Crystal structure of the entire respiratory complex I

Journal name:
Nature
Volume:
494,
Pages:
443–448
Date published:
DOI:
doi:10.1038/nature11871
Received
Accepted
Published online

Abstract

Complex I is the first and largest enzyme of the respiratory chain and has a central role in cellular energy production through the coupling of NADH:ubiquinone electron transfer to proton translocation. It is also implicated in many common human neurodegenerative diseases. Here, we report the first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3Å resolution. The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron–sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron–sulphur cluster N2. Notably, the chamber is linked to the fourth channel by a ‘funnel’ of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle.

At a glance

Figures

  1. Structure of the entire complex I from T. thermophilus.
    Figure 1: Structure of the entire complex I from T. thermophilus.

    a, An overview. FMN and Fe–S clusters are shown as magenta and red–orange spheres, respectively, with cluster N2 labelled. Key helices around the entry point (Q) into the quinone-reaction chamber, and approximate membrane position, are indicated. b, Putative proton-translocation channels in the antiporter-like subunits. Polar residues lining the channels are shown as sticks with carbon in dark blue for the first (N-terminal) half-channel, in green for the second (C-terminal) half-channel and in orange for connecting residues. Key residues—GluTM5 and LysTM7 from the first half-channel, Lys/HisTM8 from the connection and Lys/GluTM12 from the second half-channel—are labelled. Approximate proton-translocation paths are indicated by blue arrows.

  2. Fold of subunit Nqo8.
    Figure 2: Fold of subunit Nqo8.

    Coloured blue to red from N to C terminus. Neighbouring subunits Nqo7 and Nqo10 are shown in light and dark grey, respectively. a, View from the cytoplasm. TM helices are numbered, with helices corresponding to the antiporter half-channel in bold. The conserved salt bridge Arg36–Asp62, supporting amphipathic helix AH1, is shown. b, Side view. Charged residues from the conserved third cytoplasmic loop, mainly lining the Q cavity, are shown as sticks. c, Alignment of TM helices 2–6 of Nqo8 (orange) with TM helices 4–8 of Nqo13 (blue). LysTM7 from Nqo13 and Glu213 from Nqo8 TM5 are shown as sticks.

  3. E-channel and central hydrophilic axis.
    Figure 3: E-channel and central hydrophilic axis.

    a, E-channel (fourth proton-translocation channel). Charged and polar residues constituting the channel are shown as sticks. Central residues are shown with carbon in yellow, those forming a link to the Q site in magenta, those that link to the cytoplasm in blue, those that link to the periplasm in green and those interacting with the quinone headgroup in cyan. Key residues are labelled, with the Glu/Asp quartet in red. Number prefix indicates subunit (omitted for Nqo8). Approximate proton-translocation path is indicated by blue arrow. Quinone cavity is shown with surface in brown. b, Central axis of charged and polar residues. Residues shown are either central to half-channels or are forming the connection between them (charged residues have carbon in magenta, polar in cyan). Most of them are located near the breaks in key helices TM7, TM8 and TM12 (antiporters), 10(TM3) and 8(TM5). Predicted waters nearby, modelled using Dowser software44, are shown as spheres. Connecting elements are shown in solid colours: helix HL in magenta and the βH element in blue, with the C-terminal helix CH29 and the β-hairpin (β-h) from each antiporter labelled. The contacting Nqo10 helix is labelled 10(H). Subunits are coloured as in Fig. 1.

  4. Quinone-reaction chamber.
    Figure 4: Quinone-reaction chamber.

    Subunits are coloured as in Fig. 1. Fe–S cluster N2 is shown as red–orange spheres. a, b, Experimental electron density (2mFo–DFc in blue, contoured at 1σ, and mFo–DFc in green, contoured at 3σ) and models obtained from crystals with bound piericidin A (a) and decylubiquinone (b). Difference electron density was calculated before ligand modelling. Nqo4 residues interacting with the headgroup are indicated. Potential polar interactions are shown labelled with distances in ångström. c, Surface (solvent-accessible) representation of the interface between two main domains. The empty crevice (C, circled; Supplementary Discussion) between Nqo10 and 7(TM1)/Nqo8, as well as helices framing the entry point to the quinone site (Q) are indicated. d, Quinone-reaction chamber, with its internal solvent-accessible surface coloured red for negative, white for neutral and blue for positive surface charges. Charged residues lining the cavity are shown with carbon in magenta and hydrophobic residues in yellow. Residues are labelled with prefix indicating subunit (omitted for Nqo8). Ala63, the site of the primary Leber’s hereditary optic neuropathy disease mutation45, is labelled in red. e, Theoretical model of bound ubiquinone-10. Carbon atom in cyan indicates the eighth isoprenoid unit. Nqo4 residues interacting with the headgroup are indicated. The quinone chamber is shown with surface in brown and helices framing its entry point are indicated. Movable helix 6(H1)27, interacting with 8(AH1), is also labelled.

  5. Proposed coupling mechanism of complex I.
    Figure 5: Proposed coupling mechanism of complex I.

    a, Overview showing key helices and residues. Upon electron transfer from cluster N2, negatively charged quinone initiates a cascade of conformational changes, propagating from the E-channel (Nqo8, Nqo10, Nqo11) to the antiporters through the central axis (red arrows) of charged and polar residues located around flexible breaks in key TM helices. Cluster-N2-driven shifts of Nqo4 and Nqo6 helices27 (blue arrows) probably assist overall conformational changes. Helix HL and the βH element help to coordinate conformational changes by linking discontinuous TM helices between the antiporters. In the antiporters, LysTM7 from the first half-channel is assumed to be protonated (through the link to cytoplasm) in the oxidized state29. Upon reduction of quinone and subsequent conformational change, the first half-channel closes to the cytoplasm, GluTM5 moves out and LysTM7 donates its proton to the connecting Lys/HisTM8 and then onto Lys/GluTM12 from the second half-channel. Lys/GluTM12 ejects its proton into the periplasm upon return from reduced to oxidized state. A fourth proton per cycle is translocated in the E-channel in a similar manner. TM helices are numbered and key charged residues (GluTM5, LysTM7, Lys/GluTM12, Lys/HisTM8 from Nqo12–14, 11(Glu67), 11(Glu32), 8(Glu213) and some residues from the connection to Q cavity) are indicated by red circles for Glu and blue circles for Lys/His. 10(Tyr59), interacting with 11(Glu32), is indicated by the empty circle. E. coli and human subunit names are also indicated. b, Schematic drawing illustrating conformational changes between the two main (low-energy) conformations. Analysis of networks of polar residues and modelled waters in the structure suggests that in the oxidized state (as crystallized) periplasmic half-channels are likely to be open. Residues shown as black circles indicate conserved prolines from the break in TM12.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Walker, J. E. The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains. Q. Rev. Biophys. 25, 253324 (1992)
  2. Yagi, T. & Matsuno-Yagi, A. The proton-translocating NADH-quinone oxidoreductase in the respiratory chain: the secret unlocked. Biochemistry 42, 22662274 (2003)
  3. Brandt, U. Energy converting NADH:quinone oxidoreductase (complex I). Annu. Rev. Biochem. 75, 6992 (2006)
  4. Ohnishi, T. Iron–sulfur clusters/semiquinones in complex I. Biochim. Biophys. Acta 1364, 186206 (1998)
  5. Sazanov, L. A. Respiratory complex I: mechanistic and structural insights provided by the crystal structure of the hydrophilic domain. Biochemistry 46, 22752288 (2007)
  6. Hirst, J. Towards the molecular mechanism of respiratory complex I. Biochem. J. 425, 327339 (2010)
  7. Sazanov, L. A. A Structural Perspective on Respiratory Complex I: Structure and Function of NADH:Ubiquinone Oxidoreductase (Springer, 2012)
  8. Galkin, A. S., Grivennikova, V. G. & Vinogradov, A. D. H. H+/2ē stoichiometry in NADH–quinone reductase reactions catalyzed by bovine heart submitochondrial particles. FEBS Lett. 451, 157161 (1999)
  9. Galkin, A., Drose, S. & Brandt, U. The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes. Biochim. Biophys. Acta 1757, 15751581 (2006)
  10. Moser, C. C., Farid, T. A., Chobot, S. E. & Dutton, P. L. Electron tunneling chains of mitochondria. Biochim. Biophys. Acta 1757, 10961109 (2006)
  11. Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. & Walker, J. E. Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc. Natl Acad. Sci. USA 107, 1682316827 (2010)
  12. Vinogradov, A. D. Catalytic properties of the mitochondrial NADH–ubiquinone oxidoreductase (complex I) and the pseudo-reversible active/inactive enzyme transition. Biochim. Biophys. Acta 1364, 169185 (1998)
  13. Schapira, A. H. Human complex I defects in neurodegenerative diseases. Biochim. Biophys. Acta 1364, 261270 (1998)
  14. Murphy, M. P. How mitochondria produce reactive oxygen species. Biochem. J. 417, 113 (2009)
  15. Dawson, T. M. & Dawson, V. L. Molecular pathways of neurodegeneration in Parkinson’s disease. Science 302, 819822 (2003)
  16. Balaban, R. S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483495 (2005)
  17. Carroll, J. et al. Bovine complex I is a complex of 45 different subunits. J. Biol. Chem. 281, 3272432727 (2006)
  18. Balsa, E. et al. NDUFA4 is a subunit of complex IV of the mammalian electron transport chain. Cell Metab. 16, 378386 (2012)
  19. Yip, C. Y., Harbour, M. E., Jayawardena, K., Fearnley, I. M. & Sazanov, L. A. Evolution of respiratory complex I: “supernumerary” subunits are present in the α-proteobacterial enzyme. J. Biol. Chem. 286, 50235033 (2011)
  20. Efremov, R. G. & Sazanov, L. A. The coupling mechanism of respiratory complex I – a structural and evolutionary perspective. Biochim. Biophys. Acta 1817, 17851795 (2012)
  21. Efremov, R. G., Baradaran, R. & Sazanov, L. A. The architecture of respiratory complex I. Nature 465, 441445 (2010)
  22. Efremov, R. G. & Sazanov, L. A. Respiratory complex I: ‘steam engine’ of the cell? Curr. Opin. Struct. Biol. 21, 532540 (2011)
  23. 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, 279288 (2011)
  24. Hirst, J., Carroll, J., Fearnley, I. M., Shannon, R. J. & Walker, J. E. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604, 135150 (2003)
  25. Althoff, T., Mills, D. J., Popot, J. L. & Kuhlbrandt, W. Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J. 30, 46524664 (2011)
  26. Sazanov, L. A. & Hinchliffe, P. Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 311, 14301436 (2006)
  27. Berrisford, J. M. & Sazanov, L. A. Structural basis for the mechanism of respiratory complex I. J. Biol. Chem. 284, 2977329783 (2009)
  28. Hunte, C., Zickermann, V. & Brandt, U. Functional modules and structural basis of conformational coupling in mitochondrial complex I. Science 329, 448451 (2010)
  29. Efremov, R. G. & Sazanov, L. A. Structure of the membrane domain of respiratory complex I. Nature 476, 414420 (2011)
  30. Fearnley, I. M. & Walker, J. E. Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim. Biophys. Acta 1140, 105134 (1992)
  31. Mathiesen, C. & Hagerhall, C. Transmembrane topology of the NuoL, M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters. Biochim. Biophys. Acta 1556, 121132 (2002)
  32. Sekiguchi, K., Murai, M. & Miyoshi, H. Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I. Biochim. Biophys. Acta 1787, 11061111 (2009)
  33. Nouws, J., Nijtmans, L. G., Smeitink, J. A. & Vogel, R. O. Assembly factors as a new class of disease genes for mitochondrial complex I deficiency: cause, pathology and treatment options. Brain 135, 1222 (2012)
  34. Angerer, H. et al. Tracing the tail of ubiquinone in mitochondrial complex I. Biochim. Biophys. Acta 1817, 17761784 (2012)
  35. Wikström, M. & Hummer, G. Stoichiometry of proton translocation by respiratory complex I and its mechanistic implications. Proc. Natl Acad. Sci. USA 109, 44314436 (2012)
  36. Verkhovsky, M., Bloch, D. A. & Verkhovskaya, M. Tightly-bound ubiquinone in the Escherichia coli respiratory Complex I. Biochim. Biophys. Acta 1817, 15501556 (2012)
  37. Page, C. C., Moser, C. C., Chen, X. & Dutton, P. L. Natural engineering principles of electron tunnelling in biological oxidation–reduction. Nature 402, 4752 (1999)
  38. Kashani-Poor, N., Zwicker, K., Kerscher, S. & Brandt, U. A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I. J. Biol. Chem. 276, 2408224087 (2001)
  39. Ohnishi, T., Nakamaru-Ogiso, E. & Ohnishi, S. T. A new hypothesis on the simultaneous direct and indirect proton pump mechanisms in NADH-quinone oxidoreductase (complex I). FEBS Lett. 584, 41314137 (2010)
  40. Berrisford, J. M., Thompson, C. J. & Sazanov, L. A. Chemical and NADH-induced, ROS-dependent, cross-linking between subunits of complex I from Escherichia coli and Thermus thermophilus. Biochemistry 47, 1026210270 (2008)
  41. Bai, F. et al. Conformational spread as a mechanism for cooperativity in the bacterial flagellar switch. Science 327, 685689 (2010)
  42. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 19481954 (2002)
  43. Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 27282733 (2007)
  44. Zhang, L. & Hermans, J. Hydrophilicity of cavities in proteins. Proteins 24, 433438 (1996)
  45. Huoponen, K., Vilkki, J., Aula, P., Nikoskelainen, E. K. & Savontaus, M. L. A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy. Am. J. Hum. Genet. 48, 11471153 (1991)
  46. Kabsch, W. XDS. Acta Crystallogr. D 66, 125132 (2010)
  47. Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271281 (2011)
  48. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760763 (1994)
  49. Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282292 (2011)
  50. Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 80608065 (2006)
  51. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007)
  52. Krivov, G. G., Shapovalov, M. V. & Dunbrack, R. L., Jr Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77, 778795 (2009)
  53. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 21262132 (2004)
  54. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355367 (2011)
  55. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 1221 (2010)
  56. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 46734680 (1994)
  57. Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529W533 (2010)

Download references

Author information

Affiliations

  1. Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK

    • Rozbeh Baradaran,
    • John M. Berrisford,
    • Gurdeep S. Minhas &
    • Leonid A. Sazanov
  2. Present address: European Bioinformatics Institute, Cambridge CB10 1SD, UK.

    • John M. Berrisford

Contributions

R.B. purified and crystallized the intact complex; J.M.B. purified and crystallized the membrane domain; G.S.M. performed co-crystallization and soaks with quinone analogues; all authors collected and analysed X-ray data; L.A.S. designed and supervised the project, analysed data and wrote the manuscript, with contributions from all authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4HE8 (membrane domain) and 4HEA (entire complex).

Author details

Supplementary information

PDF files

  1. Supplementary Information (8.2M)

    This file contains a Supplementary Discussion, Supplementary Tables 1-9, Supplementary Figures 1-7 and Supplementary References.

Additional data