- Email: [email protected]
A novel NADH dehydrogenase family widespread in bacteria
e88
Abstracts
coverage on the electrode surface, we have increased the specific surface area of the electrode by deposition of single-walled carbon nanotubes (SWNTs). The structural stability of the proteins at high temperature was monitored by Fourier-transform infrared spectroscopy. We have established a correlation between the temperature-dependent activity of the enzymes and the occurrence of major structural changes. Finally, the possible structural factors which contribute to the thermostability of T. thermophilus SQR are discussed. References [1] C. R. D. Lancaster, In: Handbook of Metalloproteins, Vol. 1 (Ed.: A. Messerschmidt), John Wiley & Sons Ltd., 2001. [2] C. R. D. Lancaster, Biochim. Biophys. Acta 1553 (2002), 1–6. [3] E. Maklashina, G. Cecchini, Biochim. Biophys. Acta 1797 (2010) 1877–1882. [4] O. Kolaj-Robin, S. R. O'Kane, W. Nitschke, C. Léger, F. Baymann, T. Soulimane, Biochim. Biophys. Acta 1807 (2011) 68–79. [5] O. Kolaj-Robin, M. R. Noor, S. R. O'Kane, F. Baymann, T. Soulimane, PLoS One 8 (2013) e53559. doi:10.1016/j.bbabio.2014.05.099
S8.P19 Delocalised electron transport and chemiosmosis in Escherichia coli Conrad W. Mullineauxa, Tchern Lenna, Isabel Llorente-Garciab, Heiko Erhardtc, Oliver L. Harrimand, Lu-Ning Liue, Thorsten Friedrichc, Mark C. Leakef a Queen Mary University of London, UK b University College London, UK c Albert-Ludwigs-Universitat Freiburg, Germany d University of Oxford, UK e University of Liverpool, UK f University of York, UK E-mail: [email protected] The membrane organisation of electron transport and chemiosmosis remains a topic of intense debate, with current models in many bioenergetic membranes favouring the assembly of multiple chemiosmotic components into supercomplexes that could control the pathways of electron flow and utilisation of the proton-motive force. We set out to investigate the distribution and dynamics of OXPHOS components in the plasma membrane of Escherichia coli using a combination of fluorescent protein tagging and fluorescence microscopy with dynamic tracking and single-particle analysis. Complexes investigated included NDH-1, SDH, Cytochrome bd-I and the proton-translocating ATPase, which could all be tagged with a variety of fluorescent proteins, with minimal loss of function [1,2]. Fluorescence microscopy in vivo showed that all complexes tested are concentrated in mobile domains in the membrane, with dimensions of about 100–200 nm and containing 10 s to 100 s of the tagged complex. Simultaneous visualisation of pairs of tagged complexes showed that different complexes are concentrated in separate domains, with no significant co-localisation and therefore no supercomplexes [2]. Since the pairs of complexes tested include the two complexes involved in one of the major respiratory electron transport pathways, and a major source and sink for the proton-motive force, it follows that both electron transport and the proton motive force are largely delocalised over the entire membrane area in E. coli. Consistent with this model, we observed rapid long-range diffusion of a fluorescent quinone analogue. We suggest that long-range quinone diffusion serves to carry electrons between islands of distinct electron transport complexes in the membrane.
