Báo cáo khóa học: Disruption of the interaction between the Rieske iron–sulfur protein and cytochrome b in the yeast bc1 complex owing to a human disease-associated mutation within cytochrome b potx

Disruption of the interaction between the Rieske iron–sulfur protein and cytochrome b in the yeast bc 1 complex owing to a human disease-associated mutation within cytochrome b Nicholas Fisher 1 , Ingrid Bourges 1 , Philip Hill 1 , Gael Brasseur 2 and Brigitte Meunier 1 1 Wolfson Institute for Biomedical Research, University College London, UK; 2 Laboratoire de Bioe ´ nerge ´ tique et Inge ´ nierie des Prote ´ ines, CNRS, Marseille, France The mitochondrial cytochrome b missense mutation, G167E, has been reported in a patient with cardiomyopathy. The residue G167 is located in an extramembranous helix close to the hinge region of the iron–sulfur protein. In order to characterize the effects of the mutation on the structure and function of the bc 1 complex, we introduced G167E into the highly similar yeast cytochrome b. The mutation had a severe effect on the respiratory function, with the activity of the bc 1 complex decreased to a few per cent of the wild type. Analysis of the enzyme activity indicated that the mutation affected its stability, which could be the result of an altered binding of the iron–sulfur protein on the complex. G167E had no major effect on the interaction between the iron– sulfur protein headgroup and the quinol oxidation site, as judged by the electron paramagnetic resonance signal, and only a minor effect on the rate of cytochrome b reduction, but it severely reduced the rate of cytochrome c 1 reduction. This suggested that the mutation G167E could hinder the movement of the iron–sulfur protein, probably by distorting the structure of the hinge region. The function of bc 1 was partially restored by mutations (W164L and W166L) located close to the primary change, which reduced the steric hindrance caused by G167E. Taken together, these obser- vations suggest that the protein–protein interaction between the n-sulfur protein hinge region and the cytochrome b extramembranous cd2 helix is important for maintaining the structure of the hinge region and, by consequence, the movement of the headgroup and the integrity of the enzyme. Keywords: Saccharomyces cerevisiae; bc 1 complex; respir- atory chain; disease; Rieske iron–sulfur protein. The mitochondrial cytochrome bc 1 complex is a homo- dimeric, membrane-spanning enzyme. It is formed from two monomeric units consisting of 10polypeptide chains. Redox- active prosthetic groups are located within three of these polypeptides: cytochrome b, cytochrome c 1 and the Rieske iron-suphur protein (ISP). The [2Fe)2S] prosthetic group of the ISP is located within the soluble C-terminal domain on the P-side of the inner mitochondrial membrane. This domain consists of residues 93–215 (using the the yeast sequence notation) and is linked by a flexible hinge (or linker) region to a transmembrane helix anchored within the inner mitochondrial membrane. This transmembrane helix is unusually long and slants across the membrane to form a functional moiety with cytochrome b and c 1 on the opposing monomeric unit. Structural, biochemical and spectroscopic data suggest that the peripheral C-terminal domain of the ISP alternates between a position close to cytochrome b and a position close to cytochrome c 1 , shuttling electrons from quinone species bound at the quinol oxidation (Q o )siteto the haem of cytochrome c 1 . The presence of a flexible hinge region [sequence TADVLAMAK(85–93) in Saccharo- myces cerevisiae] within the ISP facilitates this movement, and this domain has been the target of extensive mutagenic investigation. Insertion or deletion of residues has a signifi- cant effect on the catalytic activity [1], whereas the enzyme can accommodate various residue replacements without loss of function. In yeast, the ISP is synthesized as a 29 kDa precursor protein with a 30 amino acid N-terminal leader sequence. It is translated on cytoplasmic ribosomes and imported into the mitochondria. Studies of the mechanism of assembly of the bc 1 complex suggest that the ISP is one of the last subunits to be integrated within the membrane-bound subcomplex [2]. The integration of the ISP is