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Cryo-EM structures define ubiquinone-10 binding to mitochondrial complex I and conformational transitions accompanying Q-site occupancy

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q10 reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the ‘active’ (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the ‘deactive’ resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.


Supplementary
Map-to-map correlation values were calculated using the Fit in Map tool in UCSF ChimeraX 7 . RMSD calculations were performed using the Align command in PyMOL 8 . Where relevant, PDB and EMDB codes are given as four-letter and five-digit codes, respectively. a) Comparisons of the maps determined here and those reported previously by our group for active, deactive, and state 3 structures of mammalian complex I. All the maps were low-pass filtered to the lowest resolution map in the table (published state 3 map at 5.6 Å resolution 3 ), and the maps determined here were fitted into the published maps to calculate map-to-map correlation values. For comparisons, cells are shaded in colour scales from blue (best fit) to white (50 th percentile) to red (worst fit). b) Comparisons of the maps and models determined here and those available in the PDB and EMDB. All the maps were low-pass filtered to the lowest resolution map in the table (published ovine closed map at 3.8 Å resolution 6 ), and the ovine maps were fitted into the maps determined here to calculate map-to-map correlation values. For comparisons, cells are shaded in colour scales from blue (best fit) to white (50 th percentile) to red (worst fit).

Supplementary Fig. 1: Purification and biochemical characterisation of Bos taurus complex I reconstituted into nanodiscs (CxI-NDs). a)
Elution of CxI-NDs from the Superose 6 increase 5/150 size-exclusion column in the final step of the preparation (see Methods for details). The CxI-NDs elute at 1.6 mL, the same as the DDM-bound enzyme. b) Specific NADH:dQ activity data on CxI-DDM and CxI-NDs show that the complex I in CxI-NDs is highly catalytically competent, but catalysis is limited by substrate access. The reference activity (DDM-CxI, before reconstitution) is conserved in CxI-NDs following addition of CHAPS (to dissociate them) and asolectin (to create a lipidic phase). Without CHAPS/asolectin the activity was much lower, so either dQ does not exchange effectively in and out of the nanodisc, its mobility is restricted, or the MSPs prevent it from entering the active site. The low observed rate of NADH oxidation, which is further decreased in the absence of dQ, may either result from dQ reduction by complex I, or dQ-mediated reoxidation of the Q10H2 generated by complex I. The data reported were recorded on the sample analysed by cryo-EM (n = 2 or 4 technical replicates), and presented as mean values ± SEM; note that inhibitor-insensitive background rates were not recorded in some cases due to lack of sample availability, and therefore all the data in the figure are reported without background subtraction to enable their comparison. 200 µM NADH was added in all cases (see Methods for experimental details).
Supplementary Fig. 2: Cryo-EM data processing -global classification. a) A flow chart of cryo-EM data processing up to the final global 3D classification leading to the three major classes -active, deactive and state 3. D1 and D2 denote Datasets 1 and 2, respectively. Where applicable, the red box denotes that this is the final map for the class. b) A representative raw micrograph from the data collections. A total of 6,780 micrographs were recorded. c) Representative 2D class averages from the data processing. The example view was selected following 2D classification of the final 3D refined particles to show classes of particles in different orientations. Fig. 3: Cryo-EM data processing -local classification. A flow chart of cryo-EM data processing from the active/deactive major classes to local substates (active-Q10, active-apo, deactive-ligand, and deactive-apo) by local classification. As substantial heterogeneity was observed in the Q-binding sites of the active and deactive maps, the major classes were first signal subtracted to retain the hydrophilic arm, focus-refined, then 3D focus-classified without alignment using class-specific local masks. A tight mask generated from a tentative Q10 model was used for local classification for the active class, and a more generous mask from a provisional protein model (ND1, NDUFS2, and NDUFS7) encapsulating the Q-binding site region proved more successful for the deactive class. The consensus deactive-ligand map showed poor densities at the distal region of the membrane arm due to subtle differences in the 'openness' of the hydrophilic and membrane arms and stronger alignment to the former. For this reason, a composite map was made for the deactive-ligand class. The consensus map was split into three by signal subtraction and focusrefinement using the three overlaid masks shown in transparent colours. Local resolution maps are shown for the distal (left) and proximal (middle) membrane domains, and the peripheral domain (right). Local resolutions were estimated using the Local Resolution function in RELION and plotted using UCSF ChimeraX 7 with map thresholds set to 0.015. The coloured key on the right indicates the resolution in Å, and the map resolution ranges are indicated in brackets for the respective maps, in blue. RELION half-map (sky blue) FSC curves are shown for each domain. The RELION map sharpening B-factors (Å 2 ) for the consensus map, the distal and proximal membrane domains, and the peripheral domain were -58, -53, -49, and -51, respectively. The resulting composite map is shown in gray at a map threshold of 6.0. Composite maps were not made for the other classes either because there was little flexibility in the distal membrane domain (active-Q10 and active-apo) or because it did not lead to a marked improvement in map densities (deactive-apo and state 3). Where applicable, red boxes denote the final maps for the classes. Lateral nanodisc widths are indicated. b) 42 phospholipids modelled in a representative CxI-ND structure (deactive-ligand), with the seven core membrane subunits represented as cartoons. All non-cardiolipin phospholipids were modelled as phosphatidylethanolamines unless density features indicated phosphatidylcholine to be more likely. All subunits are coloured as in Fig. 1.  Supplementary Fig. 8. Free energy profiles for additional structural properties obtained from metadynamics simulations. Three combinations of sidechain protonation were simulated with Asp160 NDUFS2 ionised (Asp -) or protonated (AspH) and His59 NDUFS2 neutral (His, Nδ1-protonated π tautomer) or di-protonated (HisH + , both Nδ1-and Nε2-protonated), as indicated above each pair of panel rows. CV describes the Q-headgroup position along the binding channel ( Fig. 3b-d).

