Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mitochondrial complex I structure reveals ordered water molecules for catalysis and proton translocation

Nature Structural & Molecular Biology volume 27, pages 892–900 (2020)Cite this article

Subjects

Abstract

Mitochondrial complex I powers ATP synthesis by oxidative phosphorylation, exploiting the energy from ubiquinone reduction by NADH to drive protons across the energy-transducing inner membrane. Recent cryo-EM analyses of mammalian and yeast complex I have revolutionized structural and mechanistic knowledge and defined structures in different functional states. Here, we describe a 2.7-Å-resolution structure of the 42-subunit complex I from the yeast Yarrowia lipolytica containing 275 structured water molecules. We identify a proton-relay pathway for ubiquinone reduction and water molecules that connect mechanistically crucial elements and constitute proton-translocation pathways through the membrane. By comparison with known structures, we deconvolute structural changes governing the mammalian ‘deactive transition’ (relevant to ischemia–reperfusion injury) and their effects on the ubiquinone-binding site and a connected cavity in ND1. Our structure thus provides important insights into catalysis by this enigmatic respiratory machine.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of the Y. lipolytica complex I model presented here.
Fig. 2: The location of water molecules in the Y. lipolytica model and interactions with the core transmembrane subunits.
Fig. 3: Comparison of the proposed conserved proton pathway in Y. lipolytica complex I and Desulfovibrio vulgaris NiFe hydrogenase (PDB 1WUI)41.
Fig. 4: A comparison of the water molecules and the TMH7b-Leu gate in subunits ND2, ND4 and ND5.
Fig. 5: Conserved π bulges in the transmembrane domain.
Fig. 6: Structural changes and hydration in the ND1 subunit of Y. lipolytica complex I with DDM bound, compared to the active state of mouse complex I.
Fig. 7: Quinone-binding cavities determined for our Y. lipolytica model, ubiquinone-bound Y. lipolytica model (PDB 6RFR), deactive mouse model (PDB 6G72) and active mouse model (PDB 6G2J).

Similar content being viewed by others

Data availability

The electron microscopy maps and masks and the models for complex I and ST1 have been deposited in the Electron Microscopy Data Bank (EMDB) and in the Protein Data Bank (PDB) with accession codes PDB 6YJ4 and PDB 6YJ5 and EMD-10815 and EMD-10816, respectively.

References

  1. Hirst, J. Mitochondrial complex I. Annu. Rev. Biochem. 82, 551–575 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Parey, K., Wirth, C., Vonck, J. & Zickermann, V. Respiratory complex I—structure, mechanism and evolution. Curr. Opin. Struct. Biol. 63, 1–9 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Kaila, V. R. I. Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I. J. R. Soc. Interface 15, 20170916 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Fassone, E. & Rahman, S. Complex I deficiency: clinical features, biochemistry and molecular genetics. J. Med. Genet. 49, 578–590 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Fiedorczuk, K. & Sazanov, L. A. Mammalian mitochondrial complex I structure and disease-causing mutations. Trends Cell Biol. 28, 835–867 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Balaban, R. S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483–495 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Babot, M., Birch, A., Labarbuta, P. & Galkin, A. Characterisation of the active/de-active transition of mitochondrial complex I. Biochim. Biophys. Acta Bioenerg. 1837, 1083–1092 (2014).

    Article  CAS  Google Scholar 

  8. Chouchani, E. T. et al. A unifying mechanism for mitochondrial superoxide production during ischemia-reperfusion injury. Cell Metab. 23, 254–263 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Stroud, D. A. et al. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature 538, 123–126 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Zhu, J., Vinothkumar, K. R. & Hirst, J. Structure of mammalian respiratory complex I. Nature 536, 354–358 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Morgner, N. et al. Subunit mass fingerprinting of mitochondrial complex I. Biochim. Biophys. Acta Bioenerg. 1777, 1384–1391 (2008).

    Article  CAS  Google Scholar 

  12. Kmita, K. & Zickermann, V. Accessory subunits of mitochondrial complex I. Biochem. Soc. Trans. 41, 1272–1279 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Kerscher, S., Dröse, S., Zwicker, K., Zickermann, V. & Brandt, U. Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I. Biochim. Biophys. Acta Bioenerg. 1555, 83–91 (2002).

