Research journey of respirasome

Abstract

Respirasome, as a vital part of the oxidative phosphorylation system, undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitochondrial membrane through the coupled translocation of protons. Copious research has been carried out on this lynchpin of respiration. From the discovery of individual respiratory complexes to the report of the high-resolution structure of mammalian respiratory supercomplex I₁III₂IV₁, scientists have gradually uncovered the mysterious veil of the electron transport chain (ETC). With the discovery of the mammalian respiratory mega complex I₂III₂IV₂, a new perspective emerges in the research field of the ETC. Behind these advances glitters the light of the revolution in both theory and technology. Here, we give a short review about how scientists ‘see’ the structure and the mechanism of respirasome from the macroscopic scale to the atomic scale during the past decades.

Keywords

electron transport chain / supercomplex organization / cellular respiration / structure of respirasome / cryo-EM / megacomplex

References

[1] Acin-Perez R,Bayona-Bafaluy MP, Fernandez-Silva P, Moreno-Loshuertos R, Perez-Martos A, Bruno C,Moraes CT, Enriquez JA (2004) Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol Cell 13:805–815

[2] Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32:529–539

[3] Adiga PS, Malladi R, Baxter W, Glaeser RM (2004) A binary segmentation approach for boxing ribosome particles in cryo EM micrographs. J Struct Biol 145:142–151

[4] Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature 308:32–36

[5] Agip A-NA, Blaza JN, Bridges HR, Viscomi C, Rawson S, Muench SP, Hirst J (2018) Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states. Nat Struct Mol Biol 25:548–556

[6] Agip AA, Blaza JN, Fedor JG, Hirst J(2019) Mammalian respiratory complex I through the lens of cryo-EM. Annu Rev Biophys 48:165–184

[7] Al-Azzawi A, Ouadou A, Tanner JJ, Cheng J (2019a) AutoCryo-Picker: an unsupervised learning approach for fully automated single particle picking in Cryo-EM images. BMC Bioinform 20:326

[8] Al-Azzawi A, Ouadou A, Tanner JJ, Cheng J (2019b) A superclustering approach for fully automated single particle picking in Cryo-EM. Genes (Basel). https://doi.org/10.3390/genes10090666

[9] Alcázar-Fabra M, Navas P, Brea-Calvo G (2016) Coenzyme Q biosynthesis and its role in the respiratory chain structure. Biochim Biophys Acta (BBA) 1857:1073–1078

[10] Allen RD, Schroeder CC, Fok AK (1989) An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques. J Cell Biol 108:2233–2240

[11] Althoff T, Mills DJ, Popot JL, Kuhlbrandt W (2011) Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30:4652–4664

[12] Bai XC, Fernandez IS, McMullan G, Scheres SH (2013) Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. Elife 2:e00461

[13] Bai XC, McMullan G, Scheres SH (2015) How cryo-EM is revolutionizing structural biology. Trends Biochem Sci 40:49–57

[14] Balsa E, Marco R, Perales-Clemente E, Szklarczyk R, Calvo E, Landazuri MO, Enriquez JA (2012) NDUFA4 is a subunit of complex IV of the mammalian electron transport chain. Cell Metab 16:378–386

[15] Barquera B (2014) The sodium pumping NADH:quinone oxidoreductase (Na(⁺)-NQR), a unique redox-driven ion pump. J Bioenerg Biomembr 46:289–298

[16] Bates MGD, Bourke JP, Giordano C, d’Amati G, Turnbull DM, Taylor RW (2012) Cardiac involvement in mitochondrial DNA disease: clinical spectrum, diagnosis, and management. Eur Heart J 33:3023–3033

[17] Bentinger M, Tekle M, Dallner G (2010) Coenzyme Q—biosynthesis and functions. Biochem Biophys Res Commun 396:74–79

[18] Bezawork-Geleta A, Rohlena J, Dong L, Pacak K, Neuzil J (2017) Mitochondrial complex II: at the crossroads. Trends Biochem Sci 42:312–325

[19] Biagini GA, Fisher N, Shone AE, Mubaraki MA, Srivastava A, Hill A, Antoine T, Warman AJ, Davies J, Pidathala C (2012) Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria. Proc Natl Acad Sci USA 109:8298–8303

[20] Bianchi C, Genova ML, Parenti Castelli G, Lenaz G (2004) The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis. J Biol Chem 279:36562–36569

[21] Blair PV (1967) Preparation and properties of repeating units of mitochondrial electron transfer. Methods Enzymol 10:208–212

[22] Blaza JN, Serreli R, Jones AJ, Mohammed K, Hirst J (2014) Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes. Proc Natl Acad Sci USA 111:15735–15740

[23] Blaza JN, Vinothkumar KR, Hirst J (2018) Structure of the deactive state of mammalian respiratory complex I. Structure 26:312–319. e313

[24] Bottani E, Cerutti R, Harbour ME, Ravaglia S, Dogan SA, Giordano C, Fearnley IM, D’Amati G, Viscomi C, Fernandez-Vizarra E (2017) TTC19 plays a husbandry role on UQCRFS1 turnover in the biogenesis of mitochondrial respiratory complex III. Mol Cell 67:96–105.e104

