Mitochondria Supercomplex – The Untold Story

Art and science are similar in some ways. The success of an artist depends upon the ability to realize the imagination, while the success of a scientist depends on the ability to find reality in the imagination.  The extensive work that has been done on mitochondria is a perfect example of this idea. 

Mitochondria is undoubtedly one of the most studied and referenced organelles in biology.  They are often called the ‘powerhouses of the cell’ because they are responsible for the generation of metabolic energy in all eukaryotic cells found in fungi, protists, plants, and animals including humans [1].  Unlike eukaryotes, prokaryotes such as bacteria, do not have their genes enclosed within the nuclei [2] and they also don’t have organelles like mitochondria [3]. 

The earliest records on intracellular structures that might have represented mitochondria probably dates as far back as the 1840s  [4]. Richard Altmann was the first to recognize the ubiquitous occurrence of mitochondria that carried out vital functions. Altmann called these organelles “Bioblasts” in his famous “Die elementarorganismen und ihre beziehungen zu den zellen” (1890) [5]. 

The work by Altmann was initially rejected and highly criticized by his peers. His idea that mitochondria resembling bacteria, carried out vital metabolic and genetic functions eventually led to the birth of many important theories including endosymbiosis, maternal inheritance, and energy metabolism [5]. Of all the theories on mitochondrial energy metabolism, none was more influential than that proposed by Peter D. Mitchell in 1961 [6].

Ever since Otto Warburg discovered Atmungsferment, the enzymatic basis for cellular respiration in 1928, scientists have vigorously investigated the structure, function and organization of the mitochondrial respiratory chain, responsible for energy production.  These understandings crystallized in the Mitchell’s chemiosmosis hypothesis (1961), which proposed that adenosine triphosphate (ATP) synthesized in mitochondria is produced from the electrochemical gradient across the inner membranes. This gradient is generated from the electron energy from NADH and FADH released from the catabolic metabolism of energy-rich molecules such as glucose and fatty acids. [7]

Although Mitchell’s chemiosmosis theory has been universally accepted for close to six decades, controversies that were raised initially still remain unresolved as of the end of 2019 [8].  Recent technological advances in the understanding of proton-displacements revealed that proton translocation is a lateral movement rather than a transversal one in coupling membranes. Whereas the exact species involved in the transfer of protons is still to be identified. 

The exact structure and organization of the mitochondrial respiratory chain proteins also eluded scientists for many years, until the the work by Schägger et al. in 2000 that identified the existence of mitochondrial supercomplexes (SC) using a high-resolution technique called BN-PAGE (blue native polyacrylamide gel electrophoresis) [9]

Supercomplexes – Surprising Combinations

Schägger provided strong evidence that the ETC (electron transport chain) proteins, complexes I to IV, can assemble into supramolecular structures [10].  Near-atomic resolution of ETC proteins based on cryo-electron microscopy and refinement technology allowed further understanding of the structural organization of supercomplexes [11].   The discovery of supercomplexes changes the way ETC complexes have been, and still are, being depicted.

The ETC complex proteins are found along the inner membrane of the mitochondria. In many organisms, Complex I, III and IV are found to associate into specific supercomplexes.  Complex I, III and IV are oxidoreductases that couple electron transport with the movement of protons across the inner mitochondrial membrane. The generation of this proton motive force allows complex V, ATP synthase to produce ATP from ADP and phosphate.  Electrons must enter the respiratory chain through NADH dehydrogenase, or Complex I, which is responsible for transferring electrons from NADH molecules to a lipophilic ubiquinone molecule. This reduced ubiquinone then shuttles electrons to Complex III or cytochrome c reductase. A small protein called cytochrome c then transfers the electrons from Complex III to Complex IV, cytochrome c oxidase, which is responsible for the transfer of electrons to molecular oxygen, reducing it to water [12].  

Complex II, or succinate dehydrogenase can transfer electrons from succinate to ubiquinone, and therefore connects the Krebs (citric acid) cycle to the respiratory chain.  Due to this unique feature, Complex II has seldom been seen to form part of any respiratory chain supercomplexes [12]. 

Most standard textbooks diagrams of the ETC would show the protein complex I to V lined up neatly, one after another in the following manner:


[Source: Biochimica et Biophysica Acta (BBA) – Bioenergetics Volume 1797, Issues 6–7, June–July 2010, Pages 664-670, Dudkina et al. 2009  Structure and function of mitochondrial supercomplexes]


In reality, supercomplexes, sometimes also referred to as respirasomes, have been found to be comprised of various combinations of complex I, III and IV.  This is an image using single particle electron-microscope projection of supercomplexes from bovine heart mitochondria by Dudkina et al. in 2010. 

Supercomplexes from Bovine Mitochondria

[Source: Biochimica et Biophysica Acta (BBA) – Bioenergetics Volume 1797, Issues 6–7, June–July 2010, Pages 664-670, Dudkina et al. 2009  Structure and function of mitochondrial supercomplexes]


The work of Davies et al. in 2018 further elucidated the organization and structure of supercomplexes with the application of cryo-electron and subtomogram averaging to mitochondrial membranes of three different eukaryotic lineages.  