    Article  CAS  Google Scholar 

  14. Tocilescu, M. A., Fendel, U., Zwicker, K., Kerscher, S. & Brandt, U. Exploring the ubiquinone binding cavity of respiratory complex I. J. Biol. Chem. 282, 29514–29520 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Varghese, F., Atcheson, E., Bridges, H. R. & Hirst, J. Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system. Hum. Mol. Genet. 24, 6350–6360 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cabrera-Orefice, A. et al. Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps. Nat. Commun. 9, 4500 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Galemou Yoga, E. et al. Mutations in a conserved loop in the PSST subunit of respiratory complex I affect ubiquinone binding and dynamics. Biochim. Biophys. Acta Bioenerg. 1860, 573–581 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Zickermann, V. et al. Mechanistic insight from the crystal structure of mitochondrial complex I. Science 347, 44–49 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Parey, K. et al. High-resolution cryo-EM structures of respiratory complex I: mechanism, assembly, and disease. Sci. Adv. 5, eaax9484 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Agip, A. A. et al. Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states. Nat. Struct. Mol. Biol. 25, 548–556 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Blaza, J. N., Vinothkumar, K. R. & Hirst, J. Structure of the deactive state of mammalian respiratory complex I. Structure 26, 312–319 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sharma, V. et al. Redox-induced activation of the proton pump in the respiratory complex I. Proc. Natl Acad. Sci. USA 112, 11571–11576 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Brandt, U. A two-state stabilization-change mechanism for proton-pumping complex I. Biochim. Biophys. Acta Bioenerg. 1807, 1364–1369 (2011).

    Article  CAS  Google Scholar 

  24. Warnau, J. et al. Redox-coupled quinone dynamics in the respiratory complex I. Proc. Natl Acad. Sci. USA 115, E8413–E8420 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fedor, J. G., Jones, A. J. Y., Di Luca, A., Kaila, V. R. I. & Hirst, J. Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I. Proc. Natl Acad. Sci. USA 114, 12737–12742 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Baradaran, R., Berrisford, J. M., Minhas, G. S. & Sazanov, L. A. Crystal structure of the entire respiratory complex I. Nature 494, 443–448 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Teixeira, M. H. & Arantes, G. M. Balanced internal hydration discriminates substrate binding to respiratory complex I. Biochim. Biophys. Acta Bioenerg. 1860, 541–548 (2019).

    Article  CAS  Google Scholar 

  28. Efremov, R. G. & Sazanov, L. A. Structure of the membrane domain of respiratory complex I. Nature 476, 414–421 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Jones, A. J. Y., Blaza, J. N., Varghese, F. & Hirst, J. Respiratory complex I in Bos taurus and Paracoccus denitrificans pumps four protons across the membrane for every NADH oxidized. J. Biol. Chem. 292, 4987–4995 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Galkin, A. S., Grivennikova, V. G. & Vinogradov, A. D. →H+/2e– stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles. FEBS Lett. 451, 157–161 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Di Luca, A., Gamiz-Hernandez, A. P. & Kaila, V. R. I. Symmetry-related proton transfer pathways in respiratory complex I. Proc. Natl Acad. Sci. USA 114, E6314–E6321 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Di Luca, A., Mühlbauer, M. E., Saura, P. & Kaila, V. R. I. How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics. Biochim. Biophys. Acta Bioenerg. 1859, 734–741 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Kotlyar, A. B. & Vinogradov, A. D. Slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase. Biochim. Biophys. Acta Bioenerg. 1019, 151–158 (1990).

    Article  CAS  Google Scholar 

  34. Meyerson, J. R. et al. Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports. Sci. Rep. 4, 7084 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Parey, K. et al. Cryo-EM structure of respiratory complex I at work. Elife 7, e39213 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Letts, J. A., Fiedorczuk, K., Degliesposti, G., Skehel, M. & Sazanov, L. A. Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk. Mol. Cell 75, 1131–1146 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Abdrakhmanova, A., Dobrynin, K., Zwicker, K., Kerscher, S. & Brandt, U. Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters. FEBS Lett. 579, 6781–6785 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. D’Imprima, E. et al. Cryo-EM structure of respiratory complex I reveals a link to mitochondrial sulfur metabolism. Biochim. Biophys. Acta Bioenerg. 1857, 1935–1942 (2016).

    Article  CAS  Google Scholar 

  39. Fontecilla-Camps, J. C., Volbeda, A., Cavazza, C. & Nicolet, Y. Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. Chem. Rev. 107, 4273–4303 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Szőri-Dorogházi, E. et al. Analyses of the large subunit histidine-rich motif expose an alternative proton transfer pathway in [NiFe] hydrogenases. PLoS ONE 7, e34666 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Ogata, H. et al. Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: conversion of the ready to the unready state. Structure 13, 1635–1642 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Haapanen, O. & Sharma, V. Role of water and protein dynamics in proton pumping by respiratory complex I. Sci. Rep. 7, 7747 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Djurabekova, A., Haapanen, O. & Sharma, V. Proton motive function of the terminal antiporter-like subunit in respiratory complex I. Biochim. Biophys. Acta Bioenerg. 1861, 148185 (2020).

    Article  CAS  PubMed  Google Scholar 

  44. Guo, R., Zong, S., Wu, M., Gu, J. & Yang, M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2. Cell 170, 1247–1257 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Gu, J. et al. The architecture of the mammalian respirasome. Nature 537, 639–643 (2016).

    Article  CAS  PubMed  Google Scholar 

  46. Laughlin, T. G., Bayne, A. N., Trempe, J. F., Savage, D. F. & Davies, K. M. Structure of the complex I-like molecule NDH of oxygenic photosynthesis. Nature 566, 411–414 (2019).

    Article  CAS  PubMed  Google Scholar 

  47. Tian, W., Chen, C., Lei, X., Zhao, J. & Liang, J. CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res. 46, W363–W367 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Galkin, A. et al. Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I. J. Biol. Chem. 283, 20907–20913 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gavrikova, E. V. & Vinogradov, A. D. Active/de-active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling. FEBS Lett. 455, 36–40 (1999).

    Article  CAS  PubMed  Google Scholar 

  50. Di Luca, A. & Kaila, V. R. I. Global collective motions in the mammalian and bacterial respiratory complex I. Biochim. Biophys. Acta Bioenerg. 1859, 326–332 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Banba, A., Tsuji, A., Kimura, H., Murai, M. & Miyoshi, H. Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I. J. Biol. Chem. 294, 6550–6561 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Matias, P. M. et al. [NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c3. J. Biol. Inorg. Chem. 6, 63–81 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION−3.1. IUCrJ 7, 253–267 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Vilas, J. L. et al. MonoRes: automatic and accurate estimation of local resolution for electron microscopy maps. Structure 26, 337–344 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Ramírez-Aportela, E. et al. Automatic local resolution-based sharpening of cryo-EM maps. Bioinformatics 36, 765–772 (2019).

    Google Scholar 

  59. de la Rosa-Trevín, J. M. et al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol. 195, 93–99 (2016).

    Article  PubMed  Google Scholar 

  60. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Abdrakhmanova, A. et al. Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica. Biochim. Biophys. Acta Bioenerg. 1658, 148–156 (2004).

    Article  CAS  Google Scholar 

  63. Angerer, H. et al. A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I. Biochem. J. 437, 279–288 (2011).

    Article  CAS  PubMed  Google Scholar 

  64. Fukasawa, Y. et al. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites. Mol. Cell. Proteom. 14, 1113–1126 (2015).

    Article  CAS  Google Scholar 

  65. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  67. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D Struct. Biol. 74, 519–530 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  69. Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Ploegman, J. H., Drent, G., Kalk, K. H. & Hol, W. G. J. Structure of bovine liver rhodanese. J. Mol. Biol. 123, 557–594 (1978).

