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• W1974246828 abstract "The mitochondrial bc1 complex catalyzes the oxidation 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 cd1 amphipathic helix in the quinol-oxidizing (QO) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the QO site. An intragenic suppressor mutation was selected (A144F+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 bc1 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 c1 during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd1 helix in the quinol-oxidizing QO site and the ISP via the flexible hinge region and that fine-tuning of the QO site catalysis can be achieved by subtle changes in the linker domain of the ISP. The mitochondrial bc1 complex catalyzes the oxidation 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 cd1 amphipathic helix in the quinol-oxidizing (QO) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the QO site. An intragenic suppressor mutation was selected (A144F+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 bc1 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 c1 during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd1 helix in the quinol-oxidizing QO site and the ISP via the flexible hinge region and that fine-tuning of the QO site catalysis can be achieved by subtle changes in the linker domain of the ISP. IntroductionThe membrane-bound mitochondrial bc1 complex (b6f complex in chloroplasts and cyanobacteria) is a key component of the respiratory and photosynthetic electron transfer chains (see Refs. 1Brandt U. Trumpower B.L. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 165-197Google Scholar, 2Gray K.A. Daldal F. Blankenship R.E. Madigan M.T. Bauer C. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1995: 747-774Google Scholar, 3Cramer W.A. Soriano G.M. Ponomarev M. Huang D. Zhang H. Martinez S.E. Smith J.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 477-508Google Scholar, 4Berry E.A. Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Google Scholar, 5Trumpower B.L. Microbiol. Rev. 1990; 54: 101-129Google Scholar 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) 1The abbreviations used are: ISP, iron-sulfur protein; RIP, nuclear gene coding for the iron-sulfur protein; cyt, cytochrome; bL, low potential cyt b heme; bH, high potential cyt b heme; DB, 2.3-dimethoxy-5-methyl-6-decyl-1.4-benzoquinone; DBH2, reduced form of DB; QO, ubiquinol oxidation site on the positive side of the inner mitochondrial membrane; QI, ubiquinone reduction site on the negative side of the inner mitochondrial membrane; WT, parental strain; MOPS, 4-morpholinepropanesulfonic acid. with a [2Fe-2S] cluster; the monohemic cyt c1; the dihemic mitochondrially encoded cytochrome b with a low potential bL heme (Em7 around –50 mV, where Em indicates equilibrium redox midpoint potential) located near the positive side of the membrane; and a high potential bH heme (Em7 around +90 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 (5Trumpower B.L. Microbiol. Rev. 1990; 54: 101-129Google Scholar). The bc1 complex catalytic activity is best described by the modified Q cycle (6Mitchell P. FEBS Lett. 1975; 59: 137-139Google Scholar, 7Mitchell P. J. Theor. Biol. 1976; 62: 327-367Google Scholar, 8Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Google Scholar, 9Crofts A.R. Meinhardt S.W. Jones K.R. Snozzi M. Biochim. Biophys. Acta. 1983; 723: 202-218Google Scholar). Electrons are delivered into a bifurcated pathway at the QO site. A first electron is transferred from quinol to the ISP (in the so-called high potential electron transfer pathway to cyt c1 and the soluble cyt c), resulting in the formation of an unstable semiquinone which then transfers a second electron to hemes bL and bH (in the so-called low potential pathway) across the membrane. At the QI site, on the negative side of the membrane, quinone is reduced to semiquinone by heme bH. Two molecules of ubiquinol at the QO site are thus required to reduce a quinone to quinol at the QI site. Although several models have been proposed to account for the bifurcated electron transfer at the QO site (10Ding H. Robertson D.E. Daldal F. Dutton P.L. Biochemistry. 