Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against transmembrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further studies to understand the enzyme coupling mechanism. Here, we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data, we suggest a new simple hypothetical coupling mechanism for the molecular machine.


Introduction
Complex I, NADH:ubiquinone oxidoreductase, is a multi-subunit enzyme found in many bacteria and most eukaryotes. It facilitates transfer of two electrons from NADH to ubiquinone, or its analogues, coupled reversibly with translocation of four protons across the membrane against trans-membrane potential (Galkin et al., 2006;Sazanov, 2015). Structures of the complete complex I from several eukaryotes (Fiedorczuk et al., 2016;Hunte et al., 2010;Kampjut and Sazanov, 2020;Zhu et al., 2016), one thermophilic bacterium (Baradaran et al., 2013), and the partial structure of the membrane domain of Escherichia coli complex I (Efremov and Sazanov, 2011), have been determined.
The composition of complex I differs significantly between species. Mitochondrial complex I has a molecular weight 1 MDa and comprises more than 35 subunits (Wirth et al., 2016), whereas bacterial analogues are much smaller with molecular weight approximately 500 kDa. Complex I from all characterized species contains homologues of 14 core subunits; seven subunits each assemble into peripheral and membrane arms, joined at their tips and form the complex with a characteristic L-shape.
The peripheral arm, exposed to the cytoplasm in bacteria or the mitochondrial matrix in eukaryotes, contains binding sites for NADH, ubiquinone, and flavin mononucleotide (FMN) as well as eight or nine iron-sulfur clusters, seven of which connect the NADH and ubiquinone-binding sites (Sazanov, 2015) enabling rapid electron transfer (Verkhovskaya et al., 2008).
resolving the interface between the arms; however, due to high-residual mobility of the arms, the antiporter-like subunits were resolved at below 8 Å (Figure 1-figure supplement 4).
Focused refinement of each arm separately and subtraction of nanodisc density (Figure 1-figure supplement 3) improved the resolution of peripheral and membrane arms to 2.7 Å and 3.9 Å , respectively (Figure 1-figure supplements 3 and 4, Table 1). After density modification (Terwilliger et al., 2020), the resolution of membrane arm improved to 3.7 Å .
Micrograph analysis, in contrast to mass photometry, revealed that large fraction of the particles corresponds to the peripheral arm only (Figure 1-figure supplement 3) that may have dissociated during cryo-EM sample preparation. These yielded 3D reconstruction to 2.8 Å resolution (Figure 1figure supplement 3), similar to the map of the peripheral arm of intact complex I. Joining two subsets and applying density modification improved resolution of the peripheral arm to 2.1 Å (Table 1, Figure 1-figure supplements 4 and 6). Using the resulting maps, atomic models of the peripheral and membrane arms have been built. The entire E. coli complex I was modeled by fitting models of the arms and extending additionally resolved loops and termini. Due to limited resolution, the antiporter-like subunits were refined as rigid bodies. The final models include 4618 residues that account for 94.7% of the total polypeptide constituting the complex ( Table 2).        Figure 1B) reflect long-range twisting and bending of arms observed between complex I from different species (Agip et al., 2018;Baradaran et al., 2013;Vinothkumar et al., 2014).
Comparison of E. coli complex I conformations reconstructed to better than 4 Å resolution revealed two modes of relative arm rotation ( Figure 1C): (1) rotation around an axis that passes through the NuoH-NuoB interface and is tilted around 45 degrees out of the plane formed by the arms with an amplitude of at least 13 degrees, and (2) rotation around an axis parallel to the membrane and roughly perpendicular to the long axis of the membrane arm with an amplitude of approximately 4 degrees. Although relative arm movements were observed in mammalian (Kampjut and Sazanov, 2020;Zhu et al., 2016) and T. thermophilus complex I (Gutiérrez-Fernández et al., 2020), their amplitudes were smaller and movement directionality was less diverse. Despite significant relative arm movements, the structure of each arm was rigid and did not reveal different conformations apart from the specific local dynamics discussed below.

Structure of the peripheral arm
Architecture of the peripheral arm reveals a novel evolutionary strategy to stabilize the subcomplex   Unlike other structurally characterized homologues, E. coli subunits NuoC and NuoD are joined in a single polypeptide. The 35 amino acid-long linker includes an a-helix (residues 180-194) that interacts with subunit NuoB (Figure 2A). The relative positions of all redox centers with FMN and nine iron-sulfur clusters, including off path cluster N7 (Sazanov and Hinchliffe, 2006), are particularly well conserved ( Figure 2C).
A distinctive feature of the E. coli peripheral arm is the presence of ordered C-terminal extensions in subunits NuoB, NuoI, and NuoF with a length of 22-45 residues and a large 94 residue insertion loop in subunit NuoG, referred to as the G-loop ( Figure 2A, Table 3). These extensions are unique among structurally characterized complex I homologues and have a well-defined structure. While the G-loop has a compact fold, the conformation of the C-terminal tails is extended. They line the surface of the peripheral arm subunits with high shape complementarity ( Figure 2A, Figure 2-figure supplement 1) and apart from a few helical turns, have no secondary structure elements (Table 3). They create additional inter-subunit contacts with some surface areas exceeding 1000 Å 2 (Table 3). Similarly, the G-loop fills a crevice between NuoCD, NuoI, and NuoG subunits ( Figure 2A) and together with the extensions increase the interaction surface between the electron acceptor module (NuoEFG) and connecting module (NuoICDB) by a factor of three (from 1400 to 4600 Å 2 ), thus stabilizing the peripheral arm assembly. These structural features are conserved within the Enterobacteriaceae family, are very common in the phylum Gammaproteobacteria and display high conservation of interfacial residues, particularly for the G-loop (Figure 2-figure supplement 1). They demonstrate a new evolutionary strategy for complex stabilization that was not observed in other complex I homologs.
A strong density near the NuoG surface coordinated by G Asp617, G Gln632, G Glu647, G Asp731 and four water molecules ( Figure 2B) was assigned to a Ca 2+ ion. The coordination number, geometry, and ion-ligand distances of ca 2.5 Å (Zheng et al., 2008) as well as the 2 mM concentration of Ca 2+ in the buffer support this assignment. Divalent ions are known to increase both the activity and stability of E. coli complex I (Sazanov et al., 2003). One of the calcium ligands, G Asp731, is part of  the G-loop, suggesting that Ca 2+ stabilizes the fold of the G-loop and consequently, the peripheral arm.
The extensions spatially overlap with the supernumerary subunits of complex I from T. thermophilus (Sazanov and Hinchliffe, 2006) and the structurally conserved supernumerary subunits of eukaryotic complex I (Zhu et al., 2016; Table 3), consistent with the suggestion that the primary role of supernumerary subunits is to stabilize the complex (Fiedorczuk et al., 2016).

