Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells

Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.     

NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4−/− mice and Leigh syndrome patients: A stabilizing role for NDUFAF2

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4^−/− mouse tissues. Ndufs4^−/− animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4^−/− mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4^−/− MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4^−/− mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.     

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals.     

Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency

Reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest complex of the mitochondrial respiratory chain and complex I deficiency accounts for approximately 30% cases of respiratory-chain deficiency in humans. Only seven mitochondrial DNA genes, but >35 nuclear genes encode complex I subunits. In an attempt to elucidate the molecular bases of complex I deficiency, we studied the six most-conserved complex I nuclear genes (NDUFV1, NDUFS8, NDUFS7, NDUFS1, NDUFA8, and NDUFB6) in a series of 36 patients with isolated complex I deficiency by denaturing high-performance liquid chromatography and by direct sequencing of the corresponding cDNA from cultured skin fibroblasts. In 3/36 patients, we identified, for the first time, five point mutations (del222, D252G, M707V, R241W, and R557X) and one large-scale deletion in the NDUFS1 gene. In addition, we found six novel NDUFV1 mutations (Y204C, C206G, E214K, IVS 8+41, A432P, and del nt 989-990) in three other patients. The six unrelated patients presented with hypotonia, ataxia, psychomotor retardation, or Leigh syndrome. These results suggest that screening for complex I nuclear gene mutations is of particular interest in patients with complex I deficiency, even when normal respiratory-chain-enzyme activities in cultured fibroblasts are observed.     

NDUFB7 and NDUFA8 are located at the intermembrane surface of complex I

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest protein complex of the oxidative phosphorylation. Crystal structures have elucidated the positions of most subunits of bacterial evolutionary origin in the complex, but the positions of the eukaryotic subunits are unknown. Based on the analysis of sequence conservation we propose intra-molecular disulfide bridges and the inter-membrane space localization of three Cx(9)C-containing subunits in human: NDUFS5, NDUFB7 and NDUFA8. We experimentally confirm the localization of the latter two, while our data are consistent with disulfide bridges in NDUFA8. We propose these subunits stabilize the membrane domain of complex I.     

Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits

Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.     

Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts

Background: Complex I (CI) deficiency is the most frequent cause of OXPHOS disorders. Recent studies have shown increases in reactive oxygen species (ROS) production and mitochondrial network disturbances in patients' fibroblasts harbouring mutations in CI subunits. Objectives: The present work evaluates the impact of mutations in the NDUFA1 and NDUFV1 genes of CI on mitochondrial bioenergetics and dynamics, in fibroblasts from patients suffering isolated CI deficiency. Results: Decreased oxygen consumption rate and slow growth rate were found in patients with severe CI deficiency. Mitochondrial diameter was slightly increased in patients' cells cultured in galactose or treated with 2′-deoxyglucose without evidence of mitochondrial fragmentation. Expression levels of the main proteins involved in mitochondrial dynamics, OPA1, MFN2, and DRP1, were slightly augmented in all patients' cells lines. The study of mitochondrial dynamics showed delayed recovery of the mitochondrial network after treatment with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (cccp) in patients with severe CI deficiency. Intracellular ROS levels were not increased neither in glucose nor galactose medium in patients' fibroblasts. Conclusion: Our main finding was that severe CI deficiency in patients harbouring mutations in the NDUFA1 and NDUFV1 genes is linked to a delayed mitochondrial network recovery after cccp treatment. However, the CI deficiency is neither associated with massive mitochondrial fragmentation nor with increased ROS levels. The different genetic backgrounds of patients with OXPHOS disorders would explain, at least partially, differences in the pathophysiological manifestations of CI deficiency.     

Human complex I defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns

Complex I defects are one of the most frequent causes of mitochondrial respiratory chain disorders. Therefore, it is important to find new approaches for detecting and characterizing Complex I deficiencies. In this paper, we introduce a new set of monoclonal antibodies that react with 39-, 30-, 20-, 18-, 15-, and 8-kDa subunits of Complex I. These antibodies are shown to aid in diagnosis of Complex I deficiencies and add understanding to the genotype-phenotype relationships of different mutations. A total of 11 different patients were examined. Four patients had undefined Complex I defects, whereas the other patients had defects in NDUFV1, NDUFS2 (two patients), NDUFS4 (two patients), NDUFS7, and NDUFS8. We show here that Western blotting with these antibodies, particularly when used in conjunction with sucrose gradient studies and enzymatic activity measurements, helps distinguish catalytic versus assembly defects and further distinguishes between mutations in different subunits. Furthermore, different mutations in the same gene are shown to give very similar subunit profiles, and we show that one of the patients is a good candidate for having a defect in a Complex I assembly factor.     

