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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.

Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Here we present three architectures of mammalian (ovine) supercomplexes determined by cryo-electron microscopy. We identify two distinct arrangements of supercomplex CICIII 2 CIV (the respirasome)—a major ‘tight’ form and a minor ‘loose’ form (resolved at the resolution of 5.8 Å and 6.7 Å, respectively), which may represent different stages in supercomplex assembly or disassembly. We have also determined an architecture of supercomplex CICIII 2 at 7.8 Å resolution. All observed density can be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production. To our knowledge, these are the first complete architectures of the dominant, physiologically relevant state of the electron transport chain. Respirasomes are supercomplexes of mitochondrial electron transport chain complexes that are responsible for cellular respiration and energy production; cryo-electron microscopy structures of mammalian (sheep) respirasomes are presented. Inside the mammalian respirasome supercomplex Mitochondrial electron transport chain complexes are responsible for cellular respiration and energy production. They are organized in supercomplexes called respirasomes. Two studies in this issue of Nature report cryo-electron microscopy structures of the supercomplex consisting of complex I, the dimer of complex III and complex IV at resolutions ranging from 5.4 Å to 7.8 Å. Maojun Yang and colleagues study the respirasome isolated from porcine heart, whereas Leonid Sazanov and colleagues obtain it from ovine heart. The structures provide insights into the organization of subunits within complexes and the interactions between the complexes.

The atomic structure of ovine mitochondrial complex I is solved at 3.9 Å resolution, revealing that supernumerary subunits stabilize the complex and providing insight into the molecular basis of its function and regulation. Mammalian mitochondrial complex 1 structure The first and largest enzyme in the respiratory chain in mammalian mitochondria is complex I, which transfers electrons from NADH to ubiquinone coupled to proton translocation across the membrane. Its huge size and complexity have meant that detailed structural information has been hard to come by. Here Leonid Sazanov and colleagues present the nearly complete atomic structure of ovine mitochondrial complex I, solved at a resolution of 3.9 Å. The structure, revealing 45 different protein subunits combined in a in highly interlinked assembly, provides a basis for understanding the molecular basis of mutations and mechanisms of complex I function and regulation. Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane 1 , 2 . It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons 3 . Here we present a nearly complete atomic structure of ovine ( Ovis aries ) mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are shown to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active–deactive transition of the enzyme. Our structure provides insight into the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations.

Mitochondrial complex III (CIII 2 ) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III 2 +IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII 2 , CIV, and SC III 2 +IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII 2 and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII 2 revealed long-range, coordinated movements across the complex, as well as the motion of CIII 2 ’s iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses. Most living things including plants and animals use respiration to release energy from food. Respiration requires the activity of five large protein complexes typically called complex I, II, III, IV and V. Sometimes these complexes combine to form supercomplexes. The complexes are similar across plants, animals and other living things, but there are also many differences. Detailed structures of the respiratory complexes have been determined for many species of animals, fungi and bacteria, highlighting similarities and differences between organisms, and providing clues as to how respiration works. Yet, there is still a lot to learn about these complexes in plants. To bridge this gap, Maldonado et al. used a technique called cryo electron microscopy to study the structure of complexes III and IV and the supercomplex they form in the mung bean. This is the first study of the detailed structure of these two complexes in plants. The results showed many similarities to other species, as well as several features that are specific to plants. The way the two complexes interact to form a supercomplex is different than in other species, as are several other, smaller, structural features. Further examination of complex III revealed that it is flexible and that movements are coordinated across the length of the complex. Maldonado et al. speculate that this may allow it to coordinate its role in respiration with its other cellular roles. Understanding how plant respiratory complexes work could lead to improvements in crop yields or, since respiration is required for survival, result in the development of herbicides that block respiration in plants more effectively and specifically. Further researching the structure of the plant respiratory complexes and supercomplexes could also shed light on how plants adapt to different environments, including how they change to survive global warming.