    Article  CAS  PubMed  Google Scholar 

  71. Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D Struct. Biol. 74, 814–840 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Bridges, H. R., Mohammed, K., Harbour, M. E. & Hirst, J. Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I. Biochim. Biophys. Acta Bioenerg. 1858, 197–207 (2017).

    Article  CAS  PubMed  Google Scholar 

  74. Kumar, P. & Bansal, M. Identification of local variations within secondary structures of proteins. Acta Crystallogr. D Biol. Crystallogr. 71, 1077–1086 (2015).

    Article  CAS  PubMed  Google Scholar 

  75. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).

    Article  CAS  PubMed  Google Scholar 

  76. The PyMOL Molecular Graphics System v.2.2.3 (Schrödinger, 2019).

Download references

Acknowledgements

We thank K. Dent and the staff at the UK National Electron Bio-Imaging Centre (eBIC) at the Diamond Light Source, proposal EM-17057–27, funded by the Wellcome Trust, MRC and BBSRC, for assistance with cryo-EM data collection; Z. Yin (MBU Cambridge) and D. Chirgadze (University of Cambridge cryo-EM facility) for assistance with cryo-EM grid preparation and screening; S. Ding and I. Fearnley (MBU Cambridge) for mass spectrometry analyses; and M. Hartley and A. Raine (MBU Cambridge) for IT support. This work was supported by the Medical Research Council (MC_U105663141 and MC_UU_00015/2 to J.H.).

Author information

Authors and Affiliations

  1. The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Daniel N. Grba & Judy Hirst

Authors
  1. Daniel N. Grba
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Judy Hirst
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.N.G. carried out all experimental and cryo-EM work, processed the cryo-EM data and built the models. D.N.G. and J.H. analyzed and interpreted the models and wrote the manuscript. J.H. directed the project.

Corresponding author

Correspondence to Judy Hirst.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Peer reviewer reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Classification and refinement of the cryo-EM maps for Y. lipolytica complex I.

The workflow, implemented in Relion 3.153,54, was used to obtain the map of the whole complex at 2.7 Å resolution, as well as a map from focused refinement of the sub-stoichiometric ST1 subunit (observed in 51% of the refined particles).

Extended Data Fig. 2 Resolution estimates of the cryo-EM maps for Y. lipolytica complex I.

a, the global map of Y. lipolytica complex I; b, the map from focused refinement of the sub-stoichiometric ST1 subunit. The masks used are indicated by outlines and local resolutions were estimated using Relion. The resolution estimates from the masked Fourier shell correlation curves, FSCfinal, are 2.7 and 3.5 Å for the global and ST1 maps at FSC = 0.143 (dotted lines) and 3.2 and 4.3 Å at FSC = 0.5 (solid lines). Model–map FSC curves (FSCmodel) are in magenta. Overfitting analyses71,72 were performed by refining the model with the atoms displaced by 0.5 Å against one half map, followed by FSC map–model validation (FSCwork). The output was used with the second half map for FSC map–model validation (FSCtest). For the global model the curves match well beyond the 2.7 Å data used for refinement. The ST1 model, which was generated from a homology model, shows evidence of overfitting. Residue side chains were removed in low resolution regions to reduce the overfitting, while the overall secondary structures and architecture of the homology model were retained.

Extended Data Fig. 3 The local resolution of sequential focused refinements of the ST1 subunit, and the partial ST1 subunit model docked onto the complex.

a, The improvement in map quality at the position of the ST1 subunit after focused refinement. b, The partially built ST1 subunit model (pink) docked onto the full Y. lipolytica complex I model. The model was built into its focus-refined map (lower left inset, surface coloured by local resolution), docked onto the model for the whole complex, then adjusted locally. ST1–subunit interactions are indicated.

Extended Data Fig. 4 Sequence alignments of the π1 bulges defined in Supplementary Table 2.

The residues that comprise the detected π bulges are highlighted in blue. When the exact position of the π bulge varied between the two detection algorithms, the π bulge that best corresponded to the π bulges detected in the other organisms was used. For mouse, the π bulge detected in the ND6 subunit is from the deactive state. ClustalW symbols (*:.) are used to indicate perfect alignment, strong similarity, and weak similarity, respectively. The initial residue numbering is displayed for the Y. lipolytica sequences.

