Structures of Tetrahymena's respiratory chain reveal the diversity of eukaryotic core metabolism

Abstract

Respiration is a core biological energy-converting process whose last steps are carried out by a chain of multisubunit complexes in the inner mitochondrial membrane. To probe the functional and structural diversity of eukaryotic respiration, we examined the respiratory chain of the ciliate Tetrahymena thermophila (Tt). Using cryo-electron microscopy on a mixed sample, we solved structures of a supercomplex between Tt complex I (Tt-CI) and Tt-CIII₂ (Tt-SC I+III₂) and a structure of Tt-CIV₂. Tt-SC I+III₂ (~2.3 megadaltons) is a curved assembly with structural and functional symmetry breaking. Tt-CIV₂ is a ~2.7-megadalton dimer with more than 50 subunits per protomer, including mitochondrial carriers and a TIM8₃-TIM13₃-like domain. Our structural and functional study of the T. thermophila respiratory chain reveals divergence in key components of eukaryotic respiration, thereby expanding our understanding of core metabolism.

Fig. 1.. Functional and structural divergence of T. thermophila’s electron transport chain.

(A) Relative CIV activity of isolated mitochondrial membranes from T. thermophila (ciliate symbol, red), S. scrofa (pig symbol, yellow) and V. radiata (plant symbol, green) using cyt c from horse (Eq, horse symbol, dotted) or T. thermophila (Tt, ciliate symbol, solid) in the presence or absence of 8 μM potassium cyanide (KCN). Average, SEM (n=4). (B) Representation of T. thermophila’s mitochondrial electron transport chain on a tubular crista with the width of the membrane determined by the observed phospholipids and hydrophobic regions of the complexes. Complexes shown in surface (Tt-CI, blue; Tt-CIII₂, green; Tt-CIV₂, pink; Tt-CV₂, yellow, PDB: 6YNY (9). Lipids shown in sphere, colored by heteroatom. Cyt c homology model shown in surface, colored by electrostatic potential. The arrow shows approximate binding sites for cyt c on Tt-CIII₂ and TtCIV₂. IMS, intermembrane space; pmf, protonmotive force. (C) CryoEM map of Tt-SC I+III₂ colored by individual subunits. (D) CryoEM map of Tt-CIV₂ colored by individual subunits.

Fig. 2.. Structural and functional features of Tt-CI.

(A) Tt-CI’s 17 core subunits in colored surfaces, accessory subunits in grey cartoons. Core subunits ND1B, ND2B and ND5B highlighted with black borders. (B) Hydrophilic axis along the membrane arm of Tt-CI. Core subunits that form the membrane arm shown in colored cylinders, colored as in (A). Core subunits ND1B and ND2B highlighted with black borders. Conserved charged residues along the hydrophilic axis as sticks, colored by subunit. (C) Q-site loops shown as cartoons embedded in the density, colored by subunit. Terminal iron-sulfur cluster N2 is shown as spheres, residues shown as sticks. (D) The 51 accessory subunits shown in colored surfaces, core subunits in grey transparent cartoons. (E) Tt-CI’s three γ-carbonic anhydrase subunits in colored cartoons on Tt-CI surface (blue). (F) Tt-CI’s ferredoxin bridge colored as cartoons on Tt-CI surface (blue).

Fig. 3.. Structural and functional comparison of complex I’s active-to-deactive (A/D) transition.

(A and B) Comparison of complex I’s ND3 TMH1–2 loop in T. thermophila (A) and O. aries (B) in the ordered (“closed”, PDB: 6ZKO) and disordered (“open”, PDB: 6ZKS) conformations. (C to F) Functional characterization of A/D in isolated mitochondrial membranes of S. scrofa (C and D) and T. thermophila (E and F) by spectroscopic measurement of NADH dehydrogenase activity at 340 nm in the presence of the indicated concentrations of n-ethylmaleimide (NEM) (C,E) or MgCl₂ (D,F) with pre-incubation with 5 μM NADH or water. Average, SEM, n=3–4. (G) Crowding around Tt-CI’s ND3 TMH1–2 loop. ND3 in blue surface. Secondary structure elements of various accessory subunits that pack against the ND3 loop in colored cartoon. Accessory subunits colored as in Fig. 2. (H) Superposition of T. thermophila ND1 (light purple cartoon) with O. aries ND1 in NADH-bound, open-state CI (“up” conformation, red cartoon, PDB: 6ZKH). O. aries ND1 up conformation would clash with ND3 TMH1–3 loop (blue surface) and NDUFS2 (yellow surface). Clash marked with red star.

Fig. 4. (preceding page). Tt-SC I+III₂ interactions and Tt-CIII₂ symmetry breaking.

(A) Contact sites between Tt-CI and Tt-CIII₂ in Tt-SC I+III₂, color-coded and shown in open-book configuration. Site 1, orange; site 2, yellow; site 3, blue; site 4, red; site 5, green. (B and C) T. thermophila-specific Tt-SC I+III₂ matrix bridges (NDUJ1/TX bridge and toe bridge) in the context of Tt-SC I+III₂, viewed from the matrix (B) or from the plane of the membrane (C). Key subunits in colored cartoons and highlighted in black outline. (D and E) Modeled lipids (spheres) at the interface between Tt-CI (blue surface) and Tt-CIII₂ (green surface). Lipids at the matrix (D) or intermembrane space (E) leaflet of the membrane viewed from the matrix or IMS, respectively. (F and G) Structure of Tt-CIII₂, with core (F) or accessory (G) subunits in surface and the rest of the complex in transparent cartoon. Unidentified chains (poly-ala model) in red. (H) Broken symmetry in the Tt-CIII₂ dimer. For clarity, maps were carved around the asymmetric subunits. For each protomer, density is shown in transparent grey, models in cartoon. The subunit labels indicate differences in each protomer. (I) UQCRSF1 head domain (magenta surface) constrained at the interface between Tt-CI and Tt-CIII₂. Iron sulfur (FeS) and heme co-factors (sticks) shown. (J and K) Comparison of b-hemes (J) and c-hemes (K) in respiratory CIII₂ and photosynthetic b₆f complex dimers across cyanobacteria PDB: 1VF5; Mastigocladus laminosus b₆f complex (63)) in grey, bacteria (PDB: 2YIU P. denitrificans (64)) in pink, yeast (PDB: 6HU9 S. cerevisiae (18)) in blue, plants (PDB: 7JRG V. radiata (20)) in green, mammals (PDB: 1BGY B. taurus (46)) in yellow and T. thermophila (orange). Hemes were superposed by one Tt-COB subunit in the dimer. Distances between hemes in Å, calculated edge-to-edge of the macrocyclic conjugated system. For clarity, only distances for T. thermophila and B. taurus are shown. (L) Schematic of the symmetry-broken Q cycle and Q cavity specialization of Tt-CIII₂. Tt-CIII protomers in dark green surface (proximal to Tt-CI Q tunnel, blue oval) or light green surface (distal to Tt-CI Q tunnel). The functional Q_N and Q_P sites are indicated with dark green ovals, corresponding to the dark green protomer. The functional UQCRSF1 head domain that is capable of a flexible swinging motion (double-headed grey arrow) is shown in dark green surface, with its FeS cluster in spheres. Both b_L and c₁ positions of the head domain are shown. Tt-CIII₂’s functional hemes in heteroatom-colored spheres; non-functional hemes in magenta spheres. Red crosses indicate inability of electron transfer. Tt-CI’s N2 FeS cluster in spheres. Homology model of Tt-cyt c in surface colored by electrostatic potential. Approximate position of inner mitochondrial membrane marked with black lines. b_H, heme b_H; b_L, heme b_L; c₁, heme c₁; e⁻, electron; H⁺, proton; IMS, intermembrane space;Q, oxidized quinone; QH₂, reduced quinone (quinol); Q•, semiquinone.

Fig. 5.. Structural features of Tt-CIV₂.

(A) Structure of Tt-CIV₂, with core subunits in surface over silhouette of entire complex. Viewed from the membrane (left) or intermembrane space (right). (B) Surface electrostatic potential of Tt-CIV₂. Cyt c crater marked with a black circle. (C and D) Charge reversal in CIV-cyt c binding between mammals and T. thermophila. CIVs were aligned by COX2. Key residues in stick. (C) Binding of bovine COX2 (red cylinder, cow symbol) to equine cyt c (yellow cylinder, horse symbol) (PDB: 5IY5) (74). (D) T. thermophila COX2 in grey cylinder. (E to G) Notable Tt-CIV₂ accessory subunits viewed from IMS. (E) Mitochondrial carrier subunits COXMC1–3. (F) Other subunits with annotated folds. (G) Mitochondrially encoded subunits YMF67, YMF70 and YMF75. (H) Side view of COXMC1–2 dimer (pink and green cylinders) with interfacial lipissssds (sticks). Key substrate-contacting residues, as well as conserved prolines of PX[D/E]XX[R/K] motif on odd-numbered helices in sticks. (I) Side view of COXMC3 (cyan cylinders) and the constraining amphipathic helix of COX3. (J) TIM8₃-TIM13₃-like hetero-hexamer domain with di-sulfide bonds in yellow. The Tt-specific loop of COX2 wraps around five of the COXTIM subunits and inserts itself into the central cavity of the hexamer.