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Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Jarman, Owen D 
Biner, Olivier 
Wright, John J 

Abstract

Mitochondrial 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 the inner mitochondrial membrane, creating the proton motive force for ATP synthesis. The mechanism by which the energy is captured, and the mechanism and pathways of proton pumping, remain elusive despite recent advances in structural knowledge. Progress has been limited by a lack of model systems able to combine functional and structural analyses with targeted mutagenic interrogation throughout the entire complex. Here, we develop and present the α-proteobacterium Paracoccus denitrificans as a suitable bacterial model system for mitochondrial complex I. First, we develop a robust purification protocol to isolate highly active complex I by introducing a His6-tag on the Nqo5 subunit. Then, we optimize the reconstitution of the enzyme into liposomes, demonstrating its proton pumping activity. Finally, we develop a strain of P. denitrificans that is amenable to complex I mutagenesis and create a catalytically inactive variant of the enzyme. Our model provides new opportunities to disentangle the mechanism of complex I by combining mutagenesis in every subunit with established interrogative biophysical measurements on both the soluble and membrane bound enzymes.

Description

Keywords

Catalysis, Cell Respiration, Electron Transport, Electron Transport Complex I, Operon, Paracoccus denitrificans, Recombinant Fusion Proteins, Substrate Specificity

Journal Title

Sci Rep

Conference Name

Journal ISSN

2045-2322
2045-2322

Volume Title

11

Publisher

Springer Science and Business Media LLC
Sponsorship
MRC (MC_UU_00015/2)
Medical Research Council (MC_UU_00015/7)