Respiration, an essential metabolic process, provides cells with chemical energy. In eukaryotes, respiration occurs via the mitochondrial electron transport chain (mETC) composed of several large membrane-protein complexes. Complex I (CI) is the main entry point for electrons into the mETC. For plants, limited availability of mitochondrial material has curbed detailed biochemical and structural studies of their mETC. Here, we present the cryoEM structure of the known CI assembly intermediate CI* from Vigna radiata at 3.9 Å resolution. CI* contains CI’s NADH-binding and CoQ-binding modules, the proximal-pumping module and the plant-specific γ-carbonic-anhydrase domain (γCA). Our structure reveals significant differences in core and accessory subunits of the plant complex compared to yeast, mammals and bacteria, as well as the details of the γCA domain subunit composition and membrane anchoring. The structure sheds light on differences in CI assembly across lineages and suggests potential physiological roles for CI* beyond assembly. Respiration is the process used by all forms of life to turn organic matter from food into energy that cells can use to live and grow. The final stage of this process relies on an intricate chain of protein complexes which produce the molecule that cells use for energy. Complexes in the chain are made up of specific proteins that are carefully assembled, often into discrete modules or intermediate complexes, before coming together to form the full protein complex. Understanding how these complexes are assembled provides important insights into how respiration works. The precise three-dimensional structure of these complexes has been identified for bacteria, yeast and mammals. However, less is known about how these respiration complexes form in plants. For this reason, Maldonado et al. studied the structure of an intermediate complex that is only found in plants, called Cl*. This intermediate structure goes on to form complex I – the largest complex in the respiration chain. A technique called cryo-electron microscopy was used to obtain a structure of Cl* at a near-atomic level of detail. This structure revealed how the proteins that make up Cl* fit together, highlighting differences and similarities in how plants assemble complex I compared to bacteria, yeast and mammals. Maldonado et al. also studied the activity of Cl*, leading to the suggestion that this complex may be more than just a stepping stone towards building the full complex I and could have its own role in the cell. The structure of this complex provides new insights into the respiration mechanism of plants and could help scientists improve crop production. For instance, new compounds may be able to block respiration in pests, while leaving the crop unharmed; or genetic modifications could create plants that respire more efficiently in different environments.

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway ‘deactive’ state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster . We show that although D. melanogaster complex I ( Dm -CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm -CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm -CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm -CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.

In voltage-gated Na⁺, K⁺, and Ca²⁺ channels, four voltage-sensor domains operate on a central pore domain in response to membrane voltage. In contrast, the voltage-gated proton channel (Hv) contains only a voltage-sensor domain, lacking a separate pore domain. The subunit stoichiometry and organization of Hv has been unknown. Here, we show that human Hv1 forms a dimer in the membrane and define regions that are close to the dimer interface by using cysteine cross-linking. Two dimeric interfaces appear to exist in Hv1, one mediated by S1 and the adjacent extracellular loop, and the other mediated by a putative intracellular coiled-coil domain. It may be significant that Hv1 uses for its dimer interface a surface that corresponds to the interface between the voltage sensor and pore in Kv channels.

Mitochondrial complex III (CIII.sub.2) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III.sub.2+IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII.sub.2, CIV, and SC III.sub.2+IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII.sub.2 and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII.sub.2 revealed long-range, coordinated movements across the complex, as well as the motion of CIII.sub.2's iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses.

Mutations in mitochondrial complex I can cause severe metabolic disease. Although no treatments are available for complex I deficiencies, chronic hypoxia improves lifespan and function in a mouse model of the severe mitochondrial disease Leigh syndrome caused by mutation of complex I subunit NDUFS4. To understand the molecular mechanism of NDUFS4 mutant pathophysiology and hypoxia rescue, we investigated the structure of complex I in respiratory supercomplexes isolated from NDUFS4 mutant mice. We identified complex I assembly intermediates bound to complex III , proving the cooperative assembly model. Further, an accumulated complex I intermediate is structurally consistent with pathological oxygen-dependent reverse electron transfer, revealing unanticipated pathophysiology and hypoxia rescue mechanisms. Thus, the build-up of toxic intermediates and not simply decreases in complex I levels underlie mitochondrial disease.

Mitochondrial complex III (CIII ) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III +IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII , CIV, and SC III +IV from determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII revealed long-range, coordinated movements across the complex, as well as the motion of CIII 's iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses.