Research ArticleSTRUCTURAL BIOLOGY

Cryo-EM structures of the air-oxidized and dithionite-reduced photosynthetic alternative complex III from Roseiflexus castenholzii

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Science Advances  29 Jul 2020:
Vol. 6, no. 31, eaba2739
DOI: 10.1126/sciadv.aba2739
  • Fig. 1 Overall structure of the photosynthetic ACIII from R. castenholzii.

    (A) The cryo-EM map of the air-oxidized ACIII is shown from the front (left) and back (right) view and represented with the dimensions of the TM and periplasmic region. Each of the six subunits is labeled with the length of its encoded amino acid. (B) Cartoon representation of the air-oxidized ACIII. The c-type hemes and lipids are shown as sticks, and the iron-sulfur clusters are shown as spheres. (C) Representation of the arrangement of cofactors in the air-oxidized ACIII. (D) Edge-to-edge distance between the iron-sulfur clusters and the hemes in the air-oxidized ACIII. The distances are labeled and shown in dashed lines. Color codes for all panels: lime green, ActA; slate, ActB; wheat, ActC; violet, ActD; yellow orange, ActE; aquamarine, ActF; red, c-type heme; orange brown, iron-sulfur clusters; yellow, lipids.

  • Fig. 2 Heme and iron-sulfur clusters in ActA and ActE subunits.

    (A) The air-oxidized minus dithionite-reduced electron potential difference map (orange) of the ACIII is shown from the front (left) and back (right) view. The structures of the air-oxidized and dithionite-reduced ACIII (white) are superimposed, with the iron-sulfur clusters and heme groups shown in sphere and stick models. The color code for each subunit and cofactors of the air-oxidized ACIII is the same as that in Fig. 1. (B) Ribbon representation of the ActA and ActE subunits bound with pentaheme and monoheme groups (red sticks). The N and C termini of the protein are highlighted with a black dot and labeled. (C) Spatial organization and immobilization of the pentaheme and monoheme groups in ActA and ActE subunits. The residues that axially coordinate the heme iron ions are shown as sticks and labeled; the center-to-center distances of the hemes are shown and labeled. (D) Overall structure of ActB subunit. The B1 and B2 domains are colored in blue and magenta, respectively. The iron-sulfur clusters are shown as spheres. (E) Coordination of the iron-sulfur clusters in the ActB subunit. The conserved cysteine residues that coordinate the iron-sulfur clusters are shown as sticks and labeled, and the B2 domain is shown as a ribbon with 80% transparency.

  • Fig. 3 TM region of R. castenholzii ACIII and a putative menaquinol binding pocket.

    (A) Ribbon representation of the side (left) and bottom-up (right) views of the ActC (wheat) and ActF (aquamarine) subunits. The TM helices of ActC and ActF are labeled with numbers, and the iron-sulfur clusters in ActB and heme groups in ActA subunit are shown in spheres and red sticks, respectively. The N and C termini of each subunit are highlighted with a black dot and labeled. (B) Open cavity (bright yellow) between the TM helices of ActA, ActC, and ActD subunits of ACIII, which is equivalent to the menaquinol binding pocket. The cavity inside the TM region of ACIII was calculated using the program HOLLOW (44), and it is shown as a surface model. (C) Zoomed-in view of the putative menaquinol binding pocket, with essential amino acids shown as stick models. (D) Interactions between the modeled menaquinol head (blue stick model) and the menaquinol binding pocket. Residues are shown as stick models, and the hydrogen bonding interactions are shown as dashed lines with distances labeled.

  • Fig. 4 Organization of the menaquinol binding pocket and proton translocation passage in the ActC subunit.

