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Role of water and protein dynamics in proton pumping by respiratory complex I

Membrane bound respiratory complex I is the key enzyme in the respiratory chains of bacteria and mitochondria, and couples the reduction of quinone to the pumping of protons across the membrane. Recently solved crystal or electron microscopy structures of bacterial and mitochondrial complexes have provided significant insights into the electron and proton transfer pathways. However, due to large spatial separation between the electron and proton transfer routes, the molecular mechanism of coupling remains unclear. Here, based on atomistic molecular dynamics simulations performed on the entire structure of complex I from Thermus thermophilus, we studied the hydration of the quinone-binding site and the membrane-bound subunits. The data from simulations show rapid diffusion of water molecules in the protein interior, and formation of hydrated regions in the three antiporter-type subunits. An unexpected water-protein based connectivity between the middle of the Q-tunnel and the fourth proton channel is also observed. The protonation-state dependent dynamics of key acidic residues in the Nqo8 subunit suggest that the latter may be linked to redox-coupled proton pumping in complex I. We propose that in complex I the proton and electron transfer paths are not entirely separate, instead the nature of coupling may in part be ‘direct’.


Results
Hydration and dynamics of antiporter-like subunits. Fully atomistic classical MD simulations performed on the entire crystal structure of complex I show that the root mean square deviation (RMSD) of protein stabilizes at a value of 4-5 Å in the first 50-100 ns of simulation time ( Supplementary Fig. S1). The RMSD of the membrane domain, comprising Nqo7, Nqo8, Nqo10-14 subunits, also converges to a stable value of ca. 4 Å within the same time domain. The data suggests that the system is fully equilibrated, and timescales of simulation are sufficient to analyze fast dynamics associated with the protein and the water molecules. The membraneembedded antiporter-type subunits Nqo12-14 are shown in orange, blue and magenta, respectively. Nqo8 (green), Nqo7, Nqo10 and Nqo11 (violet-indigo) are also displayed along with the hydrophilic subunits Nqo6 (light blue) and Nqo4 (pink) that form the Q-binding site scaffold. Q (purple) binds in a buried binding site. All other hydrophilic subunits are shown in grey. Covalently-bound FeS clusters (pink-yellow spheres), and FMN (orange) at the terminal end of the hydrophilic domain are also shown. NADH binds next to FMN and acts as a direct donor of two electrons to the latter. The region studied in this work is marked by a thick black box, and is shown to atomic details in the inset (box with dotted lines). The residues discussed in this work are also marked (inset).
Starting from a completely dehydrated protein, the two 500 ns simulation trajectories (setups I and II) reveal rapid hydration of the protein that is within the first 100 ns (Fig. 2). The three membrane-bound subunits Nqo12-14, which are homologous to the Na + /H + antiporters, are found to hydrate extensively (Fig. 2). In each of these subunits, the water molecules enter from both sides of the membrane, and a sigmoidal shaped pathway is transiently formed, which is in agreement with the earlier structural and simulation studies 18,19,31,35 .
After an initial steep rise, the hydration in different membrane bound subunits plateau to relatively stable values in the rest of the simulation time (100-500 ns). However, some short term fluctuations in water occupancy are observed in Nqo7/10/11 and Nqo8 subunits occurring at around 400 ns (see black curves in the lower panel of Fig. 2). These occur due to transient hydration and dehydration events in a particular region of the protein. This is more clearly displayed in Supplementary Fig. S2, which shows that the maximum change in hydration (dehydration) at around 400 ns takes place next to certain specific residues (Asn35 of Nqo10, Asn36 of Nqo11, and Glu213 and Glu35 of Nqo8). Similarly, the increase in hydration in Nqo13 (setup II) and in Nqo14 (setup I) at around 400 and 250 ns, respectively (Fig. 2), occurs partly due to the flipping of the sidechains of amino acid residues. The sidechain flip of His211 (Nqo13) and Lys216 (Nqo14), shown as a change in RMSD and sidechain dihedral in Supplementary Figs S2 and S3, respectively, coincides with the increase in hydration in the nearby region (see also Supplementary Table S1). Since, sidechain fluctuations are likely to occur in these simulation Figure 2. Hydration of the protein subunits based on simulations of setup I (black) and setup II (red) with Q ox and QH 2 , respectively (see also Table 1). Number of water molecules is plotted (ordinate) vs the simulation time (abscissa, in ns). Only water molecules (oxygen atom) within 4 Å of the selected amino acids are considered in the analysis (see Supplementary Table S1). Table 1. Model systems and simulation lengths. * The Q is modeled as Q OX (oxidized) or QH 2 (doubly reduced and doubly protonated) at the site close to N2 FeS center (Q 1 ) or at another location half-way through the Q tunnel (Q 2 ) (see Methods section). † Including five independent ~5 ns simulations. # Including one 266 ns and three independent ~80 ns simulations with QH 2 modeled at few different locations in the middle region of the Q-tunnel.
