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The coupling mechanism of mammalian mitochondrial complex I

Nature Structural & Molecular Biology volume 29, pages 172–182 (2022)Cite this article

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Abstract

Mammalian respiratory complex I (CI) is a 45-subunit, redox-driven proton pump that generates an electrochemical gradient across the mitochondrial inner membrane to power ATP synthesis in mitochondria. In the present study, we report cryo-electron microscopy structures of CI from Sus scrofa in six treatment conditions at a resolution of 2.4–3.5 Å, in which CI structures of each condition can be classified into two biochemical classes (active or deactive), with a notably higher proportion of active CI particles. These structures illuminate how hydrophobic ubiquinone-10 (Q10) with its long isoprenoid tail is bound and reduced in a narrow Q chamber comprising four different Q10-binding sites. Structural comparisons of active CI structures from our decylubiquinone–NADH and rotenone–NADH datasets reveal that Q10 reduction at site 1 is not coupled to proton pumping in the membrane arm, which might instead be coupled to Q10 oxidation at site 2. Our data overturn the widely accepted previous proposal about the coupling mechanism of CI.

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Fig. 1: Structures of two classes of CIs.
Fig. 2: Binding sites of Qs and rotenone in the Q chamber of active CI.
Fig. 3: Conformational changes of the β1–β2S2 loop at site 1 of active CI during Q reduction.
Fig. 4: Polar residues and water molecules related to proton translocation within the membrane core subunits and NDUFS2 of active CI.
Fig. 5: A Q-binding pocket in the NDUFA9 C-terminal domain of active CI.
Fig. 6: Proposed catalytic model for mammalian respiratory CI.

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Data availability

The atomic coordinates of the CI in six distinct states have been deposited in the worldwide Protein Data Bank (PDB) with the following accession codes nos.: 7V2C, 7VXP, 7VXS, 7VZV, 7VZW, 7V2D, 7VXU, 7VY1, 7W00 and 7W0H (Q10 dataset); 7V2E, 7VY8, 7VY9, 7W0R, 7W0Y, 7V2F, 7VYA, 7VYE, 7W1O and 7W1P (Q10–NADH dataset); 7V2H, 7VB7, 7VBL, 7W2R, 7W2U, 7W2Y, 7V2K, 7VBN, 7VBP, 7W31, 7W32 and 7W35 (DQ–NADH dataset); 7V2R, 7VYN, 7VYS, 7W4C, 7W4D, 7W4E, 7W4F, 7W4G, 7V30, 7VZ1, 7VZ8, 7W4J, 7W4K, 7W4L, 7W4M, 7W4N and 7W4Q (Q1–NADH dataset); 7V31, 7VYF, 7VYG, 7W1T, 7W1U, 7V32, 7VYH and 7VYI (rotenone dataset); and 7V33, 7VBZ, 7VC0, 7W1V, 7W1Z, 7W20, 7V3M, 7VWJ, 7VWL, 7W2K and 7W2L (rotenone–NADH dataset). The corresponding maps have been deposited in the Electron Microscopy Data Bank (EMDB) with the accession nos.: EMD-31640, EMD-32186, EMD-32187, EMD-32230, EMD-32231, EMD-31641, EMD-32188, EMD-32191, EMD-32232 and EMD-32242 (Q10 dataset); EMD-31643, EMD-32196, EMD-32197, EMD-32248, EMD-32249, EMD-31644, EMD-32198, EMD-32202, EMD-32253 and EMD-32254 (Q10–NADH dataset); EMD-31645, EMD-31874, EMD-31881, EMD-32265, EMD-32266, EMD-32267, EMD-31646, EMD-31883, EMD-31884, EMD-32269, EMD-32270 and EMD-32271 (DQ–NADH dataset);http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-31648EMD-31647, EMD-32210, EMD-32214, EMD-32300, EMD-32301, EMD-32302, EMD-32303, EMD-32304, EMD-31648, EMD-32218, EMD-32222, EMD-32305, EMD-32306, EMD-32307, EMD-32308, EMD-32309 and EMD-32312 (Q1–NADH dataset); EMD-31649, EMD-32203, EMD-32204, EMD-32255, EMD-32256, EMD-31650, EMD-32205 and EMD-32206 (rotenone dataset); and EMD-31651, EMD-31886, EMD-31887, EMD-32257, EMD-32259, EMD-32260, EMD-31652, EMD-32154, EMD-32155, EMD-32263 and EMD-32264 (rotenone–NADH dataset). Source data are provided with this paper.

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Acknowledgements

We thank the Cryo-EM Facility Center of Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for providing the facility support, and J. Lei, X. Li and F. Yang for technical support in EM data acquisition. The computation was completed on the Yanglab GPU workstation. This work was supported by funds from the National Key R&D Program of China (nos. 2017YFA0504600 and 2016YFA0501100 to M.Y.), the National Science Fund for Distinguished Young Scholars (no. 31625008 to M.Y.), the National Natural Science Foundation of China (nos. 32030056, 21532004 and 31570733 to M.Y., and 31800620 to J.G.) and the China Postdoctoral Science Foundation (nos. 2017M620040 and 2018T110091 to J.G.).