facilitated by Bcs1p, an AAA family member [3] that binds on the precytochrome bc 1 complex [3]. The cytochrome b missense mutation, G167E, has been detected in a patient with severe cardiomyopathy [4]. G167 is located in the extramembranous cd2 helix of cyto- chrome b, a region not thought to be directly associated with quinol binding at the Q o site. However, G167 is within 5A ˚ of residues forming the hinge region of the ISP (Fig. 1). Replacement of glycine at position 167 by a bulkier and potentially charged glutamate residue may interfere with ISP movement, altering the catalytic activity of the complex. Alternatively, the G167E mutation could affect Correspondence to B. Meunier, Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK. Fax: + 44 20 79165994, Tel.: + 44 20 76796860, E-mail: b.meunier@ucl.ac.uk Abbreviations: EPR, electron paramagnetic resonance; ISP, iron–sulfur protein; Q o , quinol oxidation site. (Received 15 December 2003, revised 6 February 2004, accepted 13 February 2004) Eur. J. Biochem. 271, 1292–1298 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04036.x the integration or stability of the ISP within the bc 1 complex. In order to investigate its effect on the bc 1 complex, we introduced this ÔhumanÕ mutation into yeast cytochrome b and studied the effects on complex assembly and activity. Secondary suppressor mutations arising from the original mutation were also identified. Experimental procedures Media and chemicals The following media were used for the growth of yeast. YPD, 1% (w/v) yeast extract, 2% (w/v) peptone, 3% (w/v)glucose;YPG,1%(w/v)yeastextract,2%(w/v) peptone, 3% (w/v) glycerol; transformation medium 0.7% (w/v) yeast nitrogen base, 3% (w/v) glucose, 2% (w/v) agar, 1 M sorbitol and 0.8 gÆL )1 of a complete supplement mixture minus uracil, supplied by Anachem. Decylubi- quinone was purchased from Sigma. Decylubiquinol was prepared as described below. Stigmatellin was purchased from Fluka. Preparation of decylubiquinol Ten milligrams of 2,3-dimethoxy-5-methyl-n-decyl-1,4-ben- zoquinone (decylubiquinone), an analogue of ubiquinone (Sigma) was dissolved in 400 lL of nitrogen-saturated hexane. An equal volume of aqueous 1.15 M sodium dithionite was added, and the mixture was shaken vigor- ously until colourless. The upper, organic phase was collected, and the decylubiquinol recovered by evaporating off the hexane under nitrogen. The decylubiquinol was dissolved in 100 lL of 96% (v/v) ethanol (acidified with 10 m M HCl) and stored in aliquots at )80 °C. The concentration of decylubiquinol was determined spectro- photometrically from absolute spectra, using e 288)320 ¼ 4.14 m M )1 Æcm )1 . Generation of the mutant strains The plasmid pBM5, carrying the wild-type intronless sequence of the CYTB gene, was constructed by blunt end cloning of a PCR product of CYTB into the pCRscript vector (Stratagene). The mutagenesis was performed using the Quickchange Site-Directed Mutagenesis Kit (Strata- gene), 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 bombard- ment was adapted from the procedure of Bonnefoy & Fox [5], as described previously [6,7]. Preparation of the 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 YPD cultures at 28 °C. The cells ( 2 g wet weight per culture) were harvested by centri- fugation (4000 g, 10 min). Cell pellets were washed by resuspension in 40 mL of 50 m M potassium phosphate, Fig. 1. Location of the mutations in the cyto- chrome bc 1 complex. The figure was prepared using the coordinates of the yeast enzyme (Protein Data Bank accession code 1KYO) with visual molecular dynamics (VMD) [20]. The cytochrome b polypeptide backbone is represented in orange, the iron–sulfur protein (ISP) in green, residue G167 is shown in light green, the compensatory mutations are shown in pink, and the hinge region of the ISP is shown in white. Ó FEBS 2004 Effect of G167E mutation in the yeast bc 1 complex (Eur. J. Biochem. 