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dc.contributor.authorOlshansky, Lisaen_US
dc.contributor.otherMassachusetts Institute of Technology. Department of Chemistry.en_US
dc.date.accessioned2016-03-03T21:08:28Z
dc.date.available2016-03-03T21:08:28Z
dc.date.copyright2015en_US
dc.date.issued2015en_US
dc.identifier.urihttp://hdl.handle.net/1721.1/101552
dc.descriptionThesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015.en_US
dc.descriptionCataloged from PDF version of thesis.en_US
dc.descriptionIncludes bibliographical references.en_US
dc.description.abstractProton-coupled electron transfer (PCET) reactions comprise a fundamental mechanism for energy transduction in nature. In catalyzing the conversion of ribonucleotides to deoxyribonucleotides, ribonucleotide reductase (RNR) performs reversible, long-range PCET over a pathway of redox active amino acids ([beta]-Y₁₂₂ >/< [beta]-Y₃₅₆ >/< [alpha]-Y₇₃₁ >/< [alpha]-Y₇₃₀ >/< [alpha]-C₄₃₉) that spans ~35 Å and two subunits. As such, RNR serves as a paradigm for the study of PCET in biology. Subunit interaction dynamics, examined by fluorescence spectroscopy, exposed mechanisms underlying allosteric control over PCET and contributed to an expanded kinetic model for turnover. Trapped meta-stable states of the active [alpha]₂[beta]₂ complex are dictated by the translocation of a single charge and attenuate dissociation 10⁴-fold. These trapped states were leveraged to resolve the stoichiometric distribution of the Y¹²²* cofactor from its ensemble average of 1.2 Y*/[beta]₂ , revealing that [beta]₂ contain either 2 or 0 Y*. Circumventing rate-limiting conformational changes that gate turnover, photoinitiated RNRs were prepared to allow photochemically driven Y₃₅₆ oxidation, and spectroscopic resolution of the ensuing reactivity. A series of photoRNRs containing unnatural FnYs (n = 2-3) and W in place of [beta]-Y₃₅₆ were prepared. All of these photo[beta]₂s give rise to transient absorption (TA) spectra consistent with their oxidized forms and undergo photochemically driven turnover. Time-resolved emission spectroscopy allowed examination of ET kinetics as a function of driving force within the [alpha]/[beta] subunit interface. Marcus-inverted kinetics were observed, providing reorganization and electronic coupling energies. Comparing ET and PCET kinetics as a function of pH, buffer concentration, oligomeric state, and buffer isotopic composition revealed new insights into biological control over PCET reactions and implicate a role of [alpha]₂ in facilitating proton transfer from [beta]-Y₃₅₆ Single wavelength TA kinetics provided direct measure of the rate constant for PCET through a, assignment of the rate-determining step as 3'-C-H bond cleavage by C₄₃₉ , and a lower bound of 7 for the associated 1° KIE. The pKa of proton acceptor(s) at the subunit interface, and the relative energies of individual radical intermediates were determined, revealing matched tuning to the surrounding environment and highlighting the subtlety of precision control underlying RNR catalysis.en_US
dc.description.statementofresponsibilityby Lisa Olshansky.en_US
dc.format.extent310 pagesen_US
dc.language.isoengen_US
dc.publisherMassachusetts Institute of Technologyen_US
dc.rightsM.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.en_US
dc.rights.urihttp://dspace.mit.edu/handle/1721.1/7582en_US
dc.subjectChemistry.en_US
dc.titleKinetics and dynamics controlling proton-coupled electron transfer in ribonucleotide reductaseen_US
dc.typeThesisen_US
dc.description.degreePh. D.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistry.en_US
dc.identifier.oclc940565653en_US


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