Mitochondria are the powerhouses of the cell, synthesizing the energy currency of the cell (ATP) by oxidative phosphorylation. Electron transfer through the chain of respiratory enzymes in the inner mitochondrial membrane is coupled to proton translocation across the membrane. The resulting proton gradient drives ATP synthase. Respiratory enzymes are large multi-subunit protein assemblies (complexes I-IV), with complex I being the largest. The basic operating principles of some components of the chain are still not understood.
Complex I (CI) is the entry point into the respiratory chains of mitochondria and bacteria. It couples the transfer of two electrons between NADH and quinone to the translocation of four protons across the membrane. The L-shaped CI consists of the hydrophilic matrix arm, where electron transfer takes place, and the membrane arm containing proton translocation channels. This arrangement implies long-range conformational coupling, but the actual mechanism is not known. In 2020 we have proposed the first experiment-based model of the coupling mechanism of mammalian complex I. However, many details remained unknown and further exploration of these ideas was required.
Furthermore, in intact mammalian mitochondria the majority of CI is found within respiratory supercomplexes (SCs), and their functional role is hotly debated. Previously we have solved structures of respirasome (CICIII2CIV) and CICIII2, but structures (and function) of other supercomplexes remained unknown.
Nicotinamide nucleotide transhydrogenase (NNT) works in tandem with the respiratory chain, coupling proton translocation to hydride transfer between NAD(H) and NADP(H), mostly forming NADPH, used to combat reactive oxygen species (ROS). We have determined the first atomic structure of the entire mammalian enzyme and proposed unique mechanism that may involve ~180 degree rotation of a large NADP(H)-binding domain. However, direct experimental evidence supporting it is still lacking.
Therefore, despite the advances in structural knowledge of the respiratory chain, there is still little understanding of the coupling mechanisms of CI and NNT, as well as of the functional role of SCs and their complete structural organisation. The ambitious overarching aim of this project is to tackle these important questions, the “grand challenges” of modern biology. It is clear that the mechanisms of both CI and NNT must involve long-range conformational changes, therefore determining (by single particle cryo-EM) the structures of these enzymes captured at different stages of the catalytic cycle will be instrumental in deducing the underlying principles. The conclusions are being verified by site-directed mutagenesis and molecular dynamics (MD) studies. The role of the SCs is studied by resolving their structures in the presence of substrates and inhibitors, to capture different aspects of their interactions, accompanied by functional assays.
As a result of this project, the mechanisms of some of the most intriguing molecular machines in biology should become clear. We will know how exactly electron transfer and proton translocation are so tightly coupled in complex I, despite being completely spatially separated. The molecular organisation, the structure and functional role of various respiratory supercomplexes will become clear. The unique “swivel-to-pump?” mechanism of transhydrogenase will be clarified. Thus, we will achieve a much more complete and rounded understanding of the workings of respiratory chain and mitochondria in general. The results will have far-reaching implications in biology and for society in general, since mitochondrial research is currently coming to the forefront in many medical research areas due to emerging role of mitochondria not only in energy production but also in biosynthesis, redox homeostasis, oncogenic signaling and apoptosis.