[25] Brandt U (2006) Energy converting NADH:quinone oxidoreductase (complex I). Annu Rev Biochem 75:69–92

[26] Brandt U (2011) A two-state stabilization-change mechanism for proton-pumping complex I. Biochim Biophys Acta 1807:1364–1369

[27] Brilot AF, Chen JZ, Cheng A, Pan J, Harrison SC, Potter CS, Carragher B, Henderson R, Grigorieff N (2012) Beam-induced motion of vitrified specimen on holey carbon film. J Struct Biol 177:630–637

[28] Brzezinski P, Adelroth P (1998) Pathways of proton transfer in cytochrome c oxidase. J Bioenerg Biomembr 30:99–107

[29] Bultema JB, Braun HP, Boekema EJ, Kouril R (2009) Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato. Biochim Biophys Acta 1787:60–67

[30] Cecchini G (2003) Function and structure of complex II of the respiratory chain. Annu Rev Biochem 72:77–109

[31] Chance B, Estabrook RW, Lee CP (1963) Electron transport in the oxysome. Science 140:379–380

[32] Chance B, Williams GR (1955) A method for the localization of sites for oxidative phosphorylation. Nature 176:250

[33] Cheng Y (2015) Single-particle cryo-EM at crystallographic resolution. Cell 161:450–457

[34] Cogliati S, Calvo E, Loureiro M, Guaras AM, Nieto-Arellano R, Garcia-Poyatos C, Ezkurdia I, Mercader N, Vazquez J, Enriquez JA (2016) Mechanism of super-assembly of respiratory complexes III and IV. Nature 539:579–582

[35] Crane FL, Hatefi Y, Lester RL, Widmer C (1957) Isolation of a quinone from beef heart mitochondria. Biochim Biophys Acta 25:220–221

[36] Crofts AR, Meinhardt SW, Jones KR, Snozzi M (1983) The role of the quinone pool in the cyclic electron-transfer chain of rhodopseudomonas sphaeroides: a modified Q-cycle mechanism. Biochim Biophys Acta 723:202–218

[37] Davies KM, Blum TB, Kuhlbrandt W (2018) Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants. Proc Natl Acad Sci USA 115:3024–3029

[38] Davies KM, Strauss M, Daum B, Kief JH, Osiewacz HD, Rycovska A, Zickermann V, Kuhlbrandt W (2011) Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc Natl Acad Sci USA 108:14121–14126

[39] De Rosier DJ, Klug A (1968) Reconstruction of three dimensional structures from electron micrographs. Nature 217:130–134

[40] Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, Schultz P (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228

[41] Dubochet J, Booy FP, Freeman R, Jones AV, Walter CA (1981) Low temperature electron microscopy. Annu Rev Biophys Bioeng 10:133–149

[42] Duchen MR, Szabadkai G (2010) Roles of mitochondria in human disease. Essays Biochem 47:115–137

[43] Dudkina NV, Kudryashev M, Stahlberg H, Boekema EJ(2011) Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography. Proc Natl Acad Sci USA 108:15196–15200

[44] Efremov RG, Sazanov LA (2012) The coupling mechanism of respiratory complex I- a structural and evolutionary perspective . Biochim Biophys Acta 1817:1785– 1795

[45] Enríquez JA (2016) Supramolecular organization of respiratory complexes. Annu Rev Physiol 78:533–561

[46] Eubel H,Heinemeyer J,Sunderhaus S, Braun HP (2004) Respiratory chain supercomplexes in plant mitochondria. Plant Physiol Biochem 42:937–942

[47] Evans DR, Guy HI (2004) Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway. J Biol Chem 279:33035–33038

[48] Fan X, Wang J, Zhang X, Yang Z, Zhang JC, Zhao L, Peng HL, Lei J, Wang HW (2019) Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution. Nat Commun 10:2386

[49] Fedor JG, Hirst J (2018) Mitochondrial supercomplexes do not enhance catalysis by quinone channeling. Cell Metab 28:525–531.e524

[50] Feng X, Fu Z,Kaledhonkar S, Jia Y, Shah B, Jin A, Liu Z, Sun M, Chen B, Grassucci RA (2017) A fast and effective microfluidic spraying-plunging method for high-resolution singleparticle cryo-EM. Structure 25:663–670.e663

[51] Feng Y, Li WF, Li J,Wang JW, Ge JP, Xu D, Liu YJ, Wu KQ, Zeng QY, Wu JW (2012) Structural insight into the type-II mitochondrial NADH dehydrogenases. Nature 491:478–482

[52] Fiedorczuk K, Letts JA, Degliesposti G, Kaszuba K, Skehel M, Sazanov LA (2016) Atomic structure of the entire mammalian mitochondrial complex I. Nature 538:406

[53] Frank J (2017a) Advances in the field of single-particle cryo-electron microscopy over the last decade. Nat Protoc 12:209–212

[54] Frank J (2017b) Time-resolved cryo-electron microscopy: Recent progress. J Struct Biol 200:303–306

[55] Frank J,Shimkin B, Dowse H (1981) Spider—a modular software system for electron image processing. Ultramicroscopy 6:343–357

