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

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.

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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).

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.

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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.).

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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.

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Correspondence to Judy Hirst.

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The authors declare no competing interests.

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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).

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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

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