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.

The canonical mitochondrial electron transport chain (mETC), composed of four integral membrane protein complexes (complexes I–IV; CI–CIV) located in the inner mitochondrial membrane (IMM), transfers electrons from NADH and succinate to molecular oxygen. The concomitant pumping of protons (H⁺) across the IMM establishes an electrochemical proton gradient that is used by ATP synthase to produce ATP (Nicholls, 2013). Whereas the atomic details of the respiratory complexes and several supercomplexes (higher-order complex assemblies) are known for yeast, mammals and bacteria (Vinothkumar et al., 2014; Fiedorczuk et al., 2016; Gu et al., 2016; Letts et al., 2016; Wu et al., 2016; Zhu et al., 2016; Zickermann et al., 2015; Blaza et al., 2018; Guo et al., 2017; Letts et al., 2019; Agip et al., 2018; Parey et al., 2018), the high-resolution structural details of the respiratory complexes and supercomplexes of plants have remained mostly unknown.

Complex III (CIII₂), also called the cytochrome bc₁ complex or ubiquinol-cytochrome c oxidoreductase, is an obligate dimer that transfers electrons from ubiquinol in the IMM (reduced by CI, CII, or alternative NADH dehydrogenases) to soluble cytochrome c in the intermembrane space (IMS) (Nicholls, 2013). This redox reaction is coupled to the pumping four H⁺ to the IMS. CIII₂ is composed of three conserved subunits present in all organisms (cytochrome b, COB; cytochrome c₁, CYC1; and the iron-sulfur ‘Rieske’ subunit, UCR1), as well as a varying number of accessory subunits present in eukaryotes (Iwata et al., 1998; Xia et al., 2013; Xia et al., 1997). Each CIII monomer contains one low-potential heme b (b_L) and one high-potential heme b (b_H) in COB, a heme c in CYC1, a 2Fe-2S iron-sulfur cluster in UCR1, as well as two quinone-binding sites (Q_P and Q_N close to the positive/IMS and negative/matrix sides respectively) in COB. Given that CIII₂ is a dimer, these sites in each CIII monomer are symmetrical within the dimer in isolation. However, the symmetry may be broken when CIII assembles into asymmetrical supercomplexes (Letts et al., 2016; Letts et al., 2019; Letts and Sazanov, 2017). CIII₂’s redox and proton pumping occur via the ‘Q-cycle’ mechanism (Cramer et al., 2011), which allows for efficient electron transfer between ubiquinol (a two-electron donor) and cyt c (a one-electron acceptor). To this end, one electron is transferred from ubiquinol in the Q_p site to the 2Fe-2S in the UCR1 head domain. The head domain then undergoes a large conformational swinging motion from its ‘proximal’ position close to the Q_P site to a ‘distal’ CYC1 binding site adjacent to heme c₁ (Zhang et al., 1998). The electron is then transferred via heme c₁ to cyt c bound to CIII₂ in the IMS. Of note, the UCR1 head domain belongs to the opposite CIII protomer relative to COB and CYC1. The second electron donated by ubiquinol is transferred via hemes b_L and b_H to a quinone in the Q_N site, reducing it to ubisemiquinone. The cycle is repeated to regenerate ubiquinol in the Q_N site, ultimately reducing two molecules of cyt c and pumping four protons.

In eukaryotes, the large CIII₂ accessory subunits exposed to the mitochondrial matrix have homology to mitochondrial processing peptidases (MPP) of the pitrilysin family (Gakh et al., 2002). These metalloendopeptidases—composed of an active β subunit and an essential but catalytically inactive α subunit—cleave mitochondrial signal sequences of proteins that are imported into the mitochondria (Gakh et al., 2002). Whereas in yeast the CIII₂ accessory subunits with MPP homology (ScCor1/Cor2) have completely lost MPP enzymatic activity, the mammalian CIII₂ homolog (UQCR1/UQCR2 heterodimer) retains basal activity to only one known substrate (the Rieske subunit) (Gakh et al., 2002; Taylor et al., 2001). Hence, in yeast and mammals this enzymatic activity is carried out by soluble MPP heterodimers in the mitochondrial matrix. In contrast, in vascular plants, there is no additional soluble MPP enzyme, and all MPP activity is provided by the CIII₂ MPP heterodimer (MPP-α/β). Thus, in plants CIII₂ serves a dual role as a respiratory enzyme and a peptidase (Braun et al., 1992; Emmermann et al., 1993; Eriksson et al., 1994; Braun et al., 1995; Eriksson et al., 1996; Braun and Schmitz, 1995a; Glaser and Dessi, 1999). This integration of respiratory and peptidase activities may have occurred early in eukaryogenesis (Braun and Schmitz, 1995b). The bioenergetic implications of this dual function of plant CIII₂ remain unknown.

Complex IV (CIV), also called cytochrome c oxidase, transfers electrons from cyt c to molecular oxygen, reducing it to water (Nicholls, 2013). The redox reaction is coupled to the pumping of four protons into the IMS. Like CIII₂, CIV is composed of three conserved subunits (COX1, COX2, COX3) and a variable number of accessory subunits, depending on the organism. Electrons are transferred from cyt c to oxygen via COX1’s dinuclear copper (Cu_A), heme a and copper-associated heme a₃ (Cu_B, binuclear center). The passage of protons from the matrix to the IMS occurs through distinct ‘channels’ formed by protonatable amino-acid residues (Rich, 2017; Rich and Maréchal, 2013; Yoshikawa and Shimada, 2015; Wikström et al., 2018). It is currently believed that, whereas yeast CIV pumps protons through the K and D transfer pathways (named after key amino-acid residues in each pathway), mammalian CIV uses an H channel in addition to the K and D channels (Rich, 2017; Rich and Maréchal, 2013; Yoshikawa and Shimada, 2015; Wikström et al., 2018; Maréchal et al., 2020). The contribution of the K, D, H pathways in plant CIV has not been characterized.

In the IMM, respiratory complexes can be found as separate entities or as higher-order assemblies known as supercomplexes (Schägger and Pfeiffer, 2000). Although it was initially hypothesized that supercomplexes would allow for direct substrate channeling between complexes, evidence has mounted against this view (Gu et al., 2016; Letts et al., 2016; Letts et al., 2019; Yu et al., 2018; Sousa et al., 2016; Blaza et al., 2014; Fedor and Hirst, 2018). Instead, supercomplexes may have roles in improving the stability of the complexes, providing kinetic advantages to the electron transfer or reducing the production of reactive oxygen species or of aggregates in the IMM (Letts and Sazanov, 2017; Milenkovic et al., 2017). Supercomplexes of various stoichiometries between CIII₂ and CIV (e.g. SC CIII₂+CIV, SC CIII₂+CIV₂) have been seen (Schägger and Pfeiffer, 2000; Eubel et al., 2004; Eubel et al., 2003). In plants, the CIII₂-CIV supercomplex of highest abundance is a single CIII dimer with a single CIV monomer (SC III₂+IV) (Eubel et al., 2004). High-resolution structures of the model yeast Saccharomyces cerevisiae and Mycobacterium smegmatis CIII₂-CIV supercomplex (SC III₂+IV₂) have recently been determined (Hartley et al., 2019; Rathore et al., 2019; Gong et al., 2018; Wiseman et al., 2018). Although there is currently no high-resolution structure for a mammalian CIII₂-CIV supercomplex, the supercomplex between CI, CIII₂ and CIV (SC I+III₂+IV, the respirasome) shows a distinct interaction interface between CIII₂ and CIV relative to the SC III₂+IV₂ from yeast and bacteria (Gu et al., 2016; Letts et al., 2016). Similar to that seen in comparative tomographic studies of plant SC I+III₂ and bovine and yeast SC I+III₂+IV (Davies et al., 2018), the above SC III₂+IV₂ studies revealed that, while the general configuration of the individual CIII₂ and CIV are conserved, the location of the binding interface between CIII₂ and CIV in the supercomplex is divergent, with different subunits involved in the different organisms. For plant CIII₂ and CIV, the only currently available structural information is from low-resolution, 2D-averages of negative-stain EM samples from A. thaliana (Dudkina et al., 2005) and potato (Bultema et al., 2009). High-resolution structures or atomic models for CIII₂, CIV or their supercomplexes are not currently available for the plant kingdom.

Here we present the cryoEM structures of CIII₂ and SC III₂+IV from the vascular plant Vigna radiata (mung bean) at nominal resolutions of 3.2 Å and 3.8 Å, respectively. Using focused refinements around CIV, we achieved a nominal resolution for CIV of 3.8 Å. The structures reveal plant CIII₂ and CIV’s active sites, as well as the plant-specific configuration of several CIII₂ and CIV subunits. The structures also show the SC III₂+IV binding interface and orientation, which is unique to plants. Additionally, using cryoEM 3D-conformation variability analysis (Punjani and Fleet, 2020), we were able to visualize the swinging motion of CIII₂’s UCR1 head domain at 5 Å resolution in the absence of substrate or inhibitors. We also observed complex-wide coupled conformational changes in the rest of CIII₂. These results question long-standing assumptions, generate new mechanistic hypotheses and provide the initial structural basis for the development of more selective agricultural inhibitors of plant CIII₂ and CIV.

Mitochondria were isolated from etiolated V. radiata hypocotyls. The electron transport chain complexes were extracted using the gentle detergent digitonin. The extracted complexes were further stabilized with amphipathic polymers (amphipol) and separated on a sucrose gradient. Given our interest in respiratory complex I, we pooled fractions containing NADH-dehydrogenase activity and set up cryoEM grids. Details on the sucrose gradient and NADH-dehydrogenase activity of the sample are available in Maldonado et al., 2020. Upon 2D classification of the particles in the micrographs, it became evident that the pooled fractions contained not only the complex intermediate CI* (Maldonado et al., 2020), but also CIII₂ and SC III₂+IV. Complex III and CIV subunits were identified in the mixed mitochondrial pooled fraction by mass spectrometry, in addition to CI subunits (Supplementary file 1a, see Materials and methods for full dataset availability). Therefore, CIII₂ and SC III₂+CIV were purified in silico from the micrographs we had previously used to solve the structure of CI* (Maldonado et al., 2020). This is an example of ‘bottom-up structural proteomics’ (Ho et al., 2020) on a partially purified sample, in which a single sample was used for the structural determination of multiple respiratory complexes. Processing of the CIII₂ and SC III₂+IV particles resulted in near-atomic reconstructions of CIII₂ at 3.2 Å, of CIV at 3.8 Å and of SC III₂+IV at 3.8 Å (Figure 1, Videos 1–3, Figure 1—figure supplements 1–3, Table 1, Supplementary file 1b).