271) 1293 2m M EDTA (pH 7.5) and centrifugation (4000 g,10min). The harvested cells were resuspended in 10 mL of 50 m M potassium phosphate, 2 m M EDTA (pH 7.5), supplemen- ted with 0.2 m M phenylmethanesulfonyl fluoride and 0.05% (w/v) BSA, prior to disruption (for 10 min at 4 °C) in a Retsch MM300 glass bead mill operating at 30 Hz. Membranes were separated from cell debris by centrifuga- tion (10 000 g, 20 min). The supernatant was centrifuged (100 000 g, 90 min) and the pelleted membranes were resuspended in 1 mL of 50 m M potassium phosphate (pH 7.5), 2 m M EDTA, containing 10% (v/v) glycerol. Resuspended membranes were stored in 100-lL aliquots at )80 °C. Cytochrome b concentration in the resuspended membranes was adjusted to 2 l M . Cytochrome c reductase activity measurements were assayed in 50 m M potassium phosphate, pH 7.5, 2 m M EDTA, 10 m M KCN, 0.025% (w/v) lauryl maltoside and 30 l M equine cytochrome c, at room temperature. Mem- branes were diluted to 2.5 n M cytochrome bc 1 complex (determined from the reduced minus oxidized difference spectra, using the bovine cytochrome b extinction coeffi- cient: e ¼ 28.5 m M )1 Æcm )1 at 562–575 nm [8]). Cyto- chrome c reductase activity was initiated by the addition of decylubiquinol (5–100 l M ). Reduction of cytochrome c was monitored in a Cary 4000 spectrophotometer at 550 vs. 542 nm. Initial rates were measured as a function of decylubiquinol concentration, and V max and K m values were derived from Eadie–Hofstee (v vs. v/[S]) plots. Spectroscopic analysis of cytochromes in whole cells Spectra were generated by scanning dithionite-reduced cell suspensions with a Cary-4000 spectrophotometer at room temperature. The cells, grown on YPD plates for 48 h, were resuspended at a concentration of  200 mgÆml )1 . Quad- ratic baseline compensation was carried out on the data, as described previously [9], to remove the distortion of the baseline. Western blotting analysis Immunodetection analyses were performed on crude mitochondrial membranes. A loading solution of 62.5 m M Tris/HCl (pH 6.8), 0.01% (w/v) bromophenol blue, 25% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoeth- anol, was added to each sample (1 : 2, v/v). The samples were then heated for 5 min at 95 °C. The analyses were performed as described previously [10]. The mitochond- rial membrane preparations (40 lg of total protein per sample) were electrophoresed on SDS/polyacrylamide gels (4–20% linear gradient polyacryamide gel) prior to transfer to poly(vinylidene difluoride) membrane by semidry electroblotting. ÔPrecision Plus Protein Dual Color standardsÕ (Bio-Rad) (10–250 kDa) were used for estimation of molecular mass. Polyclonal antisera against cytochrome c oxidase subunits VI and VIa were kindly supplied by J. W. Taanman (Royal Free and University College Medical School, London, UK). Polyclonal anti- sera against cytochrome c 1 , Rieske iron–sulfur protein and QCR7 were generously supplied by B. L. Trum- power (Dartmouth Medical School, Hanover, NH, USA). Pre-steady state cytochrome reduction kinetics The reduction kinetics of cytochrome b and cytochromes c and c 1 were monitored using a dual-wavelength Aminco DW2A spectrophotometer equipped with a rapidly stirred reaction cuvette, as described previously [11]. Electron paramagnetic resonance (EPR) analysis EPR analysis was performed as described previously [11]. Results and discussion Effect of the mutation G167E on the assembly and function of the bc 1 complex The mutation G167E was introduced into yeast cyto- chrome b by the biolistic method, as described previously [6,12]. The resulting mutant was respiratory growth defici- ent. The effect of the mutation on cytochrome b content in whole cells was monitored spectrophotometrically (Fig. 2). The aerobic spectrum of the mutant cells (Fig. 2, G167E ox) showed a peak at 562 nm, corresponding to reduced cytochrome b (presumably high potential cytochrome b haem), and a peak at 575 nm, corresponding to oxy- flavohemoprotein [13]. After a 1 min incubation, the cell suspension became anaerobic as a result of respiration. Cytochrome c and cytochrome c oxidase were reduced and the signal of oxyflavohemoprotein disappeared (Fig. 2, G167E red, lower trace). The addition of dithionite had little effect (Fig. 2, G167E red, upper trace). The wild type cells demonstrated fast O 2 consumption, with the cell suspen- sions becoming anaerobic immediately and the cytochromes becoming fully reduced (Fig. 2, WT red). The cytochrome b content of the mutant cells (based on the reduced spectra in the visible region) was decreased by  25% compared to Fig. 2. Optical spectra of the wild type and mutant cells. Optical spectra of cell suspensions of the wild type (wt) and mutant (G167E) were obtained as described in the Experimental procedures. wt red, Spec- trum of reduced (anaerobic) wild type cells; G167E red, spectra of reduced mutant cells. The lower trace shows the degree of reduction upon anaerobiosis, and the upper trace is the same sample upon addition of dithionite. G167E ox, spectrum of aerobic mutant cells. 