timescales, we suggest that the hydration in the membrane-bound subunits is stable, and small variations occur around an equilibrium value. In subunit Nqo12, the water-based connection between the middle of the subunit and the P-side of the membrane ('P-path') forms readily in both 500 ns MD simulations I and II, and comprises conserved charged or polar amino acid residues ( Fig. 3 and Supplementary S4). In addition to the putative proton exit route or the P-path, two proton uptake paths are observed ('N-path'), one closer to the interface between the Nqo12 and Nqo13 subunits in setup II, and the other one enclosed in subunit Nqo12 in setup I (Fig. 3, see also Supplementary Fig. S4). The interfacial hydration close to the N-side of the membrane was also observed in the earlier simulation study by Kaila et al. on E. coli enzyme 31 . However, in contrast to that our simulation data show substantial hydration confined to Nqo12 subunit (upper panel in Fig. 3 from simulation of setup I) that is around the conserved residue Ser138 (Supplementary Table S1). In our simulations, a transient connectivity is also observed between the two paths (N and P-paths) formed by water molecules and His241 of Nqo12 (Fig. 3). However, the water occupancy in this middle region remains low ( Fig. 3 and Supplementary S4), and may form a barrier preventing proton leak from the P-side to the N-side of the membrane. Almost all of the Nqo12 residues that participate in proton channel formation are found to be conserved in the E. coli enzyme (Supplementary Table S1), and the observed hydration in this region is also in good agreement with the earlier predictions from X-ray and simulation data 17,18,31 .
In order to further elucidate the role of water molecules in proton pumping by complex I, we analyzed the electric dipole moment of water molecules in various hydrated regions of the protein ( Supplementary Fig. S5). Remarkably, the total electric dipole moment vector of water molecules orients in such a way that would allow efficient transfer of protons from the N-phase of the membrane to the conserved proton acceptor, Glu132, in the middle of the Nqo12 subunit (see panel A in Supplementary Fig. S5). The water dipole and hydrogen bond arrangements observed further corroborate the proton transfer pathways identified in this work.
During simulations of setups I and II we observed stabilization of the ion-pair formed by two highly conserved residues, Arg163 of Nqo12 and Glu377 of neighboring Nqo13 subunit, which tightens the subunit-subunit interface ( Supplementary Fig. S6). In various crystal or cryo-EM structures, the distance between the two residues (Arg:Cζ -Glu:Cδ distance) is 6.3 Å 18 , 9.6 Å 19 , 8.5 Å 20 and 8.1 Å 21 , whereas this distance reduces to ca. 4.3 Å in the two longest simulations (setups I and II) due to the ion-pairing. Moreover, this ion-pair is placed in a particularly . Proton channels in antiporter-type subunits Nqo12-14 from simulation setups I (top) and II (bottom) with Q ox and QH 2 , respectively (see also Table 1). The putative proton transfer paths from the N-side to the P-side are marked with an S-shaped black arrow. The residues that take part in water-wire formation are also listed (acidic -red, basic -blue, and polar -green). Water molecules are shown in purple. The water positions are instantaneous, and represent a simulation snapshot. The water occupancy, calculated by averaging water positions over entire simulation trajectory, is displayed as an orange mesh plotted with an isovalue of 0.1. Water molecules within 6 Å of selected residues (Supplementary Table S1) were used to calculate the occupancy plot.