[56] Frenzel M, Rommelspacher H, Sugawa MD, Dencher NA (2010) Ageing alters the supramolecular architecture of OxPhos complexes in rat brain cortex. Exp Gerontol 45:563–572

[57] Friedrich T (2014) On the mechanism of respiratory complex I. J Bioenerg Biomembr 46:255–268

[58] Fu Z, Kaledhonkar S, Borg A, Sun M, Chen B, Grassucci RA, Ehrenberg M, Frank J (2016) Key intermediates in ribosome recycling visualized by time-resolved cryoelectron microscopy. Structure 24:2092–2101

[59] Genova ML, Lenaz G (2011) New developments on the functions of coenzyme Q in mitochondria. BioFactors 37:330–354

[60] Genova ML, Lenaz G (2014) Functional role of mitochondrial respiratory supercomplexes. Biochim Biophys Acta 1837:427–443

[61] Gomez LA, Monette JS, Chavez JD, Maier CS, Hagen TM (2009) Supercomplexes of the mitochondrial electron transport chain decline in the aging rat heart. Arch Biochem Biophys 490:30–35

[62] Gong H, Li J, Xu A, Tang Y, Ji W, Gao R, Wang S, Yu L, Tian C, Li J (2018) An electron transfer path connects subunits of a mycobacterial respiratory supercomplex. Science.https://doi.org/10.1126/science.aat8923

[63] Green DE, Tzagoloff A (1966) The mitochondrial electron transfer chain. Arch Biochem Biophys 116:293–304

[64] Greggio C, Jha P, Kulkarni SS, Lagarrigue S, Broskey NT, Boutant M, Wang X,Conde Alonso S, Ofori E, Auwerx J (2017) Enhanced respiratory chain supercomplex formation in response to exercise in human skeletal muscle. Cell Metab 25:301–311

[65] Grigorieff N (1998) Three-dimensional structure of bovine NADH: ubiquinone oxidoreductase (complex I) at 22 A in ice. J Mol Biol 277:1033–1046

[66] Gu J, Wu M,Guo R,Yan K, Lei J, Gao N, Yang M (2016) The architecture of the mammalian respirasome. Nature 537:639

[67] Gu J, Zhang L, Zong S, Guo R, Liu T, Yi J,Wang P, Zhuo W, Yang M (2019) Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1. Science 364:1068–1075

[68] Guo R, Gu J, Zong S, Wu M, Yang M (2018) Structure and mechanism of mitochondrial electron transport chain. Biomed J 41:9–20

[69] Guo R, Zong S, Wu M, Gu J, Yang M (2017) Architecture of human mitochondrial respiratory megacomplex I2III2IV2. Cell 170:1247–1257.e1212

[70] Gupte SS, Hackenbrock CR (1988) The role of cytochrome c diffusion in mitochondrial electron transport. J Biol Chem 263:5248–5253

[71] Hackenbrock CR (1977) Molecular organization and the fluid nature of the mitochondrial energy transducing membrane. In: Abrahamsson S, Pascher I (eds) Structure of biological membranes. Springer, Boston, MA,pp 199–234

[72] Harding JW Jr, Pyeritz EA, Copeland ES, White HB 3rd (1975) Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver. Biochemical Journal 146:223–229

[73] Hartley AM, Lukoyanova N, Zhang Y, Cabrera-Orefice A, Arnold S, Meunier B, Pinotsis N, Marechal A (2019) Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1. Nat Struct Mol Biol 26:78–83

[74] Hatefi Y (1985) The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 54:1015–1069

[75] Hatefi Y, Haavik AG, Fowler LR, Griffiths DE (1962) Studies on the electron transfer system. XLII. Reconstitution of the electron transfer system. J Biol Chem 237:2661–2669

[76] Hayward SB, Glaeser RM (1979) Radiation damage of purple membrane at low temperature. Ultramicroscopy 04:201–210

[77] Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:899–929

[78] Henderson R, Unwin PN (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257:28–32

[79] Heron C, Ragan CI, Trumpower BL (1978) The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinolcytochrome c oxidoreductase. Restoration of ubiquinone-pool behaviour. Biochem J 174:791–800

[80] Hill R, Keilin D (1930) The porphyrin of component c of cytochrome and its relationship to other porphyrins. Proc R Soc Lond B Biol Sci 107:286–292

[81] Hirst J (2013) Mitochondrial complex I. Annu Rev Biochem 82:551–575

[82] Hirst J (2018) Open questions: respiratory chain supercomplexes—why are they there and what do they do? BMC Biol 16:111

[83] Hochli M, Hackenbrock CR (1976) Fluidity in mitochondrial membranes: thermotropic lateral translational motion of intramembrane particles. Proc Natl Acad Sci USA 73:1636–1640

[84] Hochli M, Hochli L, Hackenbrock CR (1985) Independent lateral diffusion of cytochrome bc1 complex and cytochrome oxidase in the mitochondrial inner membrane. Eur J Cell Biol 38:1–5

[85] Hofhaus G, Weiss H, Leonard K (1991) Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I) . J Mol Biol 221:1027–1043

