cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.     

Benzothiadiazole inhibits mitochondrial NADH:ubiquinone oxidoreductase in tobacco

An inducer of acquired disease resistance in plants, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester, exhibited direct, concentration-dependent inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain of cultured tobacco cells. The complex I activity was less sensitive to inhibition by salicylic acid, an endogenous activator of acquired disease resistance. Using a dichlorodihydrofluorescein assay, it was found that benzothiadiazole, salicylic acid and the complex I inhibitor rotenone, increased reactive oxygen species production within cells in a concentration-dependent manner. The results indicate that both benzothiadiazole and salicylic acid affect the mitochondria of treated plant cells and result in increased production of reactive oxygen species. The biochemical basis of this response could be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I that results in channelling of electrons via complex II, with concomitant higher levels of superoxide production.     

Respiratory chain analysis of skin fibroblasts in mitochondrial disease

NADH:ubiquinone dehydrogenase (complex I) deficiency can be diagnosed from cultured skin fibroblasts using a number of methods, the most commonly used is a linked assay of rotenone-sensitive complex I + III activity (NADH:cytochrome c reductase). Because of interference from diaphorases, this method requires either the isolation of mitochondria (or at least partial purification). For a suitable mitochondrial preparation from skin fibroblasts, this requires the culturing of 4-20 individual 100mm tissue culture plates, depending on the purity of preparation required. These assays are therefore time-consuming, and do not assist in a rapid diagnosis. There is also no clear demarkation between the normal range of activity and the deficient range since mild mutations can produce only partial decreases in complex I activity. Equally, assaying patient cells that do not have a specific deficiency may prove to be time-wasting in the process of providing a quick, definitive clinical diagnosis. The lactate/pyruvate ratio of fibroblasts has been used to indicate the extent of respiratory chain involvement, as cells with a metabolic defect usually produce more lactate with an increased ratio from 25:1 to much higher values [Methods Enzymol. 264 (1996) 454]. This measurement may not always be conclusive, as the values can fluctuate as a result of culture medium, cell passage number, cell number and viability. In this report, we evaluate the use of pyruvate oxidation measurements from whole cells prepared from a single plate of cultured fibroblasts as an alternative to lactate/pyruvate ratios, or other methods both direct and indirect as indicators of the extent of respiratory chain involvement and the possibility of a defect within complex I. Whole cell 2-14C pyruvate oxidation appears to indicate the presence of a complex I defect in patients compared to normal controls more reliably than L/P ratios, but this has some puzzling exceptions.     

Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.     

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 design and synthesis of novel inhibitors of NADH:ubiquinone oxidoreductase

Potent new inhibitors of NADH:ubiquinone oxidoreductase (complex I) have been designed, with the help of molecular modelling, by hybridisation of known complex I inhibitors with inhibitors of cytochrome c oxidoreductase. The most interesting compound was the chromone 7 which was a selective inhibitor of complex I (IC(50) 15 nM) and showed acaricidal activity against spider mites.     

Superoxide production is inversely related to complex I activity in inherited complex I deficiency

Deficiency of NADH:ubiquinone oxidoreductase or complex I (CI) is the most common cause of disorders of the oxidative phosphorylation system in humans. Using life cell imaging and blue-native electrophoresis we quantitatively compared superoxide production and CI amount and activity in cultured skin fibroblasts of 7 healthy control subjects and 21 children with inherited isolated CI deficiency. Thirteen children had a disease causing mutation in one of the nuclear-encoded CI subunits, whereas in the remainder the genetic cause of the disease is not yet established. Superoxide production was significantly increased in all but two of the patient cell lines. An inverse relationship with the amount and residual activity of CI was observed. In agreement with this finding, rotenone, a potent inhibitor of CI activity, dose-dependently increased superoxide production in healthy control cells. Also in this case an inverse relationship with the residual activity of CI was observed. In sharp contrast, however, rotenone did not decrease the amount of CI. The data presented show that superoxide production is increased in inherited CI deficiency and that this increase is primarily a consequence of the reduction in cellular CI activity and not of a further leakage of electrons from mutationally malformed complexes.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Lack of assembly of mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase and loss of enzyme activity in a human cell mutant lacking the mitochondrial ND4 gene product.

In most eukaryotic cells, the respiratory chain NADH dehydrogenase (Complex I) is a multimeric enzyme under dual (nuclear and mitochondrial) genetic control. Several genes encoding subunits of this enzyme have been identified in the mitochondrial genome from various organisms, but the functions of these subunits are in most part unknown. We describe here a human cell line in which the enzyme lacks the mtDNA-encoded subunit ND4 due to a frameshift mutation in the gene. In this cell line, the other mtDNA-encoded subunits fail to assemble, while at least some of the nuclear-encoded subunits involved in the redox reactions appear to be assembled normally. In fact, while there is a complete loss of NADH:Q1 oxidoreductase activity, the NADH:Fe(CN)6 oxidoreductase activity is normal. These observations provide the first clear evidence that the ND4 gene product is essential for Complex I activity and give some insights into the function and the structural relationship of this polypeptide to the rest of the enzyme. They are also significant for understanding the pathogenetic mechanism of the ND4 gene mutation associated with Leber's hereditary optic neuropathy.     

