Altered Quinone Biosynthesis in the Long-lived clk-1Mutants of Caenorhabditis elegans

Mutations in theclk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ9) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ9. Instead, the UQ9 biosynthesis intermediate, demethoxyubiquinone (DMQ9), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ9 in C. elegans. Interestingly, the activity levels of NADH-cytochrome creductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ9 can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ2 was chemically synthesized. We find that DMQ2 can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ2, cannot. The accumulation of DMQ9 and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span. Mutations in theclk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ9) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ9. Instead, the UQ9 biosynthesis intermediate, demethoxyubiquinone (DMQ9), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ9 in C. elegans. Interestingly, the activity levels of NADH-cytochrome creductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ9 can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ2 was chemically synthesized. We find that DMQ2 can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ2, cannot. The accumulation of DMQ9 and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span. ubiquinone demethoxy ubiquinone or 2-methoxy-5-methyl-6-nonaprenyl-1,4-benzoquinone 2-methoxy-3-hydroxy-5-methyl-6-nonaprenyl-1,4-benzoquinone 2,3-methoxy-5-methyl-6-nonaprenyl-1,4-benzoquinone reactive oxygen species high performance liquid chromatography The understanding of the biological pathways that control life span can be studied in Caenorhabditis elegans through the identification of genes that alter the length of life when mutated (1Vanfleteren J.R. Braeckman B.P. Neurobiol. Aging. 1999; 20: 487-502Crossref PubMed Scopus (149) Google Scholar). For example, mutations in clk-1 are known to cause an extended life span, as well as the slowing of a variety of developmental and physiological events, including the cell cycle, embryogenesis, post-embryonic development, and rhythmic adult behaviors (2Wong A. Boutis P. Hekimi S. Genetics. 1995; 139: 1247-1259Crossref PubMed Google Scholar, 3Lakowski B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (422) Google Scholar). Thus, CLK-1 is expected to play a unique biological role that is necessary to determine the life span and to coordinate these various biological processes. However, the biochemical differences betweenclk-1 mutants and the wild-type strain, which might indicate the function of CLK-1, have yet to been identified (1Vanfleteren J.R. Braeckman B.P. Neurobiol. Aging. 1999; 20: 487-502Crossref PubMed Scopus (149) Google Scholar, 4Hekimi S. Lakowski B. Barnes T.M. Ewbank J.J. Trends Genet. 1998; 14: 14-19Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 5Gems D. Curr. Biol. 1999; 9: R614-R616Abstract Full Text Full Text PDF PubMed Google Scholar, 6Branicky R. Bénard C. Hekimi S. Bioessays. 2000; 22: 48-56Crossref PubMed Scopus (73) Google Scholar, 7Hekimi S. Hekimi S. The Molecular Genetics of Aging. Springer-Verlag, Berlin2000: 81-112Google Scholar). clk-1 encodes a 187-residue polypeptide that is homologous to yeast coq7/cat5 (8Ewbank J.J. Barnes T.M. Lakowski B. Lussier M. Bussey H. Hekimi S. Science. 1997; 275: 980-983Crossref PubMed Scopus (264) Google Scholar). COQ7/CAT5 is located in the inner membrane of yeast mitochondria and is necessary for the biosynthesis of ubiquinone (UQ)1 in yeast (9Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,10Jonassen T. Proft M. Randez-Gil F. Schultz J.R. Marbois B.N. Entian K.-D. Clarke C.F. J. Biol. Chem. 1998; 273: 3351-3357Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Therefore, yeast coq7/cat5 mutants, which lack UQ6, are unable to grow on nonfermentable carbon sources (9Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Orthologs of clk-1/coq7/cat5 have also been reported from mammals, including human (11Jonassen T. Marbois B.N. Kim L. Chin A. Xia Y.-R. Lusis A.J. Clarke C.F. Arch. Biochem. Biophys. 