Author information

Author notes

  1. These authors contributed equally: Jinke Gu, Tianya Liu, Runyu Guo, Laixing Zhang.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Protein Science, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Tsinghua-Peking Center for Life Sciences, Beijing, China

    Jinke Gu, Tianya Liu, Runyu Guo, Laixing Zhang & Maojun Yang

  2. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shenzhen University Health Science Center, Shenzhen, China

    Jinke Gu

  3. SUSTech Cryo-EM Facility Center, Southern University of Science & Technology, Shenzhen, China

    Maojun Yang

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  1. Jinke Gu
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Contributions

M.Y. directed the study. J.G. did the protein purification and detergent screening, and performed EM sample preparation. J.G. and T.L. performed data collection and structural determination, and built the atomic models. J.G., T.L., R.G., L.Z. and M.Y. analyzed the data, drew the figures and wrote the manuscript. All authors contributed to the discussion of the data and editing the manuscript.

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Correspondence to Maojun Yang.

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Nature Structural and Molecular Biology thanks Georgios Skiniotis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Biochemical characterization and cryo-EM analyses of SCI1III2IV1.

a, A representative trace of size-exclusion chromatography of the respiratory chain SCI1III2IV1. b, Fractions of size-exclusion chromatography were analyzed by bule native PAGE and in-gel NBT staining. Each experiment was repeated three times independently with similar results. c, A representative cryo-EM micrograph from 5,490 micrographs of Q10 dataset. Scale bar: 100nm. d, Power-spectrum of the micrograph in panel c. The white circle indicates the 2.5-Å frequency. Source Data for b are available online.

Source data

Extended Data Fig. 2 Image processing work flow of a representative dataset.

In the calculation of the porcine CI of Q1-NADH dataset, 7,064 micrographs were collected, obtaining 1,150k particles in total. 871k particles were kept after 2D classification and were subject to a 3D classification of SCI1III2IV1. 657k particles were selected to perform CI-centered particle re-extraction. Through a no-alignment 3D classification, 302k and 209k intact CI particles were categorized as ‘Class 1’ (active state) and ‘Class 2’ (deactive state), while broken particles with a low population were excluded. Different reconstructions were performed, and detailed procedures were described in Methods.

Extended Data Fig. 3 Statistics of the final density maps.

a, Local resolution estimation of the active state CI, matrix arm and membrane arm maps from the Q1-NADH dataset. Viewed along the inner membrane. The color scale from 2.2-3.1 Å is shown in left. b, Particle orientation distributions in the last iteration of the active state CI structural refinement from the Q1-NADH dataset. Red cylinders mean more particles on these orientations. Heights of cylinders represent the relative numbers of particles. c, Gold-standard Fourier Shell Correlation (FSC=0.143) curve of the final density maps. The final resolution of active and deactive CI maps from Q10, Q10-NADH, DQ-NADH, Q1-NADH, Rotenone and Rotenone-NADH datasets are 2.9 Å, 3.3 Å, 2.8 Å, 3.1 Å, 2.5 Å, 2.7 Å, 2.6 Å, 2.7 Å, 2.9 Å, 3.2 Å, 2.5 Å and 2.8 Å, respectively. d, Cross-validation with maps used in panel c and corresponding models. The reported resolution (FSC=0.5) of active and deactive CI maps from Q10, Q10-NADH, DQ-NADH, Q1-NADH, Rotenone and Rotenone-NADH datasets are 3.0 Å, 3.4 Å, 2.9 Å, 3.1 Å, 2.6 Å, 2.8 Å, 2.6 Å, 2.8 Å, 3.0 Å, 3.2 Å, 2.6 Å and 2.9 Å, respectively.

Extended Data Fig. 4 Characteristics of Q-chamber, E-channel, and NADH-binding site.

a, The connection between Q-chamber and E-channel. The red dashed lines represent hydrogen bonds (H-bonds). b, Representative NADH-binding site. Key residues of NDUFV1 that interact with NADH are shown as sticks. Density for NADH is contoured at 3 σ. This figure is generated by CIDQN structure. c, The size of Q10 headgroup. The lengths of Q10 headgroup in two axes are indicated by dashed arrows and shown. d, The size of the Q-chamber bottleneck at the exit point. Q10 and residues of the bottleneck are shown as sticks. Distances between two atoms are indicated by black dashed lines and shown. AH1, amphipathic helix 1; TMH, transmembrane helix. e, Cryo-EM map of the CIR Q-chamber. NDUFS2, NDUFS7 and ND1, forming the Q-chamber, are colored and labeled with texts in the same color. N2 FeS cluster, rotenone and Q10 are shown in surface representation for clarity. Distance between Q10 at site 2 and N2 FeS Cluster is indicated and shown.

Extended Data Fig. 5 Structural features of the Q-binding site 3.

a, Cryo-EM map of the CIQD Q-chamber. NDUFS2, NDUFS7 and ND1, forming the Q-chamber, are colored and labeled with texts in the same color. N2 FeS cluster and Q10 are shown in surface representation for clarity. Distance between Q10 at site 3 and N2 FeS cluster is indicated and shown. AH1, amphipathic helix 1; TMH, transmembrane helix. b, Density for Q10 bound at site 3 of the CIQD Q-chamber (at 2 σ contour level). c, Q10 binding site 3 in the Q-chamber of CIQD. Residues participating in Q10 binding at site 3 are shown as sticks and labeled with texts. The H-bonds are shown as red dashed lines.

