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dc.contributor.authorJones, Andrew JYen
dc.contributor.authorBlaza, James Nen
dc.contributor.authorVarghese, Febinen
dc.contributor.authorHirst, Judyen
dc.date.accessioned2018-08-15T15:26:56Z
dc.date.available2018-08-15T15:26:56Z
dc.date.issued2017-03en
dc.identifier.issn0021-9258
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/278850
dc.description.abstractRespiratory 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.
dc.format.mediumPrint-Electronicen
dc.languageengen
dc.rightsAttribution 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subjectMitochondriaen
dc.subjectAnimalsen
dc.subjectCattleen
dc.subjectParacoccus denitrificansen
dc.subjectProtonsen
dc.subjectElectron Transport Complex Ien
dc.subjectNADen
dc.subjectAdenosine Triphosphateen
dc.subjectElectron Transporten
dc.subjectOxidation-Reductionen
dc.subjectOxidative Phosphorylationen
dc.subjectProton-Motive Forceen
dc.titleRespiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized.en
dc.typeArticle
prism.endingPage4995
prism.issueIdentifier12en
prism.publicationDate2017en
prism.publicationNameThe Journal of biological chemistryen
prism.startingPage4987
prism.volume292en
dc.identifier.doi10.17863/CAM.26227
dcterms.dateAccepted2017-02-07en
rioxxterms.versionofrecord10.1074/jbc.m116.771899en
rioxxterms.versionVoR*
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2017-03en
dc.contributor.orcidBlaza, James N [0000-0001-5420-2116]
dc.contributor.orcidVarghese, Febin [0000-0003-4457-7736]
dc.contributor.orcidHirst, Judy [0000-0001-8667-6797]
dc.identifier.eissn1083-351X
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idMRC (MC_U105663141)


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International