 mg (77 K spectra) of cells/ml and reduced by dithionite. Quadratic baseline compensation was carried out on the data as described (13) to remove the distortion of the baseline. Spectral resolution was 0.2 nm.
Western Blotting Analysis-Immunodetection analyses were performed on crude mitochondrial membranes. Loading solution of 62.5 mM Tris-HCl (pH 6.8), The samples were heated for 5 min at 95°C. The analyses were performed as described in Ref. 10. The mitochondrial membrane preparations (40 g of total protein/sample) were electrophoresed on SDS-polyacrylamide gels (4 -20% linear gradient polyacryamide gel) prior to transfer to polyvinylidene difluoride membrane by semi-dry electroblotting. ''Precision Plus Protein Dual Color'' standards (Bio-Rad) (10 -250 kDa) were used for sizing. The polyclonal antisera against subunits Cox VIa and Cox VI were kindly provided by Dr. J. W. Taanman (Royal Free and University College Medical School, London, UK). The polyclonal antisera against cytochrome c 1 , iron-sulfur protein, and QCR7p were generously provided by Prof. B. L. Trumpower (Dartmouth Medical School, Hanover, NH).
EPR Analysis-The analysis was performed as described in Ref. 14.

Generation of the Yeast Mutants and Respiratory Growth
Competence-The yeast mutants harboring the mutations in the mitochondrially encoded cytochrome b gene were generated by the biolistic methods as described in Refs. 8 -10. It is interesting to note that all the strains constructed were homoplasmic and contained only one population of mitochondrial DNA. The mutants G33S, S152P, G291D, and ⌬252-259 were not able to grow on respiratory medium. Y279C had a less severe impairment of the respiratory growth. The growth rate (doubling time) in respiratory medium (YPG) at 28°C was ϳ12 h, compared with 4 h for the wild type (WT) cells. G252D showed no respiratory growth deficiency, as observed previously (10). The respiratory growth rate was 4 h, identical to WT.

Effect of the Mutations on Cytochrome b and ISP Content-
The effects of the mutations on the level of cytochrome b in whole cells were monitored (Table I). The cytochrome b content (based on the dithionite reduced spectra in the visible region, as exemplified in Fig. 2) was decreased by 25 to 50% of the wild type level, whereas the cytochrome c content was increased as observed previously for other respiratory deficient mutants (15). G252D showed the WT level of cytochrome b, as expected. It is interesting to note that in the respiratory deficient mutants, as shown for ⌬252-259 in Fig. 2, the aerobic spectrum (dotted line) showed a peak at 562 nm, corresponding to reduced cytochrome b, presumably b h , and a small peak at 575 nm, corresponding to oxyflavohemoprotein (16). Upon addition of dithionite (solid line), cytochromes c, c 1 , and oxidase (aa 3 ) were reduced and the signal of oxyflavohemoprotein disappeared. This behavior was observed for all the mutants studied here, except for G252D. WT and G252D exhibited fast O 2 consumption. The cell suspensions became anaerobic immediately, and the cytochromes became fully reduced.
To analyze further the content in bc 1 complex subunits, mitochondrial membranes were prepared and the level of ISP, cytochrome c 1 , and QCR7p monitored by Western blotting as described under ''Experimental Procedures.'' As shown in Fig.  3, a severe decrease in ISP content was observed for mutants G33S, S152P, ⌬252-259, Y279W, and G291D, whereas Y279C and -A contained the WT level of this subunit. The amount of QCR7p was also decreased in these mutants, but to a lesser degree, whereas the cytochrome c 1 content was not affected. Mutant S152P seemed the most affected as ISP was only detected after a longer exposure. The decreased level of the ISP in the G33S mutant was unexpected because this residue is located at the Q i site, far from the domains of contact between cytochrome b and the ISP. However, 77 K spectra of whole cell suspension (Fig. 4) showed that the peak position of cytochrome b was red-shifted from 561 to 562.5 nm. This is sug-  1

complex activity in membranes
