3.D.1 The H⁺ or Na⁺-translocating NADH Dehydrogenase (NDH) Family

NADH:ubiquinone oxidoreductases type I (NDH-Is) of bacteria and of eukaryotic mitochondria and chloroplasts couple electron transfer to the electrogenic transport of protons (Brandt, 2006) or Na⁺ (Gemperli, et al., 2007). The subunit, NuoL, is related to Na⁺/ H⁺antiporters of 2.A.63.1.1 (PhaA and PhaD). NuoL has been shown to transport Na⁺ and K⁺ independently of other Nuo subunits (Gemperli et al., 2007). These protein complexes are multi-subunit complexes with 13 or 14 recognized subunits in the E. coli or P. denitrificans complex and about 30-50 distinct subunits in the complexes of eukaryotic mitochondria.  The structures and mechanisms of respiratory chain complex I have been reviewed (Sazanov 2015).  There is a probable link between redox changes at heme a and modulation of intramolecular proton-transfer rates (Vilhjálmsdóttir et al. 2015).  Respiratory complexes I, III and IV form a complex, and it's archetecture has been studied (Gu et al. 2016). The mechanism of electron flow-coupled proton pumping may involve the semi-quinone free radical (Ohnishi et al. 2018). The mammalian complex I pumps protons within water wires using tightly linked conformational and electrostatic coupling principles (Röpke et al. 2020). The binding pocket for quinones in mitochondrial respiratory complex I is also the site of binding of several chemical inhibitors of complex I activity (Murai 2020). Damage to cerebral mitochondria, particularly opening of the mitochondrial permeability transition pore (MPTP), provides a key mechanism of ischemic brain injury; therefore, modulation of MPTP may be a target for a neuroprotective strategy in ischemic brain pathologies. Brain tissue necrosis can be prevented by the biguanides, metformin and phenformin, as well as Complex I inhibitors rotenone and amobarbital and by the MPTP inhibitor cyclosporine A (Skemiene et al. 2020). The structure of the Escherichia coli respiratory complex I, reconstituted into lipid nanodiscs, revealed an uncoupled conformation (Kolata and Efremov 2021). Transcriptomic and metabolomic analyses revealed that the potent antifungal pyrylium salt inhibits mitochondrial complex I in Candida albicans (Lv et al. 2023).  Quinone catalysis modulates proton transfer reactions in the membrane domain of respiratory complex I (Kim et al. 2023). 

Complex I contains a ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron-sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal beta-sheet of the 49-kDa subunit (Tocilescu et al., 2010). All 45 subunits of the bovine NDHI have been sequenced (Cardol et al., 2004; Gabaldon et al., 2005). Each complex contains noncovalently bound FMN, coenzyme Q and several iron-sulfur centers. The bacterial NDHs have 8-9 iron-sulfur centers. Electrons pass from NADH to FMN, to the various iron-sulfur centers, and finally to ubiquinone or another quinone. NDHI, a complex I in mitochondria, forms a 'supercomplex' with complex III (3.D.3) and complex IV (3.D.4) (Schafer et al., 2007). The mechanistic details are discussed by Hirst (2010). The interaction of mitochondrial complex I with complex III is discussed by Dudkina et al. (2009). The x-ray structures of the functional modules of the 40 subunit Yarrowia lipolytica Complex I have been examined, and the basis for conformational coupling has been presented (Hunte et al., 2010).  Moreover, a molecular mechanism of the proton pump has been proposed (Verkhovskaya and Bloch 2013). The cardiac mitochondria respirasome is adapted for the β-oxidation of fatty acids (Panov 2024).

The positions of all iron-sulfur clusters relative to the membrane arm were determined in the complete enzyme complex. The ubiquinone reduction site resides close to 30 Å above the membrane domain. The arrangement of functional modules suggests conformational coupling of redox chemistry with proton pumping and essentially excludes direct mechanisms. (Hunte et al., 2010) suggest that a ~60 Å long helical transmission-element is critical for transducing conformational energy to proton-pumping elements in the distal module of the membrane arm.  The x-ray structures of various complexes have been solved, and a coupling mechanism involving long range conformational changes has been proposed (Sazanov et al. 2013).  Thjere are several assembly proteins for complex 1, and NDUFAF6 (333 aas; Q3300K2) plays important roles in complex I assembly and mitochondrial disease (Sung et al. 2024).