Oxidative damage to mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by mass spectrometry

There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50-80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the beta and the d subunits of the F1F0-ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr122, while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr46, Tyr50, and Tyr51. In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I subunits (Taylor, S. W., Fahy, E., Murray, J., Capaldi, R. A., and Ghosh, S. S. (2003) J. Biol. Chem. 278, 19587-19590). These sites of oxidation and nitration may be useful biomarkers for assessing oxidative stress in neurodegenerative disorders.     

Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase

Human NADH CoQ oxidoreductase is composed of a total of 43 subunits and has been demonstrated to be a major site for the production of superoxide by mitochondria. Incubation of rat heart mitochondria with ATP resulted in the phosphorylation of two mitochondrial membrane proteins, one with a M(r) of 6 kDa consistent with the NDUFA1 (MWFE), and one at 18kDa consistent with either NDUFS4 (AQDQ) or NDUFB7 (B18). Phosphorylation of both subunits was enhanced by cAMP derivatives and protein kinase A (PKA) and was inhibited by PKA inhibitors (PKAi). When mitochondrial membranes were incubated with pyruvate dehydrogenase kinase, phosphorylation of an 18kDa protein but not a 6kDa protein was observed. NADH cytochrome c reductase activity was decreased and superoxide production rates with NADH as substrate were increased. On the other hand, with protein kinase A-driven phosphorylation, NADH cytochrome c reductase was increased and superoxide production decreased. Overall there was a 4-fold variation in electron transport rates observable at the extremes of these phosphorylation events. This suggests that electron flow through complex I and the production of oxygen free radicals can be regulated by phosphorylation events. In light of these observations we discuss a potential model for the dual regulation of complex I and the production of oxygen free radicals by both PKA and PDH kinase.     

cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I

The subunits of complex I encoded by the mammalian nuclear genes NDUFS4 (AQDQ protein) and NDUFB11 (ESSS protein) contain serine/threonine consensus phosphorylation sequences (CPS) in their presequence, the first also in the C-terminus. We have studied the impact of PKA mediated phosphorylation on the mitochondrial import of in vitro and in vivo synthesized NDUFS4 protein. The intramitochondrial accumulation of the mature form of in vitro synthesized NDUFS4 protein, but not that of ESSS protein, was promoted by PKA and depressed by alkaline phosphatase (AP). In HeLa cells, control or transfected with the NDUFS4 cDNA construct, the mitochondrial level of mature NDUFS4 protein was promoted by 8-Br-cAMP and depressed by H89. Ser173Ala mutagenesis in the C-terminus CPS abolished the appearance in mitochondria of the mature form of NDUFS4 protein. The promoting effect of PKA on the mitochondrial accumulation of mature NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol.     

Leigh syndrome associated with mitochondrial complex I deficiency due to novel mutations In NDUFV1 and NDUFS2

Leigh syndrome (LS) is a progressive neurodegenerative disease caused by either mitochondrial or nuclear DNA mutations resulting in dysfunctional mitochondrial energy metabolism. Mutations in genes encoding for subunits of the respiratory chain or assembly factors of respiratory chain complexes are often documented in LS cases. Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) enzyme deficiencies account for a significant proportion of mitochondrial disorders, including LS. In an attempt to expand the repertoire of known mutations accounting for LS, we describe the clinical, radiological, biochemical and molecular data of six patients with LS found to have novel mutations in two complex I subunits (NDUFV1 and NDUFS2). Two siblings were homozygous for the previously undescribed R386C mutation in NDUFV1, one patient was a compound heterozygote for the R386C mutation in NDUFV1 and a frameshift mutation in the same gene, one patient was a compound heterozygote for the R88G and R199P mutations in NDUFV1, and two siblings were compound heterozygotes for an undescribed E104A mutation in NDUFS2. After the novel mutations were identified, we employed prediction models using protein conservation analysis (SIFT, PolyPhen and UCSC genome browser) to determine pathogenicity. The R386C, R88G, R199P, and E104A mutations were found to be likely pathogenic, and thus presumably account for the LS phenotype. This case series broadens our understanding of the etiology of LS by identifying new molecular defects that can result in complex I deficiency and may assist in targeted diagnostics and/or prenatal diagnosis of LS in the future.     