References [1] T. Lenn T, M.C. Leake, C.W. Mullineaux. Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo, Mol. Microbiol. 70 (2008) 1397–1407. [2] I. Llorente-Garcia, T. Lenn, H. Erhardt, O. Harriman, L.-N. Liu, A. Robson, S.W. Matthews, N. Willis, C. Bray, S.-H. Lee, J.Y. Shin, C. Bustamente, J. Liphardt, T. Friedrich, C.W. Mullineaux, M.C. Leake. Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation, BBA-Bioenergetics 1837 (2014) 811–824. doi:10.1016/j.bbabio.2014.05.100
S8.P20 Structural membrane proteomics focused on respiratory protein complexes of hyperthermophilic eubacterium Aquifex aeolicus Yang Nia, Ye Gaoa, Ilka Wittigb, Julian Langera, Guohong Penga, Hartmut Michela a Max-Planck-Institut für Biophysik, Germany b Molecular Bioenergetics, Medical School, Goethe University, Germany E-mail: [email protected] The respiratory chain in eukaryotic mitochondria mainly consists of four transmembrane protein complexes. In contrast, the protein components of bacterial respiratory chains are much more diverse and complex, which enable microorganisms to cope with various living environment. Aquifex aeolicus is Gram-negative, hyperthermophilic, chemolithotrophic and microaerophilic eubacteria, and its genome has been sequenced [1]. Previously, a supercomplex composed of respiratory chain complexes III and IV was isolated in A. aeolicus [2]. In this project, A. aeolicus native membranes were solubilized using mild detergents. The solubilized A. aeolicus membrane proteins were prefractionated by gel filtration chromatography or sucrose gradient ultracentrifugation and further resolved by Blue Native PAGE (BN PAGE) and high resolution Clear Native PAGE (hrCN PAGE). The respiratory chain complexes were indicated by in-gel activity assay and identified by mass spectrometry. Preliminary results showed that complex I, as well as complex V, was detected in several bands on BN PAGE and hrCN PAGE. In those bands several other redox membrane proteins were found. More experiments are under way to investigate the potential interactions and regulations among these respiratory chain complexes. References [1] G. Deckert, P.V. Warren, et al., The complete genome of the hyperthermophilic bacterium Aquifex aeolicus, Nature 392 (1998) 353–358. [2] Y. Gao, B. Meyer, L. Sokolova, K. Zwicker, M, Karas, B. Butschy, G. Peng, H. Michel, Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 3275–3280. doi:10.1016/j.bbabio.2014.05.101
S8.P21 A novel NADH dehydrogenase family widespread in bacteria Ana R. Ramos, Sofia S. Venceslau, Fabian Grein, Gonçalo Oliveira, Inês A.C. Pereira ITQB-UNL, Portugal E-mail: sofi[email protected]
Abstracts
Recently, a new mechanism for energy coupling called FlavinBased Electron Bifurcation (FBEB) was proposed to explain energy conservation in anaerobic organisms [1]. This mechanism, which allows the thermodynamically unfavorable reduction of ferredoxin with NADH by coupling it to a favorable reduction, was most likely present in the early life forms on Earth. Here we describe a new NAD(P)H dehydrogenase (FloxABCD) likely to be involved in FBEB, which was identified in sulfate reducing organisms [2,3]. The floxABCD genes are usually found next to hdrABC genes that code for a heterodisulfide reductase [3]. The flox-hdr cluster is found in a large number of bacteria belonging to Chlorobi, Proteobacteria, Firmicutes, Bacteroidetes, Spirochaetes and Acidobacteria phyla, pointing for a general and important role in the energy metabolism of these organisms. Here, we present results on the function of the FloxABCD-HdrABC in Desulfovibrio vulgaris Hildenborough that indicate its involvement in ethanol oxidation and a possible link to sulfite reduction. References [1] R.K. Thauer, A.K. Kaster, H. Seedorf, W. Buckel, R. Hedderich, Methanogenic archaea: ecologically relevant differences in energy conservation, Nat. Rev. Microbiol. 6 (2008) 579–591. [2] S.A. Haveman, V. Brunelle, J.K. Voordouw, G. Voordouw, J.F. Heidelberg, R. Rabus, Gene expression analysis of energy metabolism mutants of Desulfovibrio vulgaris Hildenborough indicates an important role for alcohol dehydrogenase, J. Bacteriol.185 (2003) 4345–4353. [3] I.A. Pereira, A.R. Ramos, F. Grein, M.C. Marques, S.M. da Silva, S.S. Venceslau, A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea, Front. Microbiol. 2 (2011) 69. doi:10.1016/j.bbabio.2014.05.102