1294 N. Fisher et al. (Eur. J. Biochem. 271) Ó FEBS 2004 the wild type level. It appeared therefore that the G167E mutation had little effect on the folding or assembly of cytochrome b. In order to monitor the content of ISP in cells, membranes were prepared as described above, in the Experimental procedures. The steady-state levels of the ISP, cytochrome c 1 and two cytochrome oxidase subunits (cox VI and cox VIa) were monitored by immunoblotting. As shown in Fig. 3, the level of ISP seemed similar to that of the wild type sample. However, variations in the ISP content were observed between different mitochondrial membrane preparations (data not shown). This could be a result of the instability of the mutant enzyme and an increased sensitivity to degradation during membrane preparation, as discussed below. The decylubiquinol (QH 2 )-cytochrome c reductase activ- ity of the mitochondrial membranes was measured spectro- photometrically at pH 7.5, as described in the Experimental procedures. All measurements were made at room tem- perature. The bc 1 QH 2 -cytochrome c reductase activity of the G167E mutant was severely decreased, exhibiting a maximal turnover number of 11 s )1 at 40 l M QH 2 .This compares to a turnover number of 50 s )1 observed for the wild type enzyme under identical assay conditions at the same concentration of quinol. Unexpectedly, increasing the quinol concentration above 40 l M resulted in a decreased activity of the G167E mutant (Fig. 4A). At 65 l M QH 2 ,the turnover number of the mutant was decreased to 4 s )1 ,8% of the wild type rate. As such, K m and V max are not useful parameters for the kinetic description of this mutant. The probable reason for this phenomenon (namely enzyme instability) is discussed in greater detail below. Instability of the mutant enzyme Further analysis of the kinetic parameters of the G167E mutant was hindered by rapid inhibition of the activity of the bc 1 complex during the assay, which was particularly noticeable at higher quinol concentrations (as shown in Fig. 4B). The inhibition of the enzyme by its product quinone could be excluded, because the addition of an equal amount of decylubiquinone to decylubiquinol at the begin- ning of the assay had no effect on the kinetics (data not shown). It is more probable that the mutant was unstable under the conditions of the assay. This was confirmed by monitoring the sensitivity of the activity of the bc 1 complex to detergent. The mutant enzyme was inactivated by a 2 min incubation with 0.025% (w/v) lauryl maltoside, with 50% activity lost after a 90 s incubation. The wild type bc 1 complex did not show this behaviour. It seems probable that the biphasic nature of the kinetic data, presented in Fig. 4B, results from the instability of the enzyme in dilute solution in the presence of detergent. The decrease in activity observed at quinol concentrations of > 40 l M (Fig. 4A) may be a result of the weakly chaotropic nature of the substrate and the inherent partioning of quinol into the Q o site. The Fig. 3. Steady-state level of the iron–sulfur protein (ISP) in G167E. Immunoblots of denaturing gels, loaded with 40 lg of protein from mutant and wild type membrane preparations, were probed with antibodies specific for cytochrome c 1 or the ISP. The loading was monitored using antibodies against the cytochrome oxidase subunits cox VI and cox VIa. Fig. 4. QH 2 -cytochrome c reductase activity. The assays were per- formed as described in the Experimental procedures. QH 2 -cyto- chrome c reductase activity was measured spectrophotometrically, at room temperature, at 550 minus 542 nm in an assay buffer consisting of 50 m M potassium phosphate, 2 m M EDTA, 10 m M potassium cyanide, 30 l M equine cytochrome c and 0.025% (w/v) lauryl maltoside, pH 7.5. The concentration of bc 1 complex in the activity assay was 2.5 n M . (A) Activity of the mutant enzyme as a function of the substrate QH 2 concentration. (B) The rate of cytochrome c reduction by wild type (wt) and mutant (G167E) membranes at 66 l M QH 2 . Ó FEBS 2004 Effect of G167E mutation in the yeast bc 1 complex (Eur. J. Biochem. 