Extended Data Fig. 6 Conformational changes between the CIQ and CIQD.

a, Superposition of CIQ and CIQD structures aligned against the NDUFV1 subunit. Gray and blue schematic diagrams illustrate the angles between matrix arm and membrane arm of CIQ and CIQD. b, Superposition of CIQ and CIQD structures aligned against the ND5 subunit. Schematic diagram illustrates the angles between the two matrix arms of CIQ and CIQD structures viewed from ND5 to ND1 along the membrane. c, Conformational changes of NDUFS7 between CIQ and CIQD. Residues involved in Q10 binding at site 1 are shown as sticks and labeled with text. Loop 1 between helices 1 and 2 (LoopH1-H2, red text) turns into a β-strand in CIQD. And loop 2 between helix-2 and β1 (LoopH2-β1; red text) undergoes an obvious conformational change between CIQ and CIQD. H, helix. d, Superposition of NDUFS2 structures between CIQ and CIQD. Residues involved in Q10 binding at site 1 are shown as sticks and are labeled. The disordered β1-β2S2 loop in CIQD is indicated by a black arrow. e, Superposition of ND1 structures from CIQ and CIQD. The disordered loop between TMHs 5 and 6 (LoopTMHs5-6) in CIQD is indicated by a black arrow. TMH, transmembrane helix; AH1, amphipathic helix 1.

Extended Data Fig. 7 Conformational changes in the Q-chamber and E-channel during the transition between active state and deactive state.

a, The H-bond network around the Q-chamber in CIQ formed by loops of NDUFS2, NDUFS7, NDUFA9, ND1, ND3, and ND6. Residues involved in the H-bond network are shown as sticks and labeled by texts. The red dashed lines represent H-bonds. b, Conformational changes of the E-channel between CIQ and CIQD. These two structures are superposed by ND4L. Polar residues and transmembrane helices undergoing conformational changes are highlighted by red text. c-f, A close-up comparison of TMH3ND6, TMH3ND1, TMH4ND1, and TMHs1-2ND3 between CIQ and CIQD. Residues undergoing conformational changes are shown as sticks and labeled by texts.

Extended Data Fig. 8 Structural comparison of polar residues and water molecules between CIDQN and CIDQND.

a-b, Side-by-side comparison of the E-channels between CIDQN (a) and CIDQND (b). Polar residues and water molecules on the central hydrophilic axis of E-channel from DQ-NADH dataset are shown as sticks and spheres, respectively. Residues and transmembrane helices undergoing conformational changes are highlighted by red texts. The gray spheres in panel a represent water molecules missing in CIDQND (b). H-bonds are shown as black dashed lines. c, Comparison of the three antiporter-like subunits between CIDQN and CIDQND. Conserved polar residues and water molecules on the central hydrophilic axis of ND2, ND4 and ND5 from DQ-NADH dataset are shown as sticks and spheres, respectively. d, The H-bond network formed between ordered water molecules and polar residues within NDUFS2 of CIDQND. e, The H-bond network formed between ordered water molecules and polar residues at the interface of ND1 and NDUFS2 of CIDQND.

Extended Data Fig. 9 Structural comparison between porcine CI in SCI1III2IV1 and ovine CI in free form.

a, Superposition of CIQ and CIClosed structures aligned against the ND5 subunit. Left panel: gray and green schematic diagrams illustrate the angles between matrix arm and membrane arm of CIQ and CIClosed, respectively. Right panel: schematic diagram illustrates the angles between the two matrix arms of CIQ and CIClosed structures viewed from ND1 to ND5 along the membrane. CIClosed, ovine closed state CI structure in free form from native dataset (PDB: 6ZKO). b, Superposition of CIQD and CIOpen structures aligned against the ND5 subunit. Left panel: blue and wheat schematic diagrams illustrate the angles between matrix arm and membrane arm of CIQD and CIOpen, respectively. Right panel: schematic diagram illustrates the angles between the two matrix arms of CIQD and CIOpen structures viewed from ND1 to ND5 along the membrane. CIOpen, ovine open state CI structure in free form from native dataset (PDB: 6ZKP).

Supplementary information

Supplementary Information

Supplementary text.

Reporting Summary

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Supplementary Table 1

Statistics for CI density maps of six distinct datasets.

Supplementary Table 2

NADH- and Q-binding sites of CI from different datasets.

Supplementary Table 3

All the subunits of porcine active state CI.

Supplementary Table 4

All the subunits of porcine deactive state CI.

Source data

Source Data Extended Data Fig. 1

Unprocessed NBT staining blue native PAGE gel.

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Gu, J., Liu, T., Guo, R. et al. The coupling mechanism of mammalian mitochondrial complex I. Nat Struct Mol Biol 29, 172–182 (2022). https://doi.org/10.1038/s41594-022-00722-w

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