Investigating the Qn Site of the Cytochrome bc1 Complex in Saccharomyces cerevisiae with Mutants Resistant to Ilicicolin H, a Novel Qn Site Inhibitor

Abstract

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex. The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex. The cytochrome bc1 complex of Saccharomyces cerevisiae is a homodimeric protein embedded in the inner membrane of mitochondria (1Trumpower B.L. Encycl. Biol. Chem. 2004; 1: 528-534Crossref Google Scholar). Each monomer consists of three catalytic subunits carrying prosthetic groups, which are cytochrome b with two b-type hemes, cytochrome c1 and the Rieske iron-sulfur protein with its [2Fe-2S] cluster (2Berry E.A Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (390) Google Scholar). Additionally, there are 7 supernumerary subunits lacking any cofactors (3Trumpower B.L. Microbiol. Rev. 1990; 54: 102-129Crossref Google Scholar). Cytochrome b is the only protein of the complex that is encoded by the organelle DNA; all the other subunits are encoded by the nucleus (4Sidhu A. Beattie D.S. J. Biol. Chem. 1983; 258: 10649-10656Abstract Full Text PDF PubMed Google Scholar). The bc1 complex transfers two electrons from ubiquinol to two molecules of cytochrome c, while translocating four protons from the negative to the positive side of the membrane via the protonmotive Q cycle mechanism (5Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar). There are two catalytic sites involved in the Q cycle mechanism. The site where ubiquinol is oxidized, center P (Qp site), and the site where quinone is reduced, center N (Qn site), are located near the electropositive and electronegative sides of the membrane, respectively. Both sites are located on cytochrome b and have different heme prosthetic groups, heme bL at center P and heme bH at center N. There are several compounds that inhibit specifically the Qp or the Qn site (6Von Jagow G. Link T.A. Methods Enzymol. 1986; 126: 253-271Crossref PubMed Scopus (312) Google Scholar). One of the Qp site inhibitors, atovaquone, is of pharmaceutical relevance because of its anti-protozoal and anti-fungal properties (7Kessl J.J. Lange B.B. Merbitz-Zahradnik T. Zwicker K. Hill P. Meunier B. Pálsdóttir H. Hunte C. Meshnick S. Trumpower B.L. J. Biol. Chem. 2003; 278: 31312-31318Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The Qp site has been characterized by applying Qp site inhibitors, which show more structural homology to the Qp site substrate ubiquinol than the few Qn site inhibitors known to date. It was shown recently that ilicicolin H, a new inhibitor isolated from the imperfect fungus Cylindrocladium ilicicola, acts at the Qn site of the S. cerevisiae bc1 complex (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Previously, two other inhibitors produced by microorganisms have been known that act at the Qn site; antimycin, which is a natural product of various species of Streptomyces (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 9Endo T. Yonehara H. J. Antibiot. (Tokyo). 1970; 23: 91-95Crossref PubMed Scopus (16) Google Scholar, 10Kim H. Esser L. Hossain M.B. Xia D. Yu C.-A. Rizo J. van der Helm D. Deisenhofer J. J. Am. Chem. Soc. 1999; 121: 4902-4903Crossref Scopus (40) Google Scholar), and funiculosin, an antibiotic produced by Penicillium funiculosum Thom (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 11Raper K.B. Thom C. A Manual of the Penicillia. 1949; (The Williams & Wilkins Co.): 616-621Google Scholar, 12Tsai A.L. Kauten R. Palmer G. Biochim. Biophys. Acta. 1985; 806: 418-426Crossref PubMed Scopus (31) Google Scholar, 13Degli Esposti M. Ghelli A. Crimi M. Baracca A. Solaini G. Tron T. Meyer A. Arch. Biochem. Biophys. 1992; 295: 198-204Crossref PubMed Scopus (12) Google Scholar, 14Ando K. Suzuki S. Saeki T. Tamura G. Arima K. J. Antibiot. 1969; 22: 189-194Crossref PubMed Scopus (41) Google Scholar). Ilicicolin H is a 5-(4-hydroxyphenyl)-α-pyridone with a decalin ring system. The 5-(4-hydroxyphenyl)-pyridone is the chromophore of tenellin, produced by the insect-pathogenic fungus Beumeria bassiana (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Even though the structures of the three Qn site inhibitors are clearly different, there are some similarities (see Fig. 1 in Ref. 8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar for a comparison of the structures). Ilicicolin H possesses a phenol ring, a feature that is also present in antimycin. There is also a resemblance with funiculosin, which shares the pyridone ring system with ilicicolin H. Similar effects of ilicicolin H and funiculosin on the cytochrome b optical spectrum have been observed. It was therefore concluded that the pyridone ring system plays an important role for ilicicolin H and funiculosin binding (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In this study we wanted to shed some light on the Qn site by applying the novel center N inhibitor ilicicolin H to isolate cytochrome b mutants in S. cerevisiae. From previous work, we know that antimycin displaces semiquinone from center N in the bovine bc1 complex (15Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar), and more recently we have found that all three of the center N inhibitors eliminate the EPR detectable signal attributed to the stable semiquinone at center N in the yeast enzyme. We thus anticipate that the location of resistance conferring mutations might identify amino acids that contribute to semiquinone stability at center N. Seven