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.     

AIF suppresses chemical stress-induced apoptosis and maintains the transformed state of tumor cells

Apoptosis-inducing factor (AIF) exhibits reactive oxygen species (ROS)-generating NADH oxidase activity of unknown significance, which is dispensable for apoptosis. We knocked out the aif gene in two human colon carcinoma cell lines that displayed lower mitochondrial complex I oxidoreductase activity and produced less ROS, but showed increased sensitivity to peroxide- or drug-induced apoptosis. AIF knockout cells failed to form tumors in athymic mice or grow in soft agar. Only AIF with intact NADH oxidase activity restored complex I activity and anchorage-independent growth of aif knockout cells, and induced aif-transfected mouse NIH3T3 cells to form foci. AIF knockdown in different carcinoma cell types resulted in lower superoxide levels, enhanced apoptosis sensitivity and loss of tumorigenicity. Antioxidants sensitized AIF-expressing cells to apoptosis, but had no effect on tumorigenicity. In summary, AIF-mediated resistance to chemical stress involves ROS and probably also mitochondrial complex I. AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function. On both counts, AIF represents a novel type of cancer drug target.     

Lambda Red-mediated mutagenesis and efficient large scale affinity purification 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. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.     

Inhibition of mitochondrial NADH:ubiquinone oxidoreductase by ethoxyformic anhydride

The NADH:ubiquinone, but not the NADH:ferricyanide, reductase activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) is inhibited by incubation of the enzyme at pH 6.0 and 0 degree C with ethoxyformic anhydride (EFA), and the inhibition is partially reversed by subsequent incubation of EFA-treated complex I with hydroxylamine. These results and spectral changes of EFA-treated complex I in the u.v. region are consistent with modification of essential histidyl or tyrosyl residues between the primary NADH dehydrogenase and the site of ubiquinone reduction. Treatment of complex I with EFA in the presence of high concentrations of Seconal or Demerol did not protect against EFA inactivation, suggesting that the site of EFA modification may not be the same as the inhibiton sites of Seconal and Demerol. However, the presence of NADH during incubation of complex I with EFA greatly enhanced the inhibition rate, indicating that the reduced conformation of complex I is more susceptible to attack by EFA.     

Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.     

C6ORF66 is an assembly factor of mitochondrial complex I

Homozygosity mapping was performed in five patients from a consanguineous family who presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (complex I) deficiency. This resulted in the identification of a missense mutation in a conserved residue of the C6ORF66 gene, which encodes a 20.2 kDa mitochondrial protein. The mutation was also detected in a patient who presented with antenatal cardiomyopathy. In muscle of two patients, the levels of the C6ORF66 protein and of the fully assembled complex I were markedly reduced. Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. Interestingly, the C6ORF66 gene product was previously shown to promote breast cancer cell invasiveness.     

The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.     

Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae

A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.     

Mitigation of NADH: ubiquinone oxidoreductase deficiency by chronic Trolox treatment

Deficiency of mitochondrial NADH:ubiquinone oxidoreductase (complex I), is associated with a variety of clinical phenotypes such as Leigh syndrome, encephalomyopathy and cardiomyopathy. Circumstantial evidence suggests that increased reactive oxygen species (ROS) levels contribute to the pathogenesis of these disorders. Here we assessed the effect of the water-soluble vitamin E derivative Trolox on ROS levels, and the amount and activity of complex I in fibroblasts of six children with isolated complex I deficiency caused by a mutation in the NDUFS1, NDUFS2, NDUFS7, NDUFS8 or NDUFV1 gene. Patient cells displayed increased ROS levels and a variable decrease in complex I activity and amount. For control cells, the ratio between activity and amount was 1 whereas for the patients this ratio was below 1, indicating a defect in intrinsic catalytic activity of complex I in the latter cells. Trolox treatment dramatically reduced ROS levels in both control and patient cells, which was paralleled by a substantial increase in the amount of complex I. Although the ratio between the increase in activity and amount of complex I was exactly proportional in control cells it varied between 0.1 and 0.8 for the patients. Our findings suggest that the expression of complex I is regulated by ROS. Furthermore, they provide evidence that both the amount and intrinsic activity of complex I are decreased in inherited complex I deficiency. The finding that Trolox treatment increased the amount of complex I might aid the future development of antioxidant treatment strategies for patients. However, such treatment may only be beneficial to patients with a relatively small reduction in intrinsic catalytic defect of the complex.     

Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas

Defects in complex I (NADH:ubiquinone oxidoreductase (EC 1.6.5.3)) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which may hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (i) respiratory complex-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (ii) transformation of the mitochondrial genome is feasible, and (iii) Chlamydomonas complex I is similar to that of humans. To illustrate this proposal, we introduced a Leu157Pro substitution into the Chlamydomonas ND4 subunit of complex I in two recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defects when present in the heteroplasmic state in a patient with chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent assembly of whole complex I (950 kDa) and the NADH dehydrogenase activity of the peripheral arm of the complex is mildly affected. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect binding of ubiquinone to the membrane domain. The in vitro defects correlate with a decrease in dark respiration and growth rate in vivo.     

Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae.

In contrast to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed.