Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and the mitochondria of most eukaryotes. In general, the bacterial complex consists of 14 different subunits. In addition to the homologues of these subunits, the mitochondrial complex contains approximately 31 additional proteins. While it was shown that the mitochondrial complex is assembled from distinct intermediates, nothing is known about the assembly of the bacterial complex. We used Escherichia coli mutants, in which the nuo-genes coding the subunits of complex I were individually disrupted by an insertion of a resistance cartridge to determine whether they are required for the assembly of a functional complex I. No complex I-mediated enzyme activity was detectable in the mutant membranes and it was not possible to extract a structurally intact complex I from the mutant membranes. However, the subunits and the cofactors of the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of some of the nuo-mutants. It is discussed whether this fragment represents an assembly intermediate. In addition, a membrane-bound fragment exhibiting NADH/ferricyanide oxidoreductase activity and containing the iron-sulfur cluster N2 was detected in one mutant.     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

A novel, enzymatically active conformation of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Electron microscopy has demonstrated the unusual L-shaped structure of the respiratory complex I consisting of two arms, which are arranged perpendicular to each other. We found that the Escherichia coli complex I has an additional stable conformation, with the two arms arranged side by side, resulting in a horseshoe-shaped structure. The structure of both conformations was determined by means of electron microscopy of gold thioglucose-stained single particles. They were distinguished from each other by titration of the complex with polyethylene glycol and by means of analytical ultracentrifugation. The transition between the two conformations is induced by the ionic strength of the buffer and is reversible. Only the horseshoe-shaped complex I exhibits enzyme activity in detergent solution, which is abolished by the addition of salt. Therefore, it is proposed that this structure is the native conformation of the complex in the membrane.     

Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichia coli is composed of 13 different subunits. The corresponding genes are organized in the nuo-operon (from NADH:ubiquinone oxidoreductase) at min 51 of the E. coli chromosome. To study the structure and function of this complex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promotor region with the phage-T7 RNA polymerase promotor and by expressing the genes with the T7 RNA polymerase coded on an inducible plasmid. It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membranes that complex I is overproduced 4-fold after induction. Complex I was isolated by chromatographic steps performed in the presence of dodecyl maltoside. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability over a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.     

Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I)

Three-dimensional structures of NADH:ubiquinone oxidoreductase (or complex I) from the respiratory chain of mitochondria and bacteria have been recently studied by electron microscopy. The low-resolution structures all reveal a characteristic L shape for complex I; however, some of the differences among these structures may have important implications for the location of the functional elements of complex I, for example, the ubiquinone-binding site.     

NADH/NAD+ interaction with NADH: ubiquinone oxidoreductase (complex I)

The quantitative data on the binding affinity of NADH, NAD(+), and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.     

Na+ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli

Following on from our previous discovery of Na+ pumping by the NADH:ubiquinone oxidoreductase (complex I) of Klebsiella pneumoniae, we show here that complex I from Escherichia coli is a Na+ pump as well. Our study object was the Escherichia coli mutant EP432, which lacks the Na+/H+ antiporter genes nhaA and nhaB and is therefore unable to grow on LB medium at elevated Na+ concentrations. During growth on mineral medium, the Na+ tolerance of E. coli EP432 was influenced by the organic substrate. NaCl up to 450 mM did not affect growth on glycerol and fumarate, but growth on glucose was inhibited. Correlated to the Na+ tolerance was an increased synthesis of complex I in the glycerol/fumarate medium. Inverted membrane vesicles catalysed respiratory Na+ uptake with NADH as electron donor. The sodium ion transport activity of vesicles from glycerol/fumarate-grown cells was 40 nmol mg-1 min-1 and was resistant to the uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), but was inhibited by the complex I-specific inhibitor rotenone. With an E. coli mutant deficient in complex I, the Na+ transport activity was low (1-3 nmol mg-1 min-1), and rotenone was without effect.     

Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.     

Iron-sulfur cluster N2 of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is located on subunit NuoB

The proton-pumping NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to 9 iron-sulfur (Fe/S) clusters participate in the redox reaction. There is discussion that the EPR-detectable Fe/S cluster N2 is involved in proton pumping. However, the assignment of this cluster to a distinct subunit of the complex as well as the number of Fe/S clusters giving rise to the EPR signal are still under debate. Complex I from Escherichia coli consists of 13 polypeptides called NuoA to N. Either subunit NuoB or NuoI could harbor Fe/S cluster N2. Whereas NuoB contains a unique motif for the binding of one Fe/S cluster, NuoI contains a typical ferredoxin motif for the binding of two Fe/S clusters. Individual mutation of all four conserved cysteine residues in NuoB resulted in a loss of complex I activity and of the EPR signal of N2 in the cytoplasmic membrane as well as in the isolated complex. Individual mutations of all eight conserved cysteine residues of NuoI revealed a variable phenotype. Whereas cluster N2 was lost in most NuoI mutants, it was still present in the cytoplasmic membranes of the mutants NuoI C63A and NuoI C102A. N2 was also detected in the complex isolated from the mutant NuoI C102A. From this we conclude that the Fe/S cluster N2 is located on subunit NuoB.     

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F(420)H(2) as electron donors. Short-chain analogues of ubiquinone like decyl-ubiquinone and ubiquinone-2 were suitable electron acceptors. The affinities towards NADH and ubiquinone-2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80 degrees C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.     

Production of reactive oxygen species by complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli and comparison to the enzyme from mitochondria

The generation of reactive oxygen species by mitochondrial complex I (NADH:ubiquinone oxidoreductase) is considered a significant cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Defining its mechanism is important for the formulation of causative connections between complex I defects and pathological effects. Oxygen is probably reduced at two sites in complex I, one associated with NADH oxidation in the mitochondrial matrix and the other associated with ubiquinone reduction in the membrane. Here, we study complex I from Escherichia coli, exploiting similarities and differences in the bacterial and mitochondrial enzymes to extend our knowledge of O2 reduction at the active site for NADH oxidation. E. coli and bovine complex I reduce O2 at essentially the same rate, with the same potential dependence (set by the NAD (+)/NADH ratio), showing that the rate-determining step is conserved. The potential dependent rate of H2O2 production does not correlate to the potential of the distal [2Fe-2S] cluster N1a in E. coli complex I, excluding it as the point of O2 reduction. Therefore, our results confirm previous proposals that O2 reacts with the fully reduced flavin mononucleotide. Assays for superoxide production by E. coli complex I were prone to artifacts, but dihydroethidium reduction showed that, upon reducing O2, it produces approximately 20% superoxide and 80% H2O2. In contrast, bovine complex I produces 95% superoxide. The results are consistent with (but do not prove) a specific role for cluster N1a in determining the outcome of O2 reduction; possible reaction mechanisms are discussed.     

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.     

The Campylobacter jejuni NADH:ubiquinone oxidoreductase (complex I) utilizes flavodoxin rather than NADH

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 Cj1575c and Cj1574c. A series of mutants was generated in which each of the 12 nuo genes (homologues to known complex I subunits) was disrupted or deleted. Each of the nuo mutants will not grow in amino acid-based medium 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 compared to the wild type. In C. jejuni, α-ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase; mutagenesis of this enzyme abolishes α-ketoglutarate-dependent O₂ uptake and fails to reduce the electron transport chain. The electron acceptor for 2-oxoglutarate:acceptor oxidoreductase was determined to be flavodoxin, which was also determined to be an essential protein in C. jejuni. A model is presented in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I.     

Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli

The proton-pumping NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. In Escherichia coli the complex is made up of 13 different subunits encoded by the so-called nuo-genes. Mutants, in which each of the nuo-genes was individually disrupted by the insertion of a resistance cartridge were unable to assemble a functional complex I. Each disruption resulted in the loss of complex I-mediated activity and the failure to extract a structurally intact complex. Thus, all nuo-genes are required either for the assembly or the stability of a functional E. coli complex I. The three subunits comprising the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of several nuo-mutants as one distinct band after BN-PAGE. It is discussed that the fully assembled NADH dehydrogenase fragment represents an assembly intermediate of the E. coli complex I. A partially assembled complex I bound to the membrane was detected in the nuoK and nuoL mutants, respectively. Overproduction of the ΔNuoL variant resulted in the accumulation of two populations of a partially assembled complex in the cytoplasmic membranes. Both populations are devoid of NuoL. One population is enzymatically active, while the other is not. The inactive population is missing cluster N2 and is tightly associated with the inducible lysine decarboxylase. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

The inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by Zn2+

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, membrane-bound enzyme. It is central to energy transduction, an important source of cellular reactive oxygen species, and its dysfunction is implicated in neurodegenerative and muscular diseases and in aging. Here, we describe the effects of Zn2+ on complex I to define whether complex I may contribute to mediating the pathological effects of zinc in states such as ischemia and to determine how Zn2+ can be used to probe the mechanism of complex I. Zn2+ inhibits complex I more strongly than Mg2+, Ca2+, Ba2+, and Mn2+ to Cu2+ or Cd2+. It does not inhibit NADH oxidation or intramolecular electron transfer, so it probably inhibits either proton transfer to bound quinone or proton translocation. Thus, zinc represents a new class of complex I inhibitor clearly distinct from the many ubiquinone site inhibitors. No evidence for increased superoxide production by zinc-inhibited complex I was detected. Zinc binding to complex I is mechanistically complicated. During catalysis, zinc binds slowly and progressively, but it binds rapidly and tightly to the resting state(s) of the enzyme. Reactivation of the inhibited enzyme upon the addition of EDTA is slow, and inhibition is only partially reversible. The IC50 value for the Zn2+ inhibition of complex I is high (10-50 microm, depending on the enzyme state); therefore, complex I is unlikely to be a major site for zinc inhibition of the electron transport chain. However, the slow response of complex I to a change in Zn2+ concentration may enhance any physiological consequences.