cytochrome b mutations, differing from the wild-type protein by only one amino acid exchange, were obtained. The sequenced mutations were confirmed and assigned to the cytochrome b gene using genetic complementation techniques. Six of the seven mutations obtained in this study have not been described before. The effects of these mutations on the activity of the enzyme, as well as the sensitivity of the altered cytochrome b proteins toward ilicicolin H, were determined enzymatically. We also checked the mutant yeast strains for cross-resistance using antimycin and funiculosin. Yeast and wild-type for the mutant was The strains for genetic were for the of S. cerevisiae were yeast with yeast with where yeast amino a amino were to the amino was was yeast and was from was a of Ilicicolin H was obtained from the The inhibitors were as to at to of and ilicicolin H, funiculosin, and antimycin. of of the wild-type was for single on were and was with and for at from were on and ilicicolin H. Each produced a different of resistant at were on and checked for ilicicolin H resistance and resistance to This of mutations The ilicicolin H resistant strains were with the to that the was The were and the were for to on with ilicicolin H. The mutant strains were by with the strains and which have different of the cytochrome b The strains were for at in to and for single on The of was that the were where The were on and with ilicicolin H to the of toward the by PubMed Scopus Google Scholar, D. P. C. Tron T. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were with the strains from the of the resistant strains with because the strains the mitochondrial of the resistant for and 1 of the cytochrome b gene The for and in mutants were to in and the were for the of to a of in were of membranes were as described Trumpower B.L. J. Biol. Chem. Full Text PDF PubMed Google with the One of was with the and for membrane the bovine and 1 were to the The was two the of the of was The was with were at in of bc1 b was determined from the spectrum of the using an of at c and 1 and were with membranes to a of cytochrome bc1 complex in with 1 and cytochrome c. The was by to a of the in the or of inhibitor. of cytochrome c was at with the in the of was to cytochrome c reduction at Methods Enzymol. Scopus Google Scholar). The activity of the bc1 complex inhibitor was determined to inhibitor The activity of the membranes from mutant was determined with that of wild-type inhibitor were in and the was were on the of cytochrome b The activity was which was at was with a using the the The of the of center N was from the of the yeast cytochrome bc1 complex with at center P C. J. Lange C. T. H. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). for S. cerevisiae Cytochrome b to the Qn Ilicicolin novel inhibitor ilicicolin H was to select for mutants resistance in S. cerevisiae. a an inhibitor for to determined that the of wild-type mutants to was and ilicicolin H. described were and on and ilicicolin H to screen for resistant To mutations that from the mutations in the cytochrome b the mutants were for resistance toward J. S. A. 1986; PubMed Scopus Google Scholar). the from the ilicicolin H were resistant to to 1 not were the of cytochrome bc1 complex in S. cerevisiae is of with cytochrome b the only protein encoded by the mitochondrial To that the was in one of the other subunits and of the ilicicolin were with the The the of the resistant were and the of the were for on strains showed a mitochondrial of the ilicicolin H resistance not The from these were for of the 1 and of the Cytochrome b from the was known that previously mutations conferring resistance to Qn site inhibitors are located in 1 or of the cytochrome b Both of these were thus sequenced from two in the ilicicolin were found in 1 or 1 the different amino acid mutations, in the by the that was The seven different at to or at to or to or and to b mutations conferring ilicicolin H resistance in S. cerevisiae are the different mutations obtained for ilicicolin H resistance on the location and of the and the of the yeast strains on a in a new The was which was obtained three from different was obtained two from while and were obtained two from the of the mutant strains was with one for The at of the yeast cytochrome b to a location for mutations, mutations at this conferring resistance to antimycin and funiculosin have been described previously J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. 1990; PubMed Scopus (31) Google Scholar). The described previously was to which been obtained in isolated It was concluded that at the to at seven different amino acids as a of a single that some might not found because a J. Biol. Chem. Full Text PDF PubMed Google Scholar). One of the two mutations in this an on a carrying the also an on there was one at and for which on is not In the of the is with a side has a more than the with which has an side In the of is a more to an amino acid with an side with an amino acid in and than with an amino acid with a side The resistant mutant strains show to that of the wild-type on The has been described as a funiculosin J. 1990; PubMed Scopus (31) Google Scholar). of the other mutations are described for the of the mutant strains was with one for of the that the mutations by are for the and to the of mutations in the cytochrome b the ilicicolin strains and strains were as described previously PubMed Scopus Google Scholar). using mutant strains only of the cytochrome b mutations to of the strains are of of and the wild-type is the its D. P. C. Tron T. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This for the different for the the the the A in Fig. 1 the by of the wild-type cytochrome b and the that are by the strains in this the three and of and consists of the four The of the are shown in in Fig. The four the of the three strains and one with the resistant in to only a of was on and on and on with ilicicolin H. In with only the resistant strains carrying the in 1 of the cytochrome b gene showed from to of is lacking the where is was In with which only consists of 1 of the cytochrome b the from to for the strains carrying the in that of the for the of on the of the There was the resistant were with for the carrying the an that is by this shown are the of the with the The of the that the mutations found by the cytochrome b of the resistant strains were for the resistance toward ilicicolin H. of the in the Ilicicolin location of the amino acids that are in the resistant strains at the Qn site some the for the inhibitor resistance. The of the yeast cytochrome b is shown in Fig. The protein the inner mitochondrial membrane with N and in the mitochondrial of the mutations are near the N the at which is located in D. The of cytochrome b in Fig. the sites and in to and the bH heme at center N. This of the that and are to the ring in the center N In which also resistance to several other center N inhibitors J. Biol. Chem. Full Text PDF PubMed Google Scholar), is from the quinone ring on the mutations by genetic the mutant strains were for of the strains on on is shown in Fig. this the resistant strains from the with were strains were on with ilicicolin H, funiculosin, or antimycin. the two inhibitors, the for was determined as described for ilicicolin H The of the in Fig. the of the of the resistant at the while the show the of strains at to the mutations the bc1 complex The on the show the of the various strains on the in the of any inhibitor. The the of for the antimycin which are shown because for the resistant to with antimycin of the strains there was not were at or The not on antimycin at at The resistant one of the which at on all not at all at the and resistant strains at a to the wild-type on was also not on ilicicolin H, of the were at or for the which ilicicolin H and funiculosin resistance at and there was other that resistance to funiculosin or antimycin. The two strains with and cytochrome b mutations on at the more than the The showed ilicicolin H resistance and cross-resistance to funiculosin. the better at funiculosin was not at inhibitors were or The wild-type only on the inhibitors were present not in the of the resistant were in the at The in confirmed the obtained with and showed in the on the of the was more than The other mutant strains at to the wild-type of the Cytochrome b on the of the bc1 the of the mutations on the activity of the bc1 c reductase activity was in membranes isolated from the various The activity of the bc1 complex from the wild-type in the of inhibitor was The activities for the bc1 from the resistant strains in the of inhibitors are shown in The activities of the bc1 in membranes from the and mutants by the from the and mutants showed a of activity of The two strains that in and to of that of the wild-type c reductase activities of mitochondrial membranes and sensitivity to ilicicolin H are the c reductase activities of mitochondrial membranes from the resistant strains and the wild-type as a of wild-type which was shown are the for of the activities by ilicicolin in a new of the Cytochrome b on toward Ilicicolin that the resistance conferring mutations altered the of ilicicolin of bc1 activity and to the of any c reductase activities of mitochondrial membranes from the mutants were in the of of ilicicolin H. The inhibitor are shown in Fig. and the are in the ilicicolin H to inhibit of the activity of the in membranes from the wild-type was the for the strains carrying the and mutations to and respectively. The four carrying the or mutations than ilicicolin H. The of the cytochrome c reductase activities with the inhibitor thus in the altered inhibitor sensitivity of these strains in It was shown previously that ilicicolin H is a inhibitor of the Qn site of the cytochrome bc1 complex in yeast (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). cytochrome b mutations conferring resistance to Qn site inhibitors, antimycin J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. PubMed Scopus Google Scholar, G. B. B. Biochem. Biophys. PubMed Scopus Google Scholar), funiculosin Esposti M. Ghelli A. Crimi M. Baracca A. Solaini G. Tron T. Meyer A. Arch. Biochem. Biophys. 