Supplementary
Properties correspond to atom-pair distances shown on top of each column and symbols correspond to distances observed in active-Q10 cryo-EM models with primary (star) and flipped  Improvements in resolution of the third state of complex I, state 3, now enable a more comprehensive consideration of this poorly understood class of particles ( Supplementary Fig. 5). Previously, for state 3 we described a loss of clear density for the C-terminal half of the ND5 transverse helix and its anchor helix (TMH16), much of the adjacent NDUFA11 subunit, and the Nterminal loop of NDUFS2 3,15 . The resolution of our early data was low (5.6 Å), but the same characteristics are conserved in our current higher resolution map (3.0 Å), so it is clear that these structural elements are disordered and/or have dissociated from the structured binding locations that they occupy on the complex in the active and deactive states. Our data now reveals also that ND6-TMH4 has lost its α-helical secondary structure in state 3, instead appearing as a poorly ordered loop, on the same exposed side of the complex as NDUFA11 and ND5-TMH16, extending the region affected by the disorder. Comparison of the nanodisc structures also shows clear differences in this region ( Supplementary Fig. 6). Instead of clear densities stretching around NDUFA11 (as observed in the active and deactive states), the MSP2N2 helices in state 3 are not clearly resolved, consistent with substantial disorder in subunit NDUFA11 and/or with the MSP2N2 helices contracting inwards to occupy the space where NDUFA11 is usually observed. This change in nanodisc structure suggests that state 3 was present in the preparation before the enzyme was reconstituted into the nanodiscs. Therefore, it may result from destabilisation of the detergentsolubilised enzyme during its purification, but is not an artefact from cryo-EM grid preparation due to instability induced at the air-water interface [16][17][18] . Interestingly, NDUFA11 has been reported to be the only subunit in the membrane arm of complex I that undergoes rapid protein turnover in C2C12 myotubes 19 , consistent with a higher propensity to dissociate in vivo. Furthermore, in the mammalian respirasome NDUFA11 is sandwiched in-between complex III and the core subunits of complex I [20][21][22] , and the removal of complex III during solubilisation of the membrane may introduce additional instability to NDUFA11 and adjacent structures.
Comparing the three states of complex I resolved here shows clearly that state 3 has more in common with the deactive state than the active state. Globally, the apparent angle between the hydrophilic and hydrophobic domains of the complex is most acute in the active state, considerably more obtuse in the deactive state and similar, or greater still, in state 3. Key structural features that distinguish the deactive state from the active state are also present in state 3, including the π-bulge in ND6-TMH3, disordered ND3 TMH1-2 loop and structures around NDUFS7-Arg77. In contrast, the NDUFS2-β1-β2 and ND1 TMH5-6 loop conformations differ from those observed in both the active and deactive states. However, as features of the Q-binding site are known to be remodelled by ligands, it is unclear whether their conformations are a feature of state 3 or determined by ligand binding. Finally, the features discussed above that are disordered in state 3 are clearly ordered (and equivalent) in both the active and deactive states, and the ND2-ND4 interface is closed in both those states. Therefore, state 3 shares a substantial number of distinguishing features with the deactive state -but it shares none of them with the active state.
Due to the extra-relaxed and disordered characteristics of state 3, we propose to name this state 'slack' complex I in the future.
The three 'open' states described for native ovine complex I 6 exhibit all the key structural features observed in our deactive states, but also features that are specific to our state 3 structure. In particular, the state named open3 displays extensive disorder (evident as weak or absent densities) for NDUFA11, the C-terminal half of the ND5 transverse helix and its anchor helix (TMH16), the ~40 N-terminal residues of NDUFS2, and ND6-TMH4. These comparisons suggest that the open conformations of ovine complex I exist on a structural spectrum between our deactive state and state 3. Similar state 3-like characteristics are also observed in maps for ovine complex I reported to be in the deactive state, which we note was prepared by incubating a concentrated aliquot of the detergent-solubilised complex I at 32 °C for 30 min after anion exchange 6 , rather than by deactivating the enzyme in its stabilising native membrane environment 2,5,23 .
In state 3 the extended interface between subunits ND2 and ND4 contains an ordered Q10 molecule, accommodated by a π-bulge formed in ND4-TMH6. It is possible that the ND4-TMH6 π-bulge forms during catalysis, such that state 3 represents a relaxed state of a hitherto-uncharacterised intermediate, in which the π-bulge causes Tyr148 ND4 to pivot away from its usual hydrogen bond to Glu559 ND5 . By doing so it may destabilise the ND2-ND4 interface, propagating instability to the transverse helix and associated structures, resulting in the observed loss of structural integrity. The interfaces between NDUFA10, ND4, and ND2 have also been observed to accumulate strain in simulation work, and proposed to modulate conformational changes within the membrane domain 24 . Intriguingly, the position of the Q10-headgroup bound at the ND2-ND4 interface in state 3 coincides with the position of a rotenone inhibitor molecule detected previously in ovine complex I open conformations 6 . Although it is difficult to conceive of any functional role for Q10 binding at this site, so distant from any of the enzyme's redox cofactors, it is possible that any molecule binding here could lock the position of Tyr148 ND4 and thereby be inhibitory -if reorganisation of ND4-TMH6 really does occur on the catalytic cycle.
Loosening the structural constraints of the transverse helix in state 3 and opening up of the ND2-ND4 interface may alternatively allow the Q10/rotenone molecule to enter in adventitiously. The propensity of the enzyme to fracture at this interface is highlighted by treatment of bovine complex I with the zwitterionic detergent N,N-dimethyldodecylamine N-oxide (LDAO) to form the ND4-ND5containing subcomplex of the distal membrane domain known as subcomplex Iβ 25 . The same fragmentation occurred during crystallisation trials on the DDM-solubilised enzyme 26 , yielding a structure of subcomplex Iβ 25 in which the C-terminal section of ND5 is highly disordered. The tendency of the ND4-ND5 module to dissociate was also illustrated by Y. lipolytica knock-out strain nb8m∆ (where NB8M is a homologue of subunit NDUFB7, adjacent to the C-terminus of ND5), in which the distal membrane domain was absent 27 . These observations argue that state 3 may represent an enzyme in the initial stages of degradation, not a catalytically relevant state.
To define the catalytic capability of state 3 it will be necessary to produce a homogeneous sample of it and characterise it both functionally and structurally. It is currently unclear how to achieve this for bovine complex I, but we note that a structure we solved previously for complex I from rhesus macaque (Macaca mulatta) 15 was dominated by particles in this state (lacking clear densities for NDUFA11, the C-terminal half of the ND5 transverse helix and its anchor helix (TMH16), the N-terminus of NDUFS2, and parts of ND6-TMH4) -and the preparation exhibited only a very low activity of 0.5 µmol min -1 mg -1 15 . Current evidence to suggest that state 3-like states do exist on the catalytic cycle is limited to their presence in a sample of ovine complex I treated with the substrates for turnover -however, similar states were present in the starting mixture and may simply not have responded to the addition of substrates 6 . Further investigations of complex I samples under established turnover conditions and in defined biochemical states will be necessary to finally establish the relevance of this intriguing enzyme state.

