Mitochondrial Respiratory Chain Supercomplexes: From Structure to Function

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F0F1-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.

Stress is a primary contributor to fatty liver syndrome (FLS) in chickens. Mitochondrial functionality is pivotal in FLS progression, with diminished supercomplex (SC) formation disrupting electron transport and escalating reactive oxygen species (ROS) production. However, the impact of stress on mitochondrial SC in chicken FLS remains elusive. This study employed corticosterone (CORT) to model chronic stress and examined its consequences on mitochondrial performance and SC configuration in both vivo and vitro FLS models. Notably, the CORT-treated hepatocytes exhibited elevated triglyceride content (P < 0.05), accompanied by increased mitochondrial ROS (P < 0.01). Moreover, CORT-exposed broilers displayed reduced body weight (P < 0.05) alongside heightened liver-to-body weight ratio (P < 0.01), indicative of liver steatosis with increased triglyceride levels in both liver and plasma (P < 0.01). Mitochondrial alterations in reduced ATP content (P < 0.05). Gene expression analysis revealed enrichment in the mitochondrial respiratory chain pathway, with down-regulated mRNA expression of Complex I-associated SC assembly factors NDUFAF5, NDUFAF7, and TIMMDC1 (P < 0.05). Meanwhile, the glucocorticoid receptor (GR) protein level and its specific binding to the NDUFAF5 gene promoter were reduced in the CORT group (P < 0.01 and P < 0.05, respectively), accompanied by a decrease in NDUFAF5 protein expression in liver, primary hepatocytes, and AML12 cells (P < 0.05). GR knockdown in AML12 cells reduced NDUFAF5 protein expression (P < 0.05). Thus, these findings imply that GR-mediated transcriptional regulation of Complex I assembly factor NDUFAF5 may influence SC assembly, shedding light on stress-induced FLS mechanisms in broilers.

NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1) is a nuclear-encoded subunit of mitochondrial Complex I. It provides a conserved FMN binding site and is responsible for transferring electrons from NADH to FMN to facilitate the entrance of electrons into the electron transport chain (ETC). NDUFV1 also has an iron sulfur cluster [4Fe-4S] (N3) that is the first of that kind in the Complex I. The defect in NDUFV1 could cause neural disease like psychomotor retardation, and some point mutations on FMN binding site have been found in patients with Leigh syndrome. The assembly of intact OXPHOS complexes (complex I~V) is very important in mitochondrial energy production. In addition, the assembly of several respiratory complexes to form a larger supercomplex may improve the efficiency of electron transport. The supercomplex composed by complex I, complex III and complex IV has been confirmed and the defect in supercomplex assembly is associated with encephalomyopathies and neurodegenerative disorders. In present study, we applied a RNA interference system to suppress the NDUFV1 expression in human embryonic kidney cell line (HEK293), and generated three knockdown cell lines A9, B5 and E12 with 55~70% reduction in NDUFV1 protein level. We also performed oxygen consumption assay, dynamic complex I activity assay, ATP determination assay and cell growth rate measurement to evaluate the effects of NDUFV1 suppression. The results showed that the metabolic activity and growth rate were significantly decreased in NDUFV1 knockdown cell lines. In the respiratory complex assembly study, we solubilized mitochondria and applied a high resolution clear native electrophoresis (HrCNE) approach to investigate whether NDUFV1 knockdown affects individual respiratory complex assembly. The results III indicated that NDUFV1 knockdown would significantly decrease the level of complex I, III and IV. Further investigation of OXPHOS supercomplex formation also showed that while NDUFV1 was suppressed, CI/CIII2/CIVn supercomplexes were significantly reduced in cells. These finding suggests that NDUFV1 might play an important role in the assembly/stability of mitochondrial OXPHOS complexes and supercomplexes.

Individual electron transport chain complexes have been shown to assemble into the supramolecular structures known as the respiratory chain supercomplexes (RCS). Several studies reported an associative link between RCS disintegration and human diseases, although the physiological role, structural integrity, and mechanisms of RCS formation remain unknown. Our previous studies suggested that the adenine nucleotide translocase (ANT), the most abundant protein of the inner mitochondrial membrane, can be involved in RCS assembly. In this study, we sought to elucidate whether ANT knockdown (KD) affects RCS formation in H9c2 cardiomyoblasts. Results showed that genetic silencing of ANT1, the main ANT isoform in cardiac cells, stimulated proliferation of H9c2 cardiomyoblasts with no effect on cell viability. ANT1 KD reduced the ΔΨm but increased total cellular ATP levels and stimulated the production of total, but not mitochondrial, reactive oxygen species. Importantly, downregulation of ANT1 had no significant effects on the enzymatic activity of individual ETC complexes I–IV; however, RCS disintegration was stimulated in ANT1 KD cells as evidenced by reduced levels of respirasome, the main RCS. The effects of ANT1 KD to induce RCS disassembly was not associated with acetylation of the exchanger. In conclusion, our study demonstrates that ANT is involved in RCS assembly.

Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.

Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.

The assembly of complex I (CI) with complexes III (CIII) and IV (CIV) of the mitochondrial respiratory chain (MRC) to configure I-III- or I-III-IV-containing supercomplexes (SCs) regulates mitochondrial energy efficiency and reactive oxygen species (mROS) production. However, whether the occurrence of SCs impacts on CI specific activity remains unknown to our knowledge. To investigate this issue, here we determined CI activity in primary neurons and astrocytes, cultured under identical antioxidants-free medium, from two mouse strains (C57Bl/6 and CBA) and Wistar rat, i.e. three rodent species with or without the ability to assemble CIV into SCs. We found that CI activity was 6- or 1.8-fold higher in astrocytes than in neurons, respectively, from rat or CBA mouse, which can form I-III2-IV SC; however, CI activity was similar in the cells from C57Bl/6 mouse, which does not form I-III2-IV SC. Interestingly, CII-III activity, which was comparable in neurons and astrocytes from mice, was about 50% lower in astrocytes when compared with neurons from rat, a difference that was abolished by antioxidants- or serum-containing media. CIV and citrate synthase activities were similar under all conditions studied. Interestingly, in rat astrocytes, CI abundance in I-III2-IV SC was negligible when compared with its abundance in I-III-containing SCs. Thus, CIV-containing SCs formation may determine CI specific activity in astrocytes, which is important to understand the mechanism for CI deficiency observed in Parkinson's disease.

Mitochondria are being recognized as key factors in many unexpected areas of biomedical science. In addition to their well-known roles in oxidative phosphorylation and metabolism, it is now clear that mitochondria are also central to cell death, neoplasia, cell differentiation, the innate immune system, oxygen and hypoxia sensing, and calcium metabolism. Disruption to these processes contributes to a range of human pathologies, making mitochondria a potentially important, but currently seemingly neglected, therapeutic target. Mitochondrial dysfunction is often associated with oxidative damage, calcium dyshomeostasis, defective ATP synthesis, or induction of the permeability transition pore. Consequently, therapies designed to prevent these types of damage are beneficial and can be used to treat many diverse and apparently unrelated indications. Here we outline the biological properties that make mitochondria important determinants of health and disease, and describe the pharmacological strategies being developed to address mitochondrial dysfunction.

Cardiac surgery is often performed by cardiopulmonary by-pass (CPB), generally associated with organ dysfunction. The aim of this work was to determine if and how this phenomenon is related to mitochondrial damage. To this purpose, the effect of the addition of serum from CPB patients to human fibroblasts cultures on mitochondrial respiratory chain and oxidative phosphorylation (OXPHOS) activities was investigated. Serum samples of five patients were obtained before (pre-CPB) and after 6 h CPB weaning (CPB). Mitochondrial OXPHOS activities were examined by polarographic and spectrophotometric assays, and reactive oxygen species (ROS) production was measured by a spectrofluorimeter. Addition of CPB serum to fibroblasts determined a decrease of mitochondrial oxygen consumption due to an inhibition of mitochondrial respiratory chain and some OXPHOS enzymes activities. This inhibition seems to be mainly related to a reduced activity of complex I. Our data represent the first translational research evidence showing that CPB determines mitochondrial dysfunction which leads to impairment of OXPHOX activities and to an increase in ROS production, compromising tissue bioenergetic efficiency.

Coenzyme Q10 is an important component of the mitochondrial electron transfer chain. A supercomplex of mitochondrial electron transfer system proteins exists. This complex also contains coenzyme Q10. The concentrations of coenzyme Q10 in tissues decrease with age and pathology. Coenzyme Q10 is given as a supplement. It is unknown whether coenzyme Q10 is transported to the supercomplex. We develop a method for measuring coenzyme Q10 in the mitochondrial respiratory chain supercomplex in this study. Blue native electrophoresis was used to separate mitochondrial membranes. Electrophoresis gels were cut into 3 mm slices. Hexane was used to extract coenzyme Q10 from this slice, and HPLC-ECD was used to analyze coenzyme Q10. Coenzyme Q10 was found in the gel at the same site as the supercomplex. Coenzyme Q10 at this location was thought to be coenzyme Q10 in the supercomplex. We discovered that 4-nitrobenzoate, a coenzyme Q10 biosynthesis inhibitor, reduced the amount of coenzyme Q10 both within and outside the supercomplex. We also observed that the addition of coenzyme Q10 to cells increased the amount of coenzyme Q10 in the supercomplex. It is expected to analysis coenzyme Q10 level in supercomplex in various samples by using this novel method.

Mitochondrial membranes harbor the electron transport chain (ETC) that powers oxidative phosphorylation (OXPHOS) and drives the synthesis of ATP. Yet, under physiological conditions, the OXPHOS proteins operate as higher-order supercomplex (SC) assemblies, although their functional role remains poorly understood and much debated. By combining large-scale atomistic and coarse-grained molecular simulations with analysis of cryo-electron microscopic data and statistical as well as kinetic models, we show here that the formation of the mammalian I/III2 supercomplex reduces the molecular strain of inner mitochondrial membranes by altering the local membrane thickness and leading to an accumulation of both cardiolipin and quinone around specific regions of the SC. We find that the SC assembly also affects the global motion of the individual ETC proteins with possible functional consequences. On a general level, our findings suggest that molecular crowding and strain effects provide a thermodynamic driving force for the SC formation, with a possible flux enhancement in crowded biological membranes under constrained respiratory conditions.

Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells

Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F1FO-ATP synthase to produce ATP for cellular metabolism. In baker's yeast, Saccharomyces cerevisiae, the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron-sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability. The cysteine desulfurase Nfs1 provides sulfur for ISC assembly and forms with partner proteins the ISC-biogenesis desulfurase complex (ISD complex). Here, we report an unexpected interaction of the active ISD complex with the cytochrome bc1 complex and cytochrome c oxidase. The individual deletion of complex III or complex IV blocks the association of the ISD complex with respiratory chain components. We conclude that the ISD complex binds selectively to respiratory chain supercomplexes. We propose that this molecular link contributes to coordination of iron-sulfur cluster formation with respiratory activity.

Today breast cancer represents the most frequent of all cancer pathologies in the world, with more than one million newly diagnosed cases and about 370000 cancer-related deaths in women each year, even with all the significant progress in its diagnosis and treatment (Ott et al., 2011). The development of breast tumor is a multistep process. It is generally thought that the initiation of breast cancer occurs after accumulation of genetic alterations that result in either activation of oncogenes and/or inactivation of tumor suppressor genes, leading to an abnormal cellular proliferation and promoting the development of tumor in mammary gland. A number of risk factors such as reproductive and hormonal factors, alcohol consumption, cigarette smoking, dietary factors and chronic inflammation have been identified for breast cancer, whose mechanisms by which they increase risk of the disease are not always clear (Mitrunen and Hirvonen, 2003). It has been proposed that the production of reactive oxygen species (ROS) leading to an oxidative stress is the linking factor between these carcinogens. The oxidative stress is defined as an imbalance between production of ROS, and their elimination by antioxidant defense system (redox imbalance). This imbalance leads to damage of important biomolecules and cells, with potential impact on the whole organism. ROS are products of a normal cellular metabolism and play vital roles in the stimulation of signalling pathways in cells in response to changes in intraand extracellular environmental conditions. During endogenous metabolic reactions, aerobic cells produce ROS such as superoxide anion (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (OH•), and organic peroxides as normal products of the biological reduction of molecular oxygen. Most ROS are generated in cells by the mitochondrial respiratory chain. Under hypoxic conditions following reoxygenation, the electron transfer at the level of complex III of mitochondrial respiratory chain to molecular oxygen occurs directly, leading to a high level of ROS. Beside oxidative phosphorylation, low levels of ROS are continuously formed by oxidase activities in peroxisomes, and by the cytochrome P450 system in endoplasmic reticulum. ROS as OH• is produced in vivo in the presence of reduced transition metals (ions of Fe, Cu) mainly via the Fenton reaction in contact with H2O2. Under normal metabolic conditions, these ROS are eliminated rapidly in normal cells by the antioxidant defense system. However, ROS may be overproduced when normal cells are exposed to a sustained environmental stress, including ionizing radiation and many

The oxidative phosphorylation (OXPHOS) system couples the transfer of electrons to oxygen with pumping of protons across the inner mitochondrial membrane, ensuring the ATP production. Evidence suggests that respiratory chain complexes may also assemble into supramolecular structures, called supercomplexes (SCs). The SCs appear to increase the efficiency/capacity of OXPHOS and reduce the reactive oxygen species (ROS) production, especially that which is produced by complex I. Studies suggest a mutual regulation between complex I and SCs, while SCs organization is important for complex I assembly/stability, complex I is involved in the supercomplex formation. Complex I is a pacemaker of the OXPHOS system, and it has been shown that the PKA-dependent phosphorylation of some of its subunits increases the activity of the complex, reducing the ROS production. In this work, using in ex vivo and in vitro models, we show that the activation of cAMP/PKA cascade resulted in an increase in SCs formation associated with an enhanced capacity of electron flux and ATP production rate. This is also associated with the phosphorylation of the NDUFS4 subunit of complex I. This aspect highlights the key role of complex I in cellular energy production.