Eukaryotic cytochrome c oxidase (CcO), the terminal component of the mitochondrial electron transport chain is a heterooligomeric complex that belongs to the superfamily of heme-copper containing terminal oxidases. The enzyme, composed of both mitochondrially and nuclear encoded subunits, is embedded in the inner mitochondrial membrane, where it catalyzes the transfer of electrons form reduced cytochrome c to dioxygen, coupling this reaction with vectorial proton pumping across the inner membrane. Due to the complexity of the enzyme, the biogenesis of CcO involves a multiplicity of steps, carried out by a number of highly specific gene products. These include mainly proteins that mediate the delivery and insertion of copper ions, synthesis and incorporation of heme moieties and membrane-insertion and topogenesis of constituent protein subunits. Isolated CcO deficiency represents one of the most frequently recognized causes of respiratory chain defects in humans, associated with severe, often fatal clinical phenotype. Here we review recent advancements in the understanding of this intricate process, with a focus on mammalian enzyme.

The biogenesis of eukaryotic COX (cytochrome c oxidase) requires several accessory proteins in addition to structural subunits and prosthetic groups. We have analysed the assembly state of COX and SCO2 protein levels in various tissues of six patients with mutations in SCO2 and SURF1. SCO2 is a copper-binding protein presumably involved in formation of the Cu(A) centre of the COX2 subunit. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated that COX holoenzyme is reduced to 10-20% in skeletal muscle and brain of SCO2 and SURF1 patients and to 10-30% in heart of SCO2 patients, whereas liver of SCO2 patients' contained normal holoenzyme levels. The steady-state levels of mutant SCO2 protein ranged from 0 to 20% in different SCO2 patient tissues. In addition, eight distinct COX subcomplexes and unassembled subunits were found, some of them identical with known assembly intermediates of the human enzyme. Heart, brain and skeletal muscle of SCO2 patients contained accumulated levels of the COX1.COX4.COX5A subcomplex, three COX1-containing subcomplexes, a COX4.COX5A subcomplex and two subcomplexes composed of only COX4 or COX5A. The accumulation of COX1.COX4.COX5A subcomplex, along with the virtual absence of free COX2, suggests that the lack of the Cu(A) centre may result in decreased stability of COX2. The appearance of COX4.COX5A subcomplex indicates that association of these nucleus-encoded subunits probably precedes their addition to COX1 during the assembly process. Finally, the consequences of SCO2 and SURF1 mutations suggest the existence of tissue-specific functional differences of these proteins that may serve different tissue-specific requirements for the regulation of COX biogenesis.

Tissue distribution and segregation and the functional consequences of heteroplasmic mitochondrial DNA mutation A3243G were studied in 30 carriers. The mutation load in hair follicles was higher in 20 patients with a broad spectrum of clinical symptoms than in 10 nonaffected carriers. The onset of the disease negatively correlated with the mutation load in blood and muscle. The activities of respiratory chain complexes in isolated muscle mitochondria did not decrease in all patients and were normal in isolated platelets. Changes in the heteroplasmy level between pairs of mothers and offspring suggest that random genetic drift is the mechanism associated with the intergenerational transmission of the A3243G mutation. In conclusion, detailed clinical investigations and mitochondrial DNA analyses in several tissues are of the highest diagnostic value for the prognosis of the disease in carriers of the A3243G mutation.

Inheritance and expression of mitochondrial DNA (mtDNA) mutations are crucial for the pathogenesis of Leber hereditary optic neuropathy (LHON). We have investigated the segregation and functional consequences of G3460A mtDNA mutation in 27 members of a three-generation family with LHON syndrome. Specific activity of respiratory chain complex I in platelets was reduced in average to 56%, but no direct correlation between the mutation load and its biochemical expression was found. Heteroplasmy in blood, platelets and hair follicles varied from 7% to 100%. Segregation pattern exhibited tissue specificity and influence of different nuclear backgrounds in four branches of the pedigree. Longitudinal analysis revealed a significant (p=0.02) decrease in blood mutation load. Although enzyme assay showed reduction of complex I activity, our results give additional support to the hypothesis that expression of LHON mutation depends on complex nuclear-mitochondrial interaction.

BACKGROUND:Kearns-Sayre syndrome is a multisystem disorder caused by rearrangements of mitochondrial genome including various deletions and/or duplications. The aim of the study is to analyse the impact of mitochondrial DNA (mtDNA) deletions on the mitochondrial energetic metabolism in five patients with Kearns-Sayre syndrome.METHODS AND RESULTS:The course of the disease is progressive in all patients. All of them have bilateral ptosis and external opthalmoplegia, four have retinitis pigmentosa, three have progressive muscle weakness and three have pacemaker because of complete A-V heart block. One patient underwent renal transplantation at the age of 12 because of a chronic renal failure. Southern blot analysis in muscle tissue revealed large scale heteroplasmic mtDNA deletions (3-7.4 kb) in all patients, the number of mutated copies of mtDNA ranged from 50 to 70%. Spectrophotometric measurements of respiratory chain complexes activities in muscle tissue revealed various combinations of defects of complex III, IV and I + III activities in all patients. Nevertheless, the lactic acidosis was permanently present only in one patient. Ragged-red fibers were found in two patients.CONCLUSIONS:Although the diagnostic of Kearns-Sayre syndrome is based on clinical features, molecular analysis of mtDNA is necessary to confirm the diagnosis. The prognosis of the disease is unfavourable and co-operation between the patient and various specialists is necessary for the treatment, which is currently only symptomatic.

BACKGROUND:Dihydrolipoamide dehydrogenase (DLD) deficiency is a rare cause of primary lactic acidosis in infancy. MATERIAL AND METHODS:This article presents the results of biochemical and molecular analyses and metabolic response to treatment procedures in a 10-week old boy presenting with vomiting, progressive hypotonia, lactic acidosis (pH 7.04; BE - 20; B-lactate 6.6 mmol/l, controls <2.1; CSF-lactate 4.8 mmol/l, controls <2.0), increased levels of branched chain amino acids in blood, and increased urinary excretion of branched chain oxo-acids due to DLD deficiency. RESULTS:DLD activity was less than 5% of control values in lymphocytes, muscle mitochondria and fibroblasts. Western blot analysis in muscle tissue showed a decrease in the quantity of DLD protein to 40% in comparison to control. A high-fat, low-protein diet supplemented with MCT oils and sodium dichloroacetate resulted in normalization of lactate, amino acids and organic acids in body fluids, but there was no improvement in psychomotor development. Novel heterozygous mutations were found in the DLD gene: A1081G and G1123A. Both mutations affect the same region of the binding site for FAD. The G1123A mutation, resulting in the substitution of Glu 375 > Lys, breaks down the possible interaction of glutamic acid with neighboring lysine and causes electrostatic and steric repulsion, which is likely to destabilize structure in this part of the protein. In case of the A1081G mutation, resulting in substitution of Met 361 > Val, no important intermolecular interactions are broken and the reason for destabilization of the protein is not as clear. CONCLUSIONS:The prognosis for children with DLD deficiency is unfavorable, although long-term normalization of most metabolites in body fluids may be achieved with the proper diet and the administration of sodium dichloroacetate.