Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Binding of malate dehydrogenase and NADH channelling to complex I

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.     

Exploring the Catalytic Core of Complex I by Yarrowia lipolytica Yeast Genetics

We have developed Yarrowia lipolytica as a model system to study mitochondrial complex I that combines the application of fast and convenient yeast genetics with efficient structural and functional analysis of its very stable complex I isolated by his–tag affinity purification with high yield. Guided by a structural model based on homologies between complex I and [NiFe] hydrogenases mutational analysis revealed that the 49 kDa subunit plays a central functional role in complex I. We propose that critical parts of the catalytic core of complex I have evolved from the hydrogen reactive site of [NiFe] hydrogenases and that iron–sulfur cluster N2 resides at the interface between the 49 kDa and PSST subunits. These findings are in full agreement with the semiquinone switch mechanism according to which coupling of electron and proton transfer in complex I is achieved by a single integrated pump comprising cluster N2, the binding site for substrate ubiquinone, and a tightly bound quinone or quinoid group.     

Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I)

The increasing availability of sequenced genomes enables the reconstruction of the evolutionary history of large protein complexes. Here, we trace the evolution of NADH:ubiquinone oxidoreductase (Complex I), which has increased in size, by so-called supernumary subunits, from 14 subunits in the bacteria to 30 in the plants and algae, 37 in the fungi and 46 in the mammals. Using a combination of pair-wise and profile-based sequence comparisons at the levels of proteins and the DNA of the sequenced eukaryotic genomes, combined with phylogenetic analyses to establish orthology relationships, we were able to (1) trace the origin of six of the supernumerary subunits to the alpha-proteobacterial ancestor of the mitochondria, (2) detect previously unidentified homology relations between subunits from fungi and mammals, (3) detect previously unidentified subunits in the genomes of several species and (4) document several cases of gene duplications among supernumerary subunits in the eukaryotes. One of these, a duplication of N7BM (B17.2), is particularly interesting as it has been lost from genomes that have also lost Complex I proteins, making it a candidate for a Complex I interacting protein. A parsimonious reconstruction of eukaryotic Complex I evolution shows an initial increase in size that predates the separation of plants, fungi and metazoa, followed by a gradual adding and incidental losses of subunits in the various evolutionary lineages. This evolutionary scenario is in contrast to that for Complex I in the prokaryotes, for which the combination of several separate, and previously independently functioning modules into a single complex has been proposed.     

Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction

The effects of Tinopals (cationic benzoxazoles) AMS-GX and 5BM-GX on NADH-oxidase, NADH:ferricyanide reductase, and NADH APAD⁺ transhydrogenase reactions and energy-linked NAD⁺ reduction by succinate, catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in submitochondrial particles (SMP), were investigated. AMS-GX competes with NADH in NADH-oxidase and NADH:ferricyanide reductase reactions (K _i = 1 M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K _i = 5 M) mechanism. Neither compound affects reverse electron transfer from succinate to NAD⁺. The type of the Tinopals' effect on the NADH APAD⁺ transhydrogenase reaction, occurring with formation of a ternary complex, suggests the ordered binding of nucleotides by the enzyme during the reaction: AMS-GX and 5BM-GX inhibit this reaction uncompetitively just with respect to one of the substrates (APAD⁺ and NADH, correspondingly). The competition between 5BM-GX and APAD⁺ confirms that NADH is the first substrate bound by the enzyme. Direct and reverse electron transfer reactions demonstrate different specificity for NADH and NAD⁺ analogs: the nicotinamide part of the molecule is significant for reduced nucleotide binding. The data confirm the model suggesting that during NADH APAD⁺ reaction, occurring with ternary complex formation, reduced nucleotide interacts with the center participating in NADH oxidation, whereas oxidized nucleotide reacts with the center binding NAD⁺ in the reverse electron transfer reaction.     