Figure 1 with 4 supplements see all

Download asset Open asset

Video 1

Download asset

Video 2

Download asset

Video 3

Download asset

Further examination using spectroscopic activity assays (Belt et al., 2017) confirmed that pooled fractions of the preparation contained CIII₂ respiratory activity (electron transfer from reduced decylubiquinone to cytochrome c) that was inhibited by CIII₂ inhibitors antimycin A and myxothiazol (Figure 1—figure supplement 4). The sample also showed CIV activity from reduced cytochrome c to oxygen that was inhibited by CIV inhibitor potassium cyanide (Figure 1—figure supplement 4). Although MPP activity of CIII₂ was recovered from isolated V. radiata mitochondrial membranes (not shown), MPP activity assays (Braun et al., 1992; Teixeira et al., 2015) of the pooled fractions were inconclusive. Owing to research restrictions during the 2020 COVID-19 pandemic, we were not able to further optimize the peptidase assay for the pooled fractions. However, given the high superposition between the V. radiata MPP domain shown here and structures of active MPP (see below), we believe our inability to confirm MPP activity in this case does not significantly impact our interpretation of the data.

Complex III dimer (CIII₂)

Overall structure and ligands

The V. radiata (Vr) structure confirms that VrCIII₂ contains 10 subunits per CIII monomer (three conserved, mitochondrially encoded subunits and seven accessory subunits), similar to yeast and mammals (Figure 2, Figure 2—figure supplement 1). As in mammals and yeast, VrCIII₂ contains 13 transmembrane helices per protomer: eight from COB (cyt b) and one from each of CYC1 (cyt c₁), UCR1, QCR8, QCR9, and QCR10 (Supplementary file 1b). The MPP domain, composed of two MPP-α/β heterodimers (one per CIII protomer), extends into the matrix. The C-terminus of CYC1 (six α-helices and a 2-strand β-sheet), the entire QCR6 subunit and UCR1’s head domain extend into the intermembrane space (IMS). Consistent with their known flexibility and conformational heterogeneity, the linker and head domains of UCR1 (Rieske iron-sulfur subunit) were disordered in our reconstruction and an atomic model was not produced for this region of UCR1. (Throughout the manuscript, we use plant subunit nomenclature unless otherwise stated; see Supplementary file 1c for details and name equivalence in other organisms.)

Figure 2 with 3 supplements see all

Download asset Open asset

All heme groups (heme b_H, b_L and heme c₁) were clearly visible and modelled into each cyt b and cyt c₁ subunit of the dimer. The distances between the hemes were consistent with that previously seen in other organisms (Figure 2B and C; Cramer et al., 2011). A catalytic Zn²⁺ ion was also visible and modelled into each MPP-β subunit (Figure 2B). Given that we were not able to build an atomic model for the head domain of UCR1, we did not model the iron-sulfur clusters (see 3DVA analysis below for more details).

Density consistent with cardiolipin, phosphatidylethanolamine and phosphatidylcholine lipids was found and modelled into the VrCIII₂ map (Figure 2E). Similar to that previously seen in yeast, the CIII₂ lipids concentrate in lipophilic cavities in COB, CYC1, UCR1, QCR8 and QCR10 close to the Q_N site, both on the surface of CIII₂ and at the interface between the CIII protomers. We also observed additional lipids on the exterior of COB close to the Q_P site.

The vast majority of the residues that form the Q_P and Q_N sites and inhibitor-binding pockets in yeast and bovine CIII₂ (Xia et al., 2013; Esser et al., 2004; Gao et al., 2003) are conserved in V. radiata (Figure 2—figure supplement 2). In particular, the COB residues that have been shown to form H-bonds with ubiquinone at the Q_N site of yeast and bovine CIII₂ are conserved in V. radiata (His208, Ser212, D235), as are the key residues of COB’s cd1 helix (Gly149-Ile253) and the PEWY motif (Pro277-Tyr280). Nevertheless, no clear density for endogenous quinone was visible in any of the binding sites of the V. radiata dimer.

It is well established that the mRNAs of several subunits of plant respiratory complexes, including several CI subunits, CIII’s COB and CIV’s COX1-3, undergo cytidine-to-uridine RNA editing at several sites (Covello and Gray, 1989; Gualberto et al., 1989; Hiesel et al., 1989). This mitochondrial RNA editing is widely conserved across vascular and non-vascular plants (Takenaka et al., 2013; Sper-Whitis et al., 1996), and most generally acts to restore consensus conserved sequences (Small et al., 2020). Inspection of the cryoEM density for the VrCOB subunits showed that the A. thaliana COB RNA-editing sites (Bentolila et al., 2008; Giegé and Brennicke, 1999) are conserved in V. radiata. Moreover, V. radiata COB contains additional RNA edit sites present in wheat, potato and rice (Ito et al., 1996; Zanlungo et al., 1993; Supplementary file 1d). Given the conservation pattern and the fact that the cryoEM density at these sites was unambiguous, residues were mutated to the edited residues in our model. RNA editing was also identified in V. radiata’s COX1 and 3 (see CIV section), as well as in CI subunits (Maldonado et al., 2020).

Differences in mitochondrially encoded subunits and non-MPP accessory subunits

Although the sequence conservation of the V. radiata (Vr) mitochondrially encoded CIII₂ subunits (COB, CYC1, UCR1) with respect to Saccharomyces cerevisiae (Sc) and bovine (Bos taurus, Bt) homologs is modest (~50%), their structural conservation is high (0.75–0.95 Å main chain RMSD, Figure 2—figure supplements 2–3). For UCR1, we are only able to compare the regions for which we built an atomic model, that is the N-terminal loop and the main helix but not the head domain. Whereas the helix is highly structurally conserved, VrUCR1 shows an extended N-terminal unstructured loop and short helix that provide more extensive contacts with MPP-β (see MPP section below).

Several of the accessory subunits of VrCIII₂ show significant differences with their yeast and bovine homologs. VrMPP-α and -β, which show the most notable differences, are described in detail in the following section. Here, we discuss VrQCR7 and VrQCR8 (Figure 2—figure supplement 3H–J). As in mammals and yeast, VrQCR7 (ScQcr7p, BtUQCRB) contains a helix that contacts the MPP-β anchoring β-sheet (via VrCYC1 and VrQCR8), a helix that contacts MPP-β in the opposite CIII monomer and a helix that contacts COB’s surface on the matrix side (COB's BC, DE and FG loops, and helices G and H; see Figure 2—figure supplement 2 for COB helix nomenclature). Nevertheless, similar to the bovine homolog, VrQCR7 is missing an N-terminal helix that in yeast provides several additional contacts with cyt b’s helix H and FG loop (Figure 2—figure supplement 3H). Similar to mammals and yeast, VrQCR8 (ScQcr8, BtUQCRQ) provides one strand to the multi-subunit β-sheet that helps anchor the MPP domain to the rest of CIII₂ (Figure 2—figure supplement 3I and J). Additionally, VrQCR8’s transmembrane helix contacts VrCOB’s helices G and H. Nevertheless, like BtUQCRQ but in contrast to ScQcr8, VrQCR8 lacks a short perpendicular helix that stacks below cyt b’s helix H in yeast (Figure 2—figure supplement 3I). Moreover, ScQcr8 also has a longer unstructured N-terminus that extends into the cavity of the MPP domain, contacting helix a and DE loop of COB in the same CIII protomer, as well as the helix a of COB of the opposite protomer. Therefore, the interaction interface between VrQCR8 and VrCOB is reduced relative to that of yeast (Figure 2—figure supplement 3J).

Differences in the MPP domain

Each CIII monomer contains an MPP-α/β heterodimer (plant MPP-α/β, yeast Cor1/2 and mammalian UQCR1/2). Given that both MPP subunits have concave surfaces that face each other, the MPP-α/β heterodimer contains a large central cavity. The VrMPP-α/β dimer shows this overall ‘clam shell’ configuration, with a highly negative surface in the interior of the cavity (Figure 3A, Figure 3—figure supplement 1A–B). This negative surface has been shown to interact with the generally positively charged pre-sequences of the MPP substrates (Taylor et al., 2001). VrMPP-β contains the characteristic inverse Zn-binding HxxEH motif of pitrilysin endopeptidases, as well as all the conserved catalytic residues (Figure 3—figure supplement 1; Gakh et al., 2002). Our cryoEM map shows density for the Zn²⁺ ion, which is coordinated by residues His137, His141, Glu217 (Figure 3B). The fourth Zn²⁺-coordinating atom—the oxygen of the water molecule that exerts the nucleophilic attack on the carbonyl carbon of the peptide bond (Gakh et al., 2002)—was not modelled. The glutamate that acts as a general base catalyst of this water molecule (Gakh et al., 2002; Taylor et al., 2001) (Glu140) is also conserved in V. radiata. MPP-β residues that were confirmed in the substrate-bound yeast soluble MPP structure (Taylor et al., 2001) to be critical for substrate recognition are conserved both in the sequence and structural location in VrMPP-β (Glu227, Asp231, Phe144, Asn167, Ala168, Tyr169; Figure 3B). Additionally, VrMPP-α contains the flexible glycine-rich stretch believed to be involved in substrate binding and/or product release in soluble ScMPP-α (Taylor et al., 2001). Consistent with this conformational flexibility, our cryoEM map shows weak density for VrMPP-α residues 340–344. Despite this structural conservation, the MPP enzymatic activity of our preparation could not be confirmed at this time.

Figure 3 with 2 supplements see all

Download asset Open asset

VrMPP-α/β’s sequences are more similar to the yeast (and bovine) soluble MPP subunits than to the respective CIII₂ subunits (Figure 3—figure supplement 1C). Consistently, the VrMPP-α/β dimer shows secondary-structure elements that are present in ScMPP-α/β and BtMPP-α/β but not in ScCor1/2. For instance, on the posterior surface of the cavity, yeast, bovine and mung bean MPP-α fold into six additional helices compared to ScCor2. Additionally, they contain an extra strand in the β-sheet underneath this helical bundle (Figure 3C, Figure 3—figure supplement 2).

