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A universal coupling mechanism of respiratory complex I

An Author Correction to this article was published on 31 October 2022

This article has been updated

Abstract

Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria1. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane2, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues3, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a ‘domino effect’ series of proton transfers and electrostatic interactions: the forward wave (‘dominoes stacking’) primes the pump, and the reverse wave (‘dominoes falling’) results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes.

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Fig. 1: The overall structure of E. coli complex I in different states.
Fig. 2: Conformational changes are induced on EcCI closing.
Fig. 3: Proton translocation pathways.
Fig. 4: The domino effect coupling mechanism of complex I.

Data availability

Cryo-EM maps and corresponding atomic models have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB), respectively, with the following accession numbers (PDB, EMDB): EcCI DDM datasets: apo resting (7P62, EMD-13215) and NADH resting (7P61, EMD-13214); EcCI DDM/LMNG datasets: apo open (7P7C, EMD-13235), apo resting (7P7E, EMD-13236), DQ open (7P7J, EMD-13237), DQ resting (7P7K, EMD-13238), NADH + FMN open (7P7L, EMD-13239), PieA + NADH open (7P7M, EMD-13240), turnover pH 6 closed (7P63, EMD-13216), turnover pH 6 open (7P64, EMD-13217), turnover pH 6 resting (7P69, EMD-13222), turnover pH 8 closed (7Z80, EMD-14540), turnover pH 8 open (7Z83, EMD-14541), turnover pH 8 open-ready (7Z84, EMD-14542) and turnover pH 8 resting (7ZC5, EMD-14620); EcCI LMNG datasets: apo open-ready (7Z7R, EMD-14535), turnover pH 6 open (7Z7T, EMD-14537), turnover pH 6 open-ready (7Z7V, EMD-14538), turnover pH 6 closed (7Z7S, EMD-14536) and turnover pH 6 resting (7ZCI, EMD-14632); OaCI datasets: pH 5.5 open (7ZDJ, EMD-14651), pH 5.5 closed (7ZDM, EMD-14658), pH 7.4 open (7ZD6, EMD-14637), pH 7.4 closed (7ZDH, EMD-14648), pH 9 open (7ZDP, EMD-14664) and pH 9 closed (7ZEB, EMD-14688).

Change history

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Acknowledgements

This research was supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Electron Microscopy Facility (EMF), the Life Science Facility (LSF) and the IST high-performance computing cluster. We thank V.-V. Hodirnau from IST Austria EMF, M. Babiak from CEITEC for assistance with collecting cryo-EM data and A. Charnagalov for the assistance with protein purification. V.K. was a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Institute of Science and Technology, Austria. V.K. and O.P. are funded by the ERC Advanced Grant 101020697 RESPICHAIN to L.S. This work was also supported by the Medical Research Council (UK).

Author information

Authors and Affiliations

Authors

Contributions

V.K. performed biochemical procedures with EcCI, prepared cryo-EM grids, acquired and processed cryo-EM data, built and analysed atomic models and analysed data. D.K. prepared cryo-EM grids of EcCI, acquired DDM datasets and analysed data. O.P. purified OaCI, prepared cryo-EM grids with OaCI, acquired and processed cryo-EM data, built and analysed atomic models and analysed data. A.W.-B. and Z.B. performed mutagenesis studies. L.S. designed and supervised the project, acquired funding, analysed data and models and wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to Leonid Sazanov.

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The authors declare no competing interests.

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Nature thanks Georgios Skiniotis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM density examples.

A) Composite LMNG_Turnover dataset open-ready state map combined from focus-refined PA and MA maps filtered to local resolution. B) Density examples of various regions of EcCI, including helices and β-strands from PA and MA. C) Densities for the key NuoCD loop. D) Density for ligands, including waters. E) Density for the NuoJ TM3 helix in open and closed states of LMNG_Turnover dataset. F) Density for the key NuoA loop in open and closed states. G) Density for the key NuoH loop in resting (“up” conformation), Open-ready and closed (“down” conformation) states. H) Density of the Ca2+ binding site. I–K) Densities in the Fe-S clusters’ environment.

Extended Data Fig. 2 Features of the Peripheral Arm structure.