1996; 330: 285-289Crossref PubMed Scopus (41) Google Scholar, 12Vojo Z. King L.M. Jonassen T. Wilkin D.J. Ho N. Munnich A. Clarke C.F. Francomano C.A. Mamm. Genome. 1999; 10: 1000-1004Crossref PubMed Scopus (64) Google Scholar, 13Asaumi S. Kuroyanagi H. Seki N. Shirasawa T. Genomics. 1999; 58: 293-301Crossref PubMed Scopus (25) Google Scholar), and appear to be highly conserved among species. Recently, a green fluorescent protein fusion to C. elegans CLK-1 was shown to localize to the mitochondria of all the somatic cells of the worm (14Felkai S. Ewbank J.J. Lemieux J. Labbe J.-C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (223) Google Scholar). However, in contrast to the situation in yeast, which is defective in respiratory growth, C. elegans clk-1 mutants are able to respire almost normally. In fact, the metabolic capacities and the ATP levels of adult clk-1mutants are unchanged or even higher than those of the wild-type strain (15Braeckman B.P. Houthoofd K. De Vreese A. Vanfleteren J.R. Curr. Biol. 1999; 9: 493-496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and clk-1 mutants mitochondria exhibit succinate-cytochrome c reductase activity that is comparable with that of wild-type mitochondria (14Felkai S. Ewbank J.J. Lemieux J. Labbe J.-C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (223) Google Scholar). These observations suggest that CLK-1 is not exclusively involved in UQ biosynthesis inC. elegans. In this report, we analyzed the quinone composition of clk-1mutants mitochondria, to elucidate the effect of clk-1mutation on the biosynthesis of UQ in C. elegans, and found that UQ biosynthesis is dramatically altered in clk-1mutants. That is, clk-1 mutants mitochondria do not possess detectable levels of UQ9 and instead contain a UQ biosynthesis intermediate, demethoxy ubiquinone (DMQ9). We further analyzed the respiratory activities of mutants mitochondria and found that DMQ can functionally replace UQ to maintain active respiration in clk-1 mutant mitochondria, despite the absence of UQ9. The wild-type strain used was the N2 (Bristol) strain. Mutant strains used were CB4876 clk-1(e2519), MQ438 clk-1 (qm51), and MQ50clk-1 (qm30). MQ50 clk-1(qm30) was supplied from the Caenorhabditis Genetics Center. Nematodes were grown at 20 °C on NGA plates, which contain 3-fold bacto-peptone with the supplement of Escherichia coli OP50. Nematodes were collected in M9 buffer and were sedimented in a 200-ml cylinder. The sedimented worms were washed with M9 buffer until the buffer become clear and were applied to Baermann's Device and left overnight. The nematodes were collected and further purified by centrifugation on 30% (w/v) sucrose, at 750 × g for 5 min at 4 °C (16Murfitt R. Vogel K. Sanadi D.R. Comp. Biochem. Physiol. 1976; 53B: 423-430Google Scholar). The worms were homogenized in 0.21 m mannitol, 0.07m sucrose, 0.1 mm EDTA (MSE), containing 1 mm phenylmethylsulfonyl fluoride, using glass-glass homogenizer (Iwaki, Tokyo) with the inclusion of glass beads (0.10–0.11 mm, B. Brown Melsungen AG). The degree of breakage was checked under a light microscope. The homogenates were then centrifuged at 1,080 × g for 10 min at 4 °C. The pellet, containing glass beads, was washed with MSE and was centrifuged at 1,080 × g for 10 min. The supernatants were then centrifuged at 23,500 × g for 10 min, and the pelleted mitochondrial fraction was resuspended in MSE. Quinones were extracted from lyophilized mitochondria (3.0 mg of protein). The mitochondria were vortexed in EtOH/n-hexane (2/5, v/v) for 10 min and centrifuged at 15,000 rpm for 5 min at room temperature. The supernatants were pooled, and the extraction of quinones was repeated two times. After drying the pooled extracts under a stream of nitrogen gas, the residue was redissolved in ethanol and analyzed by HPLC. Quinones were applied to a reverse-phase column (Inertsil ODS-3, C-18, 5 μm, 4.6 × 250 mm, GL Science, Tokyo) and was eluted in isocratic condition (1 ml/min), with diisopropyl ether/MeOH (1/4, v/v) as described previously (17Hiraishi A. Hoshino Y. J. Gen. Appl. Microbiol. 