Bound water molecules
At 2.1 Å resolution, 1170 water molecules associated with the peripheral arm were modeled ( Figure 2D). The positions of 180 water molecules are conserved with those identified in the peripheral arm of ovine complex I (Kampjut and Sazanov, 2020; Figure 2D, red spheres). Most of conserved waters are buried in the interior of the subunits, shielded from the solvent, and likely play a structural role in stabilizing the subunit fold. Only a few waters interact closely with iron-sulfur  clusters and may influence their potential (Tables 4 and 5). The water molecules close to or between iron-sulfur clusters are not more conserved than those in the other parts of the complex, suggesting that they were not evolutionary selected to optimize the rate of electron transfer as was suggested (Schulte et al., 2019). At 2.1 Å resolution, several unusual density features were observed next to some surface-exposed histidines and between some cysteine-methionine pairs as listed in Table 6 and depicted in Fig

Electron input and output sites
The FMN conformation and key water molecules in the NADH-binding pocket are conserved (Kampjut and Sazanov, 2020;Schulte et al., 2019). This includes W1060, hydrogen bonded to the isoalloxazine ring N5 atom of FMN and to F Glu92, which likely acts as the activating group during catalysis of hydride transfer from NADH (Fraaije et al., 2000; Figure 3A). Schulte et.al. (Schulte et al., 2019) suggested a mechanism for regulation of reactive oxygen species (ROS) generation by E. coli complex I that involves flipping the carbonyl oxygen of F Glu93 upon enzymatic reduction. Our structure unambiguously places the corresponding carbonyl oxygen in a Table 5. Differences in the hydrogen bond network of iron-sulfur clusters in complex I structures solved at high resolution and water molecules in the immediate cluster environment.
(Only the clusters for which such comparison could have been done and clusters displaying differences in the environment are listed).  conformation that points away from FMN ( Figure 3A) similar to conformations found in the reduced and oxidized ovine complex I (Kampjut and Sazanov, 2020), which does not support its involvement in ROS regulation. E. coli-specific features in the FMN-binding pocket include F His400 that replaces the Leu residues found in other homologues. F His400 is in Van der Waals contact with the isoalloxazine ring of FMN; its imidazole ring interacts directly with the N3 cluster iron atom and forms a hydrogen bond with Sg of F Cys357 coordinating N3 ( Figure 3A). F His400 is solvent-accessible even in the presence of NADH, and therefore, may become protonated upon N3 reduction. F Arg320 is positioned such that it can form hydrogen bonds with the ribose moiety of the NADH nicotinamide group and may stabilize bound dinucleotide ( Figure 3A). Both Arg320 F and His400 F may serve to counter-balance the negative charges of electrons on N1a and N3 clusters and to increase protein stability. The structure does not reveal specific features explaining the decreased affinity for FMN in the reduced enzyme (Holt et al., 2016). This can be attributed to minor conformational changes in the pocket upon enzyme reduction.
The Q-binding site in complex I is formed at the end of a crevice between NuoD and NuoB subunits (Baradaran et al., 2013). In E. coli, this wedge is formed by the 58-69 stretch of NuoB and the tip of the 220-225 loop from subunit NuoD. Both D Tyr273 and D His224, found in the proximity of bound decylubiquinone (Baradaran et al., 2013) are conserved in E. coli and point toward the quinone binding site ( Figure 3D), whereas the tip of the 218-223 loop is flexible as in many complex I structures.

Environment and potentials of iron-sulfur clusters
At a resolution of 2.1 Å the atoms constituting the iron-sulfur clusters are resolved as independent density blobs. The conformation of side chains as well as the positions of hydrating waters in the primary and secondary interaction spheres are mostly unambiguously resolved ( Figure 3). In E. coli complex I, cluster N1a can be reduced by NADH due to its uniquely high potential (~À0.3 V), differentiating it from other characterized species in which N1a cannot be reduced by NADH (Birrell et al., 2013;Zu et al., 2002). The potential of iron-sulfur clusters in proteins among other factors depends on solvent exposure, proximity of charged residues, and the number of hydrogen bonds formed between the cluster environment and sulfur atoms of clusters and coordinating cysteines (Denke et al., 1998;Fritz et al., 2002). Comparison of the chemical environment of N1a with other high-resolution structures of complex I revealed three specific differences explaining the higher potential of the N1a cluster (Table 4): (1) E. coli-specific E Asn142 forms a hydrogen bond with Sg of E Cys97 coordinating the N1a cluster and with N1a S1 ( Figure 3B), consistent with its mutation to Met decreasing potential by 53 mV (Birrell et al., 2013). (2) In E. coli, water molecule  W74 forms a hydrogen bond with Sg of E Cys97. This water resides in a hydrophilic cavity created by E. coli specific E Gly140, replacing the alanine residue found in other species.
(3) Because of small differences in the backbone conformation of NuoF, the backbone nitrogen of F Gly97 forms a hydrogen bond with Sg of E Cys133 in E. coli and Aquifex aeolicus but not in Ovis aries ( Figure 3B, Table 4).
The environment of the other iron-sulfur clusters is mainly conserved. The differences in hydrogen donors to the clusters, cysteine sulfur atoms, and water molecules in the cluster vicinity are listed in Table 5 Clusters N3 and N2 are briefly discussed below as being the most interesting.
Cluster N3 interacts with E. coli-specific F His400 ( Figure 3A); however, the potential of N3 is very similar between the species (Leif et al., 1995;Yagi and Matsuno-Yagi, 2003). The effect of proximal His residue is likely compensated by F Trp363 replacing the hydrogen bond donors (Glu or Gln) found in other species ( Table 5).
The potential of cluster N2, the electron donor to quinone, varies in different species (Hirst and Roessler, 2016) notably being lower in E. coli compared to its mammalian analogues (À220 mV vs. À140 mV, respectively). However, the structure shows that the polar environment of N2 is very conserved ( Figure 3C), including two water molecules, W211 and W438. Two arginines found in close proximity to the N2 cluster, Arg270 D and Arg250 D , have conserved positions despite 49kDa Arg85 in the mammalian homologue ( D Arg250) being dimethylated (Carroll et al., 2013). This modification prevents it from forming a hydrogen bond with B Cys63, which should decrease N2 potential in the mitochondrial enzyme. Therefore, computational modeling, now enabled by the high-resolution structure, will be required to explain the difference in the cluster potentials.

