The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes1, whose precise role is debated. Supercomplexes CIII2CIV1-2 (refs. 2,3), CICIII2 (ref. 4) and CICIII2CIV (respirasome)5,6,7,8,9,10 exist in mammals, but in contrast to CICIII2 and the respirasome, to date the only known eukaryotic structures of CIII2CIV1-2 come from Saccharomyces cerevisiae11,12 and plants13, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII2CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII2CIV from the CIII2 precursor to the final CIII2CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. 14) deep into CIII2, while its C terminus is integrated into CIV. Our structures (which include CICIII2 and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII2CIV and has no role in the assembly of the respirasome. We show that CIII2 is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII2, explaining the presence of one copy of CIV in CIII2CIV in mammals. Finally, we show that CIII2 and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII2CIV in fine tuning the efficiency of electron transfer in the electron transport chain.
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Structures of the CIII2CIV intermediate, assembled locked, mature unlocked and CIII2 from CICIII2 were deposited in the PDB (access IDs 7O3E, 7O37, 7O3C and 7O3H, respectively) and corresponding cryo-electron microscopy density maps in the EMDB (IDs 12705, 12702, 12703 and 12706, respectively). Any other relevant data and information are available from the corresponding author on request. The following models, deposited in the PDB (https://www.rcsb.org), have been cited throughout the paper: 1NTZ, 3CX5, 3L75, 5IY5, 5J4Z, 5Z62, 6HWH, 6HU9, 6QBX and 7JRP.
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We thank the pre-clinical facility of the IST Austria and A. Venturino for assistance with the animals; and V.-V. Hodirnau for assistance during the Titan Krios data collection, performed at the IST Austria. The data processing was performed at the IST high-performance computing cluster. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 754411.
The authors declare no competing interests.
Peer review information Nature thanks Werner Kühlbrandt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
a, Purification of the mouse sample: on top, chromatograms of the MonoQ (left), the first SEC (middle) and the second SEC (right). The pooled fractions are highlighted in blue. At the bottom, native PAGE and western blot of the purification steps: from left to right, Coomassie-stained solubilised material (SM), then Coomassie staining, complex I in-gel activity assay, complex IV in-gel activity assay, western blot for Core2, western blot for SCAF1 of the purification steps. b, Purification of the ovine sample: the same steps as shown in a. For Gel source data, please see Supplementary Fig. 1. The mouse purification was repeated at least 5 times, the ovine preparation was repeated at least 10 times, all attempts of replication were successful, and one representative preparation per sample type is shown in the figure.
Scheme of the processing pipeline, with representative micrograph, pre-processing, classification steps and final maps, coloured by local resolution, with relative angular distribution plot.
Extended Data Fig. 3 Processing pipeline for the mouse CICIII2 data, FSC curves of the mouse maps and representative densities from the cryo-EM maps.
a, Processing pipeline as in Extended Data Fig. 2: the same set of micrographs was used, but the classification focused on CICIII2. b, Gold-standard half-map and model-map FSC curves for the mouse dataset. c–h, Representative densities of CIII2 (c–e) and CIV (f–i) for α-helices -contoured at 2.7σ- (c and f), β-sheets -contoured at 2.7σ- (d and g), cofactors -contoured at 1.9σ- (e and i) and lipids -contoured at 1.8σ- (h, from CIII2).
a, Processing pipeline of the ovine CIII2CIV dataset, as in Extended Data Figs. 2, 3. b, Final maps of the ovine CIII2CIV locked (left, 2.8σ contour) and unlocked (right, 3σ contour for CIII and 6σ for CIV), shown from the side (top) and from the matrix (bottom). The respective mouse models, coloured as in Figs. 1, 3 , were superimposed on CIII2, to show the conservation of the conformations across mammalian species.
a, Cryo-EM map density (grey mesh) carved around Sub9 (orange cartoon) and SCAF1 (blue) positions in supercomplexes CIII2CIV, CICIII2 and CICIII2CIV. For CIII2CIV and the mouse CICIII2, the unique positions of a single copy each of Sub9 and SCAF1 were used, while for respirasomes, where the limited resolution prevented Sub9-focused classification and so CIII2 adopts a random orientation, two-fold symmetry-related positions of these proteins were also added. Contour levels are 2, 1.9 and 2.1σ for mouse CIII2CIV mature unlocked, assembled locked and intermediate locked respectively; 8σ for ovine CIII2CIV mature unlocked; 2.9σ for mouse CICIII2; 6.7σ for mouse CICIII2CIV; 6.7 and 5.3σ for ovine CICIII2CIV tight and loose respectively. b, Sub9 (orange) integration into the MPP walls: UQCRC1 (Core1) and UQCRC2 (Core2) are shown in grey (Core1 light and Core2 dark), except for the strands of Core2 forming continuous sheets with Sub9, which are shown in yellow. c, SCAF1 (blue), Sub7 (yellow) and ISP (red) arrangement in the intermediate (left) and assembled locked (right) classes. The rest of CIII is depicted in grey. The density for Sub7 is carved in grey, to show the ordering of the loop in the assembled versus intermediate class. In the insets at the bottom, the densities for SCAF1 and ISP are shown in the two classes. 2.9σ contour for CIII2CIV intermediate and 2.3σ for assembled.