Furthermore, some structural features of VrMPP-α and -β appear to be specific to plants, compared to the available structures (Figure 3C–D, Figure 3—figure supplements 1–2, Videos 4–5). Firstly, VrMPP-α shows a short two-strand β-sheet on its posterior surface. Secondly, the extended N-terminus of VrMPP-α wraps over the posterior surface of VrMPP-β in the same CIII monomer. Thirdly, a ~ 50 amino-acid N-terminal extension on VrMPP-β folds into an alpha helix that forms extensive contacts with MPP-α of the same CIII protomer as well as with VrQCR7 of the opposite protomer. A further difference in the overall configuration of the VrMPP domain comes from VrUCR1, whose longer N-terminus forms extensive plant-specific contacts with MPP-β’s helical bundle and β-sheet (Figure 3—figure supplement 2).

Video 4

Download asset

Video 5

Download asset

These additional contacts and secondary-structure elements may serve to stabilize plants’ MPP domain. Furthermore, they strengthen the connection between the VrMPP domain and the rest of VrCIII₂. These plant-specific features of the MPP domain could play functional roles in plant CIII₂’s peptidase activity and, potentially, on the coupling between CIII₂’s respiratory and non-respiratory functions (see Discussion).

Conformational heterogeneity analysis of CIII₂

Given CIII₂’s known conformational flexibility, for example the essential (Xiao et al., 2000; Obungu et al., 2000; Nett et al., 2000) motion of UCR1’s head domain during the Q-cycle, we decided to explore the conformational heterogeneity of our CIII₂ particles using cryoSPARC’s 3D variability analysis (3DVA) (Punjani and Fleet, 2020). The 3DVA algorithm uses probabilistic principal component analysis to produce distinct 3D volumes (one per principal component) that reveal the sample’s conformational heterogeneity as a continuous motion. Analyzing the individual frames of the motion of the volume, one can reconstruct discrete and continuous conformational changes of the protein sample.

We first analyzed the conformational heterogeneity of the entire CIII₂ low-pass filtered at 6 Å. This allowed for the observation of overall changes at the level of secondary structure. Incorporating data at resolutions higher than 5 Å led to the dominance of high-resolution noise (in particular from the lipid-amphipol belt), precluding clear results. The analysis at 6 Å revealed the lack of QCR10 in some particles, as well as a change in QCR9 (principal components 0–1, Videos 6–7), indicating that these subunits only have partial occupancy in the complex. QCR10 was previously found missing in preparations of mammalian CIII₂, likely due to de-lipidation during purification (Letts et al., 2019; Huang et al., 2005). Given that our purification is very gentle and the supercomplex interactions are maintained, it is unclear whether the changes in QCR10 and QCR9 in a sub-population of particles have biological significance or are simply due to the purification procedure. More importantly, 3DVA revealed that CIII₂ exhibits coordinated ‘breathing’ motions within and between the protomers of the dimer (components 0–3, Figure 4A–B, Videos 6–9). The motions, which may be parallel or anti-parallel across the dimer (compare Videos 6–8 with Video 9), extend from the top of matrix-exposed MPP domain to the bottom of the IMS-exposed regions of CYC1, UCR1, and QCR6. This finding contrasts with previous assumptions on CIII₂’s potential for long-range conformational coupling and has implications for the potential interplay between plant CIII₂’s respiratory and peptide-processing functions (see Discussion).

Figure 4

Download asset Open asset

Video 6

Download asset

Video 7

Download asset

Video 8

Download asset

Video 9

Download asset

We then examined the conformational heterogeneity of the UCR1 head domain by using a mask around the IMS-exposed domains of CIII₂ at 5 Å. The largest variability component revealed a near-continuous conformational motion in the position of the head domain of UCR1, demonstrating its swinging motion from the proximal b position (close to COB’s Q_P-site) to its distal c position (close to CYC1) during the Q-cycle (Xia et al., 2013; Cooley, 2013; Berry and Huang, 2011; Figure 4C). Moreover, the motions of the UCR1 head domains of the CIII dimer in this variability component were anti-parallel: that is, when one domain is in the proximal position, the other one is in the distal position and vice versa (Figure 4C, Video 10). However, weaker variability components also suggested that the position of UCR1 head domains may also be equivalent in some instances.

Video 10

Download asset

Given that the conformational heterogeneity precluded us from building an atomic model of the UCR1 head domain, we rigid-body fit a homology model into the extreme locations of the 3DVA volume and confirmed these corresponded to the Q-cycle proximal and distal positions (Figure 4C, Video 10). At the distal position, the FeS cluster of VrUCR1 was ~11 Å away from CYC1’s heme c₁ (measured edge-to-edge to the ring system). This is in agreement with previous structures of the UCR1 head domain and within electron-transfer distance (14 Å) (Huang et al., 2005; Palsdottir et al., 2003). Given that we did not model quinone in our structure, we could not measure its distance to the UCR1 FeS cluster at the proximal position. However, we measured the distance between UCR1’s FeS-coordinating residue Cys235 and COB’s Tyr285, which is within H-bonding distance to quinone (Palsdottir et al., 2003) (ScRip1-Cys180 and ScCyb-Tyr279 in yeast). This distance (~4.5 Å) was also consistent with those previously seen (Huang et al., 2005; Palsdottir et al., 2003), placing the V. radiata FeS cluster within electron-transfer distance to quinone when the head domain is at the proximal position. This confirms that the motion revealed by 3DVA is the expected swinging motion of UCR1. Moreover, given that this flexibility was observed in the absence of substrates or inhibitors, it confirms that UCR1’s head domain is intrinsically mobile. Further studies are needed to examine to what extent the movement of the UCR1 head domains are correlated across the CIII dimer and what the mechanistic implications of parallel or anti-parallel movements may be.

Complex IV

Overall structure and ligands

The initial resolution of CIV in SC III₂+IV was lower than that of CIII₂ (Figure 1—figure supplement 1). This is due to the fact that during 3D-refinement the particle poses are dominated by the larger CIII₂ and that there is conformational flexibility at the supercomplex interface. This flexibility between CIII₂ and CIV within SC III₂+IV has been previously seen in cryoEM reconstructions of CIII₂-CIV supercomplexes in bacteria and yeast (Hartley et al., 2019; Rathore et al., 2019; Gong et al., 2018; Wiseman et al., 2018). Nonetheless, focused refinements around CIV resulted in an improved reconstruction with a nominal resolution of 3.8 Å for CIV, which allowed for atomic model building (Figure 5, Figure 1—figure supplement 2, Video 2).

Figure 5 with 1 supplement see all

Download asset Open asset

At this resolution, all prosthetic groups (heme a, heme a₃, dinuclear copper A and copper B at the binuclear center), as well as a Mg²⁺ ion and a Zn²⁺ ion were visible and modelled into the structure (Figure 5B–C). The residues that coordinate the prosthetic groups, including the covalent bond between Cε-Tyr247 and the Nε-His243 that coordinates copper B in VrCOX1, are clearly visible and hence conserved in V. radiata. The distances between the prosthetic groups are consistent with those seen in other organisms (Figure 5C; Rich, 2017). Additionally, density consistent with cardiolipin, phosphatidylethanolamines and phosphatidylcholines was seen and modelled into the CIV map (Figure 5D). These lipids and acyl chains are located in hydrophobic cavities of VrCOX1 and VrCOX3, in similar locations to those seen in yeast and bovine CIV. Moreover, conserved RNA-editing sites were unambiguously identified in VrCOX1 and VrCOX3 and thus changed in the model (Supplementary file 1d).

Plant CIV composition

The subunit composition of plant CIV has remained unclear, with different mass spectrometry studies suggesting up to 13 subunits, including some putative plant-specific subunits (Millar et al., 2004; Klodmann et al., 2011; Senkler et al., 2017; Elina Welchen and Mansilla, 2018). Our structure shows that VrCIV is composed of 10 subunits (three conserved, mitochondrially encoded subunits and seven accessory subunits). In contrast, mammalian CIV contains 11 accessory subunits and yeast CIV contains 9. Thus, although VrCIV’s overall architecture is similar to other organisms', given the lack of certain homologs (ScCox26, ScCox6/BtCOX5a, BtCOX7b, BtCOX8, BtNDUFA4), there are significant differences (Figure 6, Figure 6—figure supplement 1).

Figure 6 with 1 supplement see all

Download asset Open asset

The presence of the V. radiata CIV subunits in the structure was confirmed by mass spectrometric analysis of our cryoEM sample (Supplementary file 1a). Our structure confirms the identity of plant COX-X2 as the homolog of mammalian COX4 (ScCox5), COX-X3 of mammalian COX7c (ScCox8) and COX-X4 of mammalian COX7a (ScCox7) (Millar et al., 2004). The putative plant-specific subunits COX-X5, GLN2, ABHD18, AARE and PRPK (Millar et al., 2004; Klodmann and Braun, 2011) were not observed in our mass spectrometry sample or our structure (Supplementary file 1a). Although two peptides of the putative subunit COX-X1 were identified in our mass spectrometry sample, the lack of unassigned density in our cryoEM reconstruction large enough to constitute an additional subunit demonstrates that this protein is not present in SC III₂+IV of etiolated V. radiata tissues. The possibility remains that free CIV and/or CIV in supercomplexes of non-etiolated tissues may have a different subunit composition.

Differences in conserved and accessory subunits

Among the VrCIV subunits that have mammalian and yeast homologs, structural conservation is generally high, particularly for the mitochondrially encoded subunits (Figure 5—figure supplement 1). The conserved covalent bond between Nε-His243 and Cε-Tyr247 on the HPEVY ring of VrCOX1 is clearly seen in our density (Figure 5—figure supplement 1J). Moreover, VrCOX2 contains the highly conserved residues Tyr255, Met362 (BtCOX2-Tyr105, Met207) believed to be part of the electron transport path from cyt c to CU_A of CIV (Shimada et al., 2017).

