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. 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).
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 Vibrio cholerae homologue has been detergent solubilized, affinity chromatographically purified, reconstituted in liposomes and characterized (Barquera et al., 2002). The purified enzyme complex includes the six subunits encoded within the nqr operon and has a H+-dependent turnover number of 720 electrons per second. It pumps H+ and generates a membrane potential, ΔΨ. It contains one ubiquinone, covalently bound flavin linked to subunits NqrB and NqrC, and at least four redox centers including at least three flavins and a 2Fe-2S center. The Klebsiella and E. coli complexes probably also transport H+ (Gemperli et al., 2003). 4H+ are believed to be pumped per NADH oxidized by the mitochondrial complex. The mechanistic relationship between these two complexes is not clear.
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+.
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).
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 enzyme NADH ubiquinone oxidoreductase (also called 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 the enzyme 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 2-oxoglutarate:acceptor oxidoreductase was determined to be flavodoxin, which was also found to be an essential protein in C. jejuni. A model is 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+. 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. Complex I energy transduction by proton pumping and Na+/H+ antiport activity is apparently not exclusive to the R. marinus enzyme. The Na+/H+ antiport activity seems not to be a general property of complex I.
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 generalized transport reactions catalyzed by NDH family members are:
NADH + ubiquinone + 4H+ (in) → NAD+ + ubiquinol + 4H+ (out)