Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 μmol NADH min⁻¹ mg⁻¹ were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.     

Superoxide radical formation by pure complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica

Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I (NADH:ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex I from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.     

Proton pumping by complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes

The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of DeltapH and Deltapsi. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-beta-D-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,DeltapH and Deltapsi, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na+ translocation by Y. lipolytica complex I.     

Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.     

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.     

Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.     

Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase)

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.     

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.     

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.     

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.     

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.     

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.     

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.