In all cases studied, Davies found complex I to form an assembly with complex III.  This in situ combination of complex I and III is conserved across all phyla. In mammals and fungi, up to two copies of complex IV can be attached to the I-III core, and interestingly, no complex IV were identified in plant supercomplexes.  Most importantly, a VARIETY of supercomplexes can COEXIST in the same membrane, with the structure and composition varying between species and even within one species [13]. 

For example, Davies et al. found that in bovine heart mitochondria, 44% of the supercomplexes contained only one single copy of Complex I (A); 16% are formed by one copy of Complex I and III each (B); 30% contained one copy each of Complex I, Complex III and IV (C); while 10% contained one complex I, one complex III and two separate complex IV (D)! 

[Source: PNAS March 20, 2018 115 (12) 3024-3029; March 8, 2018 Davies et a. Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants]

The Fluid, Solid and Plastic Models of Supercomplexes

The ability to observe supercomplexes at atomic resolution spurred numerous research that attempt to explain these novel structural organization of ETC complexes.  Currently, there are three accepted models for the organization of ETC complexes 

Fluid-State Model 

The fluid, or fluid-state model states that the ETC complexes do not interact; that these respiratory complexes freely diffuse in membranes and that the electron transfers are based on random collisions of the single complexes [17].  The fluid model was generally accepted until the invention of blue native polyacrylamide gel electrophoresis (BN-PAGE), which allowed the separation and clear visualization of supercomplexes. 

Solid-State Model

The solid-state model challenges the fluid model in that complexes form supercomplexes that interact with each other. This model has been supported by a wide variety of experimental findings [14].  These solid-state complexes contain proteins that transfer electrons along pre-defined, enclosed pathways between their component complexes and do not exchange them with the outside [18].

Plasticity Model

The plasticity model presented by Acín-Peréz et al. (2008) proposed that free individual units of respiratory complexes and supercomplexes maintain a dynamic equilibrium through constant dissociation and reassembly [15].  The plasticity model is by far, the most widely accepted theory about respiratory chain organization. In this model, most of Complex II, Complex IV and a relevant proportion of Complex II stand alone and seem to move freely along the inner mitochondrial membrane while the majority of Complex I is stabilized by Complex III, with or without several copies of Complex IV.  Electron transfers are believed to be carried out by ubiquinone shuttling within the I-III-IV supercomplexes [19].  

This constant assembly and disassembly of supercomplexes serve vital biological regulatory functions.

Biological Significance of Supercomplexes

Since the introduction of the concept of supercomplex, extensive studies have been performed to unravel the reasons for the formation of supercomplex organization in OXPHOS (oxidative phosphorylation).  It is now widely acknowledged that the stability of Complex I is significantly enhanced by the formation of supercomplexes with Complex III and IV [20].  

Studies have shown that the formation of supercomplex can dramatically lower the generation of reactive oxygen species [21].  Supercomplexes have been seen to enhance the catalytic activity of individual components [22], and it is also possible that supercomplex can increase efficiency of electron transfer through substrate channeling, although structural data do not convincingly support the hypothesis of substrate-channeling [23].

In general, supercomplexes are ubiquitously accepted by the scientific community. However, questions regarding the rationale for their existence, and whether they confer functional and/or structural advantages, or whether they actually contribute to the pathogenesis of human disease remain unanswered.   Several insightful studies managed to shed some light on how nature uses supercomplexes in the most unexpected ways. 

Supercomplex Assembly in the Brain – A Tale of Neurons and Astrocytes

Neurons and astrocytes are known for the difference in their redox and bioenergetic characteristics. Neurons depend upon oxidative phosphorylation to generate energy to support neurotransmission and survival [24];  whereas astrocytes do not have that requirement. In 2016, Lopez-Fabuela et al. made the startling discovery that neurons show a much greater proportion of complex I that are assembled into supercomplexes, whereas astrocytes have predominantly free complex I that are not assembled into supercomplexes [25].  This unique feature was also correlated with the several-fold higher rates of reactive oxygen species found to be generated by astrocytes compared to neurons. 

The lower proportion of Complex I assembled into supercomplexes in astrocytes also explained their lower respiration rate and higher glycolytic metabolism [26].  The large difference in mitochondrial ROS generated in neurons and astrocytes is tightly correlated with the amount of free complex I that is not assembled into supercomplexes.  The authors concluded that free unassembled Complex I in astrocytes is the reason for the higher ROS production and that is also why astrocytes are protected by a robust redox antioxidant system [27]. 

The plasticity model proposes that supercomplexes maintain a dynamic equilibrium through constant assembly and disassembly. Therefore, it is not surprising that increased energy requirements can increase the rate of supercomplex assembly.  