1984; 30: 435-448Crossref Scopus (104) Google Scholar). The eluted quinones were identified by comparing their retention times with authentic UQ9 (Sigma). The spectral characteristics of each quinone were monitored using photodiode array UV-visible detector (Shimadzu SPD10-A). The concentration of quinones was determined spectrophotometrically using coefficients of E 1cm1% = 185 for UQ (18Lester R.L. Hatefi Y. Widmer C. Crane F.L. Biochim. Biophys. Acta. 1959; 33: 169-185Crossref PubMed Scopus (42) Google Scholar), and Emax = 14.5 mm−1cm−1 at 271 nm for DMQ (19Trumpower B.L. Opliger C.E. Olson R.E. Chem. Phys. Lipids. 1974; 13: 123-132Crossref PubMed Scopus (4) Google Scholar). The mass spectrum of the quinone accumulated in clk-1 (qm51) was analyzed by Hitachi M-8000 LC/MS 3DQ system with atmospheric pressure chemical ionization. NADH-cytochrome c reductase activity and succinate-cytochrome c reductase activity were assayed as described previously (20Takamiya S. Furushima R. Oya H. Mol. Biochem. Parasitol. 1984; 13: 121-134Crossref PubMed Scopus (32) Google Scholar) in 50 mmpotassium phosphate buffer (pH 7.7), 200 μm NADH, or 10 mm potassium succinate, 2 mm KCN, and 50 μm horse heart ferricytochrome c. NADH-quinone reductase activity was assayed in 50 mm potassium phosphate buffer (pH 7.7), 200 μm NADH, 2 mm KCN, and 90 μm quinone analogues. The oxidation of NADH was monitored at 340 nm, using a millimolar extinction coefficient of 6.2 for NADH. Succinate-quinone reductase activities were measured as described previously (21Kita K. Vibat C.R.T. Meinhardt S. Guest J.R. Gennis R.B. J. Biol. Chem. 1989; 264: 2672-2677Abstract Full Text PDF PubMed Google Scholar). DMQ2 and 3-hydroxy-UQ2 were synthesized as described previously (22Ohshima M. Miyoshi H. Sakamoto K. Takegami K. Iwata J. Kuwabara K. Iwamura H. Yagi T. Biochemistry. 1998; 37: 6436-6445Crossref PubMed Scopus (46) Google Scholar). All the assays were performed at 20 °C. Quinones were extracted from the mitochondria of N2 andclk-1 mutant strains, and the quinone composition was directly analyzed by reverse-phase HPLC (Fig.1). Three different mutant strains, including a missense mutant (e2519), a deletion mutant (qm30), and a splice acceptor mutant (qm51), were used for the analysis. The major peak at 18.3 min from N2 mitochondria is identical to standard UQ9 for both elution time and absorption property (Fig. 1, A andB, and Fig. 2 A). However, a corresponding peak was not observed in clk-1mutants. The mutant mitochondria instead exhibited a major peak eluting 1 min earlier than UQ9 (Fig. 1, C–E). The slightly polar nature and the absorption property (absorption peak at 270 nm, Fig. 2 B) of this compound coincide well with those reported for the ubiquinone biosynthesis intermediate, DMQ9(see Fig. 3) (19Trumpower B.L. Opliger C.E. Olson R.E. Chem. Phys. Lipids. 1974; 13: 123-132Crossref PubMed Scopus (4) Google Scholar, 23Kwon O. Kotsakis A. Maganathan R. FEMS Microbiol. Lett. 2000; 186: 157-161Crossref PubMed Google Scholar). A mass spectrometry analysis of the accumulated quinone in clk-1(qm51) mutant detected a molecular ion peak atm/z 765, which corresponds to the molecular mass of DMQ9 (theoretical mass [C53H80O3] = 765.2005) (Fig.4). For comparison, the mass spectrum of the standard UQ9 (Sigma) showed a molecular ion peak atm/z 795 (theoretical mass [C54H82O4] = 795.2264) (data not shown).Figure 2Absorption spectra of quinones in N2 andclk-1 mutants mitochondria. The spectra of the quinones from N2 (eluted at 18.31 min in Fig. 1 B) (A) and clk-1 mutant (qm51) (eluted at 17.11 min in Fig. 1 E) (B) are monitored by photodiode array UV-visible detector. Maximum absorption of each compound (λmax) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Pathway for biosynthesis of UQ9. The proposed pathway of UQ biosynthesis in eukaryotes (9Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) is shown. The accumulation of DMQ9 inclk-1 mutants indicates that CLK-1 is necessary for the step converting DMQ9 to 3-hydroxy-UQ9. The intermediates indicated are (from the top) 3-nonaprenyl-4-hydroxybenzoate, DMQ9, 3-hydroxy-UQ9, and UQ9.