Electron Transport Chain Supercomplex Research Articles

Architecture of Human Mitochondrial Respiratory Megacomplex I2III2IV2

The respiratory megacomplex represents the highest-order assembly of respiratory chain complexes, and it allows mitochondria to respond to energy-requiring conditions. To understand its architecture, we examined the human respiratory chain megacomplex-I2III2IV2 (MCI2III2IV2) with 140 subunits and a subset of associated cofactors using cryo-electron microscopy. The MCI2III2IV2 forms a circular structure with the dimeric CIII located in the center, where it is surrounded by two copies each of CI and CIV. Two cytochrome c (Cyt.c) molecules are positioned to accept electrons on the surface of the c1 state CIII dimer. Analyses indicate that CII could insert into the gaps between CI and CIV to form a closed ring, which we termed the electron transport chain supercomplex. The structure not only reveals the precise assignment of individual subunits of human CI and CIII, but also enables future in-depth analysis of the electron transport chain as a whole.

Abstract 427: Alterations in the Mitochondrial Supercomplex in Pediatric Dilated Cardiomyopathy

Organization of the mitochondrial electron transport chain (ETC) into a protein “supercomplex” has been shown to be critical for optimal mitochondrial respiration, and is dependent on the phospholipid composition of the inner mitochondrial membrane. A close physical interaction between the ETC supercomplex and the fatty acid beta-oxidation system (FAO, which provides necessary reducing equivalents for ETC) has also been proposed. We have previously demonstrated that content of the primary mitochondrial phospholipid, cardiolipin, is altered in pediatric dilated cardiomyopathy (DCM), with evidence for its dysregulated biosynthesis. We hypothesized that altered cardiolipin content in pediatric DCM is correlated with altered supercomplex-associated ETC activity and mitochondrial fatty acid β-oxidation. A cross-sectional investigation was performed using myocardium from 16 children with DCM and 15 non-failing (NF) controls from the University of Colorado Heart Tissue Bank. Using blue native (BN) -PAGE with in-gel activity staining we demonstrated lower activity of supercomplex-associated complexes I (DCM 80% of NF, P<0.05) and IV (DCM 72% of NF, P<0.05) in pediatric DCM compared with NF controls. Using BN-PAGE and Western blot, as well as proteomic analysis of isolated supercomplex bands, we demonstrated interaction of the ETC supercomplex with FAO enzymes. Quantification of fatty acyl-CoAs was also performed in tissue from pediatric patients with DCM which demonstrated altered content of a subset of acyl-CoAs when compared to NF controls. We detected higher content of some C8, C10 and C12 CoAs in DCM compared with NF (P<0.05), with depletion of C18:1, C18:2, and C16 species (P<0.05). There was no difference between groups in free CoA or Acetyl-CoA. Taken together, these data suggest a potentially important interaction between the ETC supercomplex and long-chain β-oxidation enzymes, which may be altered on heart failure. We provide preliminary evidence for disrupted energy utilization in the failing pediatric heart.

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

Elucidating Mitochondrial Electron Transport Chain Supercomplexes in the Heart During Ischemia–Reperfusion