1992; 31: 3144-3158Google Scholar, 11Brandt U. FEBS Lett. 1996; 387: 1-6Google Scholar, 12Link T.A. FEBS Lett. 1997; 412: 257-264Google Scholar, 13Junemann S. Heathcote P. Rich P.R. J. Biol. Chem. 1998; 273: 21603-21607Google Scholar, 14Crofts A.R. Guergova-Kuras M. Huang L. Kuras R. Zhang Z. Berry E.A. Biochemistry. 1999; 38: 15791-15806Google Scholar, 15Snyder C.H. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2000; 275: 13535-13541Google Scholar), the mechanism at the molecular level is not completely understood. Several three-dimensional structures of eukaryotic bc1 complexes have been obtained in the presence or absence of different inhibitors (16Xia D. Yu C.A. Kim H. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Google Scholar, 17Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Google Scholar, 18Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Google Scholar, 19Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Google Scholar), and more recently, the yeast bc1 complex structure was obtained at 2.3 Å, including bound water molecules (20Hunte C. Koepke J. Lange C. Robmanith T. Michel H. Structure. 2000; 8: 669-684Google Scholar). These different structures as well as biochemical and spectroscopic data (21Brasseur G. Sled V. Liebl U. Ohnishi T. Daldal F. Biochemistry. 1997; 36: 11685-11696Google Scholar, 22Tian H. Yu L. Mather M.W. Yu C.A. J. Biol. Chem. 1998; 273: 27953-27959Google Scholar, 23Tian H. White S. Yu L. Yu C.A. J. Biol. Chem. 1999; 274: 7146-7152Google Scholar, 24Nett J.H. Hunte C. Trumpower B.L. Eur. J. Biochem. 2000; 267: 5777-5782Google Scholar, 25Darrouzet E. Valkova-Valchanova M. Daldal F. Biochemistry. 2000; 39: 15475-15483Google Scholar, 26Obungu V.H. Wang Y. Amyot S.M. Gocke C.B. Beattie D.S. Biochim. Biophys. Acta. 2000; 1457: 36-44Google Scholar, 27Darrouzet E. Valkova-Valchanova M. Moser C.C. Dutton P.L. Daldal F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4567-4572Google Scholar, 28Darrouzet E. Moser C.C. Dutton P.L. Daldal F. Trends Biochem. Sci. 2001; 26: 445-451Google Scholar, 29Brugna M. Rodgers S. Schricker A. Montoya G. Kazmeier M. Nitschke W. Sinning I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2069-2074Google Scholar) 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 c1 (distal conformation or “c1” state), thus solving the apparent incompatibility between distances and rate of electron transfer between the redox centers (16Xia D. Yu C.A. Kim H. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Google Scholar, 17Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Google Scholar, 18Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Google Scholar, 19Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Google Scholar). This movement is made possible by the presence of a flexible “hinge” region (sequence 85TADVLAMAK93 in the yeast Saccharomyces cerevisiae) located after the transmembrane domain of the ISP. However, recent data (30Yan J. Cramer W.A. J. Biol. Chem. 2003; 278: 20925-20933Google Scholar, 31de Vitry C. Finazzi G. Baymann F. Kallas T. Plant Cell. 1999; 11: 2031-2044Google Scholar) obtained with the cyt b6f 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 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 QO site. Studies of mutations in the region Trp-142 → Thr-145 in the cd1 helix in different organisms have shown the importance of this region for the electron transfer at the QO 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 bc1 activity because of quinol-binding deficiency. Analysis of suppressors obtained from this non-functional mutant showed that an intragenic suppressor mutation in the QO 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 QO site in cyt b and the ISP via the hinge region.MATERIALS AND METHODSMedia, Growth Conditions, in Vivo Phosphorylation Efficiency, and Preparation of Mitochondria—Growth conditions have been described previously (32Meunier-Lemesle D. Chevillotte-Brivet P. Pajot P. Eur. J. Biochem. 1980; 111: 151-159Google Scholar). Growth yield, phosphorylation efficiency, and growth rate were determined as outlined in Refs. 33Chevillotte-Brivet P. Meunier-Lemesle D. Eur. J. Biochem. 1980; 111: 161-169Google Scholar and 34Coppee J.-Y. Brasseur G. Brivet-Chevillotte P. Colson A.-M. J. Biol. Chem. 1994; 269: 4221-4226Google Scholar. Preparation of mitochondria was performed according to the method of Guerin et al. (35Guerin B. Labbe P. Somlo M. Methods Enzymol. 