[86] Hofmann AD, Beyer M, Krause-Buchholz U, Wobus M, Bornhauser M, Rodel G (2012) OXPHOS supercomplexes as a hallmark of the mitochondrial phenotype of adipogenic differentiated human MSCs. PLoS ONE 7:e35160

[87] Hu M, Yu H, Gu K, Wang Z, Ruan H,Wang K, Ren S, Li B, Gan L, Xu S (2018) A particle-filter framework for robust cryo-EM 3D reconstruction. Nat Methods 15:1083–1089

[88] Hunte C, Zickermann V, Brandt U (2010) Functional modules and structural basis of conformational coupling in mitochondrial complex I. Science 329:448–451

[89] Ikeda K, Shiba S, Horie-Inoue K, Shimokata K, Inoue S (2013) A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle. Nat Commun 4:2147

[90] Iverson TM, Luna-Chavez C,Cecchini G, Rees DC (1999) Structure of the Escherichia coli fumarate reductase respiratory complex. Science 284:1961–1966

[91] Iwata S, Lee JW, Okada K, Lee JK, Iwata M, Rasmussen B, Link TA, Ramaswamy S, Jap BK (1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science 281:64–71

[92] Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376:660–669

[93] Kadenbach B (2017) Regulation of mammalian 13-subunit cytochrome c oxidase and binding of other proteins: role of NDUFA4. Trends Endocrinol Metab 28:761–770

[94] Kalckar HM (1974) Origins of the concept oxidative phosphorylation. Mol Cell Biochem 5:55–62

[95] Kalckar HM (1991) 50 years of biological research—from oxidative phosphorylation to energy requiring transport regulation. Annu Rev Biochem 60:1–38

[96] Keilin D, Hartree EF (1947) Activity of the cytochrome system in heart muscle preparations. Biochem J 41:500–502

[97] Kerscher S, Drose S, Zickermann V, Brandt U (2008) The three families of respiratory NADH dehydrogenases. Results Probl Cell Differ 45:185–222

[98] Kerscher SJ (2000) Diversity and origin of alternative NADH: ubiquinone oxidoreductases. Biochim Biophys Acta 1459:274–283

[99] Konstantinov AA, Siletsky S, Mitchell D, Kaulen A, Gennis RB (1997) The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer. Proc Natl Acad Sci USA 94:9085–9090

[100] Krause F (2006) Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes. Electrophoresis 27:2759–2781

[101] Krause F, Reifschneider NH, Vocke D, Seelert H, Rexroth S, Dencher NA (2004a) “Respirasome”-like supercomplexes in green leaf mitochondria of spinach. J Biol Chem 279:48369–48375

[102] Krause F, Scheckhuber CQ, Werner A, Rexroth S, Reifschneider NH, Dencher NA, Osiewacz HD (2004b) Supramolecular organization of cytochrome c oxidase- and alternative oxidasedependent respiratory chains in the filamentous fungus Podospora anserina. J Biol Chem 279:26453–26461

[103] Kroger A, Klingenberg M (1973) The kinetics of the redox reactions of ubiquinone related to the electron-transport activity in the respiratory chain. Eur J Biochem 34:358–368

[104] Kuijper M, van Hoften G, Janssen B, Geurink R,De Carlo S, Vos M, van Duinen G,van Haeringen B, Storms M (2015) FEI’s direct electron detector developments: embarking on a revolution in cryo-TEM. J Struct Biol 192:179–187

[105] Lamantea E, Carrara F,Mariotti C, Morandi L, Tiranti V, Zeviani M (2002) A novel nonsense mutation (Q352X) in the mitochondrial cytochrome b gene associated with a combined deficiency of complexes I and III. Neuromuscul Disord 12:49–52

[106] Langlois R, Pallesen J, Ash JT, Nam Ho D, Rubinstein JL, Frank J (2014) Automated particle picking for low-contrast macromolecules in cryo-electron microscopy. J Struct Biol 186:1–7

[107] Lapuente-Brun E, Moreno-Loshuertos R, Acin-Perez R, Latorre-Pellicer A, Colas C,Balsa E, Perales-Clemente E, Quiros PM, Calvo E, Rodriguez-Hernandez MA (2013) Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Science 340:1567–1570

[108] Lax NZ, Turnbull DM, Reeve AK (2011) Mitochondrial mutations: newly discovered players in neuronal degeneration. Neuroscientist 17:645–658

[109] Leigh KE, Navarro PP, Scaramuzza S, Chen W, Zhang Y, Castano-Diez D, Kudryashev M (2019) Subtomogram averaging from cryo-electron tomograms. Methods Cell Biol 152:217–259

[110] Lenaz G, Genova ML (2007) Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling. Am J Physiol Cell Physiol 292:C1221–1239

[111] Lenaz G, Genova ML (2012) Supramolecular organisation of the mitochondrial respiratory chain: a new challenge for the mechanism and control of oxidative phosphorylation. Adv Exp Med Biol 748:107–144

[112] Lenaz G,Tioli G,Falasca AI, Genova ML (2016) Complex I function in mitochondrial supercomplexes. Biochim Biophys Acta 1857:991–1000