Structure of the membrane arm
The model of complete membrane arm, including the previously missing subunit NuoH (Efremov and Sazanov, 2011), was built into the density map with local resolution better than 3.5 Å at the arm center and approximately 4.0 Å at its periphery ( Figure 1A, Figure 1-figure supplement 4). An additional density belt corresponding to the lipid nanodisc is clearly visible around the membrane-embedded region ( Figure 1A, Video 1). It is flat in the plane of the membrane with a thickness of approximately 30 Å , and closely matches hydrophobic surface of the membrane arm. The belt locally bends next to the subunit NuoL at the region where it interacts with the long amphipathic helix and amphipathic helix connecting H TMH1-TMH2 ( H AH1) protrudes into the nanodisc (Video 1).
The structure of the membrane arm in the lipid nanodisc is very similar to the crystal structure of the detergent-solubilized membrane arm (Efremov and Sazanov, 2011) [RMSD of 1.1 Å (1888 Ca)] ( Figure 4-figure supplement 1). The curvature of the membrane arm observed previously (Efremov and Sazanov, 2011) was unchanged in the lipid environment, and therefore, is not an artifact of crystallization or solubilization (Verkhovskaya and Bloch, 2013). Local structural differences in crystal structure include expected repositioning of A TMH1 next to H TMH2 ( Baradaran et al., 2013), and a change in conformation of the M TMH5-TMH6 loop (Figure 4-figure supplement 1).
The fold of subunit NuoH is similar to the structures of T. thermophilus and of eukaryotic complexes with one important exception. The density for the N-terminus of NuoH (residues 1-52) that includes H TMH1 and a part of H TMH1-TMH2 loop, is completely missing in the reconstructions of the membrane fragment and of the complete complex ( Figure 1, Video 1).
The structures visualize a complete chain of charged residues connecting the Q-site with charged residues in antiporter-like subunits ( Figure 4A). We analyzed the environment of ionizable residues found within the 'E-channel' (Baradaran et al., 2013), a region situated between the Q-cavity and antiporter-like subunit NuoN, to evaluate the presence of a continuous proton translocation path linking the Q-cavity with the antiporter-like subunits suggested for ovine complex I (Kampjut and Sazanov, 2020).
The trans-membrane region of E. coli NuoH contains fewer charged residues than its structurally characterized homologs (Figure 4-figure supplement 2). Here, only E. coli-specific H His208, separated from H Glu157 by 12 Å , is found in the center of NuoH ( Figure 4A). A large hydrophilic cavity stretches from the Q-site towards the center of subunit NuoH and ends next to the invariant H Glu157 (hereafter, invariant residues are marked in bold), suggesting that this glutamic acid can exchange protons with a bound ubiquinone.
The region between NuoH and NuoN includes six ionizable side chains located in the middle of the membrane bilayer, four of which are invariant ( Figure 4A). The distances between the residues vary from 5 Å to 12 Å which requires either displacement of the side chains or bridging water molecules to facilitate proton exchange between them. Analysis of cavities and potential hydration sites using DOWSER++ (Morozenko and Stuchebrukhov, 2016) shows that the residues H Glu157 and A Asp79 along with the carbonyl oxygen of J Gly61 on the p-bulge of J TM3 (Efremov and Sazanov, 2011) point into a hydrophilic cavity that can accommodate several water molecules enabling proton exchange between the ionizable residues. Similarly, cavities that can be hydrated link a chain of ionizable residues J Glu55-K Glu36-K Glu72-N Glu133 potentially enabling proton exchange between its ends. The residues A Asp79 and J Glu55/ K Glu36 are separated by a distance exceeding 12 Å and a region packed with hydrophobic residues, making proton exchange between the Q-site and NuoN unlikely. A Glu81, located opposite A Asp79 on A TMH2, apparently does not participate in linking A Asp79 with N Glu133. However, it faces hydrophilic environment of J Ser145, E. coli-specific J Glu142, and A Glu102, potentially linking it to the periplasmic surface ( Figure 4A). Interestingly, mutation of any individual carboxylic groups in the A Glu81/ A Asp79 pair does not affect pumping, while mutation of both residues completely abolishes the protein activity (Kao et al., 2004), suggesting that they are functionally important but interchangeable. Thus, analysis of the protein translocation pathways indicates that in E. coli, no continuous proton path exists between the Q-site and NuoN.  Curiously, H Lys274, almost universally conserved in complex I and related hydrogenases, is found in the H TMH7 off the main pathways proposed for proton translocation ( Figure 4A), however, the length and flexibility of the side chain allow it to switch between the extracellular surface and center of the membrane.
The cytoplasmic half of J TMH3 found to assume two alternative conformations in eukaryotic complex I (Agip et al., 2018;Kampjut and Sazanov, 2020) is very well-resolved in our reconstructions, suggesting the absence of alternative conformations in E. coli complex I. A peculiar feature is observed in subunit NuoM instead.
The density of the cytoplasmic half of M TM8 is poor and fragmented between residues 255 and 265, indicating the existence of multiple conformations ( Figure 4B, Video 2). This region is buried in the middle of NuoM and the density of surrounding helices is very well-resolved indicating local character of the disorder. This region spans the invariant M Lys265, includes the p-bulge, and M Asp258 in some bacteria. Interestingly, in T. thermophilus the cytoplasmic region of 13 TM8 is rotated by two residues relative to E. coli structure ( Figure 4B) which results in repositioning of T. thermophilus 13 Lys235 ( M Lys265 in E. coli) from the center of the second structural repeat (TM9-TM13) towards the interface between the structural repeats facing 13 His218 ( M His248 in E. coli). Thus, higher mobility of the helical fragment situated at a critical position at the interface of symmetry-related modules may indicate p-bulge-enabled helical rotation and its potential role in proton translocation.