Supplementary Note 2: Minor differences between the structures of nanodisc-reconstituted and DDM-solubilised complex I
First, in all CxI-ND maps, the tip of the β-hairpin in the ND6 TMH4-5 loop on the intermembrane space face is shifted by 3-3.5 Å towards subunit ND2, so that it makes contact with the ND2-TMH4-5 helix-loop-helix motif and ND4L-TMH1. In the DDM-solubilised reference active-state structure 1 , there is an unmodelled density between the two structural elements that resembles a DDM molecule displacing the ND6-β-hairpin. The β-hairpin motif is not present in M. musculus complex I 5,23 , while in the lauryl maltose neopentyl glycol (LMNG)-solubilised ovine enzyme 6 its conformation matches the CxI-ND conformation.
Second, a density for a steroid molecule (modelled as a cholate, which was added at the reconstitution step) is present in all CxI-ND maps at the interface between subunits ND5 and NDUFB6. The nearby NDUFB6 matrix helix/loop is resolved for the first time here, perhaps as a result of the structure being stabilised by incorporation of the additional cholate molecule and enclosed by the adjacent MSP2N2 helices -although the previously unstructured region makes remarkably few direct contacts with any other subunits.
Third, the C-terminal peptide of mammalian subunit NDUFS1 (ca. 10 residues) is also resolved for the first time here, with the terminal Cys704 NDUFS1 residue lodged between subunits NDUFS1, NDUFS6, and NDUFS8. The absence of DDM in the sample buffer may be responsible for the improved resolution of this peptide in the nanodisc structures, on the outside of the hydrophilic domain.