Mutagenesis investigations into proton transfer pathways and control points in respiratory complex I

Respiratory complex I (NADH:ubiquinone oxidoreductase) is a large multi-subunit membrane protein that uses the energy from electron transfer from NADH to ubiquinone to transport four protons across an energy transducing membrane. The protons contribute to building the proton-motive force that powers oxidative phosphorylation, and the oxidation of NADH is crucial for NADH/NAD+ homeostasis, positioning complex I at a key control point in aerobic metabolism. Despite extensive structural characterisation, the mechanism of complex I catalysis remains poorly understood, with proposed mechanisms lacking both molecular detail and biochemical support. In this thesis the genetically tractable α-proteobacterium Paracoccus denitrificans and its accompanying toolkit of biochemical, biophysical and structural techniques was used to investigate proton translocation in the membrane domain, using amino acid substitutions to target, disrupt and identify proton pathways and control points. In the first major results chapter, the proton uptake pathways in the membrane domain of P. denitrificans complex I are defined. Structural and computational analyses have previously been used to propose uptake pathways in each of three antiporter-like subunits (ND2, ND4 and ND5), connecting the negatively charged face of the membrane to the ‘central axis’. In this chapter, by systematically substituting common residues in each pathway, only the pathways in subunits ND4 and ND5 are revealed to be active, while the pathway in ND2 is not. This fundamental advance in knowledge of complex I catalysis answers the conundrum of how three subunits pump four protons and restricts the opportunity space for future mechanistic proposals. Three further results chapters describe the use of amino acid substitutions to probe different aspects of complex I catalysis: i) strategies devised to interrogate candidate proton output pathways in subunits ND2 and ND4 proved inconclusive; ii) a set of variants in subunits ND6 and ND3 provided initial data to challenge the formation of a π-bulge (observed in structures of resting states and widely exploited in mechanistic proposals) during catalysis; and iii) structural analysis of an inactive variant of P. denitrificans complex I by cryo-electron microscopy, to attempt to capture a stalled intermediate of catalysis, revealed a small number of discrete changes but also highlighted the limitations of the experimental approach. These three chapters reveal new and promising strategies for future explorations of complex I catalysis using the power of the P. denitrificans system.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Waddell, Robert Alexander

Advisor dc:contributor.advisor

• Hirst, Judy

• cryo-EM
• mutagenesis
• Paracoccus denitrificans
• respiratory complex I

Licence

• https://www.repository.cam.ac.uk/bitstreams/d068301c-d044-4333-8008-ad74b847dff5/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.129564

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/402031