Cytochromes were monitored in whole cell suspension reduced by dithionite (a) at room temperature as described under "Experimental Procedures." Cytochrome b content was estimated by using 562-575 ϭ 28 mM Ϫ1 cm Ϫ1 (12). The concentration in WT was 3.7 nmol g Ϫ1 cells. The QH 2 -cytochrome c reductase activity was measured in crude membrane preparations. The preparation of mitochondrial membranes and details of the QH 2 -cytochrome c reductase assay are described under "Experimental Procedures." Initial rates were measured as a function of decylubiquinol concentration, and V m and K m values derived from Eadie- gestive of distortion of the local environment around heme b h . Total loss of heme b h in the G33S mutant could be excluded as the bc 1 complex retained a low level of catalytic activity ( Table  I). The resulting alteration in the structure of the cytochrome b polypepeptide is apparently responsible for the destabilization and loss of the ISP from the complex. Effect of the Mutations on the bc 1 Activity-The cytochrome c reductase activity was monitored spectrophotometrically as a function of decylubiquinol (QH 2 ) concentration, as described under ''Experimental Procedures.'' The apparent V m and K m for QH 2 were calculated from initial rate measurements using derived Eadie-Hofstee plots (Table I). No bc 1 activity could be detected in mitochondrial membranes prepared from mutants ⌬252-259 and Y279W. It seemed likely therefore that the small population of ISP-containing enzyme observed in Fig. 3 was non-functional. G33S, S152P, and G291D showed a severely decreased catalytic activity: V m 10, 5, and 9 s Ϫ1 , respectively, compared with 80 s Ϫ1 for the WT. The decreased level of ISP in the mutants could account for the low turnover rate observed. Because the mutated residues are located in the catalytic domain of the enzyme, it is likely that they would also hinder the functioning of a fully assembled complex. Y279A and -C exhibited a different behavior because the enzyme was assembled at near WT level, as judged by the amount of ISP. However, the catalytic activity of these two mutants was diminished compared with WT (V m values: 21 and 59% of the WT rates for Y279A and -C, respectively, under the assay conditions). The mutation Y279C increased the K m for quinol from 18 to 23 M, which is suggestive of a less efficient binding interaction at Q o . The mutation Y279A decreased the K m for quinol 2-fold, indicating that the Q o site was becoming saturated at lower concentrations of quinol than the WT, and may also be indicative of a decreased quinol ''on'' rate. It should be noted that the k min (an apparent second-order rate constant equal to V m /K m ) values for the Y279A and -C mutants are very similar at 1.89 and 2.04 M Ϫ1 s Ϫ1 (cf. 4.44 M Ϫ1 s Ϫ1 for the WT). EPR analyses were performed to obtain more information on quinol binding in these two mutants.
EPR Analysis of Y279C and -A-Residue Tyr-279 is located near the docking site of the ISP [2Fe-2S] center and is likely to be involved in the stabilization and/or positioning of the quinol in the active site (17). It was therefore interesting to monitor the Q o site occupancy by EPR using the characteristic interaction between the [2Fe-2S] cluster of the ISP and the quinone/ quinol binding 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 and that this signal shifts to a lower g x value of around 1.77 when the Q pool is fully reduced (18,19). When the Q o site is empty, because of either a mutation or extraction of quinones, the value of the g x signal shifts to a lower value of around 1.76. The EPR spectrum of the WT sample reduced with ascorbate showed a g y peak at g ϭ 1.90 and a g x trough centered at g ϭ 1.80, which is characteristic of a Q o site fully occupied with oxidized quinone (Fig. 5A). In Y279A and -C, the total level of [2Fe-2S] cluster as indicated by the g y signal was identical to that of the WT. However, the g x trough was less pronounced and shifted to a lower value of g x ϭ 1.76 -1.77, thus indicating a partially empty Q o site. This result is in agreement with the lower catalytic efficiency for quinol (k min ) observed in the mutants. Addition of stigmatellin fixes the ISP [2Fe-2S] cluster in a position close to cytochrome b (Fig. 5C). The mutants were still able to bind the inhibitor as shown by the g x trough. However, Y279C exhibited a shifted g x signal (g x ϭ 1.80) in comparison with that of the WT (g x ϭ 1.775). This indicates a modified binding of stigmatellin within the Q o site or an altered interaction between ISP and stigmatellin.