The peripheral-membrane arm interface
The interface between membrane and peripheral arms mediates the coupling of ubiquinone reduction to proton translocation across the membrane. It is highly conserved between complex I homologs and related membrane-bound hydrogenases (Baradaran et al., 2013;Grba and Hirst, 2020;Kampjut and Sazanov, 2020;Yu et al., 2020;Yu et al., 2018; Figure 5-figure supplement 1) and forms through interaction between subunits NuoB and NuoD of the peripheral arm with NuoH and the A TMH1-TMH2 loop of the membrane domain.
Apart from several interfacial regions of subunits NuoB, NuoD, and NuoI that become more ordered upon complex formation ( Figure 5-figure supplement 2, Table 2), the membrane-facing surface of E. coli peripheral arm, including the residues lining the Q-cavity, is highly mobile in both dissociated and complexed arms (Figure 1-figure supplement 4, Figure 5-figure supplement 2). This suggest that the interfacial region of the peripheral arm is inherently flexible and likely responsible, at least in part, for the high relative mobility of the arms. Similar to complex I from T. thermophilus (Baradaran et al., 2013), there are no specific conformational changes at the interface upon association of the arms.
Structure of the NuoH surface and relative arrangement of subunits NuoB and NuoD in E. coli complex I is similar to that of complex I from other species, however, their relative positions differ. In E. coli complex I, NuoB and NuoD are rotated around an axis passing through the center of NuoH and the interface between NuoF and NuoG by approximately 15 degrees anticlockwise when observed from the top of the peripheral arm ( Figure 5A). This results in over 10 Å shift of fourhelical interfacial region of NuoD, and its separation from NuoH ( Figure 5B). The highly conserved fragment of the A TMH1-TMH2 loop (residues 46-53), that plugs the crevice between NuoD and NuoB ( Figure 5-figure supplement 1B) and interacts with the ubiquinone-coordinating loop D 221-228, is also disordered ( Figure 5B).
On the opposite side of the interface, structural rearrangements include a 7-degree tilt of A TMH1 that becomes more perpendicular to the membrane plane and approximately 15-degree rotation of H AH1 in the direction of H TMH1 and towards the membrane center ( Figure 5C) Video 2. The fragmented density of the NuoM TM8 is surrounded by well-resolved TMHs. https://elifesciences.org/articles/68710#video2 reducing opening of the Q-cavity entrance ( Figure 5D). Homology modeling indicates that the observed rearrangements are still compatible with H TMH1 occupying its expected position without any steric clashes ( Figure 5D).
Rotation of the NuoB/NuoD module creates multiple openings in the interface between the arms ( Figure 5B). They are large enough to allow water and proton exchange between the Q-cavity and   bulk solvent, suggesting that the ubiquinone bound within the Q-site can receive protons directly from the solvent. Therefore, we think the resolved conformations of complex I represent uncoupled states in which redox reaction is not coupled to proton translocation.
To better understand the reasons for the observed uncoupled conformation and the missing density for H TMH1, we purified E. coli complex I in detergent LMNG, showed that it can catalyze redox reactions ( Figure 6-figure supplement 1) and solved its structure to resolution of 6.7 Å (Figure 6figure supplement 2). The detergent-solubilized complex also displays high relative mobility of the arms (Figure 6-figure supplement 3) and has uncoupled conformation ( Figure 6). Its peripheral arm is rotated even further away from the expected coupled state position than in the nanodiscreconstituted structures. Both the cryo-EM sample preparation conditions and more homogeneous distribution of particle orientations indicate that interaction of the complex with air-water interface was significantly reduced when compared with the complex in nanodiscs. This allows us to conclude that neither air-water interface nor reconstitution into nanodiscs cause the uncoupled conformations.
The H TMH1 helix is resolved in the detergent-solubilized complex ( Figure 6A). Its density is weaker than that of the surrounding helices and it is strongly bent ( Figure 6B). Simultaneously, H AH1 takes the conformation resembling other complex I homologs while A TMH1 bends towards the arm core. The arrangement of helices in detergent-solubilized reconstruction appears to be more compact and more bent than in the lipid environment which may restrain the otherwise more flexible H TMH1.

Discussion
Structural features of E. coli complex I E. coli complex I is composed of the smallest number of subunits among all structurally characterized complex I homologs. Yet, it still evolved a strategy to stabilize peripheral arm assembly without involving additional subunits. The interactions between subunits are stabilized by extended C-termini and a large G-loop (Figure 2)    the complex stability (Sazanov et al., 2003). This indicates existence of evolutionary pressure on maintaining the peripheral arm integrity, which was 'solved' in a species-dependent manner.

