A novel variant in COX16 causes cytochrome c oxidase deficiency, severe fatal neonatal lactic acidosis, encephalopathy, cardiomyopathy, and liver dysfunction.

COX16 is involved in the biogenesis of cytochrome-c-oxidase (complex IV), the terminal complex of the mitochondrial respiratory chain. We present the first report of two unrelated patients with the homozygous nonsense variant c.244C>T(p. Arg82*) in COX16 with hypertrophic cardiomyopathy, encephalopathy and severe fatal lactic acidosis, and isolated complex IV deficiency. The absence of COX16 protein expression leads to a complete loss of the holo-complex IV, as detected by Western blot in patient fibroblasts. Lentiviral transduction of patient fibroblasts with wild-type COX16 complementary DNA rescued complex IV biosynthesis. We hypothesize that COX16 could play a role in the copper delivery route of the COX2 module as part of the complex IV assembly. Our data provide clear evidence for the pathogenicity of the COX16 variant as a cause for the observed clinical features and the isolated complex IV deficiency in these two patients and that COX16 deficiency is a cause for mitochondrial disease.

Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive.

COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca(2+)]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy.

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.

Effect of resveratrol on cultured skin fibroblasts from patients with oxidative phosphorylation defects.

Few therapeutic options are available to patients with oxidative phosphorylation disorders. Administering pharmacological agents that are able to stimulate mitochondrial biogenesis have been put forward as a possible treatment, yet the approach remains in need of thorough testing. We investigated the effect of resveratrol in an in vitro setting. Mitochondrial enzymatic activities were tested in cultured skin fibroblasts from patients harboring a nuclear defect in either complex II or complex IV (n = 11), and in fibroblasts from healthy controls (n = 11). In the latter, preincubation with resveratrol resulted in a significant increase of citrate synthase, complex II and complex IV enzyme activity. In patients with complex II or complex IV deficiency, however, activity of the deficient complex could not be substantially augmented, and response was dependent upon the residual activity. We conclude that resveratrol is not capable of normalizing oxidative phosphorylation activities in deficient cell lines.

Beneficial effects of resveratrol on respiratory chain defects in patients' fibroblasts involve estrogen receptor and estrogen-related receptor alpha signaling.

Mitochondrial respiratory chain (RC) disorders are the most prevalent inborn metabolic diseases and remain without effective treatment to date. Up-regulation of residual enzyme activity has been proposed as a possible therapeutic approach in this group of disorders. As resveratrol (RSV), a natural compound, was proposed to stimulate mitochondrial metabolism in rodents, we tested the effect of this compound on mitochondrial functions in control or in Complex I (CI)- or Complex IV (CIV)-deficient patients' fibroblasts. We show that RSV stimulates the expression of a panel of proteins representing structural subunits or assembly factors of the five RC complexes, in control fibroblasts. In moderate RC-deficient patients' cells, RSV treatment increases the amount of mutated proteins and stimulates residual enzyme activities. In these patients' cells, we establish that up-regulation of RC enzyme activities induced by RSV translates into increased cellular O2 consumption rates and results in the correction of RC deficiencies. Importantly, RSV also prevents the accumulation of lactate that occurred in RC-deficient fibroblasts. Different complementary approaches demonstrate that RSV induces a mitochondrial biogenesis that might underlie the increase in mitochondrial capacities. Finally, we showed that, in human fibroblasts, RSV stimulated mitochondrial functions mainly in a SIRT1- and AMPK-independent manner and that its effects rather involved the estrogen receptor (ER) and estrogen-related receptor alpha (ERRα) signaling pathways. These results represent the first demonstration that RSV could have a beneficial effect on inborn CI and CIV deficiencies from nuclear origin, in human fibroblasts and might be clinically relevant for the treatment of some RC deficiencies.

Diagnosis and treatment of mitochondrial myopathies.

Mitochondrial disorders are a heterogeneous group of disorders resulting from primary dysfunction of the respiratory chain. Muscle tissue is highly metabolically active, and therefore myopathy is a common element of the clinical presentation of these disorders, although this may be overshadowed by central neurological features. This review is aimed at a general medical and neurologist readership and provides a clinical approach to the recognition, investigation, and treatment of mitochondrial myopathies. Emphasis is placed on practical management considerations while including some recent updates in the field.

Copper and bezafibrate cooperate to rescue cytochrome c oxidase deficiency in cells of patients with SCO2 mutations.

BACKGROUND: Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients' cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene. METHODS: We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production.. RESULTS: Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 µM BZF, and peaked at 400 µM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 µM CuCl2 and 200 µM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells. CONCLUSIONS: These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.