1979; 55: 149-159Google Scholar) with slight modifications (32Meunier-Lemesle D. Chevillotte-Brivet P. Pajot P. Eur. J. Biochem. 1980; 111: 151-159Google Scholar).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 (36Beckmann J.D. Ljungdahl P.O. Trumpower B.L. J. Biol. Chem. 1989; 264: 3713-3722Google Scholar) 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 (37Meunier B. Biochem. J. 2001; 354: 407-412Google Scholar). 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 (36Beckmann J.D. Ljungdahl P.O. Trumpower B.L. J. Biol. Chem. 1989; 264: 3713-3722Google Scholar), 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 ρ0 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 (38Dujardin G. Pajot P. Groudinsky O. Slonimski P.P. Mol. Gen. Genet. 1980; 179: 469-482Google Scholar) 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.Table IGenetic background and growth characteristics of the strains used in this studyStrainsmtDNA, cyt bNuclear genotypeGrowth on respiratory substrates (glycerol, EtOH)Doubling time on EtOHPhosphorylation efficiency on EtOHDoubling time on galactosePhosphorylation efficiency on galactoseBM2Ala-144Diploid RIP/RIP++3 h, 40 min1001 h, 55 min100B78A144FDiploid RIP/RIP-NANA3 h25R28A144F+F179LDiploid RIP/RIP+5 h, 55 min1002 h, 20 min100R20/B78A144FDiploid ripT85A/ripT85A+6 h, 10 min982 h, 20 min99R20/BMAla-144Diploid ripT85A/ripT85A++3 h, 50 min981 h, 55 min97R8/B78A144FDiploid ripA90G/ripA90G+5 h, 30 min1012 h, 30 min97R8/BMAla-144Diploid ripA90G/ripA90G++3 h, 20 min981 h, 55 min96R3/B78A144FDiploid ripA92D/ripA92D+6 h, 10 min992 h, 20 min98R3/BMAla-144Diploid ripA92D/ripA92D++4 h992 h, 10 min100R26/B78A144FDiploid ripA92T/ripA92T+6 h, 20 min100ND96R26/BMAla-144Diploid ripA92T/ripA92T++3 h, 30 min102ND100 Open table in a new tab Activities of the Whole Respiratory Chain and of Its Various Segments—Activities have been monitored as described previously (39Brasseur G. Coppée J.-Y. Colson A.-M. Brivet-Chevillotte P. J. Biol. Chem. 1995; 270: 29356-29364Google Scholar, 40Brasseur G. di Rago J.-P. Slonimski P.P. Lemesle-Meunier D. Biochim. Biophys. Acta. 2001; 1506: 89-102Google Scholar). The I50 value for inhibitors is defined as the concentration required to decrease the DBH2-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 I50r is the ratio between the I50 in a mutated strain and the I50 obtained with the WT strain and indicates the resistance of a strain to the inhibitor in comparison with the WT strain (41Brasseur G. Brivet-Chevillotte P. FEBS Lett. 1994; 354: 23-29Google Scholar). Extinction coefficients of 6.22, 16, 18, and 24 mm–1 cm–1 were used for calculation of NADH, DB, cyt c + c1, 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 (32Meunier-Lemesle D. Chevillotte-Brivet P. Pajot P. Eur. J. Biochem. 1980; 111: 151-159Google Scholar).Redox Titrations—Optical redox titrations were carried out at 25 °C as described by Dutton (42Dutton P.L. Methods Enzymol. 1978; 54: 411-435Google Scholar), 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 (20 μm); ferrocene-monocarboxylic acid (20 μm); 1,4-benzoquinone (20 μm); 2,5-dimethyl-p-benzoquinone (20 μm); 1,2-naphthoquinone (20 μm); phenazine methosulfate (20 μm); tetramethyl-p-benzoquinone (20 μm); 2-methyl-1,4-naphthoquinone (20 μm); 2,5-dihydroxy-p-benzoquinone (20 μm); indigo carmine (5 μm); 2-hydroxy-1,4-naphthoquinone (20 μm); and anthraquinone-2-sulfonate (20 μm).Presteady-state Cytochrome Reduction Kinetics by the Center O and Center I Pathways—Cyt b and cyt cc1 reduction kinetics were monitored with a dual wavelength Aminco DW2A spectrophotometer equipped with a rapidly stirred reaction cuvette, as described previously (40Brasseur G. di Rago J.-P. Slonimski P.P. Lemesle-Meunier D. Biochim. Biophys. Acta. 2001; 1506: 89-102Google Scholar).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 (43Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Google Scholar). The x-ray coordinates of the yeast mitochondrial bc1 complex in the presence of the inhibitor stigmatellin (20Hunte C. Koepke J. Lange C. Robmanith T. Michel H. Structure. 