NuoB B B A A A A A A A TMH1 1 1 1 1 1 H H H AH1
The membrane arm structure reveals no apparent continuous proton translocation path between the Q-cavity and subunit NuoN. Further, there are no indications for the existence of different conformations in the cytoplasmic half of J TMH3 observed in mammalian complex I (Agip et al., 2018;Kampjut and Sazanov, 2020) attributed to deactive-active transition (Agip et al., 2018) or more recently, to different catalytic intermediates (Kampjut and Sazanov, 2020). This suggests that these states are either suppressed in the resting state of the bacterial complex or do not occur at all. Conversely, M TMH8 displays localized disorder next to the p-bulge, indicating possible involvement of this helix in the structural rearrangements associated with proton translocation, and to our knowledge, this represents the first indication of specific conformational changes in antiporter-like subunits.
Purified E. coli complex I is known to be more flexible and fragile than its homologs from other organisms (Morgan and Sazanov, 2008;Sazanov et al., 2003). Our cryo-EM reconstructions reveal the reasons for its high flexibility. The peripheral and membrane arms are mainly rigid, whereas the connection between arms is flexible ( Figure 1C, Figure 1-figure supplement 5). The high mobility of the interfacial regions and the relative rotation of the arms disrupts conserved interfacial interactions and exposes Q-cavity to the solvent ( Figure 5A). This differentiates E. coli complex I from its structurally characterized homologs in which the Q-cavity is sealed from the solvent. Thus, we interpret the observed conformation as an uncoupled state.
The reasons for such conformation are not clear. 3D cryo-EM reconstructions obtained for the detergent-solubilized and the nanodiscs-reconstituted complex I allow us to exclude the air-water interface or the nanodisc as the cause. In turn, the lipid bilayer mimetics provided by detergent micelle or lipid nanodiscs might not be sufficiently close to the native lipid bilayer, causing uncoupling. Alternatively, this conformation may have a functional origin and correspond to a resting state of E. coli complex I (Belevich et al., 2017) analogous to the deactive state in eukaryotic complex I (Babot et al., 2014). However, the latter have been associated with local conformational changes (Agip et al., 2018;Parey et al., 2018), rather than displacement of the complete domains. Figure 7. Proposed coupling mechanism in respiratory complex I. Ubiquinone reduction decreases the proton potential in the Q-cavity, generating/enhancing potential difference between the Q-cavity and periplasmic space. It is subsequently neutralized by protons translocated through NuoH from the periplasm into the Q-cavity and this translocation is coupled with the reversible translocation of three protons into the periplasm. Color coding of the schematic subunits in the membrane arm is similar to that described in Figure 1, negatively charged states of ubiquinone are shown in red.
The absence of H TMH1 density in nanodiscs, but not in detergent, is another unique feature of E. coli complex I. H TMH1 is exposed to the lipid environment and the width of the nanodisc next to H TMH1 is similar to other regions around the membrane arm (Video 1). Moreover, homology modeled H HTM1 fits the empty space without steric clashes suggesting that H HTM1 is dynamic rather than displaced or unfolded. By comparing the detergent-solubilized and reconstituted complexes we can conclude that position and dynamics of this helix is neither the cause of the uncoupled conformation nor of the high relative mobility of the arms.

Hypothetical coupling mechanism
The absence of a continuous proton-translocation pathway between the Q-site and subunit NuoN, as well as high flexibility of the peripheral arm interface are not consistent with the recently proposed coupling mechanisms relying on specific movements of the interfacial loops (Cabrera-Orefice et al., 2018;Kampjut and Sazanov, 2020). This led us to ask whether a coupling mechanism consistent with known complex I properties, but without the movements of interfacial loops is conceivable.
Imposing the microscopic reversibility constraints (Astumian et al., 2016;Onsager and Machlup, 1953) and the requirement for applicability of the mechanism to the entire class of evolutionarily related complexes we came up with a new, simple mechanism that to the best of our knowledge has not been considered previously. It is briefly outlined below with more details provided in Appendix.
Coupling can be enabled by the formation of a cavity isolated from external protons that is accessible to electron acceptors in their neutral form only. The requirement for a tightly coupled cavity is consistent with high conservation of the interface between the arms. Due to its small size, the redox potential of ubiquinone will be strongly modulated by the extraction/addition of single protons from/to the cavity (Lemmer et al., 2011), and reciprocally, activity of the protons in the cavity will be modulated by the changes of the ubiquinone redox state. Upon ubiquinone reduction, the decreased proton activity is rectified by proton transfer through NuoH from the extracellular compartment as shown in Figure 7. This proton transfer is coupled to the transfer of three protons through three antiporter-like subunits in the opposite direction resulting in a net transfer of 2H + per 1 e -. Consequently, the entire membrane module of complex I functions as a proton antiporter with stoichiometry 1H + in /3H + out and most likely operates by a classical alternating access mechanism (Jardetzky, 1966). The mechanism is applicable to evolutionary-related hydrogenases. It suggests that a pH jump creates a proton motive force between the sealed Q-cavity and periplasmic surface and might be used to trap equilibrium conformations associated with proton translocation by the membrane domain of complex I.

Conclusions
Here we described the cryo-EM structures of E. coli respiratory complex I that reveal unique structural features. We discovered an evolutionary strategy specific to mesophilic bacteria for stabilizing the peripheral arm assembly through the extended C-termini, the G-loop, and the bound Ca 2+ ion. In the membrane arm, M TMH8 displays dynamics unseen in the other complex I homologues which may reflect conformational flexibility associated with proton translocation. We also observed the relative rotation of the membrane and peripheral arms disrupting the conserved interface and trapping the complex in an uncoupled conformation. Whether this conformation is biologically relevant or is a result of protein purification is to be clarified by further research. Generation of an E. coli strain expressing Twin-Strep-tagged respiratory complex I

