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Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1

Nature Structural & Molecular Biology volume 26, pages 78–83 (2019)Cite this article

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Abstract

Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in an SC with cytochrome bc1 (complex III, CIII). Here, we present the cryogenic electron microscopy (cryo-EM) structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologs is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A—involved in allosteric inhibition by ATP—may arise from its association with CIII. The CIII–CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.

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Fig. 1: Overall structure of the S. cerevisiae III2IV2 SC.
Fig. 2: Structure of S. cerevisiae CIV.
Fig. 3: Interactions of Qcr10 with other subunits of CIII, and that of Rip1 with a lipid at the interface with CIV.
Fig. 4: Protein–protein interactions between CIV and CIII.
Fig. 5: Alignment of the mammalian I1III2IV1 respirasome with the III2IV2 SC from S. cerevisiae.

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Data availability

All relevant data are included in the manuscript and/or are available from the corresponding authors upon reasonable request. Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-0262 (III2IV2 SC), EMD-0269 (CIVa) and EMD-0268 (CIVb). The coordinates of the atomic model of the III2IV2 SC built from a combination of the three maps have been deposited in PDB under accession code 6HU9.

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Acknowledgements

We would like to thank Diamond Light Source for access and support to the cryo-EM facilities at the UK National Electron Bio-Imaging Centre (eBIC, proposal no. EM14704-36) funded by the Wellcome Trust, the Medical Research Council, UK and the Biotechnology and Biological Sciences Research Council. We would also like to thank D. Houldershaw and the computer support group at Birkbeck. This work was supported by the Medical Research Council (Career Development Award no. MR/M00936X/1 to A.M.), the Birkbeck Wellcome Trust Institutional Strategic Support Fund (grant no. 105628/Z/14/Z to A.M.) and a Wellcome Trust grant to the Birkbeck EM facility (no. 079605/2/06/2).

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Authors and Affiliations

  1. Institute of Structural and Molecular Biology, Birkbeck College, London, UK

    Andrew M. Hartley, Natalya Lukoyanova, Nikos Pinotsis & Amandine Maréchal

  2. Institute of Structural and Molecular Biology, University College London, London, UK

    Yunyi Zhang & Amandine Maréchal

  3. Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands

    Alfredo Cabrera-Orefice & Susanne Arnold

  4. Institute for Integrative Biology of the Cell, Université Paris-Saclay, Gif-sur-Yvette, France

    Brigitte Meunier

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Contributions

A.M. designed and supervised the research. B.M. produced the yeast mutant strain. A.M.H. did all the protein work with contribution from Y.Z. A.C.-O. and S.A. performed mass spectrometry analysis. N.L. and A.M.H. performed all microscopy work. A.M.H. and N.P. processed the cryo-EM images. N.P. built the model with inputs from A.M. and A.M.H. A.M., A.M.H. and N.P. wrote the manuscript with contributions from all authors.

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Correspondence to Nikos Pinotsis or Amandine Maréchal.

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Supplementary Figure 1 Biochemical characterization of S. cerevisiae III2IV2 SC.

a, UV-visible redox spectrum of the purified yeast III2IV2 SC. Peaks are labeled and correspond to the A-type hemes present in CIV (445/604 nm) and the B- and C-type hemes present in CIII (432/562 nm and 554 nm). b, Gel filtration profile of the yeast III2IV2 SC. The fraction eluting between 12 and 12.5 mL was used for cryo-EM. c, BN-PAGE gel of the purified yeast III2IV2 SC. d, Oxygen consumption rate of the III2IV2 SC (red line) in the presence of 50 μM cytochrome c, initiated by addition of 40 μM decylubiquinol (decylUQH2). A baseline (green) is recorded after addition of 1 mM KCN, an inhibitor of CIV. e, Oxygen consumption rate of the III2IV2 SC without exogenous cytochrome c (experiment details are as in d). Data in a–c are representative of three independent experiments with similar results.