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Mass spectrum of the quinone biosynthesis intermediate from clk-1 (qm51) mutant mitochondria. The mass spectrum of the quinone accumulated inclk-1 (qm51) mitochondria, which corresponds to the peak eluting at 17.1 min in Fig. 1 E, is shown. The molecular structure of DMQ9 is also shown. The peak atm/z 765 corresponds to the molecular ion of DMQ9.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The amount of DMQ9 in all the three mutant strains was in the same range as UQ9 content in N2 mitochondria (TableI). In all clk-1 mutants, the peak corresponding to UQ9 was undetectable by UV absorbance, indicating that the levels of UQ9 inclk-1 mutants mitochondria are less than 0.1 nmol/mg. Sinceclk-1 mutants show normal levels of oxygen consumption (15Braeckman B.P. Houthoofd K. De Vreese A. Vanfleteren J.R. Curr. Biol. 1999; 9: 493-496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and succinate-cytochrome c reductase activity (14Felkai S. Ewbank J.J. Lemieux J. Labbe J.-C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (223) Google Scholar), it has been suggested that CLK-1 may not be critically involved in UQ biosynthesis in nematodes (4Hekimi S. Lakowski B. Barnes T.M. Ewbank J.J. Trends Genet. 1998; 14: 14-19Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 5Gems D. Curr. Biol. 1999; 9: R614-R616Abstract Full Text Full Text PDF PubMed Google Scholar, 6Branicky R. Bénard C. Hekimi S. Bioessays. 2000; 22: 48-56Crossref PubMed Scopus (73) Google Scholar, 7Hekimi S. Hekimi S. The Molecular Genetics of Aging. Springer-Verlag, Berlin2000: 81-112Google Scholar). Our findings, however, clearly demonstrate that clk-1 encodes a protein that is absolutely required for the biosynthesis of UQ in C. elegans (Fig. 3).Table IQuinone content and enzymatic activities of N2 and clk-1 mutant mitochondriaStrainQuinone content 1-an = 3 for N2,e2519, and qm51; n = 4 forqm30.NADH-cytochromecreductase 1-bn = 3 for qm51;n = 5 for e2519 and qm30;n = 6 for N2.Succinate-cytochromec reductase 1-bn = 3 for qm51;n = 5 for e2519 and qm30;n = 6 for N2.DMQ9UQ9nmol mg−1 (±S.D.)nmol min−1 mg−1 (±S.D.)N2ND 1-cND, not detectable.1.04 content was analyzed using mg of mitochondria, as described under NADH-cytochrome creductase and succinate-cytochrome c reductase activities were assayed at with n = 3 for N2,e2519, and qm51; n = 4 n = 3 for qm51;n = 5 for e2519 and qm30;n = 6 for not in a content was analyzed using mg of mitochondria, as described under NADH-cytochrome creductase and succinate-cytochrome c reductase activities were assayed at with mitochondria. UQ is known as an of the respiratory electron chain and is required for mitochondrial In fact, UQ in mitochondrial S. D. D. S. A. 1989; PubMed Scopus Google Scholar), and yeast which not UQ6, is defective in (9Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). the absence of and the normal of respiration observed inclk-1 mutants S. Hekimi S. The Molecular Genetics of Aging. Springer-Verlag, Berlin2000: 81-112Google Scholar, S. Ewbank J.J. Lemieux J. Labbe J.-C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (223) Google Scholar, B.P. Houthoofd K. De Vreese A. Vanfleteren J.R. Curr. Biol. 1999; 9: 493-496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) the of electron is in clk-1 a amount of DMQ9 is accumulated in clk-1 mutants mitochondria, might be that DMQ9 as an electron carrier in mutants mitochondria. To test this possibility, we measured various electron activities in clk-1mutants mitochondria creductase activity of mutants mitochondria was measured and found to be slightly the (14Felkai S. Ewbank J.J. Lemieux J. Labbe J.