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The mitochondrial oxidative phosphorylation system is central to cellular metabolism. It comprises five enzymatic complexes and two mobile electron carriers that work in a mitochondrial respiratory chain. By coupling the oxidation of reducing equivalents coming into mitochondria to the generation and subsequent dissipation of a proton gradient across the inner mitochondrial membrane, this electron transport chain drives the production of ATP, which is then used as a primary energy carrier in virtually all cellular processes. Minimal perturbations of the respiratory chain activity are linked to diseases; therefore, it is necessary to understand how these complexes are assembled and regulated and how they function. In this Review, we outline the latest assembly models for each individual complex, and we also highlight the recent discoveries indicating that the formation of larger assemblies, known as respiratory supercomplexes, originates from the association of the intermediates of individual complexes. We then discuss how recent cryo-electron microscopy structures have been key to answering open questions on the function of the electron transport chain in mitochondrial respiration and how supercomplexes and other factors, including metabolites, can regulate the activity of the single complexes. When relevant, we discuss how these mechanisms contribute to physiology and outline their deregulation in human diseases.Mitochondrial respiration relies on five enzymatic complexes that couple electron transport with proton pumping, leading to ATP synthesis. Recent studies have shed new light on the organization, assembly and mechanisms of the respiratory complexes, including the formation of their larger assemblies — respiratory supercomplexes — and their roles in physiology.

Five protein complexes, CI–CV, form the oxidative phosphorylation electron transport chain in the mitochondrial membrane and can be found organized into supercomplexes (SCs): I+III2+IV, or respirasome; I+III2; III2+IV; and CV2. Letts and Sazanov review current knowledge on the structure, assembly and function of respiratory SCs. The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI–CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII 2 and CIV (SC I+III 2 +IV, known as the respirasome), as well as with CIII 2 alone (SC I+III 2 ). CIII 2 forms a supercomplex with CIV (SC III 2 +IV) and CV forms dimers (CV 2 ). Recent cryo-EM studies have revealed the structures of SC I+III 2 +IV and SC I+III 2 . Furthermore, recent work has shed light on the assembly and function of the SCs. Here we review and compare these recent studies and discuss how they have advanced our understanding of mitochondrial electron transport.

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13 C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization. Production of inflammatory mediators by M1-polarized macrophages is thought to rely on suppression of mitochondrial metabolism in favor of glycolysis. Refining this concept, here the authors define metabolic targets of nitric oxide as responsible for the mitochondrial rewiring resulting from polarization.

Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.

Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels. In human, plant and other eukaryotic cells, fats are an important source of energy and also play many other roles including waterproofing, thermal insulation and energy storage. Eukaryotic cells have two systems that make the building blocks of fats (known as fatty acids) and one of these systems, called the mtFAS pathway, operates in small compartments known as mitochondria. This pathway only has one known product, a small fat molecule called lipoic acid, which mitochondria attach to several enzymes to allow them to work properly. The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells. They achieve this using large groups of proteins known as ETC complexes. To build these complexes, families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures. However, it remains unclear precisely how this process works. Here, Nowinski et al. used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells. The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than ‘normal’ cells. Furthermore, a family of ETC assembly factors were less stable in the mutant cells. These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes. Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another, as yet unidentified, product that regulates this process, instead. MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement, slurred speech and impaired vision in children. Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway, therefore, a better understanding of how the pathway works may help researchers develop new treatments in the future. More broadly, these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified.

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III2, and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III2 and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

Respiration is one of the most vital and basic features of living organisms. In mammals, respiration is accomplished by respiratory chain complexes located on the mitochondrial inner membrane. In the past century, scientists put tremendous efforts in understanding these complexes, but failed to solve the high resolution structure until recently. In 2016, three research groups reported the structure of respiratory chain supercomplex from different species, and fortunately the structure solved by our group has the highest resolution. In this review, we will compare the recently solved structures of respirasome, probe into the relationship between cristae shape and respiratory chain organization, and discuss the highly disputed issues afterwards. Besides, our group reported the first high resolution structure of respirasome and medium resolution structure of megacomplex from cultured human cells this year. Definitely, these supercomplex structures will provide precious information for conquering the mitochondrial malfunction diseases.

Mitochondria are recognized as the main source of ATP to meet the energy demands of the cell. ATP production occurs by oxidative phosphorylation when electrons are transported through the electron transport chain (ETC) complexes and develop the proton motive force across the inner mitochondrial membrane that is used for ATP synthesis. Studies since the 1960s have been concentrated on the two models of structural organization of ETC complexes known as “solid-state” and “fluid-state” models. However, advanced new techniques such as blue-native gel electrophoresis, mass spectroscopy, and cryogenic electron microscopy for analysis of macromolecular protein complexes provided new data in favor of the solid-state model. According to this model, individual ETC complexes are assembled into macromolecular structures known as respiratory supercomplexes (SCs). A large number of studies over the last 20 years proposed the potential role of SCs to facilitate substrate channeling, maintain the integrity of individual ETC complexes, reduce electron leakage and production of reactive oxygen species, and prevent excessive and random aggregation of proteins in the inner mitochondrial membrane. However, many other studies have challenged the proposed functional role of SCs. Recently, a third model known as the “plasticity” model was proposed that partly reconciles both “solid-state” and “fluid-state” models. According to the “plasticity” model, respiratory SCs can co-exist with the individual ETC complexes. To date, the physiological role of SCs remains unknown, although several studies using tissue samples of patients or animal/cell models of human diseases revealed an associative link between functional changes and the disintegration of SC assembly. This review summarizes and discusses previous studies on the mechanisms and regulation of SC assembly under physiological and pathological conditions.

Oxidative phosphorylation (OXPHOS) is vital for the regeneration of the vast majority of ATP in eukaryotic cells 1 . OXPHOS is carried out by large multi-subunit protein complexes in the cristae membranes, which are invaginations of the mitochondrial inner membrane. The OXPHOS complexes are a mix of subunits encoded in the nuclear and mitochondrial genomes. Thus, the assembly of these dual-origin complexes is an enormous logistical challenge for the cell. Using super-resolution microscopy (nanoscopy) and quantitative cryo-immunogold electron microscopy, we determined where specific transcripts are translated and where distinct assembly steps of the dual-origin complexes in the yeast Saccharomyces cerevisiae occur. Our data indicate that the mitochondrially encoded proteins of complex III and complex IV are preferentially inserted in different sites of the inner membrane than those of complex V. We further demonstrate that the early, but not the late, assembly steps of complex III and complex IV occur preferentially in the inner boundary membrane. By contrast, all steps of complex V assembly occur mainly in the cristae membranes. Thus, OXPHOS complex assembly is spatially well orchestrated, probably representing an unappreciated regulatory layer in mitochondrial biogenesis. Using super-resolution microscopy and cryo-electron microscopy, Stoldt et al. show that mitochondrial transcript translation and OXPHOS complex assembly are spatially partitioned within the mitochondrial membrane.

Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans , however, the positions of heme b 595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism. Cytochrome bd oxidases couple quinol oxidation and the release of protons to the periplasmic side with proton uptake from the cytoplasmic side to reduce dioxygen to water and they are the terminal reductases in bacterial and archaeal respiratory chains. Here the authors present the cryo-EM structure of Escherichia coli bd oxidase and discuss mechanistic implications.