271) 1295 activity of the human G167E mutant bc 1 complex has been assayed in postmortem heart homogenates, and was found to be 23% of the control rate [4]. The addition of lauryl maltoside, which stimulated the bc 1 activity of control samples, inhibited the activity in samples from the patient. This was in agreement with the data obtained in this study, using the mutant yeast enzyme. It has been reported that a mutation in the hinge region of the Rhodobacter sphaeroides bc 1 complex, which reduced the flexibility of the neck region, increased the sensitivity of the enzyme to detergent, with activity lost as a result of destabilization of the ISP [14]. It seems probable that this is also the case for the G167E mutant, and that activity is lost owing to the destabilization of the complex (loss of the ISP) on dilution or exposure to detergent. Weakened ISP binding, as a result of the introduction of a bulky and potentially charged glutamate residue at position 167, may also explain the variations in the ISP content observed in different membrane prepara- tions. The integrity of the complex appears to be sensitive to the protein/lipid ratio. It is also possible that the G167E mutation increased the sensitivity of the ISP to proteolytic cleavage. Study of the R. sphaeroides bc 1 complex has shown that the hinge region of the ISP is sensitive to proteolytic cleavage by various proteases. As the degree of proteolysis varied in mutants, it was suggested that muta- tions altered the conformation of the hinge region, increas- ing its accessibility to proteases [15]. As G167 is located close to residues in the hinge region (Fig. 1), the introduction of glutamate could alter the structure of this domain, facilita- ting proteolytic attack. Note that further analyses of the bc 1 complex were performed using the membrane preparations with a high ISP content, as shown in Fig. 3. Selection and characterization of reversions From the respiratory deficient mutant, G167E, revertants (respiratory growth competent clones) were selected on respiratory medium. The cytochrome b gene of the rever- tants was sequenced. Three compensatory mutations were found: a mutation at the same codon restoring G167; and two mutations in close proximity to G167E, namely W164L and W166L. These two mutations only partially compensa- ted for the respiratory defect because the doubling time of the revertants in respiratory medium was  15 h (the doubling time is 4 h in the wild-type strain, at 28 °C). The bc 1 QH 2 - cytochrome c reductase activity of the revertants, assayed as described above in the Experimental procedures, showed only a 2.7-fold increase compared to the primary mutant. It is probable that the replacement of tryptophan by the smaller aliphatic residue, leucine, reduced the steric (or electrostatic) hindrance caused by G167E. However, the secondary mutations were not fully compensatory, because, in the revertants, the bc 1 activity remained low and the instability of the complex persisted, as observed for the primary mutant. In order to monitor the effect of the secondary mutations (W164L and W166L) alone on the function of the bc 1 complex, we introduced these changes into wild type cytochrome b by the biolistic method (as described in the Experimental procedures). These mutations had no effect on the respiratory growth or bc 1 assembly and activity compared to wild type cells (data not shown). Thus, the replacement of tryptophan with a smaller residue (leucine) at positions 164 or 166 can be accommodated by the enzyme. Pre-steady state reduction of cytochromes b and c 1 In order to investigate which step was modified in the overall steady-state electron transfer from ubiquinol to cytochrome c, the kinetics of reduction of cytochromes b and c 1 were measured in the mutant and revertant strains. The rates of reduction of cytochromes c + c 1 ,inmutants and revertants, was decreased to  35% of the wild type rate (Fig. 5A). The cytochrome b reduction kinetic (Fig. 5B), by the center P pathway (in