Structural Studies into the Mechanism and Organisation of Mammalian Respiratory Complex I

Abstract

Respiratory complex I (NADH:ubiquinone oxidoreductase) is the major entry point of electrons into the electron transport chain and couples its redox activity with proton pumping across the inner mitochondrial membrane, thus contributing to the proton motive force which drives ATP synthesis. With single particle cryo-EM as a routine approach for high-resolution structure determination, there are extensive complex I structures available across multiple species, with conflicting proposals for how it couples ubiquinone reduction to proton translocation over a long range. However, there is a lack of structural data for complex I in different induced protonation states and, consequently, in support of proposals for how proton translocation is regulated across its hydrophilic central axis. Secondly, structural information for complex I and its constituent supercomplexes from immortalised human cell lines remains limited despite its potential as a system for studying human complex I disease mutations and its unique metabolic adaptations. Chapter 2 uses structural data from two high-resolution cryo-EM datasets (each up to 2.2 Å) of mammalian complex I in induced protonated and deprotonated states at a pH range where the enzyme retains biochemical stability to identify key molecular ‘switches’ that likely move to regulate proton flow between putative input and output channels and the central axis during a catalytic cycle. Finally, biochemical analyses in energy-transducing membranes suggests that pH does not impact enzyme global state proportions, in contrast with previous proposals. Chapter 3 characterises structures of the I1:III2 respiratory supercomplex from immortalised human cell lines, with a catalytically active ubiquinone-bound complex I in biochemically defined states at up to 2.6 Å and a dissociated strictly C2 symmetric complex III2 at 2.2 Å. The complex I:III interface is highly flexible and undergoes a continuous motion likely to support the structural or mechanistic costabilisation of respiratory complexes. In summary, this work explores complex I and III2 structure and interplay in a pathologically relevant and metabolically unique system. Chapter 4 examines potential future perspectives from Chapters 2 and 3, including the use of the affinity-tagged complex I cell line system from Chapter 3 to isolate human complex I biogenesis intermediates for structural characterisation by cryo-EM.