[113] Leonard K, Haiker H, Weiss H (1987) Three-dimensional structure of NADH: ubiquinone reductase (complex I) from Neurospora mitochondria determined by electron microscopy of membrane crystals. J Mol Biol 194:277–286

[114] Lepault J, Dubochet J, Baschong W, Kellenberger E (1987) Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples. EMBO J 6:1507–1512

[115] Letts JA, Fiedorczuk K, Degliesposti G, Skehel M, Sazanov LA (2019) Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk. Mol Cell 75:1131–1146

[116] Letts JA, Fiedorczuk K, Sazanov LA (2016) The architecture of respiratory supercomplexes. Nature 537:644–648

[117] Letts JA, Sazanov LA (2017) Clarifying the supercomplex: the higher-order organization of the mitochondrial electron transport chain. Nat Struct Mol Biol 24:800–808

[118] Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB, Gubbens S, Agard DA, Cheng Y (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590

[119] Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504:107–112

[120] Lim Y-A, Rhein V, Baysang G, Meier F, Poljak AJ, Raftery M, Guilhaus M, Ittner LM, Eckert A, Götz J (2010) Aβ and human amylin share a common toxicity pathway via mitochondrial dysfunction. Proteomics 10:1621–1633

[121] Lobo-Jarne T, Ugalde C (2018) Respiratory chain supercomplexes: structures, function and biogenesis. Semin Cell Dev Biol 76:179–190

[122] Luo F, Gui X, Zhou H, Gu J, Li Y, Liu X, Zhao M, Li D, Li X, Liu C (2018) Atomic structures of FUS LC domain segments reveal bases for reversible amyloid fibril formation. Nat Struct Mol Biol 25:341–346

[123] Maranzana E, Barbero G, Falasca AI, Lenaz G, Genova ML (2013) Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I. Antioxid Redox Signal 19:1469–1480

[124] Marques I,Dencher NA, Videira A, Krause F (2007) Supramolecular organization of the respiratory chain in Neurospora crassamitochondria. Eukaryot Cell 6:2391–2405

[125] McMullan G, Faruqi AR, Clare D, Henderson R (2014) Comparison of optimal performance at 300 keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147:156–163

[126] McMullan G, Faruqi AR, Henderson R (2016) Direct electron detectors. Methods Enzymol 579:1–17

[127] Melo AM, Bandeiras TM, Teixeira M (2004) New insights into type II NAD(P)H: quinone oxidoreductases. Microbiol Mol Biol Rev 68:603–616

[128] Merk A, Bartesaghi A, Banerjee S, Falconieri V, Rao P, Davis MI, Pragani R, Boxer MB, Earl LA, Milne JLS (2016) Breaking cryo-EM resolution barriers to facilitate drug discovery. Cell 165:1698–1707

[129] Milenkovic D, Blaza JN, Larsson N-G, Hirst J (2017) The enigma of the respiratory chain supercomplex. Cell Metab 25:765–776

[130] Mileykovskaya E, Penczek PA, Fang J, Mallampalli VK, Sparagna GC, Dowhan W (2012) Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy. J Biol Chem 287:23095–23103

[131] Mitchell P(1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144

[132] Mitchell P (1975a) The protonmotive Q cycle: a general formulation. FEBS Lett 59:137–139

[133] Mitchell P (1975b) Protonmotive redox mechanism of the cytochrome b-c1 complex in the respiratory chain: protonmotive ubiquinone cycle. FEBS Lett 56:1–6

[134] Mitchell P (2011) Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biochim Biophys Acta (BBA) 1807:1507–1538

[135] Mourier A, Matic S, Ruzzenente B, Larsson NG, Milenkovic D (2014) The respiratory chain supercomplex organization is independent of COX7a2l isoforms. Cell Metab 20:1069–1075

[136] Muller F, Crofts AR, Kramer DM (2002) Multiple Q-cycle bypass reactions at the Qo site of the cytochrome bc1 complex. Biochemistry 41:7866–7874

[137] Nicholson WV, Glaeser RM (2001) Review: automatic particle detection in electron microscopy. J Struct Biol 133:90–101

[138] Nogales E,Scheres SH (2015) Cryo-EM: a unique tool for the visualization of macromolecular complexity. Mol Cell 58:677–689

[139] Nubel E, Wittig I, Kerscher S, Brandt U, Schagger H (2009) Twodimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica. Proteomics 9:2408–2418

[140] Ogura T, Sato C (2004) Automatic particle pickup method using a neural network has high accuracy by applying an initial weight derived from eigenimages: a new reference free method for single-particle analysis. J Struct Biol 145:63–75

[141] Ohnishi T, Kawaguchi K, Hagihara B (1966) Preparation and some properties of yeast mitochondria. J Biol Chem 241:1797–1806

[142] Ohnishi T,Ohnishi ST, Shinzawa-Itoh K, Yoshikawa S, Weber RT (2012) EPR detection of two protein-associated ubiquinone components (SQ(Nf) and SQ(Ns)) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I. Biochim Biophys Acta 1817:1803–1809

[143] Osuda Y, Shinzawa-Itoh K, Tani K, Maeda S, Yoshikawa S, Tsukihara T, Gerle C (2016) Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes. Microscopy (Oxf) 65:263–267