Activation of the PPAR/PGC-1alpha pathway prevents a bioenergetic deficit and effectively improves a mitochondrial myopathy phenotype.

Neuromuscular disorders with defects in the mitochondrial ATP-generating system affect a large number of children and adults worldwide, but remain without treatment. We used a mouse model of mitochondrial myopathy, caused by a cytochrome c oxidase deficiency, to evaluate the effect of induced mitochondrial biogenesis on the course of the disease. Mitochondrial biogenesis was induced either by transgenic expression of peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator alpha (PGC-1alpha) in skeletal muscle or by administration of bezafibrate, a PPAR panagonist. Both strategies successfully stimulated residual respiratory capacity in muscle tissue. Mitochondrial proliferation resulted in an enhanced OXPHOS capacity per muscle mass. As a consequence, ATP levels were conserved resulting in a delayed onset of the myopathy and a markedly prolonged life span. Thus, induction of mitochondrial biogenesis through pharmacological or metabolic modulation of the PPAR/PGC-1alpha pathway promises to be an effective therapeutic approach for mitochondrial disorders.

Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts.

Mitochondria are dynamic organelles with continuous fusion and fission, the equilibrium of which results in mitochondrial morphology. Evidence points to there being an intricate relationship between mitochondrial dynamics and oxidative phosphorylation. We investigated the bioenergetics modulation of mitochondrial morphology in five control cultured primary skin fibroblasts and seven with genetic alterations of oxidative phosphorylation. Under basal conditions, control fibroblasts had essentially filamentous mitochondria. Oxidative phosphorylation inhibition with drugs targeting complex I, III, IV or V induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked to mitochondrial oxidative phosphorylation. Its alteration by glycolysis inhibition points to a severe oxidative phosphorylation defect.

[Chronic progressive external ophthalmoplegia: clinical and electromyographic manifestations in a series of cases]. / Oftalmoplejía externa progresiva crónica: manifestaciones clínicas y electromiográficas en una serie de casos.

INTRODUCTION: Chronic progressive external ophthalmoplegia (CPEO) is a common mitochondrial disease. The different conditions in this group of diseases overlap clinically, enzymatically and genetically. There is no effective treatment. Ptosis improves with corrective surgery involving tarsorrhaphy as a palliative measure. CASE REPORTS: Code numbers were examined in a retrospective study conducted in order to search for patients with ptosis or ophthalmoplegia who had either visited or been admitted to the neurology department over the last 10 years. Data concerning these patients' clinical features and results of complementary tests were collected. Six patients with CPEO were identified, five of whom were females. Ages ranged from 44 to 72 years. All the patients had ptosis, although 50% were asymmetric. Half of them reported mild dysphagia while swallowing liquids. Levels of creatine phosphokinase and acetylcholine antireceptor antibodies were normal. Half the patients showed increased jitter and a muscle biopsy revealed that five of them had ragged red fibres. The most frequent enzyme deficit was complex I and IV deficiency. There were no familial forms; the most common genetic anomaly was single deletion in the mitochondrial deoxyribonucleic acid. CONCLUSIONS: In cases of ptosis and ophthalmoplegia that do not respond to anticholinesterases, knowledge of this condition makes it possible to avoid the use of immunosuppressant drugs, which have important side effects.

Oftalmoplejía externa progresiva crónica: manifestaciones clínicas y electromiográficas en una serie de casos / Chronic progressive external ophthalmoplegia: clinical and electromyographic manifestations in a series of cases

Introducción. La oftalmoplejía externa progresiva crónica(CPEO) es una enfermedad mitocondrial común. Este grupode enfermedades presenta solapamiento clínico, enzimático y genéticoentre las diferentes entidades. No existe un tratamiento eficaz.La ptosis mejora con cirugía correctora de tarsorrafia comouna medida paliativa. Casos clínicos. Estudio retrospectivo en elque se busca por codificación a pacientes con ptosis u oftalmoplejíaen consultas o ingresados en neurología durante los últimos 10años. Se recogieron datos de la clínica y pruebas complementariasde estos pacientes. Se identificó a seis pacientes con CPEO; cincode ellos fueron mujeres. Sus edades estaban comprendidas entrelos 44 y los 72 años. Todos los pacientes presentaban ptosis, aunqueel 50% era asimétrica. La mitad refería disfagia leve paralíquidos. Los niveles de creatinfosfocinasa y de anticuerpos antirreceptoresde acetilcolina fueron normales. Existía un aumentodel jitter en la mitad de los pacientes y fibras rojas rasgadas en labiopsia muscular de cinco de ellos. El déficit enzimático más frecuentefue el de los complejos I y IV. No existieron formas familiares;la anomalía genética más común fue la deleción única en elácido desoxirribonucleico mitocondrial. Conclusión. El conocimientode esta entidad permite, en casos de ptosis y oftalmoplejíaque no responden a anticolinesterásicos, evitar el uso de medicacionesinmunosupresoras con efectos secundarios importantes

Introduction. Chronic progressive external ophthalmoplegia (CPEO) is a common mitochondrial disease. Thedifferent conditions in this group of diseases overlap clinically, enzymatically and genetically. There is no effective treatment.Ptosis improves with corrective surgery involving tarsorrhaphy as a palliative measure. Case reports. Code numbers wereexamined in a retrospective study conducted in order to search for patients with ptosis or ophthalmoplegia who had eithervisited or been admitted to the neurology department over the last 10 years. Data concerning these patients' clinical featuresand results of complementary tests were collected. Six patients with CPEO were identified, five of whom were females. Agesranged from 44 to 72 years. All the patients had ptosis, although 50% were asymmetric. Half of them reported mild dysphagiawhile swallowing liquids. Levels of creatine phosphokinase and acetylcholine antireceptor antibodies were normal. Half thepatients showed increased jitter and a muscle biopsy revealed that five of them had ragged red fibres. The most frequentenzyme deficit was complex I and IV deficiency. There were no familial forms; the most common genetic anomaly was singledeletion in the mitochondrial deoxyribonucleic acid. Conclusions. In cases of ptosis and ophthalmoplegia that do not respondto anticholinesterases, knowledge of this condition makes it possible to avoid the use of immunosuppressant drugs, which haveimportant side effects

Binding of copper is a mechanism of homocysteine toxicity leading to COX deficiency and apoptosis in primary neurons, PC12 and SHSY-5Y cells.

Children with hereditary severe hyperhomocysteinemia present with a variety of neurological impairment, and mild hyperhomocysteinemia has been associated with neurodegeneration in the elderly. The link of hyperhomocysteinemia to neurological dysfunction is unknown. We investigated mitochondrial mechanisms of homocysteine (HCys) neurotoxicity in rat dopaminergic pheochromocytoma cells, human neuroblastoma cells and primary rat cerebellar granule neurons. HCys dose dependently impaired cytochrome c oxidase (COX) activity as well as stability and induced reactive oxygen species and apoptotic cell death. We found that HCys binds the COX cofactor Cu(2+), and Cu(2+) supplementation prior to HCys treatment preserved COX activity and prevented cell death. The Cu(2+) chelating action of HCys and impairement of COX activity represent novel mechanisms of HCys neurotoxicity, which might be preventable by supplementation of Cu(2+).

Cardiac cytochrome-c oxidase deficiency occurs during late postnatal development in progeny of copper-deficient rats.

Although cytochrome-c oxidase (CCO) is a copper-dependent enzyme, the effect of maternal copper deficiency on the expression of CCO activity during postnatal development of the neonatal rat heart has not been investigated extensively. Here, we show that CCO activity in heart mitochondria isolated from neonates of copper-deficient dams did not exhibit significant reductions until postnatal days (PND) 15 and 21. In addition, immunoblot analysis indicated that the CCO subunit (Cox-1) was reduced on postnatal Days 10 and 21, and that Cox-4 was reduced on PND 21 in heart mitochondria of the neonates from copper-deficient dams. These findings indicate that the impairment of CCO activity in neonatal heart by maternal copper deficiency occurs late in the postnatal heart development. Furthermore, the concurrent reductions in Cox-1 and Cox-4 suggest that the impaired CCO activity reflects a CCO deficiency in heart mitochondria. CCO activity and Cox-1 in heart mitochondria were not fully restored by 6 weeks of postweaning copper repletion in the pups of copper-deficient dams. This indicates that prolonged maternal intake of moderately low dietary copper produces CCO deficiency in cardiac mitochondria of neonates during late postnatal heart development, after terminal differentiation of cardiomyocytes occurs. The resistance of CCO deficiency to repair by dietary copper supplementation may be related to the relatively slow turnover of the affected mitochondria in the terminally differentiated heart.

Congenital cataract, muscular hypotonia, developmental delay and sensorineural hearing loss associated with a defect in copper metabolism.