S8.P22 Divide to conquer: From the study of the individual subunits to the understanding of the whole alternative complex III Patrícia N. Refojo, Miguel A. Ribeiro, Miguel Teixeira, Manuela M. Pereira Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal E-mail: [email protected] In Rhodothermus marinus membranes, the quinol:electron acceptor oxidoreductase activity is performed by the alternative complex III (ACIII) [1–4]. This seven subunit complex is a member of a recently identified family of enzymes, which catalyzes an equivalent reaction to the bc1 complex, but is structurally unrelated to it. The available information on the structure and operating mechanisms on ACIII is still scarce. Therefore, the aim of this work was to characterize the ACIII subunits as individual proteins. For that, the genes coding for the subunits, namely those coding for the two cytochromes, were cloned and expressed in Escherichia coli. The biochemical characterization of the proteins was carried out and their function within the complex was explored. This knowledge provides new insights into the ACIII structure and function. References [1] M.M. Pereira, J.N. Carita, M. Teixeira, Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: a novel multihemic cytochrome bc, a new complex III, Biochemistry, 38 (1999) 1268–1275. [2] M.M. Pereira, P.N. Refojo, G.O. Hreggvidsson, S. Hjorleifsdottir, M. Teixeira, The alternative complex III from Rhodothermus marinus —
e89
a prototype of a new family of quinol:electron acceptor oxidoreductases, FEBS Lett, 581 (2007) 4831–4835. [3] P.N. Refojo, F.L. Sousa, M. Teixeira, M.M. Pereira, The alternative complex III: a different architecture using known building modules, Biochim Biophys Acta, 1797 (2010) 1869–1876. [4] P.N. Refojo, M. Teixeira, M.M. Pereira, The alternative complex III of Rhodothermus marinus and its structural and functional association with caa3 oxygen reductase, Biochim Biophys Acta, 1797 (2010) 1477–1482. doi:10.1016/j.bbabio.2014.05.103
S8.P23 Factors in culture medium enhancing amino acid production and switching branches of the respiratory chain of Corynebacterium glutamicum Junshi Sakamoto, M. Sasaki, C. Shiiba, T. Kusumoto Kyushu Institute of Technology, Japan E-mail: [email protected] Corynebacterium glutamicum is an aerobic Gram-positive bacterium of industrial importance, in the production of amino acids, e.g. glutamate and lysine, used as nutritious additives in food and feed. In a series of our early studies to understand its aerobic energy metabolism, we have identified three enzyme complexes in the respiratory chain and their gene clusters; cytochrome bd-type menaquinol oxidase [1], cytochrome “bcc”-type quinol:cytochrome c oxidoreductase (Complex III) [2], and cytochrome aa3-type cytochrome c oxidase (Complex IV) [3]. These enzymes compose two electron-transferring routes, bd route and bcc-aa3 route, which have different ratios of proton translocated/electron transferred [4]. These routes are selectively operated depending on subspecies of the organism and environmental or growth conditions such as the extent of aeration, and this switching can be monitored precisely by a newly developed assay system using the green fluorescent protein (GFP) as a reporter [5]. Recently, we also found that the selection of the respiration routes was dependent on the concentration of yeast extract contained in the growth medium. Proteomic analyses indicated that several soluble enzymes in the central metabolism, various oxidoreductases, and some transcription factors were either increased or decreased by adding yeast extract to the medium. In addition, this ingredient also markedly enhanced glutamate production by the cells. We are currently trying to isolate and identify the factors in the extract switching the respiratory enzymes and affecting the glutamate production by partial purification with hydrophobic and ion-exchange chromatographies, mass analyses and so on. References [1] T. Kusumoto, M. Sakiyama, J. Sakamoto, S. Noguchi, N. Sone, Arch. Microbiol. 