A significant difference is seen in VrCOX4, which is missing a ~100 amino-acid N-terminal helical domain compared to its homologs (ScCox5, BtCOX4; Figure 6A and C). In yeast and mammals, this N-terminal helical domain mainly interacts with ScCox6/BtCOX5a, which is one of the accessory subunits that is missing in plants (Supplementary file 1c). Moreover, in yeast SC III₂+IV₂, this helical domain of ScCox6 interacts with MPP-β of CIII₂ to provide the main CIII:CIV contacts for supercomplex formation. Hence, this interface is absent in the plant SC III₂+IV (see SC section below).

Further differences are found in VrCOX4’s C-terminus, which shows an extended loop that reaches towards the IMS side of VrCOX2 and then folds back towards the solvent-accessible face of CIV. Another difference in the vicinity of VrCOX4 is seen in VrCOX5c, which lacks the C-terminal helix that in BtCOX6c makes additional contacts to BtCOX2 and BtCOX4 (Figure 6A, Figure 6—figure supplement 1). These differences in VrCOX4 and VrCOX5c—and the lack of a homolog for ScCox5/BtCOX4—are notable, as these subunits form the majority of interactions between CIV and CIII₂ in the plant SC III₂+IV (see SC section below). Differences in VrCOX5b (shorter N-terminus), VrCOX7a (longer N-terminus with additional contacts to VrCOX3) and VrCOX7c (different path for the unstructured N-terminus) compared to their mammalian counterparts (Figure 6—figure supplement 1) are also likely related to the fact that these subunits provide SC-interface contacts in mammals not observed in plants. However, whether these differences are a cause or an effect of the divergent supercomplex binding interfaces remains to be determined.

Additional differences are seen in VrCOX6a (ScCox13, BtCOX6a). In this case, the plant subunit is more similar to the bovine homolog. Its shorter helix interacts with VrCOX1 and VrCOX3 on the IMS side but does not provide matrix-side interactions as the yeast homolog does (Figure 6—figure supplement 1).

CIV’s proton transfer pathways

Translocation of protons through CIV in different organisms occurs via the D, K, and H ‘channels’ (proton transfer pathways) of the COX1 subunit (Rich, 2017; Rich and Maréchal, 2013; Yoshikawa and Shimada, 2015; Wikström et al., 2018). The D and K channels are essential for coupled proton transfer in CIV of bacteria, yeast and mammals (Rich, 2017; Rich and Maréchal, 2013; Yoshikawa and Shimada, 2015; Wikström et al., 2018; Maréchal et al., 2020). However, whereas mutations of key H channel residues abolish proton pumping in bovine CIV (Shimokata et al., 2007; Tsukihara et al., 2003), analogous mutations have no effect on growth, respiratory rate or proton-to-electron ratios of yeast CIV (Maréchal et al., 2020). Thus, it is currently thought that the H channel only plays a role in proton transfer in mammalian CIV (although it may still act as a dielectric channel in yeast [Rich and Maréchal, 2013; Maréchal et al., 2020]). The contribution of the D, K, and H channels to CIV proton transfer in plants is unknown.

Sequence and structural analyses show that all protonatable residues of the D and K channels of yeast and mammals are conserved in VrCOX1 (Figure 7, Figure 7—figure supplement 1). In contrast, several of the residues of the mammalian H channel are not conserved in mung bean (Figure 7—figure supplement 1). In bovine’s H channel, the amide bond between Tyr440 and Ser441 and an H-bond network towards Asp51 are essential features for proton translocation (Yoshikawa and Shimada, 2015; Shimokata et al., 2007; Tsukihara et al., 1996). None of these key bovine residues are conserved in V. radiata (or S. cerevisiae) (Figure 7, Figure 7—figure supplement 1). Moreover, in the bovine H channel mutation of Ser441 to Pro441 abolishes proton pumping (Shimokata et al., 2007). The corresponding amino-acid for BtCOX1-Ser441 in both V. radiata and yeast is proline (VrCOX1-Pro443; ScCox1-Pro441). Additional amino-acid differences between V. radiata and B. taurus are seen at the entrance and exit of the H channel (Figure 7). The above suggests that, similar to yeast, the H channel is not a proton transfer pathway in plant CIV and that proton transfer in plant CIV occurs via the D and K channels. Experimental evidence is needed to confirm this hypothesis.

Figure 7 with 1 supplement see all

Download asset Open asset

Supercomplex III₂-IV (SC III₂+IV)

By docking the individually refined CIII₂ and CIV models into the SC III₂+IV composite map (Figure 1, Figure 1—figure supplement 2, Video 3), we were able to define the binding interface of the plant supercomplex. Direct contacts between the complexes occur in one site in the matrix side and one site in the IMS (Figure 8). Site 1 (matrix side), shows a single hydrophobic contact between one residue of VrQCR8 (Pro31) and one residue of VrCOX2 (Trp59) (Figure 8B). Our cryoEM reconstruction also contains a short stretch of weak, unassigned density near the first modelled residue of VrCOX5c (BtCOX6c, ScCox9), which could maximally represent an additional four amino acids. If so, the N-terminus of VrCOX5c could potentially provide additional contacts with CIII₂. However, this density may also correspond to a bridging lipid bound between the two complexes.

Figure 8 with 1 supplement see all

Download asset Open asset

The limited matrix-side contacts in V. radiata are in stark contrast to the supercomplex interface in S. cerevisiae, where binding is dominated by interactions between ScCor1 (VrMPP-β) and ScCOX5a (VrCox4) on the matrix side. Instead, V. radiata’s site 2 (IMS side) provides the bulk of the protein-protein interactions of the supercomplex, with a hydrophobic interaction between VrQCR6 and VrCOX5c (Leu26 and Leu51 respectively) as well as an interface between VrQCR6 (Pro19 and Lys20) and VrCOX4 (Arg114-Phe117) (Figure 8C). Despite the potential for lipid bridges at the matrix leaflet of the IMM, there are no direct contacts in the membrane and no protein contact at the IMS leaflet of the IMM. The overall limited binding interactions between V. radiata’s CIII₂ and CIV, and, consequently, the lower expected stability of the plant supercomplex compared to yeast's, are consistent with the fact that (CIII₂+CIV)_n supercomplexes have only been experimentally identified in a few of the plant species studied (Dudkina et al., 2006; Braun, 2020).

Unsurprisingly given the large differences in contacts, there is a significant difference in the angle between CIII₂ and CIV in V. radiata versus yeast, resulting in a more ‘open’ orientation in V. radiata (Figure 8—figure supplement 1). This orientation leads to a difference of 18° in the angle between the CIII₂ and CIV as measured by the relative positions of the b_h-hemes in CIII₂ and the a-hemes in CIV. The difference in orientation also results in a larger estimated distance between the CIII₂- and CIV-bound cyt c in V. radiata (~70 Å) than in yeast (~61 Å) (Figure 8—figure supplement 1E).

Here, we present the first structures and atomic models of CIII₂, CIV and SC III₂+IV from the plant kingdom (Figure 1, Videos 1–3). These V. radiata structures reveal atomic details of the catalytic sites and co-factor binding of plant CIII₂ (Figure 2) and CIV (Figure 5). Moreover, they show plant-specific structural features for several subunits, most notably the MPP subunits of CIII₂ (Figure 3). Conformational heterogeneity analysis of CIII₂ allowed us to observe the swinging motion of UCR1’s head domain in the absence of substrates or inhibitors, and revealed coordinated, complex-wide motions for CIII₂ (Figure 4, Videos 6–10). The CIV structure defines the subunit composition of the plant complex and suggests that the proton translocation in plants occur via the D and K, rather than the H channel (Figures 5, 6 and 7). The structures also reveal the plant-specific arrangement of the CIII₂-CIV interface in the supercomplex, which occurs mostly on the IMS side of the membrane and at a different angle from that seen in other organisms (Figure 8 and Figure 8—figure supplement 1).

PDB accession codes: 7JRG, 7JRO, 7JRP. EMBD accession codes: 22445, 22449, 22450, 22447, 22448. EMPIAR accession code: EMPIAR-10586.

1. 1. Maldonado M
   2. Guo F
   3. Letts JA

   (2020) MassIVE

   ID MSV000086237. Mass spectrometry data for Vigna radiata concentrated, pooled peak 2 fractions from sucrose gradient, Maldonado et al 2020.

   https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=22e3e0e4b0714a008d882918deda9888
2. 1. Maldonado M
   2. Guo F
   3. Letts JA

   (2020) ProteomeXchange

   ID PXD021850. Mass spectrometry data for Vigna radiata concentrated, pooled peak 2 fractions from sucrose gradient, Maldonado et al 2020.

   http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD021850
3. 1. Maldonado M
   2. Letts JA

   (2020) RCSB Protein Data Bank

   ID 7JRG. Plant Mitochondrial complex III2 from Vigna radiata.

   https://www.rcsb.org/structure/7JRG
4. 1. Maldonado M
   2. Letts JA

   (2020) RCSB Protein Data Bank

   ID 7JRO. Plant Mitochondrial complex IV from Vigna radiata.

   https://www.rcsb.org/structure/7JRO
5. 1. Maldonado M
   2. Letts JA

   (2020) RCSB Protein Data Bank

   ID 7JRP. Plant Mitochondrial complex SC III2+IV from Vigna radiata.

   https://www.rcsb.org/structure/7JRP
6. 1. Maldonado M
   2. Letts JA

   (2020) EMDataBank

   ID 22445. Plant Mitochondrial complex III2 from Vigna radiata.

   http://emsearch.rutgers.edu/atlas/22445_summary.html
7. 1. Maldonado M
   2. Letts JA

   (2020) EMDataBank

   ID 22449. Plant Mitochondrial complex IV from Vigna radiata.

   http://emsearch.rutgers.edu/atlas/22449_summary.html
8. 1. Maldonado M
   2. Letts JA

   (2020) EMDataBank

   ID 22450. Plant Mitochondrial complex SC III2+IV from Vigna radiata composite map.

   http://emsearch.rutgers.edu/atlas/22450_summary.html
9. 1. Maldonado M
   2. Letts JA

   (2020) EMDataBank

   ID 22447. Plant mitochondrial supercomplex III2+IV from Vigna radiata.

   http://emsearch.rutgers.edu/atlas/22447_summary.html
10. 1. Maldonado M
    2. Letts JA