a, Surface charge representation (as in Fig. 3) of Core1 and SCAF1 in the unlocked class (full model is grey with a sketched eye indicating the perspective). SCAF1 cartoon is green. A white asterisk indicates the MPP "mouth". b, SCAF1 (cyan) and COX7A1 (blue) models fitted to the locked (left, 2.5σ contour) and unlocked class (right, 2.7σ contour) CIV maps. The arrows in b and c indicate bulky residues used to identify the subunit based on the map density. SCAF1 model, but not COX7A1, fits the density in both classes. c, Ovine SCAF1 (cyan) and COX7A1 (blue) models fitted to CIV map from respirasome (7.3σ contour): contrary to CIII2CIV, COX7A1 model fits the density, unlike SCAF1. d, Sequence alignment of mammalian COX7A isoforms. The SCAF1 portions interacting with CIII2 are indicated above the alignment. The black arrows indicate the positions highlighted in b and c. e, Activity measurements (described in the Methods) of CIII2+CIV (light grey) and supercomplex CIII2CIV (dark grey). The electron transfer rates (in e-/s) are calculated from the CIII2-driven oxidation of DQH2 or from the CIV-driven oxidation of reduced cyt c, as indicated. The bars represent the mean, the dots depict the single measurements. The 5-10 nM ox cyt c represents 2.5-5:1 cyt c/CIII2CIV ratio (two independent experiments). All other measurements come from 3 independent experiments (two values in the CIII2+CIV sample with 50 μM red cyt c overlap, therefore only two dots are visible). f, Cofactor distances within and between CIII2 and CIV, in the unlocked conformation. Cyt c is bound to CIII2 and CIV as in Fig. 3b. The transparent cartoon represents the protein subunits, the cofactors are sticks (hemes) and spheres (FeS clusters and Cu centers), in orange for CIII2, cyan for CIV and black for cyt c. The dashed lines represent the IMM.
Extended Data Fig. 8 Structural differences among supercomplexes and comparison between CIII2 movements in CICIII2 and intermediate vs assembled CIII2CIV.
a, Top left, CIII2CIV and respirasome (ovine, PDB 5j4z) aligned by CIII2: CI of respirasome is grey, CIV of respirasome is blue, CIII2 is yellow and CIV of CIII2CIV is cyan (the colour scheme for mammalian CIII2CIV is consistent throughout the panel). CIII2CIV locked is shown on the left, unlocked on the right. The two possible orientations of CIII2CIV based on CIII2 are shown at the top and at the bottom. Top right, plant CIII2CIV (PDB 7jrp) and mammalian CIII2CIV aligned by CIII2 in the locked (top) and unlocked (bottom) conformations. The plant CIV is blue. Bottom left, yeast CIII2CIV2 (PDB 6hu9) and mammalian CIII2CIV aligned by CIII2 in the locked (left) and unlocked (right) conformations. The yeast CIV is blue. Bottom right, M. smegmatis CIII2CIV2 (PDB 6hwh) and mammalian CIII2CIV aligned by CIII2 in the locked (left) and unlocked (right) conformations. The bacterial CIV is blue. The straight arrows indicate the shift of CIV towards mammalian CIV, and the curved arrows indicate the 90 or 180o flip of CIV around its vertical axis. b, Side (top) and IMS (bottom) views of the extreme frames of CIII2 3DVA mode 0 (open on the left and closed in the middle, 5σ contour). Ovals indicate the position of the ISP Fe-S domains, only visible in the closed state. On the right, overlay of the two extremes, with closed in dark grey and open in light grey. Arrows indicate the “breathing” movement of the TM and IMS domains. c, Same as b, but with CIII2 intermediate (left) and assembled (right) models from CIII2CIV fitted to the densities and coloured as in Figs. 1, 3.
This file contains additional discussion relevant for the main text.
Supplementary Fig. 1 . All the original, uncropped scans of the native gels and western blots shown in Extended Data Fig. 1 are shown.
Supplementary Video 1 2CIV.The mature unlocked state map and model are shown, followed by a transition through the three different conformations, viewed from the side and from the top. Finally, the cytochrome c binding sites, inferred from the crystal structures cited in the manuscript, are shown. The colour scheme is the same as in the rest of the manuscript.
Supplementary Video 2 2 in CICIII2. The result of the 3DVA analysis on CIII2 is shown, viewed from the side first, then from the IMS. The fluctuating movements between a closed state, with the two ISP C-terminal domains visible, and an open state, with no density corresponding to ISP, are depicted.
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Vercellino, I., Sazanov, L.A. Structure and assembly of the mammalian mitochondrial supercomplex CIII2CIV. Nature 598, 364–367 (2021). https://doi.org/10.1038/s41586-021-03927-z
Nature Reviews Molecular Cell Biology (2021)