Extended Data Fig. 5 Comparison of the π bulges in ND4-TMH8 of complex I from Y. lipolytica and T. thermophilus (PDB 4HEA)26 and ND6-TMH3 of complex I from Y. lipolytica and M. musculus (PDB 6G2J and PDB 6G72)20.

a, The backbones of both ND4 subunits are shown in ribbon with equivalent residues in sticks. The T. thermophilus backbone deviates substantially from α-helical structure around the conserved Lys235 residue, shifting its orientation, with a π bulge observed only in the π2 position. b, The conserved proline on the loop between TMHs 7 and 8 is shifted along the sequence by one residue, resulting in a markedly different entry geometry to the π bulge helix. c, ND6 is in teal and ND3 is in green. Homologous residues are labelled. The orientations of backbone groups at the π bulge in the Y. lipolytica and deactive mouse models agree closely. The active mouse model no longer adopts the π bulge conformation; the helix is rotated and the conserved glycine residue (Gly61) in the π bulge hydrogen-bonding network is displaced. See also Supplementary Video 4.

Extended Data Fig. 6 Lipid interactions with the conserved π bulge of ND1 and the amphipathic helix of NDUFS8 in Y. lipolytica.

a, The ND1 subunit and the amphipathic helix of NDUFS8 are shown in orange and blue cartoon, respectively. Lipids (PE: phosphatidylethanolamine and CDL: cardiolipin) and relevant side chains are shown in black sticks, along with the conserved π1 bulge. The view in a is from the matrix and is the same as that in Fig. 5a. The in-membrane view in b) highlights the membrane-embedded amphipathic helix of NDUFS8 and shows how the sandwiched PE molecule helps to bridge the vertical and horizontal helices of ND1 and NDUFS8, respectively.

Extended Data Fig. 7 A comparison of residues in the ND1 subunit of our Y. lipolytica complex I with a DDM (n-dodecyl β-d-maltoside) molecule bound, the Y. lipolytica complex I with ubiquinone-9 bound (PDB 6RFR)19, and the active mouse model (PDB 6G2J)20 with nothing observed in the ubiquinone-binding site.

a, b and c show the hydrogen-bonding networks (dashed lines) present at the hydrophilic kink region of the ubiquinone-binding channel. The ND1-bound DDM molecule is displayed in orange sticks for Y. lipolytica and as an outline in the active mouse enzyme as a reference. The ubiquinone-9 molecule is shown similarly. Residues of ND1 and NDUFS7 are coloured orange and purple sticks, respectively. Water molecules are in red spheres. Equivalent residues where displayed are as follows: Q34(MmQ32), R36(MmR34), D99(MmD78), D101(MmD80), R27(MmR25), T23(MmT21), D53(MmD51), W77(MmW56), Q113(MmQ92), R108(MmR87), Y232(MmY228), E206(MmE202), E231(MmE227), E208(MmE204), D203(MmD199), R199(MmR195), R297(MmR274), R302(MmR279), E26 (MmE24).

Supplementary information

Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Peer Review Information

Supplementary Video 1

Hydration of complex I and the antiporter-like (ND) subunits of complex I.

Supplementary Video 2

Differences between ND1, ND3 and ND6 NDUFS2 of our DDM-bound Yarrowia lipolytica model and the active mouse model (PDB 6G2J).

Supplementary Video 3

Ubiquinone-binding and ND1 cavities of our DDM-bound Yarrowia lipolytica model, the ubiquinone-bound Yarrowia lipolytica model (PDB 6RFR), the active mouse model (PDB 6G2J) and the deactive mouse model (PDB 6G72).

Supplementary Video 4

Movement of ND6-π1 in the transition from our DDM-bound Yarrowia lipolytica model to that of the active mouse model (PDB 6G2J).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Grba, D.N., Hirst, J. Mitochondrial complex I structure reveals ordered water molecules for catalysis and proton translocation. Nat Struct Mol Biol 27, 892–900 (2020). https://doi.org/10.1038/s41594-020-0473-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-020-0473-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing