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.
Highlights
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
We introduced an unmarked insertion of the His6-tag into the chromosomal DNA of P. denitrificans strain Pd1222-∆Hy35 by suicide vector-mediated homologous recombination, an approach previously used to create unmarked deletions in the P. denitrificans genome[35,40,41]
The α-proteobacterium P. denitrificans, a close relative of the protomitochondrion, is a prime candidate for a model system that could be exploited to unlock the catalytic mechanism of mitochondrial complex I, as it enables all three approaches
Summary
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 ‘resolution revolution’ has resulted in a surge of complex I structural data, especially resulting from cryoEM analyses, including native enzyme structures from bacteria, yeast, plants and mammals[10,11,13,14,15,16], and substrate and inhibitor bound s tates[17,18,19,20] These structures, along with molecular dynamic simulations[2,21,22], have identified intriguing structural elements and key residues that are likely involved in the coupling mechanism and proton transfer pathways. Probing the catalytic roles of these elements and sites further is hindered by lack of model systems that first, are suitable for both functional and structural analyses, and second, allow genetic manipulation of the core membrane domain subunits, which are encoded on the mitochondrial DNA in eukaryotes. Development of an experimentally versatile and fully genetically tractable bacterial model system has a crucial role to play in addressing mechanistic questions
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