The mammalian enzyme complexes contain several water soluble peripheral membrane proteins which are anchored to the integral membrane constituents. The seven most hydrophobic proteins of the complex are encoded within mammalian or fungal mitochondrial genomes while the remainder are nuclearly encoded (Brandt, 2006; Gray et al., 2004). All thirteen of the E. coli proteins, which comprise NADH dehydrogenase I and are encoded within the nuo operon, are homologous to mitochondrial complex I subunits. Fearnley and Walker (1992) have provided multiple alignments and hydropathy plots for many of the NDH complex subunits, but phylogenetic analyses are not included. NDH-I complexes contrast with NDH-II complexes that do not transport H⁺ or Na⁺. Nelfinavir, and anticancer drug, induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport via metabolic rewiring and activation of the unfolded protein response (Besse et al. 2021).

Efremov et al. (2010) have determined the structure of the E. coli NDH-I transmembrane structure at 3.9 A resolution. The antiporter-like subunits NuoL/M/N each contains 14 conserved transmembrane (TM) helices. Two of them are discontinuous, but subunit NuoL contains a 110-A long amphipathic α-helix, spanning the entire length of the domain. The structure of the entire complex I from Thermus thermophilus at 4.5 A resolution reveals an L-shaped assembly with 63 TM helices. The architecture of the complex suggests that the conformational changes at the interface of the two main domains drives the long amphipathic α-helix of NuoL in a piston-like motion, tilting nearby discontinuous TM helices, resulting in proton translocation (Efremov et al., 2010). This work has been reviewed and evaluated (Efremov and Sazanov, 2011b). Terminal respiratory oxidases allow microorganisms to adapt to low-oxygen conditions, survive in chemically aggressive environments and acquire antibiotic resistance (Siletsky and Borisov 2021). Three-dimensional structures with atomic resolution of members of all major groups of terminal respiratory oxidases, heme-copper oxidases, and bd-type cytochromes, have been obtained. These groups of enzymes have different origins, but all of them are united by a catalytic reaction of four-electron reduction in oxygen into water which proceeds without the formation and release of potentially dangerous ROS from active sites. The review by Siletsky and Borisov 2021 analyzes structural and functional studies of oxygen reduction intermediates in the active sites of terminal respiratory oxidases, the features of catalytic cycles, and the properties of the active sites of these enzymes.

The NDH family includes complexes in bacteria, archaea and eukaryotic organelles (mitochondria and chloroplasts) that may use different electron donors. Thus, the bacterial and mitochondrial complexes function as NADH dehydrogenases, but some of the archaeal complexes function as F₄₂₀H₂ dehydrogenases (see TC# 3.D.9). The electron donor(s) for the cyanobacterial and plastid complexes is (are) not yet known. Despite the potentially different electron input sites, eleven polypeptide chains constitute the structural framework for proton translocation and quinone binding. At least six of these subunits are also present in a family of membrane-bound multisubunit [NiFe] hydrogenases. One of these hydrogenases (from Paracoccus furiosus) has been shown to couple electron transfer to H⁺ translocation (Friedrich and Scheide, 2000; Sapra et al., 2003). In E. coli these hydrogenases are hydrogenases 3 and 4 of the formate hydrogen lyase system. They also include the CO-induced hydrogenase of Rhodospirillum rubrum and the Ech hydrogenase of Methanosarcina barkerei. The archaeal hydrogenase transfers reducing equivalents generated by the oxidation of the low potential electron donor ferridoxin to protons, yielding H₂, coupled to the generation of a pmf (Sapra et al., 2003). Several archaea have two such enzymes of unknown physiological function.