1992; 295: 198-204Crossref PubMed Scopus (12) Google Scholar, J. 1990; PubMed Scopus (31) Google Scholar, G. Scopus Google Scholar, G. P. PubMed Scopus Google Scholar), and J. Biol. Chem. Full Text PDF PubMed Google Scholar, 1986; PubMed Scopus Google been of the strains carrying these mutations are We to ilicicolin H for a mutant screen in S. cerevisiae to identify of ligand and to at center N. acids by ilicicolin H binding also the semiquinone binding site, this antimycin (15Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar), displaces the R. and B. L. an mutant the seven different of the mutations obtained are for which been previously characterized as conferring funiculosin resistance J. 1990; PubMed Scopus (31) Google Scholar). It that there are several the cytochrome b gene where mutations at at and at different were found at of these The for these three amino acid are located in 1 of the cytochrome b the for is in complementation were to the that there were mutations to the ilicicolin H resistance. The with ilicicolin H confirmed that the cytochrome bc1 of the strains were resistant to the inhibitor. The for of cytochrome c reductase activity from in mitochondrial membranes from the wild-type to than with membranes from the strains with mutations to or of the of the yeast bc1 complex confirmed that and are in to the at the Qn site, while is more from the Qn site than the other three of this in of a A the center N The strains with the and mutations on a In the and mutant strains In the bc1 activities in the membranes from the and mutants were to of that of the wild-type The of the on catalytic activity is not the of the side to of the ring, and its with the more side might to to the Qn site by It is to that the bovine bc1 complex has at the and is resistant to ilicicolin H (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), the activity of the bovine is not This differences in is at center N in the yeast and bovine In the yeast there is a the quinone ring and C. J. Lange C. T. H. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and is that of with this and thus with In the bovine the a to the quinone ring, and the not with this Esser L. B. Yu L. Yu C.-A. Xia D. 2003; PubMed Scopus Google Scholar). reduction at center N is than ubiquinol at center P (2Berry E.A Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (390) Google Scholar), we the that the has structural effects on center P in the yeast enzyme. It is also that the to well the of the side is of the quinone ring (see Fig. and one that a in an semiquinone at the Qn In we to the stability of the EPR detectable semiquinone and the of cytochrome b reduction by ubiquinol at center N center P is in the bc1 complex isolated from this The bc1 activities for the two mutations that on the are a activity of the bc1 complex of to in that these mutations to an or the bc1 complex The mutant strains were for by at The at with an or stability in this the of this mutant at was better funiculosin was This which was that this this inhibitor to to the bc1 complex in a that the not inhibit its the not to in the of more at ilicicolin H was that this also bc1 The of this mutant at was also better ilicicolin H was a and binding from this to a for mutations, which has been as for some J. Biol. Chem. Full Text PDF PubMed Google and its effects by a structural a to the Qn at seven different amino acids are at this by single J. Biol. Chem. Full Text PDF PubMed Google Scholar). to this known are the to J. Biol. Chem. Full Text PDF PubMed Google Scholar), and B. L. toward ilicicolin H and from this and It is that one amino acid at this to not the bc1 activity the other exchange, to has effects on activity and of the on The is for an amino acid with an side with also carrying a is a more than with an amino acid with a side other mutations at these two we to the of center N. of the in this study also for the Qn site as a for and To only the Qp site has been in this (7Kessl J.J. Lange B.B. Merbitz-Zahradnik T. Zwicker K. Hill P. Meunier B. Pálsdóttir H. Hunte C. Meshnick S. Trumpower B.L. J. Biol. Chem. 2003; 278: 31312-31318Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, A.L. Hill PubMed Scopus Google Scholar). differences in the amino acids that are for the of center N a and its is an in the of and this is by the of the cytochrome b of a of several Esposti M. S. Crimi M. Ghelli A. T. Meyer A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). There are some of Qn site inhibitor the yeast cytochrome bc1 complex is more to by ilicicolin H than its bovine (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). It is also known that some mitochondria are resistant to funiculosin Esposti M. Ghelli A. Crimi M. Baracca A. Solaini G. Tron T. Meyer A. Arch. Biochem. Biophys. 1992; 295: 198-204Crossref PubMed Scopus (12) Google Scholar), while this Qn site inhibitor is the yeast enzyme. and B. L. cross-resistance of Qn site mutations with several Qn site inhibitors the to identify similarities and differences in binding This is also for the of specifically the Qn Ilicicolin H and funiculosin have the on the heme bH in which the a of (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). was concluded that the pyridone ring, a structural feature that ilicicolin H and funiculosin (8Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), plays a role in binding to the Qn site, and these two inhibitors were Qn site have been previously the to resistance to antimycin and funiculosin in cytochrome b N. K. J. Biol. PubMed Scopus Google Scholar), and to in yeast cytochrome b is resistant to antimycin R. M. A. Microbiol. Scopus (12) Google and G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). all resistant yeast mutants are to a quinone J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, M. A. J. Biochem. PubMed Scopus Google Scholar). We thus that the mutations found in this study cross-resistance to the other Qn site The resistance to funiculosin and the resistance to all three ilicicolin H, funiculosin, as described J. 1990; PubMed Scopus (31) Google Scholar), and antimycin, a resistance that not been to this before. there was a of cross-resistance for the other mutations This that there differences in the binding for the Qn site