interesting location in partly non-polar surroundings in the middle of the membrane (Supplementary Fig. S6). Due to the presence of charged residues (see also Supplementary Table S2), the region gets rapidly hydrated and water molecules are exchanged between the two subunits. The stabilized subunit-subunit interface may play a role in the long-range coupling between the Q-reduction and proton pumping in distant subunit Nqo12.
In contrast to Nqo12, rather large number of water molecules are found to diffuse in the middle of subunits Nqo13 and Nqo14 ( Fig. 3 and Supplementary S4), most likely due to the presence of charged residues Lys235 and Lys216 of Nqo13 and Nqo14, respectively (cf. neutral His241 in Nqo12 in Supplementary Table S2). A closer look to the hydration in the middle of the subunits Nqo13 and Nqo14 reveal preferred orientation of water dipoles that is favorable for Grotthuss-type proton transfer 36 (Supplementary Fig. S5). The water-based connectivity between the middle of the subunit and the N-side of the membrane is also established in both the subunits (Fig. 3). However, the hydration close to the P-side of the membrane is found to be low in both the simulation setups I and II (see also Supplementary Fig. S4). The relatively low water occupancy in this region may represent a conformation in which proton 'back-leak' is minimized.
The two residues (Lys235 in Nqo13, and Lys216 in Nqo14) that are seen to participate in proton channel formation in our simulations are conserved in the E. coli complex I (Lys265 in NuoM and Lys247 in NuoN, respectively, see Supplementary Table S1). These residues were also found to attract water molecules in their vicinity in the earlier simulation study by Kaila et al. (ref. 31). Overall, in agreement with earlier work, our simulation data reveal high and low water occupancies in the regions close to the N-and the P-side of the membrane, respectively.
In stark contrast to the Nqo12/13 subunit-subunit interface ( Supplementary Fig. S6), the hydration of Nqo13/14 interface is not observed, primarily due to the missing inter-subunit ion pairing. However, despite this the interface is stabilized by the formation of transient H-bonds in the loops of Nqo13 and Nqo14 subunits ( Supplementary Fig. S7). Similar stabilization is also observed for the Nqo12/13 and Nqo14/7/10 interfaces suggesting that in addition to the long horizontal helix, loop-loop interactions may also keep antiporter-type subunits together.
Even though several similarities are observed between the two longest simulations (setups I and II), some differences are also apparent. First is the presence of water molecules in the lower part of Nqo12 subunit in simulation of setup II (lower panel of Fig. 3). However, when hydration data from other setups are analyzed ( Supplementary Fig. S4), virtually there is no water occupancy in this region of the enzyme, suggesting that the observed hydration is transient, and is probably due to the stochastic nature of the simulations. Similarly, when data from other simulations are taken into account ( Supplementary Fig. S4), sufficient water density is found in the middle of the Nqo14 subunit, similar to Nqo13. Overall, the sequence and structural homology between antiporter-type subunits, partial overlap of water distribution in the three subunits ( Fig. 3 and Supplementary S4) as well as similarity with the earlier simulation work on E. coli enzyme (see Supplementary Table S1), further strengthen our results and conclusions.