    (2020) EMDataBank

    ID 22448. Plant mitochondrial complex III2 from Vigna radiata supercomplex III2+IV, focused refinement.

    http://emsearch.rutgers.edu/atlas/22448_summary.html
11. 1. Maldonado M
    2. Padavannil A
    3. Zhou L
    4. Guo F
    5. Letts JA

    (2020) Electron Microscopy Public Image Archive

    ID EMPIAR-10586. Cryo electron micrographs of digitonin-solubilized, amphipol-stabilized, sucrose-gradient-purified V. radiata mitochondrial membranes - mixed fraction containing CI*, CIII2 and SC III2+IV.

    https://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10586/

1. 1. Agip AA
   2. Blaza JN
   3. Bridges HR
   4. Viscomi C
   5. Rawson S
   6. Muench SP
   7. Hirst J

   (2018) Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states

   Nature Structural & Molecular Biology 25:548–556.

   https://doi.org/10.1038/s41594-018-0073-1

   • PubMed
   • Google Scholar
2. 1. Barrientos A
   2. Fontanesi F
   3. Díaz F

   (2009) Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays

   Current Protocols in Human Genetics 19:63.

   https://doi.org/10.1002/0471142905.hg1903s63

   • Google Scholar
3. 1. Belt K
   2. Huang S
   3. Thatcher LF
   4. Casarotto H
   5. Singh KB
   6. Van Aken O
   7. Millar AH

   (2017) Salicylic Acid-Dependent plant stress signaling via mitochondrial succinate dehydrogenase

   Plant Physiology 173:2029–2040.

   https://doi.org/10.1104/pp.16.00060

   • PubMed
   • Google Scholar
4. 1. Bentolila S
   2. Elliott LE
   3. Hanson MR

   (2008) Genetic architecture of mitochondrial editing in Arabidopsis thaliana

   Genetics 178:1693–1708.

   https://doi.org/10.1534/genetics.107.073585

   • PubMed
   • Google Scholar
5. 1. Berry EA
   2. Huang LS

   (2011) Conformationally linked interaction in the cytochrome bc(1) complex between inhibitors of the Q(o) site and the rieske iron-sulfur protein

   Biochimica Et Biophysica Acta 10:1349–1363.

   https://doi.org/10.1016/j.bbabio.2011.04.005

   • Google Scholar
6. 1. Blaza JN
   2. Serreli R
   3. Jones AJ
   4. Mohammed K
   5. Hirst J

   (2014) Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes

   PNAS 111:15735–15740.

   https://doi.org/10.1073/pnas.1413855111

   • PubMed
   • Google Scholar
7. 1. Blaza JN
   2. Vinothkumar KR
   3. Hirst J

   (2018) Structure of the deactive state of mammalian respiratory complex I

   Structure 26:312–319.

   https://doi.org/10.1016/j.str.2017.12.014

   • PubMed
   • Google Scholar
8. 1. Braun HP
   2. Emmermann M
   3. Kruft V
   4. Schmitz UK

   (1992) The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain

   The EMBO Journal 11:3219–3227.

   https://doi.org/10.1002/j.1460-2075.1992.tb05399.x

   • PubMed
   • Google Scholar
9. 1. Braun HP
   2. Emmermann M
   3. Kruft V
   4. Bödicker M
   5. Schmitz UK

   (1995) The general mitochondrial processing peptidase from wheat is integrated into the cytochrome bc1-complex of the respiratory chain

   Planta 195:396–402.

   https://doi.org/10.1007/BF00202597

   • PubMed
   • Google Scholar
10. 1. Braun HP

    (2020) The oxidative phosphorylation system of the mitochondria in plants

    Mitochondrion 53:66–75.

    https://doi.org/10.1016/j.mito.2020.04.007

    • PubMed
    • Google Scholar
11. 1. Braun HP
    2. Schmitz UK

    (1995a) The bifunctional cytochrome c reductase/processing peptidase complex from plant mitochondria

    Journal of Bioenergetics and Biomembranes 27:423–436.

    https://doi.org/10.1007/BF02110005

    • PubMed
    • Google Scholar
12. 1. Braun HP
    2. Schmitz UK

    (1995b) Are the 'core' proteins of the mitochondrial bc1 complex evolutionary relics of a processing protease?

    Trends in Biochemical Sciences 20:171–175.

    https://doi.org/10.1016/S0968-0004(00)88999-9

    • PubMed
    • Google Scholar
13. 1. Bultema JB
    2. Braun H-P
    3. Boekema EJ
    4. Kouřil R

    (2009) Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1787:60–67.

    https://doi.org/10.1016/j.bbabio.2008.10.010

    • Google Scholar
14. 1. Burke PV
    2. Raitt DC
    3. Allen LA
    4. Kellogg EA
    5. Poyton RO

    (1997) Effects of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast

    Journal of Biological Chemistry 272:14705–14712.

    https://doi.org/10.1074/jbc.272.23.14705

    • Google Scholar
15. 1. Cooley JW

    (2013) Protein conformational changes involved in the cytochrome bc1 complex catalytic cycle

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1827:1340–1345.

    https://doi.org/10.1016/j.bbabio.2013.07.007

    • Google Scholar
16. 1. Covello PS
    2. Gray MW

    (1989) RNA editing in plant mitochondria

    Nature 341:662–666.

    https://doi.org/10.1038/341662a0

    • PubMed
    • Google Scholar
17. 1. Cramer WA
    2. Hasan SS
    3. Yamashita E

    (2011) The Q cycle of cytochrome bc complexes: a structure perspective

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1807:788–802.

    https://doi.org/10.1016/j.bbabio.2011.02.006

    • Google Scholar
18. 1. Davies KM
    2. Blum TB
    3. Kühlbrandt W

    (2018) Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants

    PNAS 115:3024–3029.

    https://doi.org/10.1073/pnas.1720702115

    • PubMed
    • Google Scholar
19. 1. Dudkina NV
    2. Eubel H
    3. Keegstra W
    4. Boekema EJ
    5. Braun HP

    (2005) Structure of a mitochondrial supercomplex formed by respiratory-chain complexes I and III

    PNAS 102:3225–3229.

    https://doi.org/10.1073/pnas.0408870102

    • PubMed
    • Google Scholar
20. 1. Dudkina NV
    2. Heinemeyer J
    3. Sunderhaus S
    4. Boekema EJ
    5. Braun HP

    (2006) Respiratory chain supercomplexes in the plant mitochondrial membrane

    Trends in Plant Science 11:232–240.

    https://doi.org/10.1016/j.tplants.2006.03.007

    • PubMed
    • Google Scholar
21. 1. Elina Welchen
    2. Mansilla N

    (2018) The complexity of mitochondrial complex IV: an update of cytochrome c oxidase biogenesis in plants

    International Journal of Molecular Sciences 19:662.

    https://doi.org/10.3390/ijms19030662

    • Google Scholar
22. 1. Emmermann M
    2. Braun HP
    3. Arretz M
    4. Schmitz UK

    (1993)

    Characterization of the bifunctional cytochrome c reductase-processing peptidase complex from potato mitochondria

    The Journal of Biological Chemistry 268:18936–18942.

    • PubMed
    • Google Scholar
23. 1. Emsley P
    2. Cowtan K

    (2004) Coot: model-building tools for molecular graphics

    Acta Crystallographica. Section D, Biological Crystallography 60:2126–2132.

    https://doi.org/10.1107/S0907444904019158

    • PubMed
    • Google Scholar
24. 1. Eriksson A
    2. Sjöling S
    3. Glaser E

    (1994) The ubiquinol cytochrome c oxidoreductase complex of spinach leaf mitochondria is involved in both respiration and protein processing

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1186:221–231.

    https://doi.org/10.1016/0005-2728(94)90181-3

    • Google Scholar
25. 1. Eriksson AC
    2. Sjöling S
    3. Glaser E

    (1996) Characterization of the bifunctional mitochondrial processing peptidase (MPP)/bc1 complex in Spinacia oleracea

    Journal of Bioenergetics and Biomembranes 28:285–292.

    https://doi.org/10.1007/BF02110702

    • PubMed
    • Google Scholar
26. 1. Esser L
    2. Quinn B
    3. Li YF
    4. Zhang M
    5. Elberry M
    6. Yu L
    7. Yu CA
    8. Xia D

    (2004) Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

    Journal of Molecular Biology 341:281–302.

    https://doi.org/10.1016/j.jmb.2004.05.065

    • PubMed
    • Google Scholar
27. 1. Esser L
    2. Gong X
    3. Yang S
    4. Yu L
    5. Yu CA
    6. Xia D

    (2006) Surface-modulated motion switch: capture and release of iron-sulfur protein in the cytochrome bc1 complex

    PNAS 103:13045–13050.

    https://doi.org/10.1073/pnas.0601149103

    • PubMed
    • Google Scholar
28. 1. Esser L
    2. Yu CA
    3. Xia D

    (2014) Structural basis of resistance to anti-cytochrome bc₁ complex inhibitors: implication for drug improvement

    Current Pharmaceutical Design 20:704–724.

    https://doi.org/10.2174/138161282005140214163327

    • PubMed
    • Google Scholar
29. 1. Esser L
    2. Zhou F
    3. Yu C-A
    4. Xia D

    (2019) Crystal structure of bacterial cytochrome bc 1 in complex with azoxystrobin reveals a conformational switch of the Rieske iron–sulfur protein subunit

    Journal of Biological Chemistry 294:12007–12019.

    https://doi.org/10.1074/jbc.RA119.008381

    • Google Scholar
30. 1. Eubel H
    2. Jänsch L
    3. Braun HP

    (2003) New insights into the respiratory chain of plant mitochondria supercomplexes and a unique composition of complex II

    Plant Physiology 133:274–286.

    https://doi.org/10.1104/pp.103.024620

    • PubMed
    • Google Scholar
31. 1. Eubel H
    2. Heinemeyer J
    3. Braun HP

    (2004) Identification and characterization of respirasomes in potato mitochondria

    Plant Physiology 134:1450–1459.

    https://doi.org/10.1104/pp.103.038018

    • PubMed
    • Google Scholar
32. 1. Fedor JG
    2. Hirst J

    (2018) Mitochondrial supercomplexes do not enhance catalysis by quinone channeling