the presence of antimycin), was decreased to  70% of the wild type rate in the mutants and revertants. Thus, the reduction of c +c 1 was significantly slower than in the wild type strain, whereas the reduction of b was less affected. The slow c +c 1 reduction could be caused by hindered ISP movement, as a result of G167E. Q o site occupancy, as examined by EPR spectroscopy With the quinone pool oxidized, the mutant G167E and the revertant G167E + W166L showed a wild type EPR signal (Fig. 5A), indicating a full occupancy of the site by quinone. This suggested that the ISP headgroup of the mutant enzyme interacted normally with the Q o site. When the Q Fig. 5. Pre-steady state kinetics. Pre-steady state measurements were performed using mitochondrial membranes, as described in the Experimental procedures. (A) Kinetics of c+ c 1 reduction; (B) kinetics of cytochrome b reduction. 1296 N. Fisher et al. (Eur. J. Biochem. 271) Ó FEBS 2004 pool was reduced, the g x signal in the mutant was downfield shifted (g x ¼ 1.795) compared to the wild type. The difference in the g x signal between Q pool oxidized and reduced was thus lower in the mutant (1.8–1.795) than the wild type strain (from 1.8 to 1.78). The nature of this shift in G167E is not, at present, understood, but it may reflect a slightly different positioning of the ISP with quinol in the Q o site. Addition of stigmatellin induced a g x trough centred at g ¼ 1.775 in the wild type strain. It was slightly upfield shifted in G167E (g x ¼ 1.765). More interestingly, the revertant G167E + W166L exhibited a double trough (g x ¼ 1.8 and g x ¼ 1.76) in the presence of stigmatellin (Fig. 6C), but it should be noted that this feature is also detectable in the EPR spectrum with the Q pool reduced (Fig. 6B). A similar double g x signal has already been observed in mutant forms of the Rhodobacter capsulatus bc 1 complex, when the Q pool is oxidized [16]. This feature was particularly noticeable in a revertant obtained from mutant T265S (yeast numbering) harbouring the suppressor muta- tion, V164A, in ISP. The nature of this double trough remains unclear. It may reflect subtle structural heterogen- eity in the sample, corresponding to two subpopulations of enzyme with their ISP in different conformations. The mutation G167E could distort the hinge region of the ISP and hinder the movement of the headgroup Mutations in the hinge region of the ISP, which were shown to decrease linker flexibility, have been previously studied in R. sphaeroides. The mutant enzyme, in which residues ADV (position 86–88 yeast numbering) were replaced with PPP, was inactive but showed a normal EPR signal. The addition of detergent restulted in the loss of the ISP, which suggested that the mutation weakened the binding of the subunit to the complex [14]. The replacement of residues ADV with PLP was less deleterious. The bc 1 complex showed a low activity, but without loss of the ISP. This mutant again showed a normal EPR spectrum. Arrhenius plots of temperature dependence of bc 1 activity in the mutant showed a higher activation energy. Therefore, it seemed probable that the increased rigidity of the hinge region increased the activation energy, and that the movement of the ISP would become the rate-limiting step of the Q o site reaction [14]. In yeast, the mutation A86L in the ISP hinge region caused a decrease in bc 1 activity of 60%, without loss of the ISP or change in the EPR signal. It was suggested that the mutation impeded the unwinding of the short stretch of the 3 10 helix located in the tether region necessary for the movement of the ISP headgroup. Consequently, the mutant enzyme had a dimin- ished activity. This was supported by the higher activation energies calculated for the mutant [17]. The insertion of one alanine residue in the hinge region of the R. capsulatus ISP decreased bc 1 complex activity, presumablyowing to reduced mobility of the ISP [18]. The same mutation was introduced in yeast. It also decreased the turnover, to 50% of the wild type rate [1]. It was suggested that mutation increased the ÔcleftÕ between ISP and cytochrome b, and modified the Q o -binding site. This was supported by the observed decrease in sensitivity to stigmatellin in the mutant enzyme. It is probable that G167E affects