173 (2000) 390–397. [2] N. Sone, K. Nagata, H. Kojima, J. Tajima, Y. Kodera, T. Kanamaru, S. Noguchi, J. Sakamoto, Biochim. Biophys. Acta 1503 (2001) 279– 290. [3] J. Sakamoto, T. Shibata, T. Mine, R. Miyahara, T. Torigoe, S. Noguchi, K. Matsushita, N. Sone, Microbiology 147 (2001) 2865–2871 [4] Y. Kabashima, J. Kishikawa, T. Kurokawa, J. Sakamoto, J. Biochem. 146 (2009) 845–855. [5] T. Kusumoto, M. Aoyagi, H. Iwai, Y. Kabashima, J. Sakamoto, J. Bioenerg. Biomembr. 43 (2011) 257– 266. doi:10.1016/j.bbabio.2014.05.104
Abstracts
coverage on the electrode surface, we have increased the specific surface area of the electrode by deposition of single-walled carbon nanotubes (SWNTs). The structural stability of the proteins at high temperature was monitored by Fourier-transform infrared spectroscopy. We have established a correlation between the temperature-dependent activity of the enzymes and the occurrence of major structural changes. Finally, the possible structural factors which contribute to the thermostability of T. thermophilus SQR are discussed. References [1] C. R. D. Lancaster, In: Handbook of Metalloproteins, Vol. 1 (Ed.: A. Messerschmidt), John Wiley & Sons Ltd., 2001. [2] C. R. D. Lancaster, Biochim. Biophys. Acta 1553 (2002), 1–6. [3] E. Maklashina, G. Cecchini, Biochim. Biophys. Acta 1797 (2010) 1877–1882. [4] O. Kolaj-Robin, S. R. O'Kane, W. Nitschke, C. Léger, F. Baymann, T. Soulimane, Biochim. Biophys. Acta 1807 (2011) 68–79. [5] O. Kolaj-Robin, M. R. Noor, S. R. O'Kane, F. Baymann, T. Soulimane, PLoS One 8 (2013) e53559. doi:10.1016/j.bbabio.2014.05.099
S8.P19 Delocalised electron transport and chemiosmosis in Escherichia coli Conrad W. Mullineauxa, Tchern Lenna, Isabel Llorente-Garciab, Heiko Erhardtc, Oliver L. Harrimand, Lu-Ning Liue, Thorsten Friedrichc, Mark C. Leakef a Queen Mary University of London, UK b University College London, UK c Albert-Ludwigs-Universitat Freiburg, Germany d University of Oxford, UK e University of Liverpool, UK f University of York, UK E-mail: [email protected] The membrane organisation of electron transport and chemiosmosis remains a topic of intense debate, with current models in many bioenergetic membranes favouring the assembly of multiple chemiosmotic components into supercomplexes that could control the pathways of electron flow and utilisation of the proton-motive force. We set out to investigate the distribution and dynamics of OXPHOS components in the plasma membrane of Escherichia coli using a combination of fluorescent protein tagging and fluorescence microscopy with dynamic tracking and single-particle analysis. Complexes investigated included NDH-1, SDH, Cytochrome bd-I and the proton-translocating ATPase, which could all be tagged with a variety of fluorescent proteins, with minimal loss of function [1,2]. Fluorescence microscopy in vivo showed that all complexes tested are concentrated in mobile domains in the membrane, with dimensions of about 100–200 nm and containing 10 s to 100 s of the tagged complex. Simultaneous visualisation of pairs of tagged complexes showed that different complexes are concentrated in separate domains, with no significant co-localisation and therefore no supercomplexes [2]. Since the pairs of complexes tested include the two complexes involved in one of the major respiratory electron transport pathways, and a major source and sink for the proton-motive force, it follows that both electron transport and the proton motive force are largely delocalised over the entire membrane area in E. coli. Consistent with this model, we observed rapid long-range diffusion of a fluorescent quinone analogue. We suggest that long-range quinone diffusion serves to carry electrons between islands of distinct electron transport complexes in the membrane.