Campylobacter jejuni encodes 12 of the 14 subunits that make up the respiratory NADH ubiquinone oxidoreductase (complex I). The two nuo genes not present in C. jejuni encode the NADH dehydrogenase, and in their place in the operon are the novel genes designated as Cj1575c (CAL25672) and Cj1574c (CAL35671; 24% identical and 41% similar (e^-5) to ORF1 of plasmid pIP501 (3.A.7.14.1)). Mutants were generated in which each of the 12 nuo genes (homologues to known complex I subunits) were disrupted or deleted (Weerakoon and Olson, 2008). Each of the nuo mutants do not grow in amino acid-based media unless supplemented with an alternative respiratory substrate such as formate. Unlike the nuo genes, Cj1574c is an essential gene and could not be disrupted unless an intact copy of the gene was provided at an unrelated site on the chromosome. A nuo deletion mutant can efficiently respire formate but is deficient in α-ketoglutarate respiratory activity when compared to wild type cells. In C. jejuni, α-ketoglutarate respiration is mediated by 2-oxoglutarate:acceptor oxidoreductase. Mutagenesis of this enzyme abolished α-ketoglutarate-dependent O₂ uptake and failed to reduce the electron transport chain. The electron acceptor for this 2-oxoglutarate oxidoreductase was determined to be flavodoxin, which was also found to be an essential protein in C. jejuni. A model was presented by Weerakoon and Olson (2008) in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I to ubiquinone.

Oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na⁺ uptake, indicating that a redox-driven, primary Na⁺ pump is operative in the inner mitochondrial membrane. A respiratory NADH dehydrogenase couples NADH-dependent reduction of ubiquinone to Na⁺ translocation. NADH-driven Na⁺ transport was sensitive rotenone, a specific inhibitor of complex I (Lin et al., 2008).

E. coli complex I (NADH dehydrogenase) is capable of proton translocation in the same direction to the established Δψ, showing that in the tested conditions, the coupling ion is H⁺ (Batista and Pereira 2011). Na⁺ transport in the opposite direction was observed, and although Na⁺ was not necessary for the catalytic or proton transport activities, its presence increased the latter. H⁺ was translocated by the P. denitrificans complex I, but in this case, H⁺ transport was not influenced by Na⁺, and Na⁺ transport was not observed. Possibly, the E. coli complex I has two energy coupling sites (one Na⁺ independent and the other Na⁺ dependent), as observed for the Rhodothermus marinus complex I, whereas the coupling mechanism of the P. denitrificans enzyme is completely Na⁺ independent. It is also possible that another transporter catalyzes the uptake of Na⁺.  Complex I energy transduction by proton pumping may not be exclusive to the R. marinus enzyme. The Na⁺/H⁺ antiport activity seems not to be a general property of complex I (Batista and Pereira 2011). 

The NADH-driven respiratory Na⁺ pump in Klebsiella pneumoniae was first reported by Peter Dimroth and co-workers (Steffen and Steuber 2013). The 3D structures of complex I from different organisms support the idea that the mechanism of cation transport by complex I involves conformational changes of the membrane-bound NuoL, NuoM and NuoN subunits. In vitro methods to follow Na⁺ transport were compared with in vivo approaches to test whether complex I, or its individual NuoL, NuoM or NuoN subunits, extrude Na⁺ from the cytoplasm to the periplasm of bacterial host cells. A truncated NuoL subunit of the E. coli complex which comprises amino acids 1-369 exhibits Na⁺ transport activity in vitro. This observation, together with an analysis of putative cation channels in NuoL, suggests that there exists in NuoL at least one continuous pathway for cations lined by amino acid residues from transmembrane segments 3, 4, 5, 7 and 8. Finally, Na⁺ transport by mitochondrial complex I has been discussed with respect to its possible role in the cycling of Na⁺ across the inner mitochondrial membrane (Steffen and Steuber 2013).

Three of the conserved, membrane-bound subunits in NADH dehydrogenase are related to each other, and to Mrp sodium-proton antiporters. Structural analysis of two prokaryotic complexes I revealed that the three subunits each contain fourteen transmembrane helices that overlay in structural alignments: the translocation of three protons may be coordinated by a lateral helix connecting them together (Efremov et al., 2010). The architecture of respiratory complex I. Nature 465, 441-447). Birrel & Hirst (2010) showed that in higher metazoans the threefold symmetry is broken by the loss of three helices from subunit ND2.

M. mazei and M. barkeri belong to the group of aceticlastic methanogens and converts acetate into the potent greenhouse gases CO₂ and CH₄. The aceticlastic respiratory chain involved in methane formation comprises the three transmembrane proteins Ech hydrogenase, F(420) nonreducing hydrogenase and heterodisulfide reductase. All three contribute to the proton motive force. ATP synthesis was observed in a cytoplasm-free vesicular system that was dependent on the oxidation of reduced ferredoxin and the formation of molecular hydrogen (Welte et al., 2010). ATP formation was not observed in a deletion mutant strain. Proton protonophores led to complete inhibition of ATP formation without inhibiting hydrogen production, whereas sodium ion ionophores did not affect ATP formation. Welte et al. (2010) concluded that the Ech hydrogenase complex acts as a primary proton pump in a ferredoxin-dependent electron transport system.