A) Novel elements stabilizing the PA. The NuoG insertion loop (magenta) interacts with NuoG and the rest of the complex, increasing the interaction surface area. Ca2+ binds at the interface of the insertion loop and core NuoG structure. Loop-helix-loop connecting element of NuoCD subunit (CDLHL, yellow) is located on the surface and interacts with the NuoG insertion loop. B) Unique C-terminal extensions in EcCI increase surface interacting area and stabilize the minimal CI version. C) Position of Fe-S clusters along the peripheral arm. Electrons are transferred as a hydride from NADH to FMN then one by one via seven Fe-S clusters to quinone. The edge-to-edge distance (Å) between the clusters is indicated. N1a and N7 are off-path clusters. The density of the water molecules within 10 Å from Fe-S clusters is shown in blue, and all experimentally identified waters are shown as red spheres. D) Multiple salt bridges stabilize the Q cavity and PA-MA interface in open and closed states. NuoI helix H1 is surrounded on all sides by tightly bound lipid molecules, which strengthen its binding to the rest of the complex. This interaction is likely essential for the overall stability of the Q cavity. E, F) The conformation of the backbone between FD92 and FP96 is unchanged in EcCI regardless of NADH presence. Red arrows point to the backbone oxygen atom, which was suggested to change conformation (peptide bond flip) in Aquifex aeolicus studies52. G) Comparison of NuoF subunits from different CI species. EcCI contains unique FR320, which points into the active site and interacts with NADH. H) At the PA-MA interface, in the closed state the Q cavity is sealed by the NuoA loop (in orange), stabilised by the indicated conserved salt bridges.

Extended Data Fig. 3 A–D) Cryo-EM density of the FMN binding site at different conditions in the presence of NADH.

NuoFE subunits are highlighted with a dashed circle, FMN and NADH are indicated by the orange and yellow arrows, respectively. The concentration of CI applied to EM grids was ~10 mg/ml in condition A and ~0.2 mg/ml in conditions B–D. A) When the concentration of the holoenzyme is high (above the Kd for FMN dissociation53) NuoFE, FMN and NADH all have clear densities. B) The same is true when the protein concentration is low but a constant electron flow from FMN to DQ occurs during turnover. C) When the protein concentration is low and the complex is reduced without electron acceptor present, NuoFE subunits get disordered and most of FMN completely dissociates from the active site. D) However, FMN remains bound when external excess of FMN is added, even though NuoEF subunits still get disordered. Inserts show zoom-in into FMN/NADH (A, B) and FMN (C, D) density. Taken together, this data provides additional confirmation of the true turnover condition in B. E) Nomenclature of core subunits of complex I in some reference species. 1The traditional nomenclature for Fe-S clusters (Nx, derived from initially described electron paramagnetic resonance (EPR) signatures54, as well as the nomenclature proposed55 on the basis of re-assignment of EPR signals to structurally observed clusters, is shown. In the new nomenclature, clusters are named according to their nuclearity (2Fe or 4Fe), their subunit location (using bovine nomenclature) and when necessary, as ligated by four Cys (C) or three Cys and one His (H). 2Cluster N7 is present only in some bacteria (for example, E. coli and T. thermophilus). 3Subunits NuoC and NuoD are fused in E. coli and some other bacteria. 4Number of transmembrane helices.

Extended Data Fig. 4

A) Lipids’ binding sites. Modelled molecules of phosphatidylethanolamine (PE, cyan) and eicosane (LFA, green). PDB ligand LFA was used when lipid head group was not resolved. Most lipids bind in hydrophobic crevices between subunits. The approximate boundaries of the lipid bilayer are indicated by the blue lines. B) Membrane-exposed hydrophobic belt. Top: the surface electrostatic potential of EcCI. Middle: EcCI coloured by the hydrophobicity of residues (white – hydrophobic, red - polar). Bottom: 10 Å low-pass filtered densities of EcCI. Lipid-detergent belt density is shown in grey, DDM/LMNG_Turnover_pH6 Open in light cyan and DDM Resting in dark cyan. All datasets in DDM/LMNG (except Apo) or LMNG with external lipids contain CI in an expanded lipid-detergent belt, while the datasets in DDM only contain CI in a shrunk lipid-detergent belt. C) Global conformational changes upon open (grey) to closed (coloured) state transition viewed from the cytosol. This process is assisted by the rotation of JTM3 and HTM4 helices as well as the tilt of the entire NuoH subunit. D) Key NuoH TM5-6 loop in different conformations. Structures in different states (indicated by NuoH colour as labelled) from LMNG_Turnover_pH6 dataset are aligned by subunit NuoH. Conserved E220, which forms different interactions in different states, is shown as sticks. Also shown are F212 and Y225, which indicate the borders of the variable region of the loop. NuoCD subunit is in grey. E, F) NADH:DQ oxidoreduction activity assays. Results are represented in μmol NADH min−1 mg−1 protein, as the mean ± SEM with values from three individual measurements shown as circles. E) EcCI. When present, lipids were added as 0.25 mg/ml ETL, and piericidin A (pA) inhibitor was added to 30 μmol. F) OaCI, Assays were performed in the presence of 0.25 mg/mL DOPC:cardiolipin (4:1) lipid mixture. Further details provided in Methods. G) The architecture of ALS subunits. The N- and C-terminal 5TM repeats with inverted symmetry are coloured. TM helices are numbered, with key residues indicated by circles in blue for lysines and in red for glutamates. Beta-hairpin (β−h) and C-terminal amphipathic helix (CH), forming contacts between subunits, are also indicated.

Extended Data Fig. 5 Quinone-binding site comparisons.

A) Comparison of key NuoCD loop in different CIs. Ovine CI (OaCI) is from PDBs 6ZKC for closed and 6ZKE for open state. TeNDH (Thermosynechococcus elongatus NDH complex) is from PDB 6NBY. Key conserved histidines are shown as sticks. B) Q binding sites. Left: in the resting state Q binds at the entrance to the Q cavity, consistent with the mammalian Qs binding site. Middle: in the open state Q binds in the Qm site, in between Qd and Qs sites (Qd and Qs quinones from the aligned structures are shown for comparison). Right: in the closed state DQ binds deep inside of the cavity, consistent with the mammalian Qd binding site, and also in the Qs site. Key residues interacting with quinone headgroup in each site are indicated. Quinone molecules from the aligned OaCI structures are shown as grey sticks. Qd site is narrow with a tight Q coordination, while Qs is looser, with some variability in the mode of binding. C) Extended NuoCD loop and side-chains of NuoB Helix3 block access to the Qd binding site in EcCI open state (left) and in TeNDH (PDB ID 6KHJ) (right), with plastoquinone (PQ) bound in the same site as Qm in E. coli. D) Q hydrophobic tail seals the Q entrance. Top: OaCI, DQ bound in the Qs site (PDB 6ZKE) is depicted as magenta spheres and protein atoms within 8 Å as transparent gray spheres. The arrangement is similar in the Qs-containing EcCI closed state structures. Bottom: EcCI, model of UQ8 fitted into Qd site (to illustrate the fit of native quinone) of the closed state structure is depicted as magenta spheres and protein atoms within 8 Å as transparent grey spheres, except for HM64 and HM67 (yellow), framing the entry. E) Q cavity in mammalian open state complex I (PDB 6ZKE) is exposed to the matrix via W site, consistent with EcCI. F) In the open-ready state of EcCI, although NuoA loop is partly ordered, the Q cavity is still exposed to the matrix via W site.

Extended Data Fig. 6 Density and the environment of bound native quinone, externally added DQ and inhibitor piericidin A.

The dataset and state of the enzyme are indicated. Cryo-EM density is carved within 2 Å of the model of the ligand. In the open state (A, C, D, E and G) the Qm site is occupied either by the native quinone (C and E), or DQ (D) or piericidin A (G). The headgroup interacts mainly with CQ328 and BV85-L86 residues in this position. In the DDM resting state (F) native quinone binds in the Qs site, stacking against HF238 and BW55. In the DDM/LMNG_Turnover_pH6 closed state (B) DQ binds in the Qd site, interacting with the key CDY277 and CDH228. In the DDM/LMNG_Turnover_pH8 (I) and LMNG_Turnover_pH6 (H) closed states the density for both Qd- and Qs-bound DQ is visible.

Extended Data Fig. 7 Waters and proton translocation pathways.

A) Cryo-EM densities for the experimental waters in the MA-focus-refined maps of EcCI LMNG_Turnover_pH6 open-ready (top) and closed (bottom) states. To allow clear visualization, the density is carved around modelled waters (red spheres) and is shown in light blue. The model is coloured by subunit as in Fig. 1a. Key residues from the central hydrophilic axis of EcCI are shown as sticks. B) A putative proton transfer pathway between the E-channel and the key CH228/CD329 residues, likely proton donors for quinone. Key protonatable residues, experimentally resolved waters and quinones from LMNG_Turnover_pH6 closed state are shown. Potential H-bonds are indicated by black dashes. C) Detailed analysis of cryo-EM density reveals charge of Glu and Asp residues in MA. Carboxyl side-chain densities of some key residues are absent (circled) in the closed state, suggesting their negative charge. In contrast, the same residues in the open state preserve densities suggesting their neutral charge. D) Comparison of NuoL TM8 helices from different CI species. Structures were aligned on EcCI NuoL subunit. Key LH254 residue and LS150 with which it can interact are shown as sticks. Due to flexibility of TMH8 key histidine can be preferentially linked either to key TM12 residue as in EcCI, TtCI and YlCI, or to key TM7 residue and the rest of the central axis as in OaCI, TeNDH (PDB 6KHJ) and AiCI (Arabidopsis italiana mitochondrial CI, PDB 7AR8). E) Comparison of NuoM TM8 helices from different CI species. In EcCI MTM8 is flexible and adopts different conformations. It is “linked” (green) in DDM/LMNG datasets PieA, Apo, Turnover in open states, and in resting states in Apo, Turnover and DDM_NADH. In DDM/LMNG datasets NADH+FMN and DQ in the open states, and resting states in DQ and DDM_Apo it is “flipped” (grey). However, both of these conformations are consistent with other CI structures as shown.