Identification and characterization two isoforms of NADH:ubiquinone oxidoreductase from the hyperthermophilic eubacterium Aquifex aeolicus

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain and the entry point for most electrons. Generally, the bacterial complex I consists of 14 core subunits, homologues of which are also found in complex I of mitochondria. In complex I preparations from the hyperthermophilic bacterium Aquifex aeolicus we have identified 20 partially homologous subunits by combining MALDI-TOF and LILBID mass spectrometry methods. The subunits could be assigned to two different complex I isoforms, named NQOR1 and NQOR2. NQOR1 consists of subunits NuoA₂, NuoB, NuoD₂, NuoE, NuoF, NuoG, NuoI₁, NuoH₁, NuoJ₁, NuoK₁, NuoL₁, NuoM₁ and NuoN₁, with an entire mass of 504.17?kDa. NQOR2 comprises subunits NuoA₁, NuoB, NuoD₁, NuoE, NuoF, NuoG, NuoH₂, NuoI₂, NuoJ₁, NuoK₁, NuoL₂, NuoM₂ and NuoN₂, with a total mass of 523.99?kDa. Three Fe-S clusters could be identified by EPR spectroscopy in a preparation containing predominantly NQOR1. These were tentatively assigned to a binuclear center N1, and two tetranuclear centers, N2 and N4. The redox midpoint potentials of N1 and N2 are ?273?mV and ?184?mV, respectively. Specific activity assays indicated that NQOR1 from cells grown under low concentrations of oxygen was the more active form. Increasing the concentration of oxygen in the bacterial cultures induced formation of NQOR2 showing the lower specific activity.     

Respiratory Complex I: Structure, Redox Components, and Possible Mechanisms of Energy Transduction

Structural arrangements and properties of redox components of the mitochondrial and bacterial proton-translocating NADH:quinone oxidoreductases are briefly described. A model for the mechanism of proton translocation at first coupling site, which emphasizes participation of specifically Complex I-associated ubisemiquinones, is discussed. An alternative mechanism is proposed where all redox reactions take place in a hydrophilic part of the enzyme and the free energy accumulated as conformational constraint drives the proton pump associated with the hydrophobic polypeptides.     

On Complex I and Other NADH:Ubiquinone Reductases of Neurospora crassa Mitochondria

The mitochondrial complex I is the first component of the respiratory chain coupling electron transfer from NADH to ubiquinone to proton translocation across the inner membrane of the organelle. The enzyme from the fungus Neurospora crassa is similar to that of other organisms in terms of protein and prosthetic group composition, structure, and function. It contains a high number of polypeptide subunits of dual genetic origin. Most of its subunits were cloned, including those binding redox groups. Extensive gene disruption experiments were conducted, revealing many aspects of the structure, function, and biogenesis of complex I. Complex I is essential for the sexual phase of the life cycle of N. crassa, but not for the asexual stage. In addition to complex I, the fungal mitochondria contain at least three nonproton-pumping alternative NAD(P)H dehydrogenases feeding electrons to the respiratory chain from either matrix or cytosolic substrates.     

Kinetics of the transhydrogenase reaction catalyzed by mitochondrial NADH:ubiquinone oxidoreductase (complex I)

The kinetics of the NADH3'-acetylpyridine adenine dinucleotide (APAD⁺) transhydrogenase reaction (DD-reaction) catalyzed by different preparations of mitochondrial NADH-dehydrogenase (submitochondrial particles (SMP), purified Complex I, and three-subunit fragment of Complex I (FP)) have been studied. Complex I (in SMP or in purified preparation) catalyzes two NADHAPAD⁺ reactions with different rates and nucleotide affinities. Reaction 1 has high affinity to APAD⁺ (K _m = 7 M, for SMP) and low rate (V _m = 0.2 mol/min per mg protein, for SMP) and occurs with formation of a ternary complex. Reaction 2 has much higher rate and considerably lower affinity for oxidized nucleotide (V _m = 1.7 mol/min per mg protein and K _m = 160 M, for SMP). FP catalyzes only reaction 1. ADP-ribose inhibits reaction 1 with mixed type inhibition (competitive with non-competitive) with respect to NADH and APAD⁺. Rhein competes with both substrates. The results suggest that at least two nucleotide-binding sites exist in Complex I.     

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200?Å wide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.     

Contribution of the Phosphorylable Complex I in the Growth Phase-Dependent Respiration of C6 Glioma Cells in Vitro

The energy metabolism of rat C6 glioma cells was investigated as a function of the growth phases. Three-dimensional cultures of C6 cells exhibited diminished respiration and respiratory capacity during the early growth phase, before reaching confluence. This decrease in respiration was neither due to changes in the respiratory complex content nor in the mitochondrial mass per se. Nevertheless, a quantitative correlation was found between cellular respiration and the rotenone-sensitive NADH ubiquinone oxidoreductase (i.e. complex I) activity. Immunoblot analysis showed that phosphorylation of the 18 kDa-subunit of this complex was associated with the growth-phase dependent modulation of complex I and respiratory activity in C6 cells. In addition, by using forskolin or dibutyryl cAMP, short-term activation of protein kinases A of C6 cells correlated with increased phosphorylation of the 18-kDa subunit of complex I, activated NADH ubiquinone oxidoreductase activity and stimulated cellular respiration. These findings suggest that complex I of C6 glioma cells is a key regulating step that modulates the oxidative phosphorylation capacity during growth phase transitions.     