-C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (223) Google Scholar). the activity of NADH-cytochrome c reductase inclk-1 mutants was comparable with that in the wild-type strain These indicate the that the electron between complex I and complex in clk-1mutants mitochondria might be by DMQ9, with almost the same as UQ9. To further the activity of DMQ as an electron we chemically synthesized a short side chain DMQ2 and measured the activities of NADH-quinone reductase and shown in DMQ2 was able to from complex I with a comparable with DMQ2 was also of as electron acceptor of complex the activity was than that with In contrast to 3-hydroxy-UQ2, which is a of UQ Fig. was unable to as electron acceptor at complex I at complex II indicating that not all the quinone biosynthesis intermediates are as by respiratory Interestingly, DMQ to be a for complex I than for complex II I and This has been also reported for coli mutants, which Biochim. Biophys. Acta. PubMed Scopus Google Scholar), suggesting that the structure and the of DMQ might be for by complex I than by complex The active respiration in clk-1 mutants mitochondria the as to yeast mutants, which also contain are defective in respiratory (9Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Our suggest that this is to the amount of accumulated in mutants (9Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and the lack of complex I in the S. respiratory activities of clk-1 mutant mitochondria using UQ as electron min−1 mg−1 (±S.D.)nmol min−1 mg−1 = not = = = were performed at using 90 μm short side chain quinone as electron as described under not in a were performed at using 90 μm short side chain quinone as electron as described under The of an altered quinone composition in clk-1mutants is the first of a biochemical betweenclk-1 mutants and wild-type strains and that CLK-1 is absolutely required for the step converting DMQ9 to 3-hydroxy-UQ9. However, are to that CLK-1 may not directly in the of DMQ9, clk-1 and its do not possess any in their structure (8Ewbank J.J. Barnes T.M. Lakowski B. Lussier M. Bussey H. Hekimi S. Science. 1997; 275: 980-983Crossref PubMed Scopus (264) Google Scholar, B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, T. Marbois B.N. Kim L. Chin A. Xia Y.-R. Lusis A.J. Clarke C.F. Arch. Biochem. Biophys. 1996; 330: 285-289Crossref PubMed Scopus (41) Google Scholar, 12Vojo Z. King L.M. Jonassen T. Wilkin D.J. Ho N. Munnich A. Clarke C.F. Francomano C.A. Mamm. Genome. 1999; 10: 1000-1004Crossref PubMed Scopus (64) Google Scholar, 13Asaumi S. Kuroyanagi H. Seki N. Shirasawa T. Genomics. 1999; 58: 293-301Crossref PubMed Scopus (25) Google Scholar), in contrast to coli which has been identified to be for the of from O. Kotsakis A. Maganathan R. FEMS Microbiol. Lett. 2000; 186: 157-161Crossref PubMed Google Scholar). The that a gene homologous to coli in the of C. elegans that than CLK-1, the of DMQ9 in C. elegans. In Clarke C.F. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar) reported that yeast CLK-1 is necessary for the of which in the of the UQ biosynthesis These observations suggest that may in a in the UQ biosynthesis of eukaryotes and that the of DMQ9 is of the major under the control of is the between UQ biosynthesis and the of clk-1 The normal of respiration observed inclk-1 mutants by and B.P. Houthoofd K. De Vreese A. Vanfleteren J.R. Curr. Biol. 1999; 9: 493-496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) that the of clk-1mutants is not the of as has been previously B. Hekimi S. S. A. 1998; PubMed Scopus Google Scholar). that that the of clk-1 mutants is not by the accumulation of DMQ9 in the adult mitochondria is the absence of between the of the mutant and the of the biochemical in the three clk-1 we not find a in the amount of DMQ9 between the missense mutant and the mutants and These observations suggest that UQ biosynthesis might be of the that is by of clk-1 mutants that is not very different in the different is the in life span (2Wong A. Boutis P. Hekimi S. Genetics. 1995; 139: 1247-1259Crossref PubMed Google Scholar, 3Lakowski B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (422) Google Scholar). The alteration of the content of different which is similar in all might to oxygen species as a of electron are to be an of B.N. Physiol. 1998; PubMed Scopus Google Scholar, Scholar, T. 2000; PubMed Scopus Google Scholar). of is the which is a intermediate the and the oxidation of UQ in complex I and complex S. Trends Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the chemical of the from DMQ9 for a of and to a of which in a life span. We the C. for MQ50 clk-1 (qm30), which is by the of for We of Tokyo) for authentic

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