Materials and methods
The native nuo operon encoding the 13 subunits of respiratory complex I (NuoA-N) was engineered with a Twin-Strep-tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK, IBA GmbH) at the N-terminus of NuoF using CRISPR-Cas9-enabled recombineering (Jiang et al., 2015). The DNA sequence encoding the C-terminal region of NuoE and N-terminus of NuoF was retrieved from GenBank (Acc. No. NC_012971.2 region 2288438-2289174). The tag-coding sequence followed by a TEV protease recognition site (Tropea et al., 2009) was appended upstream of the NuoF N-terminus and was codonoptimized, together with the 2288766-2288807 region of the genomic fragment. Such designed, linear DNA knock-in cassette was synthesized (GenScript). The vectors pCas and pTargetF were gifts from Sheng Yang (Addgene plasmids #62225 and #62226). The N20 sequence (GGTCAGCGGA TGCGTTTCGG) was introduced into pTargetF by inverse PCR. Genomic engineering was performed according as described by Jiang et al., 2015. Briefly, pCas vector was transformed into the chemically competent E. coli BL21AI strain (Thermo Fisher Scientific Inc). The transformants were grown in shake-flask culture at 30˚C in Lysogeny Broth (LB) medium containing 25 mg mL À1 (w/v) kanamycin monosulfate and 10 mM L-arabinose. Upon reaching OD 600 0.5, the bacteria were rendered electrocompetent and were co-electroporated with the linear DNA cassette and the mutated pTargetF vector. The transformants were selected on LB-agar plates supplemented with 25 mg mL À1 (w/v) kanamycin and 50 mg mL À1 (w/v) streptomycin, or 50 mg mL À1 (w/v) spectinomycin. The positives, identified by colony PCR and DNA sequencing, were cured of the plasmids as described previously (Jiang et al., 2015). We further refer to the modified strain as E. coli BL21FS (NuoF-Strep).
Expression and purification of respiratory complex I E. coli BL21FS was cultivated in LB medium for 48 hr at 37˚C in a microaerobic environment. The cells were harvested by centrifugation and the membrane fraction was isolated as described by Sazanov et al., 2003. All subsequent steps were performed at 4˚C. The homogenate was solubilized in 2% (w/v) n-Dodecyl b-D-maltoside (DDM, Anatrace) for 2 hr while stirring, after which the non-solubilized fraction was removed by ultracentrifugation at 225,000 Â g for 1 hr. . The purity of the eluted protein was assessed by SDS-PAGE and activity assays (Figure 1-figure supplement 2). The purified complex I was concentrated using an Amicon Ultra-4 100K centrifugal filter (Merck) to 0.5 mg mL À1 (w/v), fast-frozen in liquid nitrogen and stored at À80˚C. For the preparation in lauryl maltose neopentyl glycol (LMNG), the protocol was modified as described below. Membranes were solubilized in 2% (w/v) LMNG (Anatrace). The buffer A-LMNG consisted of 50 mM Bis-tris pH 6, 2 mM CaCl 2 , 200 mM NaCl, 0.03% (w/v) LMNG, 10% (v/v) sucrose, 0.2 mM PMSF. The buffer B-LMNG was the buffer A-LMNG supplemented with 5 mM D-desthiobiotin. The LMNG-purified complex I was concentrated using an Amicon Ultra-4 100K centrifugal filter (Merck) to 10 mg mL À1 (w/v) and loaded on the Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated in 20 mM Bis-Tris pH 6.0, 200 mM NaCl, 2 mM CaCl 2 and 0.003% (w/v) LMNG. The protein-containing fractions were pooled, concentrated to 2-3 mg mL À1 (w/v) using Amicon Ultra-0.5 100K centrifugal concentrators, and used for cryo-EM grid preparation.

Reconstitution of respiratory complex I into lipid nanodiscs
The membrane scaffold protein MSP2N2 was expressed and purified following a published protocol (Grinkova et al., 2010). Purified, lipid-containing complex I preparation at 520 nM concentration was mixed with 10.4 mM MSP2N2 (1:20 protein:MSP molar ratio, no additional lipids were added during reconstitution) and incubated for 1 hr at 4˚C. Subsequently, the detergent was removed by adding 0.5 g mL À1 (w/v) Bio-Beads (Bio-Rad) overnight at 4˚C. The reconstituted protein was further purified on the Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated in a buffer comprising 20 mM Bis-Tris pH 6.8, 200 mM NaCl and 2 mM CaCl 2 . The protein-containing fractions were pooled and concentrated to 0.1-0.2 mg mL À1 (w/v) using Amicon Ultra-0.5 100K centrifugal concentrators.
For the NADH:FeCy assay, 0.9 nM of complex I and 1 mM FeCy (Sigma Aldrich BVBA) was added to the assay buffer in a stirred quartz cuvette. For the NADH:Q1/DQ, 3-9 nM of detergent-solubilized or nanodisc-reconstituted complex I and 100 mM Q1/DQ (Sigma Aldrich BVBA) was added to the assay buffer in a stirred quartz cuvette at 30˚C and incubated for 5 min. The reactions were initiated by adding 100 mM NADH (Carl-Roth GmbH) and followed as reduction in absorbance at 340 nm using a Varian Cary 300 UV-Vis spectrophotometer (Agilent Technologies, Inc). To perform the inhibition assay, 20 mM Piericidin A (Cayman Chemical) was added during the NADH:Q1 or NADH: DQ reaction.

Mass photometry
The composition of the nanodisc-reconstituted protein preparation was assessed using mass photometry on a Refeyn OneMP instrument (Refeyn Ltd.), which was calibrated using an unstained native protein ladder (NativeMark Unstained Protein Standard A, Thermo Fisher Scientific Inc). Measurements were performed on the reconstituted complex I at a concentration of 0.015 mg ml À1 using AcquireMP 2.2.0 software and were analyzed using the DiscoverMP 2.2.0 package.

Preparation of cryo-EM samples
The cryo-EM samples were prepared using a CP3 cryoplunge (Gatan). Quantifoil R0.6/1 Cu300 holey carbon grids were cleaned with chloroform, acetone, and isopropanol as described by Passmore and Russo, 2016. The grids were glow discharged in the ELMO glow discharge system (Corduan Technologies) from both sides for 2 min at 11 mA and 0.28 mbar. For the nanodisc-reconstituted sample, four microliters of the reconstituted protein solution at 0.15 mg ml À1 concentration were applied on a grid and blotted from both sides for 2.2 s with Whatman No. 3 filter paper at 97% relative humidity. The LMNG-purified complex I was supplemented with 0.2% CHAPS (Anatrace) and applied at concentration 2-3 mg ml À1 . The grids were plunge-frozen in liquid ethane at À176˚C and stored in liquid nitrogen.