Efremov and Sazanov (2011) have reported the crystal structure of the membrane domain of complex I at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.

The architecture of the hydrophobic region of complex I shows multiple proton transporters that are mechanically interlinked. Transduction of conformational changes to drive the transmembrane transporters linked by a 'connecting rod' during the reduction of ubiquinone (Q) can account for two or three of the four protons pumped per NADH oxidized. The remaining proton must be pumped by direct coupling at the Q-binding site. Treberg et al. (2011) proposed direct and indirect coupling mechanisms to account for the pumping of the four protons.

The first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3 A resolution has been reported (Baradaran et al. 2013). The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron-sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron-sulphur cluster N2. The chamber is linked to the fourth channel by a 'funnel' of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle (Baradaran et al., 2013). 

The x-ray structure of mitochondrial complex I has been solved at a resolution of 3.6 to 3.9 angstroms, describing in detail the central subunits that execute the bioenergetic function (Zickermann et al. 2015). A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the 'deactive' form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site, providing support for a two-state stabilization-change mechanism of proton pumping (Zickermann et al. 2015).  The largest membrane-embedded subunits, ND5, ND4, and ND2, share a similar core of 14 TMSs with two repeats of five TMHs (A, TMH4–8; B, TMH9–13) in inverted topology. Each repeat features a discontinuous helix (TMH7a/b, TMH12a/b). Such helices are hallmarks of ion-translocating membrane proteins (Zickermann et al. 2015). Indeed, ND5, ND4, and ND2 are homologous to the Mrp Na⁺/H⁺ antiporter family, which suggests a role in proton pumping (Zickermann et al. 2015). ND5 has a C-terminal extension with a lateral helix, >60 Å long, lining ND5, ND4, and ND2 on the concave side of the arm close to the matrix side. The C terminus of ND5 is anchored to ND2 via a V-shaped arrangement of TMSs 16 and 17. The lateral helix was previously identified in the low-resolution analysis of mitochondrial complex I from Y. lipolytica, and is also present in the bacterial and bovine complexes in which its C terminus is anchored by one TMS only.  

Berrisford et al. 2016; have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and more recently of the intact, entire complex I from T. thermophilus (536 kDa, 16 subunits, 9 iron- sulphur clusters, 64 transmembrane helices). The 95 Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. 

Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Letts et al. 2016 presented the architectures of mammalian (ovine) supercomplexes determined by cryo-electron microscopy. The former group identified two distinct arrangements of supercomplex CICIII2CIV (the respirasome)-a major 'tight' form and a minor 'loose' form (resolved at the resolution of 5.8 A and 6.7 A, respectively), which may represent different stages in supercomplex assembly or disassembly.They also determined an architecture of supercomplex CICIII2 at 7.8 A resolution. All observed density could be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production (Letts et al. 2016). Gu et al. 2016 reported the 5.4 Å structure of the 1.7 MDa respirasome from pig heart.  The CIII dimer as well as CIV bind to the same side of the L-shaped CI with their TMSs aligned to form a transmembrane disk. Complexes I, III and IV, comprising the respirasome, have been solved by cryo-EM, showing the architecture of the respirasome with near-atomic detail. ATP synthase occurs as dimers in the inner membrane, which by its curvature is responsible for the folding of the membrane into cristae. This allows for a huge increase in available surface area that makes mitochondria the efficient energy organelles of the eukaryotic cell (Sousa et al. 2018).

Fiedorczuk et al. 2016 identified All 14 conserved core subunits and 31 mitochondrial supernumerary subunits within the L-shaped molecule. The hydrophilic matrix arm includes flavin mononucleotide and 8 iron-sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are probably involved in inter-subunit interactions. The two different conformations of the complex, may be related to the conformationally driven coupling mechanism and to the active-deactive transition of the enzyme.

In Prevotella bryantii, the Na⁺-translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction (Hau et al. 2023). P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. Hau et al. 2023 presented 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking revealed the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. These findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.

The generalized transport reactions catalyzed by NDH family members are:

NADH + ubiquinone + 4H⁺ (in) → NAD⁺ + ubiquinol + 4H⁺ (out)

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