Supplementary Figure 2 Cryo-EM data collection and workflow of 3D classification and focused refinement.

a, A representative cryo-EM micrograph of III2IV2 SC (scale bar, 30 nm). b, Representative 2D class averages of the III2IV2 SC (scale bar, 20 nm). c, Fourier shell correlation (FSC) curves between two independently refined half-maps for the III2IV2 SC map and for the two CIV maps, each individually refined. d, Surface rendering maps colored according to local resolution. Upper and middle panels present the maps of the III2IV2 SC and CIVa and CIVb after focused refinement. The lower display shows the central sections of the two CIV maps to emphasize further the improvement in resolution (scale bar, 50 Å). e, Euler angle distributions of the 44,915 particles included in the calculation of the III2IV2 SC map for two orientations rotated by 90 degrees. Data collection and structure calculation are not repeated.

Supplementary Figure 3 Fitting of the CIV model into the cryo-EM density.

a, Fitting of cofactors, metal clusters and identified lipids into the CIVa cryo-EM map density. Below each model are the names of the cofactor/metal/lipid, alongside the domain and chain as in PDB 6HU9 they belong to. The numbering refers to residues found nearby or providing coordination (PEF, palmitoyl-phosphatidylethanolamine; PCF, diacyl-glycero-phosphocholine). b, Two examples of high-resolution α-helical regions of CIV, Cox5A which forms the interface with CIII and the core domain Cox1. c, Fitting of a β-sheet from Cox2. d, Fitting of the distal subunit Cox13 (chain k) and the new subunit Cox26 (chain l) into the density. The N and C termini of the chains are indicated. The corresponding cofactors, metals, lipids and subunits from the other CIV monomer (CIVb) display identical density (data not shown).

Supplementary Figure 4 Fitting of the CIII model into the cryo-EM density.

a, Fitting of cofactors, metal clusters and identified lipids into the III2IV2 SC cryo-EM map density. Below each model are the names of the cofactor/metal/lipid, alongside the domain and chain as in PDB 6HU9 they belong to. The numbering refers to residues found nearby or providing coordination (PEF, palmitoyl-phosphatidylethanolamine; PCF, diacyl-glycero-phosphocholine). b, Fitting of the subunits Rip1 (chain E) and Qcr10 (chain J) into the density. The N and C termini of the chains are indicated. Note the lower resolution of the flexible C-terminal catalytic domain of Rip1 (zoom in). This domain, as well as Qcr10, displays higher B factors compared to the other subunits of CIII (data not shown). The corresponding domains from the second monomer of CIII display identical density (data not shown).

Supplementary Figure 5 Structural alignment of the two halves of the III2IV2 SC.

a,b, Side (a) and top (b) views of a structural alignment of the two halves of the III2IV2 SC, aligned on their core CIII subunits, Cor1, Cor2 and cytochrome b. This reveals minimal deviation of Cox5A at the SC interface (~0.3–0.5 Å), but a larger deviation of Cox13 (3.0 Å), at the extreme periphery of the SC. Cox26 and Qcr10 are displayed in green and red, respectively.

Supplementary Figure 6 Position of COX7B would not preclude interactions via COX4-1 in a mammalian CIII–CIV SC.

a,b, Side (a) and top (b) view of a putative mammalian III2IV1 SC formed after substitution of the yeast CIV monomer (CIVa) with bovine CIV (PDB 1V54) as per our III2IV2 SC structure (the other CIV is not shown). COX7B (blue), a subunit of mammalian CIV, lies near COX4-1 (pale cyan; the homolog of yeast Cox5A). However, it would not interfere with any interactions between CIII and COX4-1, such as those seen in the yeast III2IV2 SC structure. COX7A (deep red), the subunit involved in interactions with CIII in the mammalian I1III2IV1 respirasome, is also highlighted.

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Hartley, A.M., Lukoyanova, N., Zhang, Y. et al. Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1. Nat Struct Mol Biol 26, 78–83 (2019). https://doi.org/10.1038/s41594-018-0172-z

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