Paracoccus denitrificans: a genetically tractable model system

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-proteobacteriumParacoccus denitrificans as a suitable bacterial model system for mitochondrial complex I.

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Fig: Purification and subunit composition of P. denitrificans complex I. (a) Typical Ni-affinity chromatography trace produced using a HisTrap HP column. The column was washed with 80 mM imidazole and protein eluted with 200 mM imidazole. (b) Typical size exclusion chromatography trace using a Superdex 200 increase 5/150 GL column. Complex I was identified by measuring the relative NADH:APAD+ activity in eluted fractions (black trace). (c) SDS-PAGE analysis of purified P. denitrificans complex I. Two full-length lanes are shown from a single gel; the image has been cut and they have been moved to be adjacent to each other. The original image is shown in SI Fig. S6. Individual complex I subunits were identified and assigned by excising each band, treating the sample with trypsin and analyzing the resultant peptides by mass spectrometry. Peptides were assigned to a subunit/protein by peptide mass fingerprinting.

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.

Jarman, O.D., Biner, O., Wright, J.J. et al. Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I.Sci Rep 11, 10143 (2021). https://doi.org/10.1038/s41598-021-89575-9

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