    (A) Organization of the menaquinol binding pocket (highlighted with a green box), [3Fe-4S] cluster, heme_1, and the putative proton translocation passage in ActC (wheat). The distance between [3Fe-4S] and the side chain of Asp171 in the menaquinol binding pocket is 12.2 Å, and the edge-to-edge distance between the [3Fe-4S] cluster and heme_1 is 8.3 Å. The residues that constitute the proton translocation passage and menaquinol binding pocket are shown as stick models, and the TM helices of ActC are shown as ribbon with 80% transparency. (B) Zoomed-in view of the hydrogen bonding networks between the menaquinol binding pocket and middle passage residues of the proton translocation passage in the ActC subunit, as well as the residues from the ActD and ActF subunits. Residues are shown as stick models, and the hydrogen bonding interactions are shown as dashed lines with distances labeled. (C) Topology diagram of the ActC subunit. The amino acids that constitute the menaquinol binding pocket and proton translocation pathway are shown in blue and green triangles, respectively.

  • Fig. 5 Comparison of the proton translocation passages in the photosynthetic and respiratory ACIII.

    (A) Comparison of the proton translocation passage in R. castenholzii ActC (wheat) with that of the respiratory ActC from R. marinus (PDB 6f0k, white) and F. johnsoniae (PDB 6btm, pink). The [3Fe-4S] cluster, heme_1, and the amino acids are shown as sticks. (B) Middle passage residues that are capable of forming hydrogen bonding networks. (C) Putative proton translocation passage in R. castenholzii ActF subunit (aquamarine) and its superimposition with that of R. marinus (PDB 6f0k, white) and F. johnsoniae (PDB 6btm, pink). (D) The middle passage residues that are capable of forming hydrogen bonds in ActF subunits are shown in stick models.

  • Fig. 6 Proposed redox-coupled proton translocation mechanism of the photosynthetic ACIII.

    The menaquinol head group and the side chains of the essential amino acids are shown to indicate the coupling mechanism of the photosynthetic ACIII from R. castenholzii. Upon menaquinol oxidation, two electrons are sequentially transferred to the [3Fe-4S] cluster with a time interval, two protons from oxidation of menaquinol are released to periplasmic space, and one proton is pumped from the cytoplasm (colored in red) through hydrogen bonding networking with the essential amino acids in the proton translocation passage. As a result, three protons are released into the periplasm per two electrons transferred during oxidation of one instance of menaquinol.

  • Table 1 Data collection, processing, and refinement statistics.

    Air-oxidized ACIII
    (EMDB-0937)
    (PDB 6LOE)
    Dithionite-reduced
    ACIII
    (EMDB-0936)
    (PDB 6LOD)
    Data collection and processing
      Magnification22,50022,500
      Voltage (kV)300300
      Electron exposure (e/Å2)5960
      Defocus range (μm)1.2–3.31.0–3.0
      Pixel size (Å)1.041.04
      Symmetry imposedC1C1
      Initial particle images (no.)257,815488,581
      Final particle images (no.)177,489207,633
      Map resolution (Å)
    FSC threshold
    3.2
    0.143
    3.5
    0.143
    Refinement
      Model resolution (Å)3.33.5
        FSC threshold0.50.5
      Map sharpening B factor (Å2)−125−135
      Model composition
        Nonhydrogen atoms19,02419,024
        Protein residues2,3292,329
        Ligands1717
      RMSDs
        Bond lengths (Å)0.0250.025
        Bond angles (°)1.5481.420
      Validation
        MolProbity score1.952.20
        Clashscore8.5714.09
        Poor rotamers (%)0.420.21
      Ramachandran plot
        Favored (%)91.8490.03
        Allowed (%)8.169.97
        Disallowed (%)0.000.00

Supplementary Materials

  • Supplementary Materials

    Cryo-EM structures of the air-oxidized and dithionite-reduced photosynthetic alternative complex III from Roseiflexus castenholzii

    Yang Shi, Yueyong Xin, Chao Wang, Robert E. Blankenship, Fei Sun, Xiaoling Xu

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    • Figs. S1 to S10
    • Tables S1 and S2

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