Structure and function of Complex I, Complex III2, and the SCI/III2. (A) The structure of the OXPHOS complexes with lipid headgroups from atomistic MD simulations. (B, C) Overview of the structure and function of redox-driven proton pumping in CI and the Q-cycle of CIII2. (D-F) Molecular models of the (D) SCI/III2, (E) CI, and (F) CIII2 used in the atomistic MD simulations (water and ions were omitted for visual clarity).

SC formation affects the concentration of lipids and the membrane thickness.

(A-C) OXPHOS complexes viewed from the N-side of the membrane. (D-F) Membrane shift induced by (D) the SC, and (E,F) the isolated CI and CIII2. The membrane shift relative to the average membrane plane is colored by the shift in z-position viewed from the N-side. (G) Local membrane thickness around SC (top), CI and CIII2 (bottom). (H) Local cardiolipin concentration. (I) Local concentration of Q (Q+QH2). (J) Cryo-EM difference map. (K) Membrane shift determined from the cryo-EM map. (L) Membrane thickness determined from cryo-EM map. (M) Density of Q/QH2 (headgroup) from cgMD simulations. Insets: Protein-Q interactions from atomistic MD. (N) Density of cardiolipin (headgroup) from cgMD simulations. Insets: Protein-CDL interactions from atomistic MD.

Conformational dynamics of the OXHPOS proteins and the SC. (A) Subunits comprising the SC interface. Inset: Interaction near the DED-loop. (B) Normal modes derived from essential dynamics analysis for the SC (left), CI (middle), and CIII2 (right). See SI Appendix, Movies 1-5 for normal modes of the SC, CI, and CIII2. (C) Decomposition of interaction energies within the SC. (D) Distribution of the opening/closing mode (left) and the twisting mode (right) for the isolated CI (in orange) and the CI within the SC (in blue). (E) Differences in the dynamics of CIII2 for minimum and maximum values of mode 1. (F) Distribution of the CI domain angle in the SC (in blue) and CI (in orange). (G,J) Conformational changes in the Rieske subunit between SC (in blue) and CIII2 (in green) affect (G) the QH2 binding in the proximal Qo site and (J) the Rieske FeS-heme c1 distance in the distal monomer. (H) Distribution of the dihedral angle in TM3 of ND6. (I) Overview of the SC showing the locations of ND6 and Rieske subunits, as well as the proximal and distal protomers of CIII2.

Lattice model of SC formation and crowding effects in the IMM. (A) Possible specific and non-specific interactions between CI (blue/white) and CIII2 (red) and their respective energies in the lattice model. (B) SC population as a function of the specific interaction energy (Especific) and molecular strain (Estrain). (C) Specific and non-specific assemblies as a function of the protein-lipid ratio with varying specific interaction energies (Especific). (D) Representative protein arrangement in crowded IMMs. (E) Average edge-to-edge distance distributions and nearest neighbor distance (red line) for specific protein-Q/protein contacts. (F) Nearest neighbor distance between Q and CI as a function of the Q/QH2 ratio. The dashed line indicates the 7 nm distance between active sites in the SC.

Proposed thermodynamic and functional effects of SCs. (A) The individual OXPHOS complexes, CI and CIII2 induce prominent molecular strain in the surrounding lipid membrane (dark green area) due to a hydrophobic mismatch between the protein and the membrane. Condensation of locally strained membrane patches entropically drives the SC formation, and leads to an overall reduction of the membrane strain (light green area around the SC), favoring the accumulation of cardiolipin around the SC. (B) During normal respiratory conditions, each OXPHOS complex is surrounded by multiple (ca. 6) quinol/quinone molecules that can act as substrates for the proteins. At limiting QH2 concentration, the quinol diffusion between CI and CIII2 becomes rate-limiting, and leads to a kinetic advantage of the SC (see also Fig. 4F).