Eukaryotic life is made possible by energy production in mitochondria, where several large membrane protein complexes of the respiratory chain work in series to produce ATP. The structure and function of these complexes have been intensively studied over decades, but the mechanistic understanding is lacking due to their elaborate architecture. This proposal’s goal is to reveal the mechanism of energy transduction by the least understood of them: complex I, respiratory supercomplexes and transhydrogenase. Complex I is the largest respiratory enzyme, containing up to 45 subunits with a total mass of ~1 MDa. We have determined the first atomic structures of complex I from bacteria and mitochondria. Mammalian complex I usually exists as a supercomplex with complexes III2 and IV: we have determined the first architecture of this ~1.7 MDa physiological “unit” of respiration. The nicotinamide nucleotide transhydrogenase couples proton motive force to mitochondrial redox homeostasis, working in tandem with the respiratory chain. We recently determined the first structure of transhydrogenase but its coupling mechanism remains controversial. Huge conformational changes are envisaged but not yet observed. The mechanism of coupling between spatially separated electron transfer and proton translocation in complex I is also a mystery. It is likewise not known why respiratory complexes are organised into supercomplexes. We will tackle all these questions by an integrative approach, solving the atomic structures of different catalytic states of the complexes by applying the latest cryo-EM methods to these extremely challenging targets. The comparison of structures, complemented by functional and computational analyses, will reveal the mechanistic basis for the function of these molecular machines, solving fundamental questions in biology. As these enzymes are involved in many severe human disorders, the acquired knowledge will also be instrumental to tackle mitochondrial diseases.
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