[144] Osyczka A, Moser CC, Daldal F, Dutton PL (2004) Reversible redox energy coupling in electron transfer chains. Nature 427:607–612

[145] Osyczka A, Moser CC, Dutton PL (2005) Fixing the Q cycle. Trends Biochem Sci 30:176–182

[146] Papa S, Capitanio G, Papa F (2018) The mechanism of coupling between oxido-reduction and proton translocation in respiratory chain enzymes. Biol Rev 93:322–349

[147] Papa S, Martino PL, Capitanio G,Gaballo A, De Rasmo D, Signorile A, Petruzzella V (2012) The oxidative phosphorylation system in mammalian mitochondria. In: Scatena R, Bottoni P, Giardina B (eds) Advances in mitochondrial medicine. Springer, Dordrecht, pp 3–37

[148] Parey K, Brandt U, Xie H, Mills DJ, Siegmund K, Vonck J, Kuhlbrandt W, Zickermann V (2018) Cryo-EM structure of respiratory complex I at work. Elife 7:e39213

[149] Penczek PA, Grassucci RA, Frank J (1994) The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultramicroscopy 53:251–270

[150] Peng G, Fritzsch G, Zickermann V, Schagger H, Mentele R, Lottspeich F, Bostina M, Radermacher M, Huber R, Stetter KO (2003) Isolation, characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus. Biochemistry 42:3032–3039

[151] Perez-Perez R, Lobo-Jarne T, Milenkovic D, Mourier A, Bratic A, Garcia-Bartolome A, Fernandez-Vizarra E, Cadenas S, Delmiro A, Garcia-Consuegra I (2016) COX7A2L is a mitochondrial complex III binding protein that stabilizes the III2+IV supercomplex without affecting respirasome formation. Cell Rep 16:2387–2398

[152] Pieczenik SR, Neustadt J (2007) Mitochondrial dysfunction and molecular pathways of disease. Exp Mol Pathol 83:84–92

[153] Pietras R, Sarewicz M, Osyczka A (2016) Distinct properties of semiquinone species detected at the ubiquinol oxidation Qo site of cytochrome bc1 and their mechanistic implications. J R Soc Interface.https://doi.org/10.1098/rsif.2016.0133

[154] Pitceathly RDS, Taanman J-W (2018) NDUFA4 (Renamed COXFA4) is a cytochrome-c oxidase subunit. Trends Endocrinol Metab 29:452–454

[155] Powell HR (2017) X-ray data processing. Biosci Rep. https://doi.org/10.1042/BSR20170227

[156] Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290–296

[157] Radermacher M, Ruiz T, Clason T, Benjamin S, Brandt U, Zickermann V (2006) The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme. J Struct Biol 154:269–279

[158] Radermacher M, Wagenknecht T, Verschoor A, Frank J (1987) Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli . J Microsc 146:113–136

[159] Ragan CI, Heron C (1978) The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association. Biochem J 174:783–790

[160] Ramirez-Aguilar SJ, Keuthe M, Rocha M, Fedyaev VV, Kramp K, Gupta KJ, Rasmusson AG, Schulze WX, van Dongen JT (2011) The composition of plant mitochondrial supercomplexes changes with oxygen availability. J Biol Chem 286:43045–43053

[161] Rathore S, Berndtsson J, Marin-Buera L, Conrad J, Carroni M, Brzezinski P, Ott M (2019) Cryo-EM structure of the yeast respiratory supercomplex. Nat Struct Mol Biol 26:50–57

[162] Razinkov I,Dandey V, Wei H,Zhang Z, Melnekoff D, Rice WJ, Wigge C, Potter CS, Carragher B (2016) A new method for vitrifying samples for cryoEM. J Struct Biol 195:190–198

[163] Reifschneider NH, Goto S, Nakamoto H, Takahashi R, Sugawa M, Dencher NA, Krause F (2006) Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE. J Proteome Res 5:1117–1132

[164] Robinson AL (1986) Electron microscope inventors share nobel physics prize. Science 234:821–822

[165] Rubinstein JL, Brubaker MA (2015) Alignment of cryo-EM movies of individual particles by optimization of image translations. J Struct Biol 192:188–195

[166] Russo CJ, Passmore LA (2016) Progress towards an optimal specimen support for electron cryomicroscopy. Curr Opin Struct Biol 37:81–89

[167] Sazanov LA (2015) A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat Rev Mol Cell Biol 16:375–388

[168] Sazanov LA, Baradaran R,Efremov RG, Berrisford JM, Minhas G (2013) A long road towards the structure of respiratory complex I, a giant molecular proton pump. Biochem Soc Trans 41:1265–1271

[169] Schägger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19:1777–1783

[170] Schafer E, Dencher NA, Vonck J, Parcej DN (2007) Threedimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria. Biochemistry 46:12579–12585

[171] Schafer E, Seelert H, Reifschneider NH, Krause F, Dencher NA, Vonck J (2006) Architecture of active mammalian respiratory chain supercomplexes. J Biol Chem 281:15370–15375