Extended Data Fig. 8 Characterization of mutations in NuoM and NuoN subunits of E. coli complex I.

A) Growth curves of the cultures grown aerobically at 37 °C in M9+malate minimal media56. In these conditions in mutants with impaired complex I activity the lag phase before entering the exponential growth phase is extended in comparison to the wild type E. coli. The extent of the lag is roughly proportional to the degree of complex I impairment. B) Activities of the inverted membrane vesicles of WT and mutant E. coli membranes. The activities were measured with deamino-NADH (dNADH), which is used exclusively by complex I in E. coli. dNADH:FeCy activity involves only the peripheral FMN site and so it reflects the assembly and the overall content of complex I in the membranes, which is similar to WT in all mutants except for subunit deletion strains. WT activity was 1.33 μmol dNADH min−1 mg−1 total membrane protein. dNADH:O2 activity reflects the activity of the entire respiratory chain, with complex I using native E. coli quinone. WT activity was 0.62 μmol dNADH min−1 mg−1 total membrane protein. dNADH:DQ reflects activity of complex I using decyl-ubiquinone, with complex IV inhibited. WT activity was 0.77 μmol dNADH min−1 mg−1 total membrane protein. Results are represented in % of WT activity, as the mean ± SEM, with values from individual measurements shown as filled circles. C) H+ translocation activities of E. coli inverted membrane vesicles, measured by the quenching of ACMA fluorescence at room temperature with an excitation wavelength of 434 nm and an emission wavelength of 477 nm. The buffer contained 2 µM ACMA, 50 mM Bis-Tris at pH 6.0, 2 mM CaCl2, 10 mM MgCl2, 10 μM valinomycin and 50 mM KCl. The addition of 0.1 mM dNADH or 2 μM of uncoupler FCCP (which dissipates ΔpH) is indicated.

Extended Data Fig. 9 Characterization of mutations in NuoH subunit of E. coli complex I.

A) Growth curves of the cultures grown aerobically at 37 °C in M9+malate minimal media56. In these conditions in mutants with impaired complex I activity the lag phase before entering the exponential growth phase is extended in comparison to the wild type E. coli. The extent of the lag is roughly proportional to the degree of complex I impairment. B) Activities of the inverted membrane vesicles of WT and mutant E. coli membranes, measured with deamino-NADH (dNADH) as described in Extended Data Fig. 8b. C) H+ translocation activities of E. coli inverted membrane vesicles, measured by the quenching of ACMA fluorescence at room temperature with an excitation wavelength of 410 nm and an emission wavelength of 480 nm. The buffer contained 1.6 μM ACMA, 20 mM Bis-Tris at pH 6.0, 2 mM CaCl2, 20 mM KCN, 200 μM DQ, 1 μM valinomycin and 120 mM KCl. The addition of 0.1 mM dNADH or 1 μM of uncoupler CCCP (which dissipates ΔpH) is indicated. D) Inhibition of the NADH:DQ activity of the purified EcCI by rotenone. Mutant HM67A was purified similarly to WT26. Non-inhibited activity of the purified mutant enzyme is somewhat higher than in membrane vesicles (B), indicating that DQ access is facilitated in detergent.

Extended Data Table 1 Cryo-EM datasets

Supplementary information

Supplementary Information

Supplementary discussion, Figs. 1–13, Tables 1–8, and references.

Reporting Summary

Supplementary Video 1

Density overview of cryo-EM maps from the LMNG_Turnover_pH6 dataset.

Supplementary Video 2

Global conformational changes occurring upon EcCI closing. Pale surface shows the Q cavity. Disordered NuoA and NuoH loops in the open state were modelled for representation purposes.

Supplementary Video 3

Conformational changes of the key Q cavity loops upon EcCI closing. Pale surface shows the Q cavity. Disordered NuoA and NuoH loops in the open state were modelled for representation purposes.

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Kravchuk, V., Petrova, O., Kampjut, D. et al. A universal coupling mechanism of respiratory complex I. Nature 609, 808–814 (2022). https://doi.org/10.1038/s41586-022-05199-7

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