Fourth proton pathway. Consistent with the presence of three homologous subunits (Nqo12-14) is the proton pumping stoichiometry of 3 H + /2e − , which has been proposed to occur at a high proton motive force (pmf) 33 , even though it remains to be demonstrated experimentally. However, experimental data clearly shows that it may reach a maximum of 4 H + /2e − in the absence of pmf 37,38 . Therefore, a fourth proton transfer pathway may be expected. Our simulation data suggest that a fourth proton channel emerges at the interface of Nqo10 and Nqo11 ( Fig. 4 and Supplementary S8). This involves highly conserved Glu32 of Nqo11, which has been found to be indispensable for the activity of the enzyme 39,40 . Interestingly, this proton uptake route is similar to what has been proposed based on the crystal structure of Escherichia coli enzyme and eukaryotic enzyme 17,19 , but different from the path observed in T. thermophilus complex I 18 . A further analysis of the simulation trajectory of setup I reveals that the pathway transiently opens for proton uptake, from the N-side of the membrane, caused by the down flip of conserved Glu32 of subunit Nqo11 (Fig. 4). The exit path for this channel comprises polar residues from Nqo10 and Nqo11 subunits, as shown in Fig. 4. Remarkably, many of the amino acid residues that are predicted to form the fourth proton channel are also found by Kaila et al. (ref. 31) in their independent simulation study on E. coli complex I (see Supplementary Table S1). We note that the down flip of Glu32 from Nqo11 is not observed in the simulation of setup II, instead it is stabilized in a different conformation hydrogen bonding to the Tyr59 of Nqo10, similar to what is seen in the crystal structure of Thermus enzyme 18 . Nevertheless, the agreement between the current and earlier simulations results, as well as the functional importance of Glu32 39,40 , strongly advocate that the region is involved in proton pumping, and may form the fourth proton pathway.
Hydration of Nqo8 subunit. The hydration pattern of Nqo8 subunit is most interesting because of very large number of water molecules that enter this subunit during the simulations (Fig. 2). It is observed that part of the subunit hydrates very rapidly (Fig. 5A), whereas the other half remains predominantly dry, mainly due to the presence of large number of hydrophobic residues. This is also observed in longer simulations I and II ( Fig. 5 and Supplementary S9). The hydration pattern shows that the section of the Q-tunnel that is embedded in the Nqo8 subunit connects to the middle of the membrane-bound subunits via charged amino acid residues and water molecules (Fig. 5B, see also Fig. 1 inset). In the two longest simulations (setups I and II), a continuous hydrated path is not established (Supplementary Fig. S9). However, when protonation states of acidic residues along this pathway are altered (Table 1), they undergo conformational changes (Supplementary Fig. S3 and Table S3), and recruit water molecules, thereby enabling a continuous connection from the Q-tunnel to the fourth putative proton channel (Fig. 5B). This unique connection extends all the way up to Glu67 of Nqo11 that is ca 35 Å from the Q-tunnel region in the Nqo8 subunit. Interestingly, when electric dipole moment vector of water molecules in this hydrated region is plotted, it is found to align in the direction from the Q-tunnel to the antiporter type subunits (see panel D in Supplementary Fig. S5). We suggest that this connectivity between the middle of the Q-tunnel and the membrane-bound subunits, as observed from simulation data, is critical for the proton pumping function of the enzyme (see below).
Hydration and dynamics of Nqo4 and Nqo6 subunits. The two subunits Nqo4 and Nqo6 form the scaffold for the Q-binding close to the N2 FeS center 18,19 . Due to the presence of multiple charged residues in Nqo4, water molecules rapidly hydrate the subunit (Figs 2, 6 and Supplementary S10), also including the region around the Q head group. This raises an important question that what prevents the protonation of the residues that are left deprotonated after the proton-coupled electron transfer to Q from N2 30 . In an earlier work, a partial stabilization of the anionic Tyr87 by neutral His38 of Nqo4 was observed 30 . However, deprotonated Tyr87 is likely   1 (B). The figure is prepared from simulation setup X in which Glu248, Glu213, Glu163 of Nqo8, and Asp72 and Asp74 of Nqo7 are protonated. In panel A, only water molecules within 4 Å of Nqo8 subunit (green) are shown. In panel B, Nqo7, Nqo8, Nqo10 and Nqo11 are colored grey, green, blue and mauve, respectively, and shown as thin ribbons (see also Supplementary Fig. S9).
to have a much higher proton affinity, so that it may be protonated by the proton released upon QH 2 oxidation (see also Methods), which would result in a loss of free energy of proton pumping. Also, the protonation of the anionic Tyr (and His38/Asp139 ion pair) from the N-or the P-side of the membrane prior to the completion of the pump cycle may destabilize conformational states necessary for proton translocation.
We suggest that Arg83 from Nqo6 electrostatically stabilizes the anionic Tyr87, as observed in our multiple independent simulations (Fig. 6, Supplementary Fig. S11, and Table 1). We note that this arginine is not fully conserved, instead replaced by a threonine in complex I from other organisms such as Bos taurus, Escherichia coli and Yarrowia lipolytica. However, our analysis of recent cryo-EM structure of mammalian complex I 20 reveal that an arginine residue (Arg85, bovine numbering, PDB id: 5LC5) from 49 kDa subunit effectively compensates for the missing arginine in the PSST subunit, and forms a hydrogen bonding interaction with Tyr108 (bovine numbering) (Supplementary Fig. S11). Interestingly, the latter arginine residue in bovine complex I has been suggested to be functionally critical based on mass-spectroscopy data 41 . We hypothesize that the position of Arg is swapped in the two enzymes classes (e.g. Thermus and Bovine), but the functional role is shared. This viewpoint is also supported by the coevolution analysis performed on Nqo4/6 sequences using two different tools, GREMLIN 42 and EVcomplex 43 . The data from GREMLIN (normalized coupling strength >1) and EVcomplex (EVcomplex score ~0.53) suggests that Thr64 in Nqo4 and Arg83 in Nqo6 are evolutionarily coupled such that their positions are swapped in other organisms, without altering the function of Arg83 (see above). Such positional swapping of functionally critical residues is well-known in the heme-copper oxidase superfamily 44,45 . We suggest that the stabilization of the deprotonated form of Tyr87 is necessary to prevent protonic equilibration between the N-side (or the P-side) of the membrane and the latter residue, which would otherwise result in loss of proton pumping.
Protonation dependent side chain dynamics. The hydration pattern observed for Nqo8 subunit in our atomistic simulations show that the water molecules together with charged residues establish a connection between the middle of the Q-tunnel and the negatively charged residues of Nqo7-8 subunits (see Fig. 5B, Supplementary Fig. S5D and inset of Fig. 1). We propose that this pathway may be used to transfer protons (e.g. released upon proposed QH 2 oxidation, see ref. 2) to the P-side of the membrane. In order to shed light on the latter aspect, we performed MD simulations by changing the protonation states of the residues that line up along the path from the Q-tunnel to the membrane-bound subunits (see Fig. 5B, Table 1 and Methods section). We observe that change in the protonation state of key acidic residues bring up on a large change in their sidechain  (Fig. 7 and Supplementary Fig. S3). This is also displayed as the root mean square fluctuation (RMSF) of the sidechains of selected amino acids (Supplementary Table S3). It is observed that the RMSF of protonated Glu223 and Glu225 in setup V, protonated Glu223 and Glu248 in setup VI, protonated Glu35 and Glu248 in setup VII, and protonated Glu35, Glu223 and Glu248 in setup VIII is consistently higher than the RMSF of same residues in setup III (model system from which all the former systems were constructed, see Methods section). Similarly, a comparison of the RMSF values of protonated Glu163 and Asp72 in systems IX and X reveal larger fluctuations relative to the setup VIII. In particular, Glu223, Glu248, and Glu213, undergo flip of their sidechains, thereby providing a water-based connectivity between the latter site at the Nqo4/6/8 interface and the membrane-subunits, that is encompassing a distance of ca. 35 Å between the Q-tunnel and the first putative proton exit route at Nqo11 (Figs 5B and 7). We also note that flipping of the sidechain of protonated amino acid residues residues, Glu223 (setup VI), Glu225 (setup V), Glu163 (setup IX), Glu248 and Glu35 (setup VII) of Nqo8 subunit ( Supplementary Fig. S3, panels C and D) coincides with the changes in their pK a values ( Supplementary  Fig. S12).