References [1] T. Lenn T, M.C. Leake, C.W. Mullineaux. Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo, Mol. Microbiol. 70 (2008) 1397–1407. [2] I. Llorente-Garcia, T. Lenn, H. Erhardt, O. Harriman, L.-N. Liu, A. Robson, S.W. Matthews, N. Willis, C. Bray, S.-H. Lee, J.Y. Shin, C. Bustamente, J. Liphardt, T. Friedrich, C.W. Mullineaux, M.C. Leake. Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation, BBA-Bioenergetics 1837 (2014) 811–824. doi:10.1016/j.bbabio.2014.05.100
S8.P20 Structural membrane proteomics focused on respiratory protein complexes of hyperthermophilic eubacterium Aquifex aeolicus Yang Nia, Ye Gaoa, Ilka Wittigb, Julian Langera, Guohong Penga, Hartmut Michela a Max-Planck-Institut für Biophysik, Germany b Molecular Bioenergetics, Medical School, Goethe University, Germany E-mail: [email protected] The respiratory chain in eukaryotic mitochondria mainly consists of four transmembrane protein complexes. In contrast, the protein components of bacterial respiratory chains are much more diverse and complex, which enable microorganisms to cope with various living environment. Aquifex aeolicus is Gram-negative, hyperthermophilic, chemolithotrophic and microaerophilic eubacteria, and its genome has been sequenced [1]. Previously, a supercomplex composed of respiratory chain complexes III and IV was isolated in A. aeolicus [2]. In this project, A. aeolicus native membranes were solubilized using mild detergents. The solubilized A. aeolicus membrane proteins were prefractionated by gel filtration chromatography or sucrose gradient ultracentrifugation and further resolved by Blue Native PAGE (BN PAGE) and high resolution Clear Native PAGE (hrCN PAGE). The respiratory chain complexes were indicated by in-gel activity assay and identified by mass spectrometry. Preliminary results showed that complex I, as well as complex V, was detected in several bands on BN PAGE and hrCN PAGE. In those bands several other redox membrane proteins were found. More experiments are under way to investigate the potential interactions and regulations among these respiratory chain complexes. References [1] G. Deckert, P.V. Warren, et al., The complete genome of the hyperthermophilic bacterium Aquifex aeolicus, Nature 392 (1998) 353–358. [2] Y. Gao, B. Meyer, L. Sokolova, K. Zwicker, M, Karas, B. Butschy, G. Peng, H. Michel, Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 3275–3280. doi:10.1016/j.bbabio.2014.05.101
S8.P21 A novel NADH dehydrogenase family widespread in bacteria Ana R. Ramos, Sofia S. Venceslau, Fabian Grein, Gonçalo Oliveira, Inês A.C. Pereira ITQB-UNL, Portugal E-mail: sofi[email protected]
Abstracts
Recently, a new mechanism for energy coupling called FlavinBased Electron Bifurcation (FBEB) was proposed to explain energy conservation in anaerobic organisms [1]. This mechanism, which allows the thermodynamically unfavorable reduction of ferredoxin with NADH by coupling it to a favorable reduction, was most likely present in the early life forms on Earth. Here we describe a new NAD(P)H dehydrogenase (FloxABCD) likely to be involved in FBEB, which was identified in sulfate reducing organisms [2,3]. The floxABCD genes are usually found next to hdrABC genes that code for a heterodisulfide reductase [3]. The flox-hdr cluster is found in a large number of bacteria belonging to Chlorobi, Proteobacteria, Firmicutes, Bacteroidetes, Spirochaetes and Acidobacteria phyla, pointing for a general and important role in the energy metabolism of these organisms. Here, we present results on the function of the FloxABCD-HdrABC in Desulfovibrio vulgaris Hildenborough that indicate its involvement in ethanol oxidation and a possible link to sulfite reduction. References [1] R.K. Thauer, A.K. Kaster, H. Seedorf, W. Buckel, R. Hedderich, Methanogenic archaea: ecologically relevant differences in energy conservation, Nat. Rev. Microbiol. 6 (2008) 579–591. [2] S.A. Haveman, V. Brunelle, J.K. Voordouw, G. Voordouw, J.F. Heidelberg, R. Rabus, Gene expression analysis of energy metabolism mutants of Desulfovibrio vulgaris Hildenborough indicates an important role for alcohol dehydrogenase, J. Bacteriol.185 (2003) 4345–4353. [3] I.A. Pereira, A.R. Ramos, F. Grein, M.C. Marques, S.M. da Silva, S.S. Venceslau, A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea, Front. Microbiol. 2 (2011) 69. doi:10.1016/j.bbabio.2014.05.102