Reversion Analysis of Mutations-From the data obtained here, it appeared that several mutations in the Q o domain affected the assembly of the ISP. We addressed then the question whether other modifications could restore the assembly of the ISP and the enzyme activity. That may highlight domains of the enzyme important for the assembly of the ISP and its interaction with cytochrome b. To this end, revertants (respiratory growth competent clones) were selected on respiratory medium from the respiratory deficient mutants S152P and G291D. The revertants were analyzed as described under ''Experimental Procedures.'' The reversion mutations were identified by sequencing. For S152P, two compensatory mutations were observed: a mutation at the same codon in cytochrome b restoring Ser-152; and a mutation in the ISP, A90D, which was found in three independent revertants. For G291D, several secondary mutations were found: two mutations in cytochrome b, D287H located in the vicinity of the primary mutation, and H53D, more than 20 Å from the primary site; five changes in the ISP, V88A/G/D, A90T/D, located in the hinge region of this subunit (Fig. 1). A90D was found in 6 independent revertants from G291D, whereas the other reversions were observed only in one or two revertants. The change A90D was thus the most frequent reversion and suppressed both S152P and G291D mutations. The non-native reversions compensate partially the respiratory defect induced by the primary mutations. Their doubling time was between 10 and 12 h (4 h in WT). Further analyses were performed on the compensatory mutation A90D in the ISP. Membranes were prepared from three strains that combined the mutation in ISP A90D with the WT cytochrome b, the mutation S152P, or the mutation G291D. The strains were constructed as described under ''Experimental Procedures.'' The QH 2 -cytochrome c reductase activities were assayed as described under ''Experimental Procedures.'' Introduction of the ISP A90D mutation into the WT cytochrome b background had no effect on the bc 1 complex activity (not shown). Thus despite its high conservation in sequences from various organisms, residue Ala-90 can be replaced by a larger and charged residue without loss of function. Introduction of the ISP A90D mutation into the S152P and G291D cytochrome b mutants resulted in enzymatic activity 2.5-and 3.3-fold higher than the rate observed for the cytochrome b mutant background alone (assayed at 70 M QH 2 ). This was sufficient to support a slow respiratory rate. Fig. 6 shows the steady-state level of ISP in the strains combining ISP A90D with WT cytochrome b (lane 1), S152P (lane 2), or G291D (lane 3). The level of ISP in the revertants was similar to that of the control strain combining ISP A90D with the WT cytochrome b. It appears therefore that the change in A90D stabilizes the binding of ISP to the bc 1 complex. The low activity of the S152P and G291D revertants suggests that the cytochrome b mutations alone cause a severe disruption of the Q o site activity in the ISP-containing complex.
Several mutations in the hinge region of the ISP have been previously obtained as suppressors of the cytochrome b mutation A144F. 2 The mutation A144F affects the binding of quinol, presumably by altering the local structure of the Q o site. The reported suppressor mutations were likely to correct the positioning of the ISP head group on the mutant cytochrome b. It was therefore interesting to see whether these suppressor mutations could compensate the defect caused by S152P and G291D. To this end, we constructed double mutants that combined the ISP mutations with cytochrome b mutations A144F, S152P, or G291D. The respiratory growth competence of the double mutants was then monitored. As shown in Fig. 7, the compensatory effect of the ISP mutations seems specific for cytochrome b mutations. The ISP mutations at residues 85, 92, and 93 compensated A144F only, whereas mutations at residues 88, 89, and 90 corrected partially A144F and G291D. S152P was compensated by the ISP mutation A90D, and very 2 B. Meunier and G. Brasseur, unpublished data. partially by ISP V88G. We could suggest that mutations at residues 85, 92, and 93 by altering the structure of the hinge region allows a greater reach of the ISP head group and a more efficient docking on the Q o site distorted by A144F. ISP A90D, on the other hand, would stabilize the binding of ISP on the bc 1 complex.

DISCUSSION
In this work, we used yeast mutants to characterize the deleterious effect of six mutations reported in patients, namely G33S, S152P, G252D, Y279C, G291D, and a short in-frame deletion of eight residues, ⌬252-259 (yeast notation).