Behavior of the modeled quinone (Q) molecule. Earlier based on atomistic MD simulations,
redox-dependent conformational changes in Nqo4 subunit were reported 30 . The current simulation data support these observations, but in addition to that highlights the important conformational changes that occur in the Nqo6 subunit. In the simulation setup II, which represents the state formed after redox-coupled protonation of bound Q near N2 center, Arg83 from Nqo6 subunit is found to stabilize the anionic Tyr87 in multiple independent simulations (Fig. 6). This occurs together with the dissociation of His-Asp ion-pair, and migration of His38 towards anionic Tyr87, a notion also supported by the structural data (PDB id: 4WZ7, see also ref 30).
The average RMSF of the Q head group (average of six aromatic carbon atoms of the ring) is found to be 2-3 Å when modeled at the site close to N2 center. On the other hand, when Q is modeled in the middle of the Q-tunnel, the RMSF is found to be slightly lower (0.7 to 2.0 Å). At any rate there are no large scale conformational changes in the Q behavior in these simulation time scales, except that the hydrophobic tail (Q 6 ) fluctuates, either when it is partly buried or completely buried in protein interior. In contrast to the mild conformational changes in Q, the protonation of residues (Glu223, Glu225, Glu248 from Nqo8) shows large scale changes in their sidechain movements, also with respect to the relatively fixed Q head group position (Supplementary Fig. S13).

Discussion
Fully atomistic classical MD simulations performed on the entire structure of respiratory complex I from T. thermophilus reveal rapid hydration of the membrane-bound subunits, and the Q-binding site region. Based on simulation data we find that transient water-based connections form in the antiporter-like subunits (Nqo12-14), which may play an important role in proton uptake. Interestingly, a complete S-shaped connectivity encompassing the entire antiporter-like subunits is never observed, which may be necessary to minimize proton leaks from the P-side to the N-side of the membrane 46 . It is to note that many of the amino acid residues that are proposed to form part of the proton conducting pathway are known to be critical based on site-directed mutagenesis data [24][25][26][27][28] . Furthermore, consistent with the 4 H + /2e − pumping stoichiometry of complex I and structural data, the simulation results show that the fourth proton channel emerges at the interface of Nqo10 and Nqo11 subunits. Overall,  Table 1) shows QH 2 (hydrogens not shown) close to the deprotonated acidic residues Glu248 and Glu223. Protonation of acidic residues (upon QH 2 oxidation) results in flipping of the side chains of protonated Glu223 (B), protonated Glu248 (C), and protonated Glu213 (D) in simulations setups VI, VII and IV, respectively (see also Table 1). The QH 2 molecule position is same in all panels, and is based on setup III simulation.
our results on proton transfer pathways in the antiporter type subunits, and another one at the Nqo10/11 interface, agree well with the earlier simulation study on complex I from E. coli. 31 The hydration pattern of Nqo8 subunit is particularly unique in that it reveals water-based connectivity between the Q-tunnel and the fourth proton channel at the interface of Nqo10 and Nqo11, thereby encompassing a distance of more than 30 Å. The conformational dynamics of the sidechains of acidic residues in Nqo8 subunit observed in our simulations is also partly supported by the structural data; some of these acidic residues are found to undergo sidechain (or backbone) conformational transitions, as observed in the crystal or cryo-EM structures of complex I (Supplementary Fig. S14).
The functional significance of the hydration observed in Nqo8 subunit also calls for a brief note. It is well-known that the mitochondrial analogue of Nqo8 subunit (see also Supplementary Table S1) is a 'hot-spot' for point mutations 47 . The hydration pattern and sidechain dynamics observed in our simulations strongly suggest that this region of complex I plays an important role in coupling between the Q redox chemistry and proton pumping, which when disrupted by point-mutations, may result in suboptimal complex I function, causing mitochondrial disorders.