2000; 8: 669-684Google Scholar) (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.RESULTSGeneration 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 cd1 helix in the QO site (Fig. 1). The replacement of alanine by the bulky phenylalanine is likely to perturb the QO-binding pocket and alter the bc1 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 QO 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 QO 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 bc1 complex, the ISP is located the closest to the QO 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 c1 (17Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Google Scholar, 18Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Google Scholar, 19Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Google Scholar, 28Darrouzet E. Moser C.C. Dutton P.L. Daldal F. Trends Biochem. Sci. 2001; 26: 445-451Google Scholar). 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 QO 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 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 bc1 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 bc1 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 (A144F+ISP T85A, A144F+ISP 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 + c1 and aa3 content in the strains tested.Table IICytochrome and ISP contents and redox midpoint potentials of the b- and c-type cytochromes of the bc1 complexStrainsCytochrome content[2Fe-2S] cluster contentEm7(±5 mV)c + c1ba + a3c + c1bHbLWT100100100100+270+95-55A144F907011050+285+75-65A144F + F179L105105115100+275+85-70A144F + ISP T85A12012011565ISP T85A11011011095A144F + ISP A90G110110115100ISP A90G120120120100A144F + ISP A92D95908575ISP A92D100100100105A144F + ISP A92T120120ISP A92T909095 Open table in a new tab 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 A144F+F179L, and the WT. No major changes of the midpoint potential were observed both for the hemes bL and bH of the mutant A144F (–65 and +75 mV, respectively) and of the revertant A144F+F179L (–70 and +85 mV, respectively) when compared with the WT (–55 and +95 mV, respectively) (Table II). As expected, no significantly different midpoint potentials were obtained for the cyt c + c1 (between +270 and +285 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 bL and bH, 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 μmol of Q reduced min–1·mg–1) than NADH-Q reductase (1.8 μmol of NADH oxidized min–1·mg–1) and is the limiting step between succinate and O2. 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. 39Brasseur G. Coppée J.-Y. Colson A.-M. Brivet-Chevillotte P. J. Biol. Chem. 1995; 270: 29356-29364Google Scholar, 46Kröger A. Klingenberg M. Eur. J. Biochem. 1973; 34: 358-368Google Scholar, and 47Meunier D. Chevillotte-Brivet P. J. Theor. Biol. 1977; 64: 137-163Google Scholar 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 A144F+F179L. 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 (39Brasseur G. Coppée J.-Y. Colson A.-M. Brivet-Chevillotte P. J. Biol. Chem. 1995; 270: 29356-29364Google Scholar, 48Bruel C. di Rago J.-P. Slonimski P.P. Lemesle-Meunier D. J. Biol. Chem. 1995; 270: 22321-22328Google Scholar, 49Bruel C. Brasseur R. Trumpower B.L. J. Bioenerg. Biomembr. 1996; 28: 59-68Google Scholar).Table IIIRespiratory rates and enzymatic activities of the different complexesStrainsNADH oxidaseSuccinate oxidaseNADH cyt cSuccinate cyt cSegment I NADH-DBComplex II succinate-DBComplex III DBH2-cyt c (T.N.)Complex IV cyt c oxidaseWT100100100100100100100100A144F1412111510340890A144F+F179L3542701129128120A144F+ISP T85A2160811991ISP T85A107106130120115106A144F+ISP A90G274732652183ISP A90G84979010214198A144F+ISP A92D2160386722104ISP A92D91758991120A144F+ISP A92T3763367016115ISP A92T126120 Open table in a new tab As expected, the bc1 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 c1 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 c1 (Fig. 2). According to the Q cycle mechanism (6Mitchell P. FEBS Lett. 1975; 59: 137-139Google Scholar, 7Mitchell P. J. Theor. Biol. 1976; 62: 327-367Google Scholar, 8Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Google Scholar, 9Crofts A.R. Meinhardt S.W. Jones K.R. Snozzi M. Biochim. Biophys. Acta. 1983; 723: 202-218Google Scholar), 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 c1. The measurement of these reducti" @default.
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• W1974246828 title "QO Site Deficiency Can Be Compensated by Extragenic Mutations in the Hinge Region of the Iron-Sulfur Protein in the bc1 Complex of Saccharomyces cerevisiae" @default.
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