[172] Schagger H, de Coo R, Bauer MF, Hofmann S, Godinot C, Brandt U (2004) Significance of respirasomes for the assembly/stability of human respiratory chain complex I. J Biol Chem 279:36349–36353

[173] Schagger H, Pfeiffer K (2001) The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes. J Biol Chem 276:37861–37867

[174] Schapira AHV (2006) Mitochondrial disease. The Lancet 368:70–82

[175] Scharfe C, Lu HH-S, Neuenburg JK, Allen EA, Li G-C, Klopstock T, Cowan TM, Enns GM, Davis RW (2009) Mapping Gene Associations in human mitochondria using clinical disease phenotypes. PLoS Comput Biol 5:e1000374

[176] Scheres SH (2012a) A Bayesian view on cryo-EM structure determination. J Mol Biol 415:406–418

[177] Scheres SH (2012b) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180:519–530

[178] Scheres SH (2014) Beam-induced motion correction for submegadalton cryo-EM particles. Elife 3:e03665

[179] Scheres SH (2016) Processing of structurally heterogeneous cryo- EM data in RELION. Methods Enzymol 579:125–157

[180] Scheres SH, Chen S (2012) Prevention of overfitting in cryo-EM structure determination. Nat Methods 9:853–854

[181] Schur FK (2019) Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging. Curr Opin Struct Biol 58:1–9

[182] Sherer TB, Betarbet R, Greenamyre JT (2002) Environment, mitochondria, and Parkinson’s disease. Neuroscientist 8:192–197

[183] Shi Y (2014) A glimpse of structural biology through X-ray crystallography. Cell 159:995–1014

[184] Sigworth FJ (1998) A maximum-likelihood approach to singleparticle image refinement. J Struct Biol 122:328–339

[185] Sousa JS, D’Imprima E, Vonck J (2018) Mitochondrial respiratory chain complexes. In: Harris JR, Boekema EJ (eds) Membrane protein complexes: structure and function. Springer, Singapore,pp 167–227

[186] Sousa JS, Mills DJ, Vonck J, Kuhlbrandt W (2016) Functional asymmetry and electron flow in the bovine respirasome. Elife. https://doi.org/10.7554/eLife.21290

[187] Standfuss J (2019) Membrane protein dynamics studied by X-ray lasers- or why only time will tell. Curr Opin Struct Biol 57:63–71

[188] Starkov AA, Fiskum G (2001) Myxothiazol induces H2O2 production from mitochondrial respiratory chain. Biochem Biophys Res Commun 281:645–650

[189] Strauss M, Hofhaus G, Schroder RR, Kuhlbrandt W (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 27:1154–1160

[190] Strecker V, Wumaier Z, Wittig I, Schagger H (2010) Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: isolation of putative respiratory strings or patches. Proteomics 10:3379–3387

[191] Stroh A, Anderka O, Pfeiffer K, Yagi T, Finel M, Ludwig B, Schagger H (2004) Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans. J Biol Chem 279:5000–5007

[192] Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, Bartlam M, Rao Z (2005) Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121:1043–1057

[193] Taylor KA, Glaeser RM (1974) Electron diffraction of frozen, hydrated protein crystals. Science 186:1036–1037

[194] Trouillard M, Meunier B, Rappaport F (2011) Questioning the functional relevance of mitochondrial supercomplexes by timeresolved analysis of the respiratory chain. Proc Natl Acad Sci USA 108:E1027–1034

[195] Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R, Yaono R, Yoshikawa S (1995) Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A. Science 269:1069–1074

[196] Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R, Yaono R, Yoshikawa S (1996) The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 272:1136–1144

[197] Turonova B, Schur FKM, Wan W, Briggs JAG (2017) Efficient 3DCTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4A. J Struct Biol 199:187–195

[198] van Heel M, Frank J (1981) Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6:187–194

[199] van Heel M, Keegstra W (1981) IMAGIC: a fast, flexible and friendly image analysis software system. Ultramicroscopy 7:113–129

[200] Vartak R, Porras CA-M, Bai Y (2013) Respiratory supercomplexes: structure, function and assembly. Protein Cell 4:582–590

[201] Vempati UD, Han X, Moraes CT (2009) Lack of cytochrome c in mouse fibroblasts disrupts assembly/stability of respiratory complexes I and IV. J Biol Chem 284:4383–4391

[202] Verner Z, Skodova I, Polakova S, Durisova-Benkovicova V, Horvath A, Lukes J (2013) Alternative NADH dehydrogenase (NDH2): intermembrane-space-facing counterpart of mitochondrial complex I in the procyclic Trypanosoma brucei. Parasitology 140:328–337

[203] Vinothkumar KR, Zhu J, Hirst J (2014) Architecture of mammalian respiratory complex I. Nature 515:80

[204] Vonck J (2012) Supramolecular organization of the respiratory chain

[205] Wagner T, Merino F, Stabrin M, Moriya T, Antoni C, Apelbaum A, Hagel P, Sitsel O, Raisch T, Prumbaum D (2019) SPHIREcrYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun Biol 2:218

[206] Wan W, Briggs JA (2016) Cryo-electron tomography and subtomogram averaging. Methods Enzymol 579:329–367