the bc 1 complex function in away similar to the hingeregion mutations, and we suggest that G167E hinders the movement of the ISP by altering the ISP–cytochrome b interaction at the hinge region. Move- ment of the extrinsic domain of the ISP is facilitated by the unwinding of a single turn of the 3 10 helix formed from residues ADV(86–88). G167 is located 9 A ˚ from A86 (and 4A ˚ from A84) and thus it seems probable that replacement of this residue with glutamate could interfere with the movement of theISP, which would, in turn, alter the catalytic activity of the complex. The mutation could also affect the binding of the ISP to the bc 1 complex and distort the Q o site. Our data showed that, in the mutant, the bc 1 activity was severely decreased. The kinetics of reduction of cytochrome c +c 1 were significantly more affected than the kinetics of cytochrome b reduction. EPR spectra in the presence of an oxidized Q pool were normal, which showed that the site was fully occupied by quinone and that the environment of the Fig. 6. Quinol oxidation (Q o ) site occupancy probed by electron para- magnetic resonance (EPR) spectra of the iron–sulfur protein (ISP) [2Fe)2S] cluster. EPR spectra were recorded after mitochondrial membranes were reduced (A) with 10 m M ascorbate (Q pool oxidized), (B) with a few grains of dithionite (Q pool completely reduced), or (C) after the addition of stigmatellin at a final concentration of 10 l M . Arrows and vertical dotted lines indicate the g-values. Spectra obtained in the presence of dithionite (B) exhibited a lower g y ¼ 1.90 signal because of a negative contribution of the complex II iron–sulfur clusters. EPR conditions were as follows: temperature, 15 K; micro- wave frequency, 9.42 GHz; microwave power, 6.3 mW; modulation frequency, 100 kHz; and modulation amplitude, 1.6 mT. Ó FEBS 2004 Effect of G167E mutation in the yeast bc 1 complex (Eur. J. Biochem. 271) 1297 [2Fe)2S] cluster wasnot affected. This argued in favour of an effect of the G167E mutation on the movement of the ISP, and not on the Q o site structure. However, the EPR signal in the presence of reduced quinol and stigmatellin was modified, suggesting a slight alteration in the interaction between the ISP headgroup and the quinol- or sigmatellin-bound Q o site. The mutant enzyme was sensitive to detergent and unstable under the assay conditions. It is suggested that the ISP was progressively lost during the assay, or that its integration within the complex was progressively distorted, causing further inhibition of the enzyme activity. An altered protein– protein interaction between the ISP hinge region and the cytochrome b extramembranous cd2 helix may result in enzyme instability. In R. capsulatus it was suggested that the mutation T265S (located in the ef loop of cytochrome b) hindered the movement of the ISP. The enzyme function was restored by a secondary mutation, G167S [16]. G167S has also been found as a suppressor of the mutation T148F, which abolishes the enzyme assembly [19]. Thus, it seems that G167S could strengthen the assembly of the mutant complex (T148F + G167S), or modify the structure of the ISP to allow its movementin the mutant enzyme (T265S + G167S). The effect of G167S alone was also studied. The change decreased the activity ofthecomplexand the steady-statelevel of ISP [19].ResidueG167 is highlyconservedbetween species, and it can be suggested that glycine at this position is required for the proper folding of the hinge region in the assembled complex. The replacement of G167 with serine (in R. capsul- atus) or glutamate (in yeast or humans) may affect the structure of the hinge region, resulting in a hindered move- ment and enzyme instability. As suggested previously for a yeast mutant with a +1 alanine insertion in the hinge region [1], G167E (and G167S to a lesser extent) could increase the size of the cleft between the ISP and cytochrome b. 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Disruption of the interaction between the Rieske iron–sulfur protein and cytochrome b in the yeast bc 1 complex owing to a human disease-associated mutation. would, in turn, alter the catalytic activity of the complex. The mutation could also affect the binding of the ISP to the bc 1 complex and distort the Q o site. Our