The stable integration of the ISP into the bc 1 complex was defective in mutants G33S, S152P, ⌬252-259, and G291D as observed by immunodetection. ⌬252-259 and G33S are located far from the region of contact with ISP. An indirect long distance effect needs to be invoked to explain the loss of the ISP. The deletion of eight amino acids (⌬252-259) may reasonably be expected to severely disrupt the assembly of the complex. The mutant bc 1 complex was still partially assembled, as judged by the optical signal for cytochrome b, but the level of ISP was dramatically decreased. This mutation is located in a surface loop connecting helices E and ef on the P side of the membrane, and is not directly in contact with the ISP. However, the alteration of the folding of cytochrome b caused by the shortening of this loop is likely to disrupt the stable binding of the ISP to the bc 1 complex. The small population of ISP-containing enzyme was inactive. This could be explained by the major alteration of the catalytic site caused by the deletion. Surprisingly, the Q i site mutation G33S altered the binding of the ISP. Two mutations of that residue have been characterized in yeast. G33D impairs the assembly of holo-cytochrome b, as judged by the loss of cytochrome b signal, whereas G33A restores the respiratory function (20). Gly-33 is found within a hydrophobic environment at the Q i site, 4.0 Å from the porphy- rin ring of heme b h . Introducing the bulkier and potentially anionic aspartyl side chain at this position would be sterically and thermodynamically unfavorable, explaining the failure of the complex to assemble in the G33D yeast mutant. The reversion G33A re-introduces a smaller group that would not disrupt severely side chain packing or interactions around the heme (21). The serine side chain of the human mutation G33S is non-ionic and less bulky than aspartate, but could still perturb the local environment of heme b h . It was previously reported that a similar mutation, G131S, located near heme b l prevented bc 1 assembly (14). We showed here that G33S had severe effects on the bc 1 structure. The spectral properties of cytochrome b have changed, which could be caused by the perturbation or loss of heme b h , and the level of the ISP was dramatically decreased. A major alteration of cytochrome b folding could explain the long distance effect of the mutation in Q i on the ISP assembly. It has been reported that failure to insert the [2Fe-2S] cluster into the ISP altered the structure of Q i , as indicated by decreased reduction of cytochrome b through Q i and decreased binding of antimycin (22). These results suggest an interaction between the Q o and Q i sites of the complex.
Residues Ser-152 and Gly-291 are located at the entrance of the Q o site. Ser-152 is a component of a short stretch of the 3 10 helix at the C-terminal end of helix cd1, immediately preceding a ␤-turn leading to helix cd2. Mutation to proline (with the subsequent loss of backbone H-bonding capacity) might be expected to distort the local fold of this region, which is in close proximity with the [2Fe-2S] group of the ISP when this subunit is docked in the b-''proximal'' position. Such a change is likely to be deleterious to the stability of the complex. Gly-291 is located in a transmembrane helix F1, a region of the molecule in close vicinity of the Q o site. Replacing glycine by aspartate in the hydrophobic interior of the lipid bilayer is thermodynamically unfavorable and likely to impair the folding of cytochrome b at the Q o site, hindering the assembly of the ISP into the complex. The respiratory function is partially restored by a second mutation in cytochrome b, D287H. Residue Asp-287 is located in the Q o region, 3 Å from the primary mutation. It is likely that the replacement of aspartate by histidine reduces the electrostatic repulsion introduced by G291D and restores a correct folding in the Q o region. Interestingly, intra-and intersubunit long distance compensatory mutations were also found: H53D in cytochrome b, located near the hinge region of the ISP (the ISP that reacts with the other monomer), and five mutations in the hinge region of the ISP at residues Val-88 and Ala-90: V88A/G/D and A90T/D. Residue His-53, located in the loop connecting helices A and ab of cytochrome b is involved in the ''mooring'' of the hinge region of the ISP. 3 Mutation to aspartate may increase the stability of binding of the ISP to the complex. Changes in the structure of the hinge region at residues 88 and 90 may have the same effect.