[207] Wang F, Gong H, Liu G, Li M,Yan C, Xia T, Li X, Zeng J (2016) DeepPicker: a deep learning approach for fully automated particle picking in cryo-EM. J Struct Biol 195:325–336

[208] Wang HW, Wang JW (2017) How cryo-electron microscopy and X-ray crystallography complement each other. Protein Sci 26:32–39

[209] Wang Y, Zhang SXL, Gozal D (2010) Reactive oxygen species and the brain in sleep apnea. Respir Physiol Neurobiol 174:307–316

[210] Wharton DC, Tzagoloff A (1962) Studies on the electron transfer system. J Biol Chem 237:2051–2061

[211] White HD, Thirumurugan K, Walker ML, Trinick J (2003) A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J Struct Biol 144:246–252

[212] Wikstrom M, Sharma V, Kaila VR, Hosler JP, Hummer G (2015) New perspectives on proton pumping in cellular respiration. Chem Rev 115:2196–2221

[213] Williams EG, Wu Y, Jha P,Dubuis S, Blattmann P, Argmann CA, Houten SM, Amariuta T, Wolski W, Zamboni N (2016) Systems proteomics of liver mitochondria function. Science 352: aad0189

[214] Wiseman B, Nitharwal RG, Fedotovskaya O, Schafer J, Guo H, Kuang Q, Benlekbir S, Sjostrand D, Adelroth P, Rubinstein JL (2018) Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis. Nat Struct Mol Biol 25:1128–1136

[215] Wittig I, Braun HP, Schagger H (2006a) Blue native PAGE. Nat Protoc 1:418–428

[216] Wittig I, Carrozzo R, Santorelli FM, Schagger H (2006b) Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation. Biochim Biophys Acta 1757:1066–1072

[217] Wittig I, Schagger H (2009) Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes. Biochim Biophys Acta 1787:672–680

[218] Wong HC, Chen J,Mouche F, Rouiller I, Bern M (2004) Model-based particle picking for cryo-electron microscopy. J Struct Biol 145:157–167

[219] Wright JJ, Salvadori E, Bridges HR, Hirst J, Roessler MM (2016) Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex I. J Inorg Biochem 162:201–206

[220] Wu M, Gu J,Guo R, Huang Y, Yang M (2016) Structure of mammalian respiratory supercomplex I1III2IV1. Cell 167:1598–1609.e1510

[221] Xia D, Yu CA, Kim H, Xia JZ, Kachurin AM, Zhang L, Yu L, Deisenhofer J (1997) Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277:60–66

[222] Yang XH, Trumpower BL (1986) Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans. J Biol Chem 261:12282–12289

[223] Yang YQ, Yu Y, Li XL, Li J, Wu Y, Yu J, Ge JP, Huang ZH, Jiang LB, Rao Y (2017) Target elucidation by cocrystal structures of NADH-ubiquinone oxidoreductase of Plasmodium falciparum (PfNDH2) with small molecule to eliminate drug-resistant malaria. J Med Chem 60:1994–2005

[224] Yankovskaya V, Horsefield R, Tornroth S, Luna-Chavez C, Miyoshi H, Leger C,Byrne B, Cecchini G,Iwata S (2003) Architecture of succinate dehydrogenase and reactive oxygen species generation. Science 299:700–704

[225] Yano T,Rahimian M, Aneja KK, Schechter NM, Rubin H, Scott CP (2014) Mycobacterium tuberculosis type II NADH-menaquinone oxidoreductase catalyzes electron transfer through a two-site ping-pong mechanism and has two quinone-binding sites. Biochemistry 53:1179–1190

[226] Yoshikawa S, Shimada A (2015) Reaction mechanism of cytochrome c oxidase. Chem Rev 115:1936–1989

[227] Yoshikawa S, Shinzawa-Itoh K, Nakashima R, Yaono R, Yamashita E, Inoue N, Yao M, Fei MJ, Libeu CP, Mizushima T (1998) Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase. Science 280:1723

[228] Zeviani M, Di Donato S (2004) Mitochondrial disorders. Brain 127:2153–2172

[229] Zhang M, Mileykovskaya E, Dowhan W(2002) Gluing the respiratory chain together: cardiolipin is required for supercomplex formation in the inner mitochondrial membrane. J Biol Chem 277:43553–43556

[230] Zhang Z, Huang L, Shulmeister VM, Chi YI, Kim KK, Hung LW, Crofts AR, Berry EA, Kim SH (1998) Electron transfer by domain movement in cytochrome bc1. Nature 392:677–684

[231] Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331–332

[232] Zhu J,Vinothkumar KR, Hirst J (2016) Structure of mammalian respiratory complex I. Nature 536:354

[233] Zivanov J, Nakane T,Scheres SHW (2019) A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6:5–17

[234] Zong S, Gu J, Liu T, Guo R, Wu M, Yang M (2018a) UQCRFS1N assembles mitochondrial respiratory complex-III into an asymmetric 21-subunit dimer. Protein Cell 9:586–591

[235] Zong S, Wu M,Gu J, Liu T, Guo R, Yang M (2018b) Structure of the intact 14-subunit human cytochrome c oxidase. Cell Res 28:1026–1034

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