The region close to the Q-binding site, formed by Nqo4, Nqo6 and Nqo7 subunits, hydrates rapidly. In such a scenario, it is not easy to envisage how the charge accumulated in the Q-binding cavity upon Q reduction would remain uncompensated by the bulk protons that is until the completion of a single turnover (milliseconds). Based on MD simulations of the state formed immediately after reduction-coupled protonation of Q (model system # II), we find that the anionic Tyr is stabilized by Arg83 from Nqo6. We suggest that this may be the mechanism to prevent the premature protonation of tyrosinate (deprotonated Tyr), which would result in loss of free energy of proton pumping. We note that some of the key residues (Tyr87 from Nqo4 and Arg83 from Nqo6) are only 6 Å from the N2 FeS cluster, and their dynamics and acid-base properties may be influenced by the redox/acid-base properties of the FeS cluster. Such subtle aspects, including polarization effects, are not taken into account in the current study, but may play an important role in enzyme mechanism 48 .
One of the key results from our simulations is the hydration of the region between the Q-tunnel and the proton pathway closest to it. Though this connectivity is partially visible in the crystal structures, our simulations reveal that the large gaps between the charged amino acid side chains are filled with dynamic water molecules (Fig. 5B). The observed water occupancy together with the sidechain dynamics of key residues completes the network, thereby highlighting the importance of protein hydration and dynamics in enzyme function. An earlier mechanistic proposal supported the role of Q as a dynamic electron carrier permanently trapped in the Q-tunnel 2 . The data presented here points to an additional role of trapped Q in translocating protons from the N-side to the P-side of the membrane, such that the water-protein based connectivity from Nqo8 to Nqo10/11 may be used to transfer protons released upon QH 2 oxidation, thereby supporting the viewpoint that part of the coupling may be 'direct' in nature.
The basic design principles of redox-coupled proton pumping in complex I are not known 2 . Our simulation data provide a significant advancement in this regard, and show how protein and water dynamics may be important in long-ranged electron-proton coupling in complex I. Our mechanistic proposals are tentative but testable, and call for further experimental (e.g. site-directed mutagenesis of Arg83 from Nqo6) and theoretical work to prove or disprove it.

Model system construction and simulation setup.
We performed all-atom classical MD simulations on the respiratory complex I from Thermus thermophilus (PDB ID: 4HEA) 18 . Earlier MD simulation studies 31,35 were performed on the membrane domain of the E. coli enzyme. Therefore, as a next relevant step, and in continuation to earlier work 30 , we have performed atomistic simulations on the entire structure of complex I. The model systems comprised entire complex I embedded in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) lipid bilayer, TIP3 water molecules, and Na + and Cl − ions mimicking the 100 mM salt concentration. The Q-binding site was modeled with a Q 6 molecule in two different states (fully oxidized quinone and doubly-protonated quinol, QH 2 ), as described earlier 30 . The resulting system consisted of approximately ~850000 atoms. The simulation program NAMD 49 together with the CHARMM36 force field for protein, lipids, water and ions 50, 51 were used to perform the simulations. The force field parameters for modelled Q 52 , reduced FeS clusters 53 , FMN 54 and deprotonated Tyr 55 were used as described earlier 30 . The N2 FeS cluster, which is the immediate electron donor to Q, has relatively high midpoint potential among all FeS clusters, ensuring its maximal electron occupancy 8 . For this reason, as well as for simplicity, we have modeled all FeS clusters in reduced state. It is also assumed that the redox state of FeS clusters does not influence the dynamics of water molecules in the membrane-bound subunits.
For model systems I and II, a 1000 step energy minimization was performed while keeping the protein fixed. The position of Q in the Q-tunnel was refined with short minimizations and simulations (rest of the system fixed). This was followed by an energy minimization of the entire system, and a 1 ns NVT simulation in which lipid tails were allowed to relax (protein backbone and other cofactors fixed). For production runs some atoms of the FeS clusters and FMN were kept restrained to their crystallographic positions using harmonic force constants of 50 kcal mol −1 Å −2 . The pressure (1 atm) and temperature (310 K) were controlled with the Nosé-Hoover Langevin barostat and Langevin thermostat [56][57][58] , respectively, as implemented in NAMD. A timestep of 1 fs was employed. The long-range electrostatics was treated with the use of particle mesh Ewald technique (PME) 59 , as available in NAMD.