Oxidative modification of mitochondrial respiratory complexes in response to the stress of Trypanosoma cruzi infection

Previously, we have shown deficiencies in the activities of the mitochondrial respiratory complexes and reduced mitochondrial ATP generation capacity in chagasic hearts infected by Trypanosoma cruzi. In this study, we determined whether the oxidative stress that occurs in response to T. cruzi infection contributes to the catalytic impairment of respiratory complexes and to subsequent mitochondrial dysfunction in murine myocardium. Our data show that oxidative injuries, as determined by the levels of lipid peroxides and protein carbonyls, are incurred in cardiac mitochondria as early as 3 days postinfection and persist throughout the infection and disease. The individual components of the respiratory complexes were separated by two-dimensional, blue-native gel electrophoresis, and carbonyl adducts were detected by Western blotting. We observed substantial carbonylation of the specific subunits of mitochondrial respiratory complexes in infected murine hearts. Of note is the oxidative modification of NDUFS1, NDUFS2, and NDUFV1, which form the catalytic core of the CI complex; UQCRC1, UQCRC2, and UQCRQ, the subunits of the core subcomplex, and UQCRH and CYC1, which form the cyt c₁ subcomplex of CIII; and a γ chain that is essential for ATP synthesis by CV complex. The extent of oxidative modifications of the subunits correlated with the catalytic defects of the respiratory complexes in the infected myocardium. Taken together, our data demonstrate that respiratory complexes are oxidatively damaged in response to the stress of T. cruzi infection. These data also suggest involvement of the specific susceptibility of the protein subunits, and not generalized mitochondrial oxidative damage in respiratory chain impairment of chagasic hearts.     

Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt -1) in the NDUFS4 gene in Leigh syndrome

Complex I deficiency, the most common cause of mitochondrial disorders, accounts for a variety of clinical symptoms and its genetic heterogeneity makes identification of the disease genes particularly tedious. Indeed, most of the 43 complex I subunits are encoded by nuclear genes, only seven of them being mitochondrially encoded. In order to offer urgent prenatal diagnosis, we have studied an inbred/multiplex family with complex I deficiency by using microsatellite DNA markers flanking the putative disease loci. Microsatellite DNA markers have allowed us to exclude the NDUFS7, NDUFS8, NDUFV1 and NDUFS1 genes and to find homozygosity at the NDUFS4 locus. Direct sequencing has led to identification of a homozygous splice acceptor site mutation in intron 1 of the NDUFS4 gene (IVS1nt -1, G-->A); this was not found in chorion villi of the ongoing pregnancy. We suggest that genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families helps to identify the disease-causing mutation. More generally, we suggest giving consideration to a more systematic microsatellite analysis of putative disease loci for identification of disease genes in inbred/multiplex families affected with genetically heterogeneous conditions.     

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

Background

The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.

Results

More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 ?) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 ?). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.

Conclusions

The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex??s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.     

Supernumerary subunits NDUFA3, NDUFA5 and NDUFA12 are required for the formation of the extramembrane arm of human mitochondrial complex I

Mammalian complex I is composed of fourteen highly conserved core subunits and additional thirty subunits acquired in the course of evolution. At present, the function of the majority of these supernumerary subunits is poorly understood. In this work, we have studied NDUFA3, NDUFA5 and NDUFA12 supernumerary subunits to gain insight into their role in CI activity and biogenesis. Using human cell lines in which the expression of these subunits was knocked down with miRNAs, we showed that they are necessary for the formation of a functional holoenzyme. Analysis of the assembly intermediates in mitochondria depleted for these subunits further suggested that they are required for assembly and/or stability of the electron transferring Q module in the peripheral arm of the CI.     

Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene

Among the mitochondrial disorders, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, in the majority of the complex I-deficient patients mutations of nuclear genes are expected. This review attempts to summarize genetic defects affecting nuclear encoded subunits of complex I reported to date focusing on those found in the NDUFS4 gene. NDUFS4 product is 18 kDa protein which appears to have a dual role in complex I, at least: cAMP-dependent phosphorylation activates the complex; non-sense mutation of NDUFS4 prevents normal assembly of a functional complex in the inner mitochondrial membrane.     

Clinical and molecular findings in children with complex I deficiency

Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.