Amphipathic C-terminal region of Escherichia coli NADH dehydrogenase-2 mediates membrane localization

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein. Bioinformatics approaches suggested the involvement of NDH-2 C-terminal region in membrane anchorage. Here, we demonstrated that NDH-2 is a peripheral membrane protein and that its predicted C-terminal amphipathic Arg390-Ala406 helix is sufficient to bind the protein to lipid membranes. Additionally, a cytosolic NDH-2 protein (Trun-3), lacking the last 43 aminoacids, was purified and characterized. FAD cofactor was absent in purified Trun-3. Upon the addition of FAD, Trun-3 maximum velocity was similar to native NDH-2 rate with ferricyanide and MTT acceptors. However, Trun-3 activity was around 5-fold lower with quinones. No significant difference in K_m values was observed for both enzymes. For the first time, an active and water soluble NDH-2 was obtained, representing a major improvement for structural/functional characterizations.     

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, nad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)⁺ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this nad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.Communicated by R. G. Herrmann     

The nuclear encoded subunits of complex I from bovine heart mitochondria

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, Iα, Iβ and Iλ, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.     

Proton Pumping of Mitochondrial Complex I: Differential Activation by Analogs of Ubiquinone

As part of the ongoing studies aimed at elucidating the mechanism of the energy conserving function of mitochondrial complex I, NADH: ubiquinone (Q) reductase, we have investigated how short-chain Q analogs activate the proton pumping function of this complex. Using a pH-sensitive fluorescent dye we have monitored both the extent and initial velocity of proton pumping of complex I in submitochondrial particles. The results are consistent with two sites of interaction of Q analogs with complex I, each having different proton pumping capacity. One is the physiological site which leads to a rapid proton pumping and a stoichiometric consumption of NADH associated with the reduction of the most hydrophobic Q analogs. Of these, heptyl-Q appears to be the most efficient substrate in the assay of proton pumping. Q analogs with a short-chain of less than six carbons interact with a second site which drives a slow proton pumping activity associated with NADH oxidation that is overstoichiometric to the reduced quinone acceptor. This activity is also nonphysiological, since hydrophilic Q analogs show little or no respiratory control ratio of their NADH:Q reductase activity, contrary to hydrophobic Q analogs.     

Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a 1T deletion in the 3' UTR of nd5 whereas dum17 is a 1T deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160-210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity.     

Human mitochondrial complex I assembly: A dynamic and versatile process

One can but admire the intricate way in which biomolecular structures are formed and cooperate to allow proper cellular function. A prominent example of such intricacy is the assembly of the five inner membrane embedded enzymatic complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system, which involves the stepwise combination of > 80 subunits and prosthetic groups encoded by both the mitochondrial and nuclear genomes. This review will focus on the assembly of the most complicated OXPHOS structure: complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Recent studies into complex I assembly in human cells have resulted in several models elucidating a thus far enigmatic process. In this review, special attention will be given to the overlap between the various assembly models proposed in different organisms. Complex I being a complicated structure, its assembly must be prone to some form of coordination. This is where chaperone proteins come into play, some of which may relate complex I assembly to processes such as apoptosis and even immunity.     

Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging

Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the 3D structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by 3D electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions.     

Complex I: A Chimaera of a Redox and Conformation-Driven Proton Pump?

From phylogenetic sequence analysis, it can be concluded that the proton-pumping NADH:ubiquinone oxidoreductase (complex I) has evolved from preexisting modules for electron transfer and proton translocation. It is built up by a peripheral NADH dehydrogenase module, an amphipatic hydrogenase module, and a membrane-bound transporter module. These modules, or at least part of them, are also present in various other bacterial enzymes. It is assumed that they fulfill a similar function in complex I and related enzymes. Based on the function of the individual modules, it is possible to speculate about the mechanism of complex I. The hydrogenase module might work as a redox-driven proton pump, while the transporter module might act as a conformation-driven proton pump. This implies that complex I contains two energy-coupling sites. The NADH dehydrogenase module seems to be involved in electron transfer and not in proton translocation.