Cryo-EM data collection
Cryo-EM images were collected on a JEOL CryoARM 300 microscope equipped with an in-column W energy filter (Fislage et al., 2020) at 300 kV, automatically using SerialEM 3.0.8 (Mastronarde, 2005). The energy filter slit was set to 20 eV width. The nanodisc sample was collected at a nominal magnification of 60,000 and the corresponding calibrated pixel size of 0.771 Å . Five images per single stage position were collected using a cross pattern with three holes along each axis (Efremov and Stroobants, 2021). The 3 s exposures were dose-fractionated into 61 frames with an electron dose of 1.06 e-Å À2 per frame. In total, 9122 zero-loss micrographs were recorded with the defocus varying between À0.9 and À2.2 mm ( Table 1).
The LMNG-solubilized sample was collected at a 60,000 nominal magnification and the calibrated pixel size of 0.766 Å . Nine images were collected per stage position using a 3x3 hole pattern. The 3 s exposures were dose-fractionated into 60 frames with 1 e -A À2 dose per frame. The defocus varied between À1.0 and À2.0 mm. During 36 hr of data collection, 13,084 zero-loss micrographs were recorded.

EM image processing
For both datasets, the dose-fractionated movies were motion-corrected using MotionCor2 (Zheng et al., 2017) in the patch mode. The Contrast Transfer Function (CTF) parameters were estimated using CTFFIND-4.1 (Rohou and Grigorieff, 2015).
For processing of the nanodisc data, 40 micrographs of various defoci were selected, manually picked, and used to train the neural network of crYOLO 1.4 (Wagner et al., 2019). After training, 1,256,734 particles were picked automatically from the complete dataset, extracted in RELION 3.0 (Zivanov et al., 2018), and imported into cryoSPARC 2.11 (Punjani et al., 2017). Following 2D classification, six initial models were generated, among which one corresponded to the peripheral armonly and another corresponded to the complete complex I. Using hetero-refinement, 441,265 and 525,680 particles were assigned to the peripheral arm and complete complex, respectively. Further processing was performed in RELION 3.1 (Zivanov et al., 2020). After per-particle CTF estimation and Bayesian polishing, 3D auto-refinement of the complete complex produced a map at an average resolution of 3.4 Å (Figure 1-figure supplement 3). However, the map was very heterogeneous with the peripheral arm resolved at 3.0-3.6 Å whereas the membrane arm was resolved at over 10 Å .
To address this heterogeneity, both arms were refined independently using multi-body refinement ; Figure 1-figure supplement 3) and the peripheral domain signal was subtracted. After two rounds of 3D classification applied to the membrane domain and nanodisc signal subtraction, a subset of 48,745 particles was 3D refined to an average resolution of 3.6 Å . However, the density map was anisotropic. To improve the reconstruction, the original stack of 525,680 particles was refined against the masked peripheral arm, followed by subtraction of the signal from the peripheral arm. Next, membrane arm map obtained above was filtered to 9 Å and used as an initial model for the 3D refinement of all resulting membrane arm particles. Next, to prevent model bias, the refined map was low-pass filtered to 20 Å and used in the subsequent 3D classification with 10 classes, t of 12 and 24˚local angular search range and 1.8˚angular step. The best class (110,258 particles and 8 Å resolution) was auto-refined using the starting model low-pass filtered to 15 Å , which produced the reconstruction to a resolution of 4.4 Å . Next, the nanodisc density was subtracted, which further improved the resolution to 3.9 Å . Following 3D classification without alignment with t of 40, eight classes, and resolution in the E-step limited to 4 Å , a subset of 37,441 particles was identified, which after auto refinement, produced a density map at an average resolution of 3.9 Å with better resolved peripheral regions. Finally, density modification with the resolve_-cryo_em tool available in PHENIX 1.18.2 (Terwilliger et al., 2020) improved the resolution to 3.7 Å (Figure 1-figure supplements 3 and 4).
After multibody refinement of the arms described above, peripheral arm particles with the subtracted membrane arm were 3D classified into 12 classes without alignment using t of 40 and resolution of the expectation step limited to 4 Å . The best class contained 134,976 particles and was further refined to 2.9 Å resolution.
A subset of 166,580 particles was selected after a similar 3D classification procedure that was applied to the 441,265 particles of dissociated peripheral arm particles. It was further cleaned from the remaining particles of the complete complex I by 2D classification, resulting in a subset of 151,357 particles that produced a density map to a resolution of 3.0 Å . As the reconstructions of the dissociated and membrane arm-subtracted peripheral arms were virtually identical, both stacks were combined. After two cycles of per-particle CTF refinement, aberration corrections, and Bayesian particle polishing in RELION 3.1, the resolution improved to 2.4 Å . Consecutive density modification in PHENIX further improved the resolution to 2.1 Å (Figure 1-figure supplements 3 and 4, Table 1).
To resolve the conformation of entire complex I, a stack of 525,680 particles was aligned to the peripheral arm using auto-refinement with a mask around the peripheral arm in RELION 3.1. Next, 3D classification without alignment into 30 classes with resolution of the expectation step limited to 20 Å and t of 4 was performed, followed by auto-refinement of each resulting class, which produced maps to a resolution in the range of 9-20 Å (some of the classes are shown in Figure 1-figure supplement 5).
Three high-resolution conformations of complete complex I were obtained as follows. Conformation one was resolved by applying the 3D classification into 15 classes, t of 6, a 24˚local angular search range, and 1.8 o sampling interval to the subset of 110,258 particles that produced the 3.9 Å reconstruction of the membrane arm (see above). The best class consisted of 23,445 particles that were refined to a resolution of 3.9 Å .
Conformations 2 and 3 were identified by applying 3D classification without image alignment into 12 classes with t of 40 and resolution of the expectation step limited to 4 Å , to the stack of 525,680 intact complex I particles. Two of the best classes, consisting of 21,620 and 21,234 particles were refined to 4.6 Å and 4.5 Å , respectively. Following density-modification in PHENIX, the resolution of the maps was improved to 3.3, 3.8, and 3.7 Å , for conformations 1, 2, and 3, respectively (Figure 1figure supplement 4C, Table 1).
From 13,084 motion-corrected micrographs containing LMNG-solubilized complex I, a subset of 9333 was selected. Next, 1,469,948 particles were picked with crYOLO 1.7.0. After 2D classification in cryoSPARC 3.2.0 792,120 particles were retained. The particles were subjected to 3D auto-refinement in RELION 3.1, followed by focused 3D auto-refinement with a mask around the peripheral arm. Next, focused 3D classification of the peripheral arm into 12 classes with t = 6, 24˚local angular search range and 1.8˚angular step resulted in a homogeneous subset of 82,433 particles that after 3D refinement produced a 4.3 Å peripheral arm reconstruction. This stack was used for 3D classification without alignment into 15 classes with t = 15 and without a mask. 3D auto-refinement of the resulting classes converged to resolution between 6.7-13 Å and revealed large relative movements of the arms ( Figure 6-figure supplement 3).

