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

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 sy...

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Autores principales:	Owen D. Jarman, Olivier Biner, John J. Wright, Judy Hirst
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a5207d2b9c08420e8ce8c857e399ee89
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spelling	oai:doaj.org-article:a5207d2b9c08420e8ce8c857e399ee892021-12-02T15:42:59ZParacoccus denitrificans: a genetically tractable model system for studying respiratory complex I10.1038/s41598-021-89575-92045-2322https://doaj.org/article/a5207d2b9c08420e8ce8c857e399ee892021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-89575-9https://doaj.org/toc/2045-2322Abstract 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.Owen D. JarmanOlivier BinerJohn J. WrightJudy HirstNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Owen D. Jarman Olivier Biner John J. Wright Judy Hirst Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I
description	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.
format	article
author	Owen D. Jarman Olivier Biner John J. Wright Judy Hirst
author_facet	Owen D. Jarman Olivier Biner John J. Wright Judy Hirst
author_sort	Owen D. Jarman
title	Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I
title_short	Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I
title_full	Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I
title_fullStr	Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I
title_full_unstemmed	Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I
title_sort	paracoccus denitrificans: a genetically tractable model system for studying respiratory complex i
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/a5207d2b9c08420e8ce8c857e399ee89
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Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I

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