Analysis of the conformational heterogeneity of the Rieske iron-sulfur protein in complex III2 by cryo-EM

Abstract

Movement of the Rieske domain of the iron-sulfur protein is essential for intramolecular electron transfer within complex III₂ (CIII₂) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII₂ from Yarrowia lipolytica at resolutions up to 2.0 Å under different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII₂ with a reduced high-potential chain increased the occupancy of the Q_o site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c₁ subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c₁ than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd₁-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.

Keywords: Rieske domains; complex III2; conformational heterogeneity; iron–sulfur proteins.

Figure 1

Cryo-EM density maps of respiratory complex III from Y. lipolytica at 2.0 Å resolution (a) (combined data sets; the position of the lipid bilayer is indicated by grey lines) and 3.3 Å resolution (b) (+atovaquone+antimycin A) with the three subunits Cyt1, Rip1 and Cob that form the catalytic core and all seven supernumerary subunits found in yeasts. The solvent-exposed domain of the Rieske iron–sulfur protein was not resolved in the consensus refinement of all data sets of samples without added inhibitor (a), whereas it was fixed in the b position (red, with the [2Fe–2S] cluster in yellow and orange) by the P_f-type inhibitor atovaquone (b). The lower panels show ligand densities after the refinement of individual data sets (c, d). After its addition, decylubiquinol was resolved in the Q_i site, while density in the Q_o site remained poorly defined. The inhibitor antimycin A displaced decylubiquinol from the Q_i site when both were added (c). Atovaquone and antimycin A were resolved in the Q_o and Q_i sites, respectively, in the 3.3 Å resoution map (d). All maps were sharpened by a B factor of −30 Ų.

Figure 2

Typical b (red), intermediate (yellow) and c (blue) positions of the Rieske domain after focused 3D classification and refinement (a, b, c) and a superposition of the three models (d). The range of different positions of the Rieske domain in all samples is visualized in the plot of the distances between Fe2 in the [2Fe–2S] cluster and Fe of heme b _L and heme c ₁, respectively (e). The three positions in (a)–(d) are marked by dashed circles in (e).

Figure 3

Rieske domain (red) in the b position. (a) shows the cryo-EM density of the ISP. The hinge (black arrowhead) extended when atovaquone was bound (Darrouzet et al., 2000; Birth et al., 2014 ▸). When the high-potential chain was reduced by ascorbate, density at the hinge and the surface (black square) of the Rieske domain was weak, indicating local heterogeneity despite strong density for the [2Fe–2S] cluster (yellow) and the surrounding tip region. When the high-potential chain was oxidized with ferricyanide, the hinge rigidified and turned into an α-helix. Details of the docking crater, which is formed by cyt b (light green), are depicted in (b) with density for the Q_o site. Atovaquone induced tight docking of the Rieske domain, as shown by strong density for the Rieske domain in (a) and close interaction of the tip of the Rieske domain with the docking crater and atovaquone in (b). When CIII₂ was reduced with ascorbate, the Rieske domain was within hydrogen-bonding distance of cyt b and potential Q_o-site occupants. Density in the Q_o site, in a similar position to atovaquone, increased when decylubiquinone was added together with ascorbate. The Rieske domain was beyond hydrogen-bonding distance to cyt b in the b positions that were observed with an oxidized high-potential chain (+FCN) and also the other tested conditions (Supplementary Fig. S4). For better visualization, a positive B factor of +10 Ų was applied to limit the map resolution. Exemplary maps coloured by local resolution are shown in Supplementary Fig. S5.

Figure 4

His190 ligating the Fe–S cluster was 3.4 and 7.6 Å away from the propionic acid group of heme c ₁ in the c positions of the Rieske domain (blue) in ascorbate-reduced (+NaAsc) and ferricyanide-oxidized (+FCN) CIII₂, respectively.

Figure 5

Cyt b in samples of CIII₂ prepared with antimycin A (a, c), oxidized with ferricyanide (b) or reduced with ascorbate (d). (a) Density for Trp142 (dashed ellipse) of the cd ₁-helix and Ile269 of the ef-loop was missing in maps of CIII₂ with only antimycin A added when the Rieske domain (cropped out, residual yellow density) was in the intermediate position. Additional density was visible at a position near the Q_o site on the left of the cd ₁-helix, partially overlapping with the position of Ile269 of the ef-loop (arrow). Densities for Val146, Ileu147, Cys148 and Asn149 were ambiguous as in the apo sample (Supplementary Fig. S7). (c) With the Rieske domain in the b position, density for Trp142 was resolved in the expected position beneath the ef-loop, as in samples with added ferricyanide (b), ascorbate (d), DQH₂ or DQ. All side chains of the ef-loop and cd ₁-helix were also resolved with the Rieske domain in the intermediate position. The overall resolutions of the maps were 3.0 Å (a), 3.0 Å (c), 2.3 Å (b) and 3.2 Å (d). In the apo sample, density for Trp142 was also weaker when the Rieske domain was in the intermediate position (Supplementary Fig. S7).

Figure 6

Typical maps from the +FCN sample with both Rieske domains in the CIII dimer. Rieske domains are coloured red, yellow and blue for the b, intermediate and c positions. (a) Refinement of particle pairs with one protomer in the b position and one in the c position (30 714 particles, 2.7 Å resolution, C1). (b, c, d) Maps from the refinement of particle pairs with both protomers in the b position (6727 particles, 3.0 Å resolution, C2), c position (11 987 particles, 2.8 Å resolution, C2) and intermediate position (9819 particles, 2.8 Å resolution, C2). All possible permutations of positions were observed.