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dc.contributor.authorRobinson, William Een
dc.contributor.authorBassegoda, Arnauen
dc.contributor.authorBlaza, James Nen
dc.contributor.authorReisner, Erwinen
dc.contributor.authorHirst, Judyen
dc.date.accessioned2020-06-22T23:31:40Z
dc.date.available2020-06-22T23:31:40Z
dc.identifier.issn0002-7863
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/307107
dc.description.abstractMetal-dependent formate dehydrogenases (FDHs) catalyze the reversible conversion of formate into CO2, a proton and two electrons. Kinetic studies of FDHs provide key insights into their mechanism of catalysis, relevant as a guide for the development of efficient electrocatalysts for formate oxidation as well as for CO2 capture and utilization. Here, we identify and explain the kinetic isotope effect (KIE) observed for the oxidation of formate and deuterioformate by the Mo-containing FDH from Escherichia coli using three different techniques: steady-state solution kinetic assays, protein film electrochemistry (PFE) and pre-steady state stopped-flow methods. For each technique, the Mo center of FDH is reoxidized at a different rate following formate oxidation, significantly affecting the observed kinetic behavior and providing three different viewpoints on the KIE. Steady-state turnover in solution, using an artificial electron acceptor, is kinetically limited by diffusional intermolecular electron transfer, masking the KIE. In contrast, interfacial electron transfer in PFE is fast, lifting electron transfer rate limitation and manifesting a KIE of 2.44. Pre-steady state analyses using stopped-flow spectroscopy revealed a KIE of 3 that can be assigned to the CH bond cleavage step during formate oxidation. We formalize our understanding of FDH catalysis by fitting all the data to a single kinetic model, recreating the condition-dependent shift in rate-limitation of FDH catalysis between active site chemical catalysis and regenerative electron transfer. Furthermore, our model predicts the steady-state and time-dependent concentrations of catalytic intermediates, providing a valuable framework for the design of future mechanistic experiments.
dc.languageenen
dc.publisherAmerican Chemical Society (ACS)
dc.rightsAll rights reserved
dc.rights.uri
dc.titleUnderstanding how the rate of C-H bond cleavage affects formate oxidation catalysis by a Mo-dependent formate dehydrogenaseen
dc.typeArticle
prism.numberjacs.0c03574en
prism.publicationNameJournal of the American Chemical Societyen
dc.identifier.doi10.17863/CAM.54200
dcterms.dateAccepted2020-06-18en
rioxxterms.versionofrecord10.1021/jacs.0c03574en
rioxxterms.versionAM
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2020-06-18en
dc.contributor.orcidReisner, Erwin [0000-0002-7781-1616]
dc.contributor.orcidHirst, Judy [0000-0001-8667-6797]
dc.identifier.eissn1520-5126
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idBBSRC (BB/J000124/1)
pubs.funder-project-idEPSRC (EP/L015978/1)
pubs.funder-project-idECH2020 EUROPEAN RESEARCH COUNCIL (ERC) (682833)
pubs.funder-project-idMRC (MC_U105663141)
pubs.funder-project-idBBSRC (BB/I026367/1)
cam.issuedOnline2020-06-18en
datacite.issupplementedby.urlhttps://doi.org/10.17863/CAM.54060
cam.orpheus.successMon Jun 29 08:55:23 BST 2020 - Embargo updated*
rioxxterms.freetoread.startdate2021-06-18


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