Structure-function studies of respiratory complex I from Paracoccus denitrificans using membrane mimetics

Respiratory complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to translocate four protons across energy-transducing membranes, contributing to the proton motive force that powers oxidative phosphorylation. Despite recent advances in structural knowledge and extensive biochemical investigations, the mechanism of redox-coupled proton translocation by complex I remains elusive. Complex I from the α-proteobacterium Paracoccus denitrificans is the closest bacterial relative of the mammalian enzyme and provides a minimal, genetically tractable system for studying its mechanism. However, the structure of the enzyme remains unknown, due to its intrinsically flexible and fragile nature, restricting the power of the system to tackle specific mechanistic questions on the molecular level. This study employs a multifaceted approach to develop a stable, native-like preparation of P. denitrificans complex I using membrane mimetics, enabling combination of functional and genetic investigations with structural analyses through single-particle cryo-electron microscopy (cryo-EM). First, a nanodisc system was developed to provide the wild-type P. denitrificans complex I with a stable, membrane environment. Following optimisation of the reconstitution conditions, biochemical and biophysical characterisations confirmed the enhanced structural stability and catalytic competence of the nanodisc-bound enzyme. Nanodiscs thus provide a new tool to alleviate the inherent instability of the bacterial enzyme and enable the preparation of high-quality cryo-EM samples for structural investigations. Next, the first high-resolution structure of isolated P. denitrificans complex I was determined at 2.3 Å-resolution in the nanodisc-membrane bilayer. In addition to the three known supernumerary subunits of P. denitrificans complex I, a new supernumerary subunit was identified that has not been observed hitherto in any other complex I species. Importantly, the enzyme is in a single, homogenous ‘ready-to-go’ resting state, and extensive structural evaluations revealed close similarity to the mammalian enzyme. These properties allow mechanistic studies to focus on catalysis, rather than regulation, and firmly establish the P. denitrificans enzyme as a powerful model organism for complex I. Finally, with the long-term aim of capturing the structures of catalytic intermediates, the functional nanodisc system was developed further to facilitate self-assembly of a minimal respiratory chain that catalyses NADH oxidation by ubiquinone cycling between complex I and an alternative oxidase. The suitability of the system for structural studies was demonstrated by determination of a 3.1 Å resting-state structure of the enzyme reconstituted in covalently circularized nanodiscs of predefined size, paving the way for future investigations into detailed structure-function relationships in respiratory complex I.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Ivanov, Bozhidar

Advisor dc:contributor.advisor

• Hirst, Judy

• cryo-EM
• membrane mimetics
• Paracoccus denitrificans
• respiratory complex I

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/eb0217ca-317f-478b-9fb1-e194f4dcab0b/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.111808

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/373309