S8.P22 Divide to conquer: From the study of the individual subunits to the understanding of the whole alternative complex III Patrícia N. Refojo, Miguel A. Ribeiro, Miguel Teixeira, Manuela M. Pereira Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal E-mail: [email protected] In Rhodothermus marinus membranes, the quinol:electron acceptor oxidoreductase activity is performed by the alternative complex III (ACIII) [1–4]. This seven subunit complex is a member of a recently identified family of enzymes, which catalyzes an equivalent reaction to the bc1 complex, but is structurally unrelated to it. The available information on the structure and operating mechanisms on ACIII is still scarce. Therefore, the aim of this work was to characterize the ACIII subunits as individual proteins. For that, the genes coding for the subunits, namely those coding for the two cytochromes, were cloned and expressed in Escherichia coli. The biochemical characterization of the proteins was carried out and their function within the complex was explored. This knowledge provides new insights into the ACIII structure and function. References [1] M.M. Pereira, J.N. Carita, M. Teixeira, Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: a novel multihemic cytochrome bc, a new complex III, Biochemistry, 38 (1999) 1268–1275. [2] M.M. Pereira, P.N. Refojo, G.O. Hreggvidsson, S. Hjorleifsdottir, M. Teixeira, The alternative complex III from Rhodothermus marinus —
e89
a prototype of a new family of quinol:electron acceptor oxidoreductases, FEBS Lett, 581 (2007) 4831–4835. [3] P.N. Refojo, F.L. Sousa, M. Teixeira, M.M. Pereira, The alternative complex III: a different architecture using known building modules, Biochim Biophys Acta, 1797 (2010) 1869–1876. [4] P.N. Refojo, M. Teixeira, M.M. Pereira, The alternative complex III of Rhodothermus marinus and its structural and functional association with caa3 oxygen reductase, Biochim Biophys Acta, 1797 (2010) 1477–1482. doi:10.1016/j.bbabio.2014.05.103
S8.P23 Factors in culture medium enhancing amino acid production and switching branches of the respiratory chain of Corynebacterium glutamicum Junshi Sakamoto, M. Sasaki, C. Shiiba, T. Kusumoto Kyushu Institute of Technology, Japan E-mail: [email protected] Corynebacterium glutamicum is an aerobic Gram-positive bacterium of industrial importance, in the production of amino acids, e.g. glutamate and lysine, used as nutritious additives in food and feed. In a series of our early studies to understand its aerobic energy metabolism, we have identified three enzyme complexes in the respiratory chain and their gene clusters; cytochrome bd-type menaquinol oxidase [1], cytochrome “bcc”-type quinol:cytochrome c oxidoreductase (Complex III) [2], and cytochrome aa3-type cytochrome c oxidase (Complex IV) [3]. These enzymes compose two electron-transferring routes, bd route and bcc-aa3 route, which have different ratios of proton translocated/electron transferred [4]. These routes are selectively operated depending on subspecies of the organism and environmental or growth conditions such as the extent of aeration, and this switching can be monitored precisely by a newly developed assay system using the green fluorescent protein (GFP) as a reporter [5]. Recently, we also found that the selection of the respiration routes was dependent on the concentration of yeast extract contained in the growth medium. Proteomic analyses indicated that several soluble enzymes in the central metabolism, various oxidoreductases, and some transcription factors were either increased or decreased by adding yeast extract to the medium. In addition, this ingredient also markedly enhanced glutamate production by the cells. We are currently trying to isolate and identify the factors in the extract switching the respiratory enzymes and affecting the glutamate production by partial purification with hydrophobic and ion-exchange chromatographies, mass analyses and so on. References [1] T. Kusumoto, M. Sakiyama, J. Sakamoto, S. Noguchi, N. Sone, Arch. Microbiol. 173 (2000) 390–397. [2] N. Sone, K. Nagata, H. Kojima, J. Tajima, Y. Kodera, T. Kanamaru, S. Noguchi, J. Sakamoto, Biochim. Biophys. Acta 1503 (2001) 279– 290. [3] J. Sakamoto, T. Shibata, T. Mine, R. Miyahara, T. Torigoe, S. Noguchi, K. Matsushita, N. Sone, Microbiology 147 (2001) 2865–2871 [4] Y. Kabashima, J. Kishikawa, T. Kurokawa, J. Sakamoto, J. Biochem. 146 (2009) 845–855. [5] T. Kusumoto, M. Aoyagi, H. Iwai, Y. Kabashima, J. Sakamoto, J. Bioenerg. Biomembr. 43 (2011) 257– 266. doi:10.1016/j.bbabio.2014.05.104