The mutation G252D had no effect on yeast growth or bc 1 activity in crude membrane preparations under standard conditions. It has been reported that the yeast G252D mutant was thermosensitive, as the mutant bc 1 complex was found to be partly inhibited when the cells were grown at higher temperature (36°C) (10). The mutation when combined with loss of the subunit Qcr9p severely decreased the complex activity. We have also noted that the isolated mutant enzyme was unstable, with activity lost on purification. 2 Examination of the HHDBTinhibited yeast bc 1 crystal structure (Protein Data Bank code 1P84 (17)) reveals a tightly bound phospholipid molecule (probably phosphatidylcholine) at the P-side interface between cytochromes b and c 1 . The lipid head group is stabilized by polar interactions with the side chains of His-185 of cytochrome c 1 and Ser-268 of cytochrome b, with the fatty acyl chains in hydrophobic contact with Trp-273. An additional hydrogen bond is provided by the imidazole ring of His-253 (cytochrome b) to an acyl ester oxygen atom of the bound lipid. Mutation of Gly-252 to aspartate may weaken or disrupt this latter hydrogen bonding association, or otherwise distort the local fold at the lipid:protein interface, facilitating delipidation (and hence inactivation) during purification. The thermosensitivity of the G252D mutant may also be because of an altered interaction with structural lipids. This may, in part, explain the pathogenicity of the G252D mutation in human tissue. In human cardiac muscle, the G252D mutation may alter the enzyme conformation and function in a similar manner to that observed in yeast grown at higher temperature or (but to a lesser extent) after delipidation. Additionally, as noted above, the mutation G252D leads to a decrease in the steady-state level of the Qcr9p subunit of the yeast bc 1 complex (10). It was proposed that a long range interaction between Gly-252 and Qcr9p was mediated by residue Lys-182 of cytochrome c 1 , and that the mutation G252D created an illegitimate electrostatic interaction with this lysyl side chain. It is interesting to note that Lys-182 is replaced by arginine in human cytochrome c 1 , and thus it is plausible to suggest that this switch may increase the severity of the bc 1 deficiency in the human instance.
The mutation Y279C affects the quinol binding. The positioning of ubiquinol at the Q o site has been suggested to be influenced by hydrogen bonding interactions with the side chain hydroxyl group of Tyr-279 (17). In Rhodobacter sphaeroides, mutation to phenylalanine has no effect on the activity of the complex, whereas mutations to leucine, glycine, or glu-3 E. Berry, personal communication.
FIG. 6. Steady-state level of the ISP in revertants. Membranes were prepared from three strains that combined the ISP mutation A90D with WT cytochrome b (A90D), with cytochrome b mutation S152P (A90D ϩ S152P), or with cytochrome b mutation G292D (A90D ϩ G291D). Immunoblots were performed as described in the legend to Fig. 3.  FIG. 7. Respiratory growth competence of cells combining cytochrome b and ISP mutations. The strains combined the cytochrome b mutation A144F, S152P, or G291D (as indicated at the top of the figure) with the ISP mutations (as indicated on the left-and righthand sides of the figure). A drop of each mutant was inoculated on a respiratory medium plate and incubated for 3 days at 28°C. tamine decreased the enzyme activity 3-, 40-, and 50-fold, respectively (23). In addition to the Y279C mutant, we also introduced two other mutations at position 279: Y279A and Y279W. The latter two mutations are not associated with disease in man, but were introduced to further investigate the sensitivity of this site to alteration. The replacement of Tyr-279 by tryptophan was very deleterious, abolishing the activity of the complex and causing the loss of the ISP (Fig. 3), presumably because of distortion of the structure at the Q o site. The mutations Y279A and -C had no major effect on the assembly of the enzyme but affected its catalytic properties. The introduction of an alanine decreased the V m to 20% of the WT value. The K m for quinol was lowered, whereas the introduction of a cysteine increased the K m for quinol. The EPR spectra of Y279C and -A showed an altered signal, which is indicative of a lower occupancy of the Q o site. Alanine at that position therefore seems more disruptive to quinol binding than cysteine. For the Y279C mutant, we suggest that the structure of the Q o site is maintained but the binding/positioning of the quinol is less effective, resulting in a lower activity and higher K m . The Y279A mutation is more deleterious to the bc 1 complex activity than Y279C. The decreased K m for quinol suggested that the Q o site is becoming saturated with substrate more rapidly than the WT because of poor electron transfer, or may reflect a decreased ''on'' rate for quinol binding. It is interesting to note that whereas the Y279A mutation causes greater loss of activity than Y279C, the Y279A mutation has no obvious effect on stigmatellin binding, whereas stigmatellin binding appears to be altered in the Y279C mutant (Fig. 5). This is likely to be because of the differences in structure between the double-ring pharmacophore of stigmatellin, and the single ring head group structure of quinol, and the interaction of these groups with the residue at position 279.
In conclusion, using yeast as a model, we have determined the molecular basis of respiratory dysfunction caused by disease mutations in human cytochrome b. G33S, S152P, G291D, and ⌬252-259 were highly deleterious and affected the assembly of the ISP to the bc 1 complex. Y279C, which exhibited a clear, albeit less severe impairment of the respiratory function, affected quinol binding. Further disease-associated mutations will be introduced into the yeast bc 1 complex and their mode of action studied as new clinical data become available.