Deficiencies of different proteins involved in copper metabolism have been reported to cause human diseases. Well-known syndromes, for example, are Menkes and Wilson diseases. Here we report a patient presenting with congenital cataract, severe muscular hypotonia, developmental delay, sensorineural hearing loss and cytochrome-c oxidase deficiency with repeatedly low copper and ceruloplasmin levels. These findings were suggestive of a copper metabolism disorder. In support of this, the patient's fibroblasts showed an increased copper uptake with normal retention. Detailed follow-up examinations were performed. Immunoblotting for several proteins including ATP7A (MNK or Menkes protein), ATP7B (Wilson protein) and SOD1 showed normal results, implying a copper metabolism defect other than Wilson or Menkes disease. Sequence analysis of ATOX1 and genes coding for proteins that are known to play a role in the mitochondrial copper metabolism (COI-III, SCO1, SCO2, COX11, COX17, COX19) revealed no mutations. Additional disease genes that have been associated with cytochrome-c oxidase deficiency were negative for mutations as well. As beneficial effects of copper histidinate supplementation have been reported in selected disorders of copper metabolism presenting with low serum copper and ceruloplasmin levels, we initiated a copper histidinate supplementation. Remarkable improvement of clinical symptoms was observed, with complete restoration of cytochrome-c oxidase activity in skeletal muscle.

Distribution of COX-negative mitochondria in myofibers of rats intoxicated with Senna occidentalis seeds.

We have described that administration of seeds or parts of the seed of Senna occidentalis (coffee senna) for long periods, induces histochemical changes in the skeletal muscles of hens and rats that are characteristic of a mitochondrial myopathy--as decrease of SDH and COX activity, with some COX negative fibers. In this experimental model of mitochondrial myopathy, as in many human mitochondrial diseases, there is a random distribution of COX negative fibers. Some fibers are completely COX negative while others are partially negative and others are completely positive. In the present work we have studied the distribution of COX negative mitochondria at transmission electron microscopy in skeletal muscle of rats in this experimental myopathy. In myofibers of intoxicated animals the expression of COX was heterogeneous. The histochemical reaction was observed in the internal membrane (more evident in mitochondrial cristae) of all mitochondria of some myofibers, while it was almost absent in other myofibers. In these myofibers the great part of the mitochondria were negative for COX reaction while other ones had a weak expression of this enzyme (dot or focal expression of COX). Our results indicated that the COX mitochondrial activity is heterogeneously impaired in myofibers of rats intoxicated with S. occidentalis. These abnormalities remember those observed in some types of human mitochondrial myopathies.

Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations.

Human SCO2 is a nuclear-encoded Cu-binding protein, presumed to be responsible for the insertion of Cu into the mitochondrial cytochrome c oxidase (COX) holoenzyme. Mutations in SCO2 are associated with cardioencephalomyopathy and COX deficiency. Studies in yeast and bacteria have shown that Cu supplementation can restore COX activity in cells harbouring mutations in genes involving Cu transport. Therefore we investigated whether Cu supplementation could restore COX activity in cultured cells from patients with SCO2 mutations. Our data demonstrate that the COX deficiency observed in fibroblasts, myoblasts and myotubes from patients with SCO2 mutations can be restored to almost normal levels by the addition of CuCl(2) to the growth medium.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.

Cytochrome c oxidase partial deficiency-associated Leigh disease presenting as an extrapyramidal syndrome.

Leigh disease is a subacute neurodegenerative disorder characterized by symmetric necrotic lesions in the basal ganglia, cerebellum, thalamus, brain stem, and optical nerves and caused by altered oxidative phosphorylation. We describe the clinical, biochemical, neuroimaging, and molecular studies of a 19-year-old boy with early-onset Leigh disease manifesting as severe extrapyramidal disorder with generalized dystonia and choreoathetosis. He was born of healthy parents after an uneventful pregnancy and delivery. At the age of 2 1/2 years, after a minor respiratory infection, he developed unstable, broad-based gait and tremor of the hands. These symptoms persisted for the next several years, when ataxia became more prominent. Difficulty in swallowing, dysarthria, trunk dystonia, and marked dyskinesia of the arms and hands gradually developed. Nystagmus, transient ptosis, and strabismus also appeared. Abnormal laboratory findings included elevated plasma and cerebrospinal fluid lactate and pyruvate, with an abnormal lactate/pyruvate ratio. Cranial computed tomography and magnetic resonance imaging demonstrated signs of cerebellar atrophy, bilateral and symmetric hypodensities in the lentiform nucleus and thalamus, and transient hyperintensities of cerebral peduncles in T2-weighted sequences suggestive of Leigh disease. Muscle biopsy revealed isolated fiber atrophy, necrotic fibers undergoing phagocytosis, and no ragged-red fibers. The measured catalytic activity of cytochrome c oxidase in skeletal muscle homogenates demonstrated a partial cytochrome c oxidase deficiency No abnormalities in the mitochondrial genome and in the SURF-1 gene were found. The boy is currently receiving levodopa therapy, creatine monohydrate, and a high dosage of thiamine and lipoic acid, his condition is stabilized, and extrapyramidal symptoms are less pronounced.

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.