Rationale for additional simulation setups. We observed extensive hydration in the Nqo8 subunit in the two longest simulations I and II (see also Results section, and Fig. 5A), that is closer to the residues Glu163 and Glu213. A water-based connectivity is also observed between the latter residues, and the acidic residues in the middle of the Q-tunnel (Glu248, Glu223 and Glu225 of Nqo8). In an earlier work 2 , it was proposed that a quinol (QH 2 ) molecule (formed after two-electron reduction from N2, and protonation from His38 and Tyr87) slides to another position somewhat in the middle of the Q-tunnel, where it gets oxidized to another loosely-bound Q at the membrane-protein interface. Even though there is no structural information for the proposed position in the middle of the Q-tunnel, we performed additional MD simulations (system III in Table 1) by modeling a QH 2 molecule next to the acidic residues Glu223, Glu225 and Glu248 that lie in the middle of the Q-tunnel. The latter acidic residues belong to the segment (Asp220-Lys282 of Nqo8), which has been identified as the inhibitor-binding site based on photo-affinity labeling of bovine mitochondrial complex I 60 , see also ref. 2.
We also performed additional simulations by modeling an oxidized Q at the same site, but by redistributing the two protons (released upon QH 2 oxidation) on nearby acidic residues (see Table 1). We emphasize that these residues in the middle of Nqo8 and Nqo7 subunits are known to be functionally important based on the site-directed mutagenesis data 47, 61-63 . All model systems (IV-VIII) were constructed by using the snapshot from the simulation of system III. Models III and IV are exactly the same except for the protonated Glu213 in setup IV, which was suggested to be the case in an earlier work 30 . The two protons released upon proposed oxidation of QH 2 molecule (by a loosely-bound Q) 2 are redistributed on to two nearby residues Glu223 and Glu225 (system V), on Glu223 and Glu248 (system VI) and on Glu35 and Glu248 (system VII). The system VIII is same as system VII, but with Glu213 protonated, see ref. 30.
Since the region close to Glu248 and Glu223 of Nqo8 connects to the acidic residues of Nqo7, via protonated Glu163 and Glu213 of Nqo8 and water molecules, we altered the protonation states of Asp72 of Nqo7, which has been found to be important based on site-directed mutagenesis data 64 , and constructed the systems IX and X (from setup VIII coordinates). The residue adjacent to Asp72 of Nqo7 is Glu74, and is known to be functionally not critical 64 . Also, based on the crystal structure analysis, we decided to leave Glu74 protonated in both the simulations IX and X. Assuming that Glu213 and Glu74 are protonated in the ground state of the enzyme, setup IX would represent a state in which the two protons released upon QH 2 oxidation reside on Glu163 and Asp72 of Nqo8 and Nqo7, respectively, whereas setup X would represent a scenario in which two released protons reside on Glu248 of Nqo8 and Asp72 of Nqo7, and Glu213, Glu163 and Glu74 are neutral in the ground state (see also ref. 30).
Simulation data were analysed with the visualisation program VMD 65 . The total simulation time is ca. 2.8 μs. Simulation snapshots are uploaded on the open-access research data repository Zenodo, and can be downloaded from the link (http://doi.org/10.5281/zenodo.267916). Residue numbers mentioned in Supplementary Table S1 were used for the data shown in Fig. 2. First, a set of residues were chosen based on the visual analysis of the water pathways. After this, only those polar residues were selected, which were found to be highly conserved, and functionally important based on site-directed mutagenesis data (see Introduction).
Electrostatic calculations were performed using PROPKA software (version 3.1) 66,67 to calculate the pK a of titratable residues. Simulation snapshots (every ns) comprising heavy atoms of subunits Nqo4, Nqo7, Nqo8, Nqo10, Nqo11 and Nqo12-14 were used to perform the pK a calculations. The pK a data is shown in Supplementary Table S2, and is in overall agreement with the standard protonation states considered for residues in membrane-bound subunits (setups I and II). Data Availability. The datasets (simulation snapshots) generated during the current study are available in