By the same authors

From the same journal

From the same journal

Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized

Research output: Contribution to journalArticle

Full text download(s)

Published copy (DOI)

Author(s)

Department/unit(s)

Publication details

JournalThe Journal of biological chemistry
DatePublished - 24 Mar 2017
Issue number12
Volume292
Number of pages9
Pages (from-to)4987-4995
Original languageEnglish

Abstract

Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.

Bibliographical note

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

    Research areas

  • Adenosine Triphosphate/metabolism, Animals, Cattle/metabolism, Electron Transport, Electron Transport Complex I/metabolism, Mitochondria/metabolism, NAD/metabolism, Oxidation-Reduction, Oxidative Phosphorylation, Paracoccus denitrificans/enzymology, Proton-Motive Force, Protons

Discover related content

Find related publications, people, projects, datasets and more using interactive charts.

View graph of relations