Mitochondrial supercomplexes (SCs) are the large supramolecular assembly of individual electron transport chain (ETC) complexes that apparently provide highly efficient ATP synthesis and reduce electron leakage and reactive oxygen species (ROS) production. Oxidative stress during cardiac ischemia-reperfusion (IR) can result in degradation of SCs through oxidation of cardiolipin (CL). Also, IR induces calcium overload and enhances reactive oxygen species (mitROS) in mitochondria that result in the opening of the nonselective permeability transition pores (PTP). The opening of the PTP further compromises cellular energetics and increases mitROS ultimately leading to cell death. Here, we examined the role of PTP-induced mitROS in disintegration of SCs during cardiac IR. The relationship between mitochondrial PTP, ROS, and SCs was investigated using Langendorff-perfused rat hearts subjected to global ischemia (25 min) followed by short-time (5 min) or long-time (60 min) reperfusion in the presence or absence of the PTP inhibitor, sanglifehrin A (SfA), and the mitochondrial targeted ROS and electron scavenger, XJB-5-131. Also, the effects of CL deficiency on SC degradation, PTP, and mitROS were investigated in tafazzin knockdown (TazKD) mice. Cardiac IR induced PTP opening and mitROS generation, inhibited by SfA. Percent distributions of SCs were significantly affected by IR, and the effects were dependent on the reperfusion time and reversed by SfA and XJB-5-131. TazKD mice demonstrated a 40% lower SC I + III+IV with reduced basal mitochondrial PTP, ROS, and ETC complex activity. Innovation and Conclusion: Sustained reperfusion after cardiac ischemia induces disintegration of mitochondrial SCs, and PTP-induced ROS presumably play a causal role in SC disassembly. Antioxid. Redox Signal. 27, 57-69.

Mitochondrial Inner Membrane Biomimic for the Investigation of Electron Transport Chain Supercomplex Bioelectrocatalysis

Researchers have proposed that the efficiency of the electron transport chain is due to the synergy among complexes I, III, and IV in the membrane. In this paper, the enzymes of the supercomplex were isolated together, reconstituted into lipids that mimic the inner membrane of mitochondria, and immobilized in a tethered lipid bilayer on a gold electrode. The supercomplex enzymes retained their activity with the addition of their substrates and were inhibited by their respective toxins. The bioelectrocatalytic studies indicate the interdependence of the activity of the different complexes in the bioelectrocatalysis of the electron transport chain supercomplex. These fundamental studies provide a starting point to consider the use of supercomplexes and enzyme cascades for bioenergy conversion applications and biosensing through the regulation of the activity by inhibition.

Impaired mitochondrial oxidative phosphorylation and supercomplex assembly in rectus abdominis muscle of diabetic obese individuals.

Skeletal muscle mitochondrial dysfunction has been documented in patients with type 2 diabetes mellitus; however, specific respiratory defects and their mechanisms are poorly understood. The aim of the current study was to examine oxidative phosphorylation and electron transport chain (ETC) supercomplex assembly in rectus abdominis muscles of 10 obese diabetic and 10 obese non-diabetic individuals. Twenty obese women undergoing Roux-en-Y gastric bypass surgery were recruited for this study. Muscle samples were obtained intraoperatively and subdivided for multiple analyses, including high-resolution respirometry and assessment of supercomplex assembly. Clinical data obtained from referring physicians were correlated with laboratory findings. Participants in both groups were of a similar age, weight and BMI. Mitochondrial respiration rates were markedly reduced in diabetic vs non-diabetic patients. This defect was observed during maximal ADP-stimulated respiration in the presence of complex I-linked substrates and complex I- and II-linked substrates, and during maximal uncoupled respiration. There were no differences in fatty acid (octanoyl carnitine) supported respiration, leak respiration or isolated activity of cytochrome c oxidase. Intriguingly, significant correlations were found between glycated haemoglobin (HbA1c) levels and maximal respiration or respiration supported by complex I, complex I and II or fatty acid. In the muscle of diabetic patients, blue native gel electrophoresis revealed a striking decrease in complex I, III and IV containing ETC supercomplexes. These findings support the hypothesis that ETC supercomplex assembly may be an important underlying mechanism of muscle mitochondrial dysfunction in type 2 diabetes mellitus.

Initiation of Electron Transport Activity and a Decrease of Oxidative Stress Occur Simultaneously during Embryonic Heart Development

Mitochondria in early embryonic hearts are not thought to produce ATP, yet they do produce reactive oxygen species (ROS) that regulate myocyte differentiation. To assess changes in ATP and ROS generation in the developing heart, we measured mitochondrial oxygen consumption, the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC), ETC supercomplex assembly, and ROS in embryonic mouse hearts. At embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the ETC complexes are present. We show that Cx-1 is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx −1, −3, and −4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In contrast, generation of reactive oxygen species (ROS), as measured with Amplex Red, is high at E9.5 and drops significantly by E11.5, coinciding with activation of the ETC. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in a decrease of ROS generation and an increase in OXPHOS due to the activation of Cx-1 and the formation of supercomplexes.

Characterization of the Electron Transport Chain Metabolon Bioelectrocatalysis

The electron transport chain is a group of membrane redox enzymes that are the driving force for the production of energy in mitochondria. Several theories and models have circulated different structures of the enzymes in the inner membrane.1 The three enzyme complexes form a supercomplex or respirasome, where Complex I, dimer Complex III, and various copies of Complex IV are electrostatically, hydrophobically and catalytically connected. While structural, kinetic, and genetic evidence prove the formation of the supercomplex, characterization of the bioelectrocatalysis has never been attempted of the three enzymes together on an electrode.2 The supercomplex contains multiple redox cofactors of the individual complex that span the lipid membrane and can come in contact with the electrode surface. By purifying the electron transport chain supercomplex, we have been able to reconstruct the inner membrane and immobilizing it onto a gold electrode. A polymer tethered lipid bilayer allows for the study of the natural bioelectrocatalysis of the large transmembrane proteins. With the addition of oxidized cytochrome c, Complex IV gives a catalytic response indicating that Complex III and Complex IV are active. With the addition of NADH and oxidized cytochrome c, the catalytic peaks increase. This indicates that all three enzymes are working together increasing their redox activities at the surface of the electrode. The supercomplex is further confirmed by inhibition of CIII and CIV, eliminating all activity of the enzymes at the electrode surface. This evidence shows how dependent the enzymes are on each other for efficient catalytic activity. The characterization of the supercomplex of these transmembrane redox enzymes and their bioelectrocatalysis on the electrode is the first for studying a natural metabolon electrochemically.

The functions of cardiolipin in cellular metabolism–potential modifiers of the Barth syndrome phenotype

The phospholipid cardiolipin (CL) plays a role in many cellular functions and signaling pathways both inside and outside of mitochondria. This review focuses on the role of CL in energy metabolism. Many reactions of electron transport and oxidative phosphorylation, the transport of metabolites required for these processes, and the stabilization of electron transport chain supercomplexes require CL. Recent studies indicate that CL is required for the synthesis of iron–sulfur (Fe–S) co-factors, which are essential for numerous metabolic pathways. Activation of carnitine shuttle enzymes that are required for fatty acid metabolism is CL dependent. The presence of substantial amounts of CL in the peroxisomal membrane suggests that CL may be required for peroxisomal functions. Understanding the role of CL in energy metabolism may identify physiological modifiers that exacerbate the loss of CL and underlie the variation in symptoms observed in Barth syndrome, a genetic disorder of CL metabolism.

Supercomplexes of the mitochondrial electron transport chain decline in the aging rat heart

Accumulation of mitochondrial electron transport chain (ETC) defects is a recognized hallmark of the age-associated decline in cardiac bioenergetics; however, the molecular events involved are only poorly understood. In the present work, we hypothesized that age-related ETC deterioration stemmed partly from disassociation of large solid-state macromolecular assemblies termed “supercomplexes”. Mitochondrial proteins from young and old rat hearts were separated by blue native-PAGE, protein bands analyzed by LC–MALDI-MS/MS, and protein levels quantified by densitometry. Results showed that supercomplexes comprised of various stoichiometries of complexes I, III and IV were observed, and declined significantly ( p < 0.05, n = 4) with age. Supercomplexes displaying the highest molecular masses were the most severely affected. Considering that certain diseases (e.g. Barth Syndrome) display similar supercomplex destabilization as our results for aging, the deterioration in ETC supercomplexes may be an important underlying factor for both impaired mitochondrial function and loss of cardiac bioenergetics with age.