    Cell Metabolism 28:525–531.

    https://doi.org/10.1016/j.cmet.2018.05.024

    • PubMed
    • Google Scholar
33. 1. Fiedorczuk K
    2. Letts JA
    3. Degliesposti G
    4. Kaszuba K
    5. Skehel M
    6. Sazanov LA

    (2016) Atomic structure of the entire mammalian mitochondrial complex I

    Nature 538:406–410.

    https://doi.org/10.1038/nature19794

    • PubMed
    • Google Scholar
34. 1. Fuchs P
    2. Rugen N
    3. Carrie C
    4. Elsässer M
    5. Finkemeier I
    6. Giese J
    7. Hildebrandt TM
    8. Kühn K
    9. Maurino VG
    10. Ruberti C
    11. Schallenberg-Rüdinger M
    12. Steinbeck J
    13. Braun HP
    14. Eubel H
    15. Meyer EH
    16. Müller-Schüssele SJ
    17. Schwarzländer M

    (2020) Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics

    The Plant Journal 101:420–441.

    https://doi.org/10.1111/tpj.14534

    • PubMed
    • Google Scholar
35. 1. Gakh O
    2. Cavadini P
    3. Isaya G

    (2002) Mitochondrial processing peptidases

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1592:63–77.

    https://doi.org/10.1016/S0167-4889(02)00265-3

    • Google Scholar
36. 1. Gao X
    2. Wen X
    3. Esser L
    4. Quinn B
    5. Yu L
    6. Yu CA
    7. Xia D

    (2003) Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the qi site

    Biochemistry 42:9067–9080.

    https://doi.org/10.1021/bi0341814

    • PubMed
    • Google Scholar
37. 1. Giegé P
    2. Brennicke A

    (1999) RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs

    PNAS 96:15324–15329.

    https://doi.org/10.1073/pnas.96.26.15324

    • PubMed
    • Google Scholar
38. 1. Glaser E
    2. Dessi P

    (1999) Integration of the mitochondrial-processing peptidase into the cytochrome bc1 complex in plants

    Journal of Bioenergetics and Biomembranes 31:259–274.

    https://doi.org/10.1023/a:1005475930477

    • PubMed
    • Google Scholar
39. 1. Goddard TD
    2. Huang CC
    3. Meng EC
    4. Pettersen EF
    5. Couch GS
    6. Morris JH
    7. Ferrin TE

    (2018) UCSF ChimeraX: meeting modern challenges in visualization and analysis

    Protein Science 27:14–25.

    https://doi.org/10.1002/pro.3235

    • PubMed
    • Google Scholar
40. 1. Gong H
    2. Li J
    3. Xu A
    4. Tang Y
    5. Ji W
    6. Gao R
    7. Wang S
    8. Yu L
    9. Tian C
    10. Li J
    11. Yen HY
    12. Man Lam S
    13. Shui G
    14. Yang X
    15. Sun Y
    16. Li X
    17. Jia M
    18. Yang C
    19. Jiang B
    20. Lou Z
    21. Robinson CV
    22. Wong LL
    23. Guddat LW
    24. Sun F
    25. Wang Q
    26. Rao Z

    (2018) An electron transfer path connects subunits of a mycobacterial respiratory supercomplex

    Science 362:eaat8923.

    https://doi.org/10.1126/science.aat8923

    • PubMed
    • Google Scholar
41. 1. Gu J
    2. Wu M
    3. Guo R
    4. Yan K
    5. Lei J
    6. Gao N
    7. Yang M

    (2016) The architecture of the mammalian respirasome

    Nature 537:639–643.

    https://doi.org/10.1038/nature19359

    • PubMed
    • Google Scholar
42. 1. Gualberto JM
    2. Lamattina L
    3. Bonnard G
    4. Weil JH
    5. Grienenberger JM

    (1989) RNA editing in wheat mitochondria results in the conservation of protein sequences

    Nature 341:660–662.

    https://doi.org/10.1038/341660a0

    • PubMed
    • Google Scholar
43. 1. Guo R
    2. Zong S
    3. Wu M
    4. Gu J
    5. Yang M

    (2017) Architecture of human mitochondrial respiratory megacomplex I2III2IV2

    Cell 170:1247–1257.

    https://doi.org/10.1016/j.cell.2017.07.050

    • Google Scholar
44. 1. Hartley AM
    2. Lukoyanova N
    3. Zhang Y
    4. Cabrera-Orefice A
    5. Arnold S
    6. Meunier B
    7. Pinotsis N
    8. Maréchal A

    (2019) Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1

    Nature Structural & Molecular Biology 26:78–83.

    https://doi.org/10.1038/s41594-018-0172-z

    • PubMed
    • Google Scholar
45. 1. Hiesel R
    2. Wissinger B
    3. Schuster W
    4. Brennicke A

    (1989) RNA editing in plant mitochondria

    Science 246:1632–1634.

    https://doi.org/10.1126/science.2480644

    • PubMed
    • Google Scholar
46. 1. Ho CM
    2. Li X
    3. Lai M
    4. Terwilliger TC
    5. Beck JR
    6. Wohlschlegel J
    7. Goldberg DE
    8. Fitzpatrick AWP
    9. Zhou ZH

    (2020) Bottom-up structural proteomics: cryoem of protein complexes enriched from the cellular milieu

    Nature Methods 17:79–85.

    https://doi.org/10.1038/s41592-019-0637-y

    • PubMed
    • Google Scholar
47. 1. Huang LS
    2. Cobessi D
    3. Tung EY
    4. Berry EA

    (2005) Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular Hydrogen-bonding pattern

    Journal of Molecular Biology 351:573–597.

    https://doi.org/10.1016/j.jmb.2005.05.053

    • PubMed
    • Google Scholar
48. 1. Huang S
    2. Lee CP
    3. Millar AH

    (2015) Activity assay for plant mitochondrial enzymes

    Methods in Molecular Biology 1305:139–149.

    https://doi.org/10.1007/978-1-4939-2639-8_10

    • PubMed
    • Google Scholar
49. 1. Huang LS
    2. Berry E

    (2016) Rieske Iron-Sulfur protein movement and conformational changes in cytochrome bc-bf complexes

    Cytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling 41:237–251.

    https://doi.org/10.1007/978-94-017-7481-9_11

    • Google Scholar
50. 1. Ito Y
    2. Nakazono M
    3. Kadowaki K
    4. Tsutsumi N
    5. Hirai A

    (1996) RNA editing of transcripts of the gene for apocytochrome b (cob) in rice mitochondria

    Genes & Genetic Systems 71:85–89.

    https://doi.org/10.1266/ggs.71.85

    • PubMed
    • Google Scholar
51. 1. Iwata S
    2. Lee JW
    3. Okada K
    4. Lee JK
    5. Iwata M
    6. Rasmussen B
    7. Link TA
    8. Ramaswamy S
    9. Jap BK

    (1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex

    Science 281:64–71.

    https://doi.org/10.1126/science.281.5373.64

    • PubMed
    • Google Scholar
52. 1. Izrailev S
    2. Crofts AR
    3. Berry EA
    4. Schulten K

    (1999) Steered molecular dynamics simulation of the rieske subunit motion in the cytochrome bc(1) complex

    Biophysical Journal 77:1753–1768.

    https://doi.org/10.1016/S0006-3495(99)77022-0

    • PubMed
    • Google Scholar
53. 1. Kearse M
    2. Moir R
    3. Wilson A
    4. Stones-Havas S
    5. Cheung M
    6. Sturrock S
    7. Buxton S
    8. Cooper A
    9. Markowitz S
    10. Duran C
    11. Thierer T
    12. Ashton B
    13. Meintjes P
    14. Drummond A

    (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data

    Bioinformatics 28:1647–1649.

    https://doi.org/10.1093/bioinformatics/bts199

    • PubMed
    • Google Scholar
54. 1. Kelley LA
    2. Mezulis S
    3. Yates CM
    4. Wass MN
    5. Sternberg MJ

    (2015) The Phyre2 web portal for protein modeling, prediction and analysis

    Nature Protocols 10:845–858.

    https://doi.org/10.1038/nprot.2015.053

    • PubMed
    • Google Scholar
55. 1. Klodmann J
    2. Senkler M
    3. Rode C
    4. Braun HP

    (2011) Defining the protein complex proteome of plant mitochondria

    Plant Physiology 157:587–598.

    https://doi.org/10.1104/pp.111.182352

    • PubMed
    • Google Scholar
56. 1. Klodmann J
    2. Braun HP

    (2011) Proteomic approach to characterize mitochondrial complex I from plants

    Phytochemistry 72:1071–1080.

    https://doi.org/10.1016/j.phytochem.2010.11.012

    • PubMed
    • Google Scholar
57. 1. Letts JA
    2. Fiedorczuk K
    3. Sazanov LA

    (2016) The architecture of respiratory supercomplexes

    Nature 537:644–648.

    https://doi.org/10.1038/nature19774

    • PubMed
    • Google Scholar
58. 1. Letts JA
    2. Fiedorczuk K
    3. Degliesposti G
    4. Skehel M
    5. Sazanov LA

    (2019) Structures of respiratory supercomplex I+III2 Reveal Functional and Conformational Crosstalk

    Molecular Cell 75:1131–1146.

    https://doi.org/10.1016/j.molcel.2019.07.022

    • PubMed
    • Google Scholar
59. 1. Letts JA
    2. Sazanov LA

    (2017) Clarifying the supercomplex: the higher-order organization of the mitochondrial electron transport chain

    Nature Structural & Molecular Biology 24:800–808.

    https://doi.org/10.1038/nsmb.3460

    • PubMed
    • Google Scholar
60. 1. Li L
    2. Li C
    3. Sarkar S
    4. Zhang J
    5. Witham S
    6. Zhang Z
    7. Wang L
    8. Smith N
    9. Petukh M
    10. Alexov E

    (2012) DelPhi: a comprehensive suite for DelPhi software and associated resources

    BMC Biophysics 5:9.

    https://doi.org/10.1186/2046-1682-5-9

    • PubMed
    • Google Scholar
61. 1. Li C
    2. Jia Z
    3. Chakravorty A
    4. Pahari S
    5. Peng Y
    6. Basu S
    7. Koirala M
    8. Panday SK
    9. Petukh M
    10. Li L
    11. Alexov E

