Respiratory complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across an energy-transducing membrane, contributing to the proton motive force used to synthesise ATP. Although structural knowledge of complex I is now extensive, the mechanisms by which it captures the redox energy for proton translocation, and the mechanisms and pathways of the proton pumps, remain elusive. In this thesis, the α- proteobacterium Paracoccus denitrificans is developed and presented as a powerful model system for understanding mitochondrial complex I, combining interrogative biophysical and structural characterisation with the potential for mutagenesis in every subunit. First, aiming to establish conditions for studying the thermodynamic reversibility of P. denitrificans complex I, activation of ATP hydrolysis by the unidirectional F1FO-ATP synthase of P. denitrificans was investigated by the deletion of potential inhibitory subunits and treatment with potential chemical activators. While no conditions were found in which ATP hydrolysis could be sufficiently activated to drive complex I in reverse, insights were gained into the regulatory mechanism of P. denitrificans ATP synthase and the inhibitory role of Mg-ADP.

Next, a strain of P. denitrificans containing an alternative NADH dehydrogenase, a bypass enzyme, was generated to facilitate the creation of deleterious complex I variants. In addition, a purification tag was engineered onto complex I, enabling its rapid purification. The isolated complex I was thoroughly characterised and its reconstitution into proteoliposomes was optimised, expanding the toolkit available for the study of complex I variants. The structure of the enzyme was also partially resolved by cryo-EM. The well-resolved map of the hydrophilic domain revealed a novel supernumerary subunit and demonstrated that P. denitrificans complex I exists entirely in the so-called ‘active’ state. However, due to conformational heterogeneity, the cryo-EM map was poorly resolved in the membrane domain, preventing detailed structural modelling of the complete enzyme.

Finally, single point variants in the Nqo13 (ND4) subunit of complex I were generated to investigate: (1) key residues in the energy propagation pathway; (2) coordination to the lateral helix of Nqo12 (ND5); and (3) a potential hydration channel controlled by a gating mechanism. Comprehensive characterisation of the variants revealed insights into all three components of the mechanism. Furthermore, no variants were identified that pump fewer than four protons per NADH oxidised, emphasising the tight coupling between ubiquinone reduction and proton translocation, which is conserved even when the rate of catalysis is compromised.