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dc.contributor.authorLe Breton, Nolwenn
dc.contributor.authorWright, John J
dc.contributor.authorJones, Andrew JY
dc.contributor.authorSalvadori, Enrico
dc.contributor.authorBridges, Hannah R
dc.contributor.authorHirst, Judy
dc.contributor.authorRoessler, Maxie M
dc.date.accessioned2018-06-04T09:05:26Z
dc.date.available2018-06-04T09:05:26Z
dc.date.issued2017-11-15
dc.identifier.issn0002-7863
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/276489
dc.description.abstractEnergy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.
dc.format.mediumPrint-Electronic
dc.languageeng
dc.publisherAmerican Chemical Society (ACS)
dc.rightsAttribution 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectAnimals
dc.subjectCattle
dc.subjectProtons
dc.subjectUbiquinone
dc.subjectElectron Transport Complex I
dc.subjectHistidine
dc.subjectElectron Spin Resonance Spectroscopy
dc.subjectElectron Transport
dc.subjectOxidation-Reduction
dc.subjectHydrogen Bonding
dc.subjectHydrogen-Ion Concentration
dc.subjectElectrons
dc.titleUsing Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.
dc.typeArticle
prism.endingPage16326
prism.issueIdentifier45
prism.publicationDate2017
prism.publicationNameJ Am Chem Soc
prism.startingPage16319
prism.volume139
dc.identifier.doi10.17863/CAM.23788
dcterms.dateAccepted2017-10-17
rioxxterms.versionofrecord10.1021/jacs.7b09261
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2017-11-03
dc.contributor.orcidHirst, Judy [0000-0001-8667-6797]
dc.contributor.orcidRoessler, Maxie M [0000-0002-5291-4328]
dc.identifier.eissn1520-5126
rioxxterms.typeJournal Article/Review
pubs.funder-project-idMedical Research Council (MC_U105663141)
pubs.funder-project-idMRC (MC_UU_00015/2)
cam.issuedOnline2017-11-03


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