Model building
Peripheral arm subunits constituting NuoB, CD, E, F, G, and I were first homology modeled in the SWISS-MODEL server (Waterhouse et al., 2018) based on the structure of T. thermophilus (PDB ID:4HEA Baradaran et al., 2013) and were rigid-body fitted into the density map in UCSF Chimera 1.13.1 (Pettersen et al., 2004). Following manual rebuilding in Coot 0.9 (Casañal et al., 2020), the model was subjected to real-space refinement against the final 2.1 Å map of the peripheral arm in PHENIX 1.19.2 (Liebschner et al., 2019) using the default parameters. Secondary structure restrains were applied only to the interfacial region resolved at a lower resolution. The value of the nonbon-ded_weight parameter was optimized. Water molecules were added to the map and validated using the 'Check/delete waters' tool in Coot 0.9. Molecular dynamics-based model idealization was conducted in ISOLDE 1.0b5 (Croll, 2018), followed by several iterations of real-space refinement without atomic displacement parameter (ADP) restraints and manual rebuilding in Coot 0.9.
For the membrane domain, the previously obtained E. coli model (PDB ID: 3RKO) was real-spacerefined in PHENIX. The missing NuoH subunit was homology-modeled using the T. thermophilus structure (PDB ID: 4HEA) in Coot 0.9. The final model was obtained after several rounds of manual rebuilding and real-space refinement using standard parameters with Ramachandran restrains, secondary-structure restrains applied to the NuoL TMH9-13, without ADP restrains, and with the optimized nonbonded_weight parameter. To generate the model of the complete complex I, the separate peripheral and membrane arm structures were combined and the missing parts at the interface (Table 2) were built manually. As the density of NuoL, NuoM and NuoN was very poor in all the resolved full conformations, these subunits were refined as rigid-body in PHENIX, whereas the others were refined using real-space refinement with minimization_global, local_grid_search, morphing, and ADP refinement. Ramachandran, ADP, and secondary-structure restrains were used. The models were validated in MolProbity (Williams et al., 2018). Structural conservation was evaluated using the ConSurf server (Ashkenazy et al., 2016). The figures and videos were generated in UCSF ChimeraX version 1.1. (Goddard et al., 2018) and PyMOL (The PyMOL Molecular Graphics System, Version 2.4.1 Schrö dinger, LLC). had an evolutionary advantage with the proton-translocating module serving as a source of protons (Yu et al., 2018) biasing H 2 evolution towards the reaction product (Boyd et al., 2014), as follows from Equation 3 and Appendix 1-table 1.
Experiments with engineering E. coli complex I lacking subunit NuoL and Y. lipolytica complex I lacking homologs of subunits NuoM and NuoL (Drö se et al., 2011;Steimle et al., 2011) (correspond to n=2 and 1, respectively) both suggested that the engineered complexes were active and for both constructs stoichiometry was estimated as 2H + /2e -. While NuoL deletion experiments support our model, the NuoL/M deletion clearly contradicts it. Both experiments should be interpreted cautiously, however. Results of NuoL deletion for E. coli complex I were not reproducible (Verkhovskaya and Bloch, 2013). In the case of Y. lipolytica, the homologs of NuoL/M dissociated from the complex along with another 11 subunits upon deletion of supernumerary subunit NB8M located at the tip of NuoL (Zickermann et al., 2015). Since the proton-translocating modules were not deleted per se, the presence of contaminating amounts of assembled complex I in the preparations that generated observed proton pumping cannot be completely excluded. It is important to note that mutation of the conserved ionizable residues on the interface between NuoN and NuoM, i.e. M E144 (Torres-Bacete et al., 2007) or its counter ion N K395 (Amarneh and Vik, 2003), result in a completely inactive complex I suggesting that dissociation of subunits NuoL/M also should render complex I inactive (Verkhovskaya and Bloch, 2013).
From our model it follows that complex uncoupling is achieved by opening the Q-cavity to the solvent. It is consistent with an elegant experiments by Cabrera-Orefice et al., 2018 in which locking the conserved A TMH1-2 plug with the cysteine bridge reversibly uncoupled the enzyme. Close examination of the crosslinked structure indicates that crosslinking fixes the plug in a conformation rendering the Q-cavity accessible to the solvent.
The exact proton translocation mechanism within the antiporter-like module is unknown and requires further experimental and computational modeling. Here, we can only speculate that given the high conservation of H Glu157, it plays an important role in the coupling and may change its protonation state during the pumping cycle. Thus, it can influence the pK a of neighboring ionizable residues. In MBH, an equivalent M Glu141 is separated from the closest ionizable H Lys409 by distance of 20 Å , which in a hydrophobic environment with a dielectric constant of 10, allow them to mutually modulate the pKa of each other by approximately 1 pH unit, similar to the free energy conserved upon ferredoxin oxidation by the protein complex. This distance is reduced to around 13 Å in MBS and to around 6 Å in complex I, consistent with the proportionally higher free energy of catalyzed reactions. While many questions on the coupling mechanism of complex I remain open, further significant advances might be expected once structures of different conformation associated with proton translocation by membrane arm will be resolved.