No abstract

Respiration is a core biological energy-converting process whose last steps are carried out by a chain of multi-subunit 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 (CI) and Tt-CIII 2 (Tt-SC I+III 2 ) and a structure of Tt-CIV 2 . Tt-SC I+III 2 (~2.3 MDa) is a curved assembly with structural and functional symmetry breaking. Tt-CIV 2 is a ~2.7 MDa dimer with over 52 subunits per protomer, including mitochondrial carriers and a TIM8 3 -TIM13 3 -like domain. Our structural and functional study of the T. thermophila respiratory chain reveals divergence in key components of eukaryotic respiration, expanding our understanding of core metabolism.

Respiration, an essential metabolic process, provides cells with chemical energy. In eukaryotes, respiration occurs via the mitochondrial electron transport chain (mETC) composed of several large membrane-protein complexes. Complex I (CI) is the main entry point for electrons into the mETC. For plants, limited availability of mitochondrial material has curbed detailed biochemical and structural studies of their mETC. Here, we present the cryoEM structure of the known CI assembly intermediate CI* from Vigna radiata at 3.9 Å resolution. CI* contains CI’s NADH-binding and CoQ-binding modules, the proximal-pumping module and the plant-specific γ-carbonic-anhydrase domain (γCA). Our structure reveals significant differences in core and accessory subunits of the plant complex compared to yeast, mammals and bacteria, as well as the details of the γCA domain subunit composition and membrane anchoring. The structure sheds light on differences in CI assembly across lineages and suggests potential physiological roles for CI* beyond assembly.

V (vacuolar)/A (archaeal)-type adenosine triphosphatases (ATPases), found in archaea and eubacteria, couple ATP hydrolysis or synthesis to proton translocation across the plasma membrane using the rotary-catalysis mechanism. They belong to the V-type ATPase family, which differs from the mitochondrial/chloroplast F-type ATP synthases in overall architecture. We solved cryo–electron microscopy structures of the intact Thermus thermophilus V/A-ATPase, reconstituted into lipid nanodiscs, in three rotational states and two substates. These structures indicate substantial flexibility between V1 and Vo in a working enzyme, which results from mechanical competition between central shaft rotation and resistance from the peripheral stalks. We also describe details of adenosine diphosphate inhibition release, V1-Vo torque transmission, and proton translocation, which are relevant for the entire V-type ATPase family.