    (2019) DelPhi suite: new developments and review of functionalities

    Journal of Computational Chemistry 40:2502–2508.

    https://doi.org/10.1002/jcc.26006

    • PubMed
    • Google Scholar
62. 1. Liebschner D
    2. Afonine PV
    3. Baker ML
    4. Bunkóczi G
    5. Chen VB
    6. Croll TI
    7. Hintze B
    8. Hung L-W
    9. Jain S
    10. McCoy AJ
    11. Moriarty NW
    12. Oeffner RD
    13. Poon BK
    14. Prisant MG
    15. Read RJ
    16. Richardson JS
    17. Richardson DC
    18. Sammito MD
    19. Sobolev OV
    20. Stockwell DH
    21. Terwilliger TC
    22. Urzhumtsev AG
    23. Videau LL
    24. Williams CJ
    25. Adams PD

    (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

    Acta Crystallographica Section D Structural Biology 75:861–877.

    https://doi.org/10.1107/S2059798319011471

    • Google Scholar
63. 1. Madeira F
    2. Park YM
    3. Lee J
    4. Buso N
    5. Gur T
    6. Madhusoodanan N
    7. Basutkar P
    8. Tivey ARN
    9. Potter SC
    10. Finn RD
    11. Lopez R

    (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019

    Nucleic Acids Research 47:W636–W641.

    https://doi.org/10.1093/nar/gkz268

    • PubMed
    • Google Scholar
64. 1. Maldonado M
    2. Padavannil A
    3. Zhou L
    4. Guo F
    5. Letts JA

    (2020) Atomic structure of a mitochondrial complex I intermediate from vascular plants

    eLife 9:e56664.

    https://doi.org/10.7554/eLife.56664

    • PubMed
    • Google Scholar
65. 1. Maréchal A
    2. Meunier B
    3. Lee D
    4. Orengo C
    5. Rich PR

    (2012) Yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem–copper oxidase superfamily

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1817:620–628.

    https://doi.org/10.1016/j.bbabio.2011.08.011

    • Google Scholar
66. 1. Maréchal A
    2. Xu JY
    3. Genko N
    4. Hartley AM
    5. Haraux F
    6. Meunier B
    7. Rich PR

    (2020) A common coupling mechanism for A-type heme-copper oxidases from Bacteria to mitochondria

    PNAS 117:9349–9355.

    https://doi.org/10.1073/pnas.2001572117

    • PubMed
    • Google Scholar
67. 1. Meyer EH
    2. Welchen E
    3. Carrie C

    (2019) Assembly of the complexes of the oxidative phosphorylation system in land plant mitochondria

    Annual Review of Plant Biology 70:23–50.

    https://doi.org/10.1146/annurev-arplant-050718-100412

    • PubMed
    • Google Scholar
68. 1. Milenkovic D
    2. Blaza JN
    3. Larsson NG
    4. Hirst J

    (2017) The enigma of the respiratory chain supercomplex

    Cell Metabolism 25:765–776.

    https://doi.org/10.1016/j.cmet.2017.03.009

    • PubMed
    • Google Scholar
69. 1. Millar AH
    2. Eubel H
    3. J�nsch L
    4. Kruft V
    5. Heazlewood JL
    6. Braun H-P

    (2004) Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits

    Plant Molecular Biology 56:77–90.

    https://doi.org/10.1007/s11103-004-2316-2

    • Google Scholar
70. 1. Millar AH
    2. Liddell A
    3. Leaver CJ

    (2007) Isolation and subfractionation of mitochondria from plants

    Methods in Cell Biology 80:65–90.

    https://doi.org/10.1016/S0091-679X(06)80003-8

    • PubMed
    • Google Scholar
71. 1. Morin A
    2. Eisenbraun B
    3. Key J
    4. Sanschagrin PC
    5. Timony MA
    6. Ottaviano M
    7. Sliz P

    (2013) Collaboration gets the most out of software

    eLife 2:e01456.

    https://doi.org/10.7554/eLife.01456

    • PubMed
    • Google Scholar
72. 1. Nesvizhskii AI
    2. Keller A
    3. Kolker E
    4. Aebersold R

    (2003) A statistical model for identifying proteins by tandem mass spectrometry

    Analytical Chemistry 75:4646–4658.

    https://doi.org/10.1021/ac0341261

    • PubMed
    • Google Scholar
73. 1. Nett JH
    2. Hunte C
    3. Trumpower BL

    (2000) Changes to the length of the flexible Linker region of the rieske protein impair the interaction of ubiquinol with the cytochrome bc1 complex

    European Journal of Biochemistry 267:5777–5782.

    https://doi.org/10.1046/j.1432-1327.2000.01650.x

    • PubMed
    • Google Scholar
74. Book

    1. Nicholls DGF

    (2013)

    Bioenergetics 4

    Academic Press.

    • Google Scholar
75. 1. Obungu VH
    2. Wang Y
    3. Amyot SM
    4. Gocke CB
    5. Beattie DS

    (2000) Mutations in the tether region of the iron–sulfur protein affect the activity and assembly of the cytochrome bc1 complex of yeast mitochondria

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1457:36–44.

    https://doi.org/10.1016/S0005-2728(99)00116-4

    • Google Scholar
76. 1. Palsdottir H
    2. Lojero CG
    3. Trumpower BL
    4. Hunte C

    (2003) Structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion Q oSite inhibitor bound

    Journal of Biological Chemistry 278:31303–31311.

    https://doi.org/10.1074/jbc.M302195200

    • Google Scholar
77. 1. Parey K
    2. Brandt U
    3. Xie H
    4. Mills DJ
    5. Siegmund K
    6. Vonck J
    7. Kühlbrandt W
    8. Zickermann V

    (2018) Cryo-EM structure of respiratory complex I at work

    eLife 7:e39213.

    https://doi.org/10.7554/eLife.39213

    • PubMed
    • Google Scholar
78. 1. Pettersen EF
    2. Goddard TD
    3. Huang CC
    4. Couch GS
    5. Greenblatt DM
    6. Meng EC
    7. Ferrin TE

    (2004) UCSF chimera--a visualization system for exploratory research and analysis

    Journal of Computational Chemistry 25:1605–1612.

    https://doi.org/10.1002/jcc.20084

    • PubMed
    • Google Scholar
79. 1. Punjani A
    2. Rubinstein JL
    3. Fleet DJ
    4. Brubaker MA

    (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

    Nature Methods 14:290–296.

    https://doi.org/10.1038/nmeth.4169

    • PubMed
    • Google Scholar
80. Preprint

    1. Punjani A
    2. Fleet DJ

    (2020) 3d variability analysis: directly resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM images

    bioRxiv.

    https://doi.org/10.1101/2020.04.08.032466

    • Google Scholar
81. 1. Rathore S
    2. Berndtsson J
    3. Marin-Buera L
    4. Conrad J
    5. Carroni M
    6. Brzezinski P
    7. Ott M

    (2019) Cryo-EM structure of the yeast respiratory supercomplex

    Nature Structural & Molecular Biology 26:50–57.

    https://doi.org/10.1038/s41594-018-0169-7

    • PubMed
    • Google Scholar
82. 1. Rich PR

    (2017) Mitochondrial cytochrome c oxidase: catalysis, coupling and controversies

    Biochemical Society Transactions 45:813–829.

    https://doi.org/10.1042/BST20160139

    • PubMed
    • Google Scholar
83. 1. Rich PR
    2. Maréchal A

    (2013) Functions of the hydrophilic channels in protonmotive cytochrome c oxidase

    Journal of the Royal Society Interface 10:20130183.

    https://doi.org/10.1098/rsif.2013.0183

    • Google Scholar
84. 1. Rohou A
    2. Grigorieff N

    (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs

    Journal of Structural Biology 192:216–221.

    https://doi.org/10.1016/j.jsb.2015.08.008

    • PubMed
    • Google Scholar
85. 1. Schägger H
    2. Pfeiffer K

    (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria

    The EMBO Journal 19:1777–1783.

    https://doi.org/10.1093/emboj/19.8.1777

    • PubMed
    • Google Scholar
86. 1. Scheres SH
    2. Chen S

    (2012) Prevention of overfitting in cryo-EM structure determination

    Nature Methods 9:853–854.

    https://doi.org/10.1038/nmeth.2115

    • PubMed
    • Google Scholar
87. 1. Schertl P
    2. Braun HP

    (2015) Activity measurements of mitochondrial enzymes in native gels

    Methods in Molecular Biology 1305:131–138.

    https://doi.org/10.1007/978-1-4939-2639-8_9

    • PubMed
    • Google Scholar
88. 1. Schorb M
    2. Haberbosch I
    3. Hagen WJH
    4. Schwab Y
    5. Mastronarde DN

    (2019) Software tools for automated transmission electron microscopy

    Nature Methods 16:471–477.

    https://doi.org/10.1038/s41592-019-0396-9

    • PubMed
    • Google Scholar
89. 1. Senkler J
    2. Senkler M
    3. Eubel H
    4. Hildebrandt T
    5. Lengwenus C
    6. Schertl P
    7. Schwarzländer M
    8. Wagner S
    9. Wittig I
    10. Braun HP

    (2017) The mitochondrial complexome of Arabidopsis thaliana

    The Plant Journal : For Cell and Molecular Biology 89:1079–1092.

    https://doi.org/10.1111/tpj.13448

    • PubMed
    • Google Scholar
90. 1. Shimada S
    2. Shinzawa-Itoh K
    3. Baba J
    4. Aoe S
    5. Shimada A
    6. Yamashita E
    7. Kang J
    8. Tateno M
    9. Yoshikawa S
    10. Tsukihara T

    (2017) Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode

    The EMBO Journal 36:291–300.

    https://doi.org/10.15252/embj.201695021

    • PubMed
    • Google Scholar
91. 1. Shimokata K
    2. Katayama Y
    3. Murayama H
    4. Suematsu M
    5. Tsukihara T
    6. Muramoto K
    7. Aoyama H
    8. Yoshikawa S
    9. Shimada H

    (2007) The proton pumping pathway of bovine heart cytochrome c oxidase

    PNAS 104:4200–4205.

