Electrostatic interactions between FeS clusters in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

The redox properties of the cofactors of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli were studied by following the changes in electron paramagnetic resonance (EPR) and optical spectra upon electrochemical redox titration of the purified protein. At neutral pH, the FMN cofactor had a midpoint redox potential ( E m) approximately -350 mV ( n = 2). Binuclear FeS clusters were well-characterized: N1a was titrated with a single ( n = 1) transition, and E m = -235 mV. In contrast, the titration of N1b can only be fitted with the sum of at least two one-electron Nernstian curves with E m values of -245 and -320 mV. The tetranuclear clusters can also be separated into two groups, either having a single, n = 1, or more complex redox titration curves. The titration curves of the EPR bands attributed to the tetranuclear clusters N2 ( g = 2.045 and g = 1.895) and N6b ( g = 2.089 and g = 1.877) can be presented by the sum of at least two components, each with E m (app) approximately -200/-300 mV and -235/-315 mV, respectively. The titration of the signals at g = 1.956-1.947 (N3 or N7, E m = -315 mV), g = 2.022, and g = 1.932 (Nx, -365 mV) and the low temperature signal at g = 1.929 (N4 or N5, -330 mV) followed Nernstian n = 1 curves. The observed redox titration curves are discussed in terms of intrinsic electrostatic interactions between FeS centers in complex I. A model showing shifts of E m due to the electrostatic interaction between the centers is presented.     

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.     

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.     

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.     

Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.     

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.     

Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.     

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.     

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.     

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson''s disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 10⁴ min⁻¹ for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 10³ complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.     

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.     

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I)

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.     

Immunocytochemical characterization of the mitochondrially encoded ND1 subunit of complex I (NADH : ubiquinone oxidoreductase) in rat brain

In Parkinson's disease, there is a selective defect in complex I of the electron transfer chain. To better understand complex I and its involvement in neurodegenerative disease, we raised an antibody against a conserved epitope of the human mitochondrially encoded subunit 1 of complex I (ND1). Antibodies were affinity purified and assessed by ELISA, immunoblotting, and immunocytochemistry. Immunoblots of brain homogenates from mouse, rat, and monkey brain showed a single 33-kDa band consistent with the predicted molecular mass of the protein. Subcellular fractionation showed the protein to be enriched in mitochondria. Immunocytochemistry in rat brain revealed punctate labeling in cell bodies and processes of neurons. Immunoreactively generally co-localized with subunit IV of complex IV. In striatum, ND1 immunoreactively was greatly enriched in large cholinergic neurons and neurons containing nitric oxide synthase, two cell populations that are resistant to excitotoxic and metabolic insults. In substantia nigra, many dopaminergic neurons had little ND1 immunoreactivity, which may help to explain their sensitivity to complex I inhibitors. In spinal cord, ND1 immunoreactively was enriched in motor neurons. We conclude that complex I is differentially distributed across brain regions, between neurons and glia, and between types of neurons. This antibody should provide a valuable tool for assessing complex I in normal and pathological conditions.     

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.     

Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, energy transducing, membrane-bound enzyme. It contains 46 different subunits and nine redox cofactors: a noncovalently bound flavin mononucleotide and eight iron-sulfur clusters. The mechanism of complex I is not known. Mechanistic studies using the bovine enzyme, a model for human complex I, have been precluded by the difficulty of preparing complex I which is pure, monodisperse, and fully catalytically active. Here, we describe and characterize a preparation of bovine complex I which fulfills all of these criteria. The catalytic activity is strongly dependent on the phospholipid content of the preparation, and three classes of phospholipid interactions with complex I have been identified. First, complex I contains tightly bound cardiolipin. Cardiolipin may be required for the structural integrity of the complex or play a functional role. Second, the catalytic activity is determined by the amounts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are bound to the complex. They are more weakly bound than cardiolipin, exchange with PC and PE in solution, and can substitute for one another. However, their nontransitory loss leads to irreversible functional impairment. Third, phospholipids are also required in the assay buffer for the purified enzyme to exhibit its full activity. It is likely that they are required for solubilization and presentation of the hydrophobic ubiquinone substrate.