Supercomplex Assembly – Balancing Act between Health & Disease

After 4 months of exercise training, the effects on mitochondrial ETC complexes in the skeletal muscles of 26 healthy sedentary older adults were evaluated.  Results after careful analysis showed that Complex I was the most upregulated ETC protein, and it was almost exclusively found to be assembled in supercomplexes of muscle mitochondria.  There was an overall increase in supercomplex content after the exercise period, in particular, complexes I, III and IV were redistributed to form supercomplex I+III+IV [16]. 

Yet the story of the supercomplex promoting molecule COX74RP reveals a different perspective. 

COX7RP is a key molecule that promotes supercomplex assembly and regulates energy generation.  Mice bred without Cox7rp showed reduced rate of ATP synthesis in liver, and also lower blood glucose levels.  COX7RP, also known as COX7A2L and SCAF1, encodes a protein that is critical for the regulation of muscle activities and the homeostasis of brown adipose tissue (BAT).  Loss of function of COX7RP led to decreased ATP production in liver, reducing hepatic gluconeogenesis and insulin sensitivity [28].  

COX7RP is induced by cellular stress.  Cellular stress almost always demands higher energy production levels to counter stress effects in both normal and cancer cells.  A major landmark study released by Ikeda et al in September 2019, revealed that COX7RP is abundantly expressed in breast and endometrial cancers and promotes cancer growth both in vitro and IN VIVO [30].  The overexpression of COX7RP is associated with prognosis of breast cancer patients. To counter cellular stress, glutathione (GSH) levels were also significantly elevated in breast cancer cells that overexpress COX7RP.  The authors believed that enhanced glutathione synthesis contributed to the elevated growth of COX7RP overexpressing cancer cells. This theory is supported by previous studies where suppression of glutathione synthesis led to reduced tumor initiation and impaired resistance to chemotherapeutic drugs. 

Everything in nature has the potential to be a double-edged sword. Supercomplex assemblies can be beneficial or detrimental in different contexts.  How that context can be shaped and modulated is key to solving the puzzle of health and disease. 

Mitochondria are dynamic organelles that constantly change their shapes and sizes. The recent discovery of the existence of MEGACOMPLEXES provide solid evidence that connects the organization of the respiratory chain with the shape of cristae….. To Be Continued……


[1] Eukaryotic Cells | Basic Biology

[2] 22.2A: Basic Structures of Prokaryotic Cells – Biology LibreTexts

[3] Mitochondria – The Cell – NCBI Bookshelf 

[4] Mitochondria: a historical review. | The Journal of Cell Biology | Rockefeller University Press

[5] From Bioblasts to Mitochondria: Ever Expanding Roles of Mitochondria in Cell Physiology

[6] Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. – PubMed – NCBI

[7] Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. – PubMed – NCBI

[8] An update of the chemiosmotic theory as suggested by possible proton currents inside the coupling membrane | Open Biology 

[9] Supercomplexes in the respiratory chains of yeast and mammalian mitochondria 

[10] Respiratory chain supercomplexes. 

[11] The architecture of respiratory supercomplexes | Nature

[12] The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes

[13] Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants | PNAS

[14] The Enigma of the Respiratory Chain Supercomplex

[15] Respiratory Active Mitochondrial Supercomplexes – ScienceDirect

[16] Exercise increase supercomplex_Enhanced Respiratory Chain Supercomplex Formation in Response to Exercise in Human Skeletal Muscle – ScienceDirect

[17] The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport | SpringerLink 

[18] Activity of the cytochrome system in heart muscle preparations | Biochemical Journal | Portland Press

[19] Structure and mechanism of mitochondrial electron transport chain – ScienceDirect

[20] Supramolecular Organization of Respiratory Complexes | Annual Review of Physiology

[21] Mitochondrial Respiratory Supercomplex Association Limits Production of Reactive Oxygen Species from Complex I | Antioxidants & Redox Signaling

[22] Mitochondrial Complex I Plays an Essential Role in Human Respirasome Assembly – ScienceDirect 

[23] Modeling the Q-cycle mechanism of transmembrane energy conversion. – PubMed – NCBI

[24] Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection. – PubMed – NCBI

[25] Complex I assembly into supercomplexes determines differential mitochondrial ROS production in neurons and astrocytes

[26] Astrocyte-neuron metabolic relationships: for better and for worse. – PubMed – NCBI

[27] Antioxidant and bioenergetic coupling between neurons and astrocytes. – PubMed – NCBI

[28] Deficiency of COX7RP, a mitochondrial supercomplex assembly promoting factor, lowers blood glucose level in mice 

[29] Isolation of Estrogen-Responsive Genes with a CpG Island Library

[30] Mitochondrial supercomplex assembly promotes breast and endometrial tumorigenesis by metabolic alterations and enhanced hypoxia tolerance | Nature Communications  

[31] Chemotherapy triggers HIF-1-dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype. – PubMed – NCBI

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