    https://doi.org/10.1073/pnas.0611627104

    • PubMed
    • Google Scholar
92. 1. Small ID
    2. Schallenberg-Rüdinger M
    3. Takenaka M
    4. Mireau H
    5. Ostersetzer-Biran O

    (2020) Plant organellar RNA editing: what 30 years of research has revealed

    The Plant Journal 101:1040–1056.

    https://doi.org/10.1111/tpj.14578

    • PubMed
    • Google Scholar
93. 1. Sousa JS
    2. Mills DJ
    3. Vonck J
    4. Kühlbrandt W

    (2016) Functional asymmetry and electron flow in the bovine respirasome

    eLife 5:e21290.

    https://doi.org/10.7554/eLife.21290

    • PubMed
    • Google Scholar
94. 1. Sousa JS
    2. Vonck J

    (2019) Respiratory supercomplexes III2IV2 come into focus

    Nature Structural & Molecular Biology 26:87–89.

    https://doi.org/10.1038/s41594-019-0184-3

    • Google Scholar
95. 1. Sper-Whitis GL
    2. Moody JL
    3. Vaughn JC

    (1996) Universality of mitochondrial RNA editing in cytochrome-c oxidase subunit I (coxI) among the land plants

    Biochimica Et Biophysica Acta (BBA) - Gene Structure and Expression 1307:301–308.

    https://doi.org/10.1016/0167-4781(96)00041-3

    • Google Scholar
96. 1. Stuchebrukhov A
    2. Schäfer J
    3. Berg J
    4. Brzezinski P

    (2020) Kinetic advantage of forming respiratory supercomplexes

    Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1861:148193.

    https://doi.org/10.1016/j.bbabio.2020.148193

    • Google Scholar
97. 1. Takenaka M
    2. Zehrmann A
    3. Verbitskiy D
    4. Härtel B
    5. Brennicke A

    (2013) RNA editing in plants and its evolution

    Annual Review of Genetics 47:335–352.

    https://doi.org/10.1146/annurev-genet-111212-133519

    • PubMed
    • Google Scholar
98. 1. Taylor AB
    2. Smith BS
    3. Kitada S
    4. Kojima K
    5. Miyaura H
    6. Otwinowski Z
    7. Ito A
    8. Deisenhofer J

    (2001) Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences

    Structure 9:615–625.

    https://doi.org/10.1016/S0969-2126(01)00621-9

    • PubMed
    • Google Scholar
99. 1. Teixeira PF
    2. Branca RM
    3. Kmiec B
    4. Glaser E

    (2015) A flowchart to analyze protease activity in plant mitochondria

    Methods in Molecular Biology 1305:123–130.

    https://doi.org/10.1007/978-1-4939-2639-8_8

    • PubMed
    • Google Scholar
100. 1. Tsukihara T
     2. Aoyama H
     3. Yamashita E
     4. Tomizaki T
     5. Yamaguchi H
     6. Shinzawa-Itoh K
     7. Nakashima R
     8. Yaono R
     9. Yoshikawa S

     (1996) The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A

     Science 272:1136–1144.

     https://doi.org/10.1126/science.272.5265.1136

     • PubMed
     • Google Scholar
101. 1. Tsukihara T
     2. Shimokata K
     3. Katayama Y
     4. Shimada H
     5. Muramoto K
     6. Aoyama H
     7. Mochizuki M
     8. Shinzawa-Itoh K
     9. Yamashita E
     10. Yao M
     11. Ishimura Y
     12. Yoshikawa S

     (2003) The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

     PNAS 100:15304–15309.

     https://doi.org/10.1073/pnas.2635097100

     • Google Scholar
102. 1. Vinothkumar KR
     2. Zhu J
     3. Hirst J

     (2014) Architecture of mammalian respiratory complex I

     Nature 515:80–84.

     https://doi.org/10.1038/nature13686

     • Google Scholar
103. 1. Wagner T
     2. Merino F
     3. Stabrin M
     4. Moriya T
     5. Antoni C
     6. Apelbaum A
     7. Hagel P
     8. Sitsel O
     9. Raisch T
     10. Prumbaum D
     11. Quentin D
     12. Roderer D
     13. Tacke S
     14. Siebolds B
     15. Schubert E
     16. Shaikh TR
     17. Lill P
     18. Gatsogiannis C
     19. Raunser S

     (2019) SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM

     Communications Biology 2:218.

     https://doi.org/10.1038/s42003-019-0437-z

     • PubMed
     • Google Scholar
104. 1. Wagner T
     2. Raunser S

     (2020) The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

     Communications Biology 3:61.

     https://doi.org/10.1038/s42003-020-0790-y

     • PubMed
     • Google Scholar
105. 1. Wikström M
     2. Krab K
     3. Sharma V

     (2018) Oxygen activation and energy conservation by cytochrome c Oxidase

     Chemical Reviews 118:2469–2490.

     https://doi.org/10.1021/acs.chemrev.7b00664

     • PubMed
     • Google Scholar
106. 1. Wiseman B
     2. Nitharwal RG
     3. Fedotovskaya O
     4. Schäfer J
     5. Guo H
     6. Kuang Q
     7. Benlekbir S
     8. Sjöstrand D
     9. Ädelroth P
     10. Rubinstein JL
     11. Brzezinski P
     12. Högbom M

     (2018) Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis

     Nature Structural & Molecular Biology 25:1128–1136.

     https://doi.org/10.1038/s41594-018-0160-3

     • Google Scholar
107. 1. Wu M
     2. Gu J
     3. Guo R
     4. Huang Y
     5. Yang M

     (2016) Structure of mammalian respiratory supercomplex I 1 III 2 IV 1

     Cell 167:1598–1609.

     https://doi.org/10.1016/j.cell.2016.11.012

     • Google Scholar
108. 1. Xia D
     2. Yu CA
     3. Kim H
     4. Xia JZ
     5. Kachurin AM
     6. Zhang L
     7. Yu L
     8. Deisenhofer J

     (1997) Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria

     Science 277:60–66.

     https://doi.org/10.1126/science.277.5322.60

     • PubMed
     • Google Scholar
109. 1. Xia D
     2. Esser L
     3. Tang W-K
     4. Zhou F
     5. Zhou Y
     6. Yu L
     7. Yu C-A

     (2013) Structural analysis of cytochrome bc1 complexes: implications to the mechanism of function

     Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1827:1278–1294.

     https://doi.org/10.1016/j.bbabio.2012.11.008

     • Google Scholar
110. 1. Xiao K
     2. Yu L
     3. Yu C-A

     (2000) Confirmation of the Involvement of Protein Domain Movement during the Catalytic Cycle of the Cytochrome bc b>b>1b>b> Complex by the Formation of an Intersubunit Disulfide Bond between Cytochrome b and the Iron-Sulfur Protein

     Journal of Biological Chemistry 275:38597–38604.

     https://doi.org/10.1074/jbc.M007444200

     • Google Scholar
111. 1. Yoshikawa S
     2. Shimada A

     (2015) Reaction mechanism of cytochrome c oxidase

     Chemical Reviews 115:1936–1989.

     https://doi.org/10.1021/cr500266a

     • PubMed
     • Google Scholar
112. 1. Yu H
     2. Wu CH
     3. Schut GJ
     4. Haja DK
     5. Zhao G
     6. Peters JW
     7. Adams MWW
     8. Li H

     (2018) Structure of an ancient respiratory system

     Cell 173:1636–1649.

     https://doi.org/10.1016/j.cell.2018.03.071

     • PubMed
     • Google Scholar
113. 1. Zanlungo S
     2. Bégu D
     3. Quiñones V
     4. Araya A
     5. Jordana X

     (1993) RNA editing of apocytochrome b (cob) transcripts in mitochondria from two genera of plants

     Current Genetics 24:344–348.

     https://doi.org/10.1007/BF00336787

     • PubMed
     • Google Scholar
114. 1. Zhang Z
     2. Huang L
     3. Shulmeister VM
     4. Chi YI
     5. Kim KK
     6. Hung LW
     7. Crofts AR
     8. Berry EA
     9. Kim SH

     (1998) Electron transfer by domain movement in cytochrome bc1

     Nature 392:677–684.

     https://doi.org/10.1038/33612

     • PubMed
     • Google Scholar
115. 1. Zheng SQ
     2. Palovcak E
     3. Armache JP
     4. Verba KA
     5. Cheng Y
     6. Agard DA

     (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

     Nature Methods 14:331–332.

     https://doi.org/10.1038/nmeth.4193

     • PubMed
     • Google Scholar
116. Preprint

     1. Zhong ED
     2. Bepler T
     3. Berger B
     4. Davis JH

     (2020) CryoDRGN: reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks

     bioRxiv.

     https://doi.org/10.1101/2020.03.27.003871

     • Google Scholar
117. 1. Zhu J
     2. Vinothkumar KR
     3. Hirst J

     (2016) Structure of mammalian respiratory complex I

     Nature 536:354–358.

     https://doi.org/10.1038/nature19095

     • PubMed
     • Google Scholar
118. 1. Zickermann V
     2. Wirth C
     3. Nasiri H
     4. Siegmund K
     5. Schwalbe H
     6. Hunte C
     7. Brandt U

     (2015) Mechanistic insight from the crystal structure of mitochondrial complex I

     Science 347:44–49.

     https://doi.org/10.1126/science.1259859

     • Google Scholar
119. 1. Zivanov J
     2. Nakane T
     3. Forsberg BO
     4. Kimanius D
     5. Hagen WJ
     6. Lindahl E
     7. Scheres SH

     (2018) New tools for automated high-resolution cryo-EM structure determination in RELION-3

     eLife 7:e42166.

     https://doi.org/10.7554/eLife.42166

     • PubMed
     • Google Scholar

Author details

1. Maria Maldonado

   Department of Molecular and Cellular Biology, University of California Davis, Davis, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3428-1053

2. Fei Guo

   1. Department of Molecular and Cellular Biology, University of California Davis, Davis, United States
   2. BIOEM Facility, University of California Davis, Davis, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. James A Letts

   Department of Molecular and Cellular Biology, University of California Davis, Davis, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Project administration, Writing - review and editing

   For correspondence

   jaletts@ucdavis.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9864-3586

• 3,889

  views

• 454

  downloads

• 653

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.