{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/115805"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/115805","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Mechanistic studies of the radical transport pathway in aminotyrosine-substituted class Ia ribonucleotide reductase","abstract":"Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'- deoxynucleotides. The focus of this thesis is the F coli class la RNR, which is comprised of two homodimeric subunits, [alpha]2 and [beta]2, forming an active [alpha]2[beta]2 complex. The [beta]2 subunit harbors the stable diferric-tyrosyl radical cofactor (Y 122*) that reversibly oxidizes the active site cysteine (C₄₃₉) in [alpha]2. This oxidation requires a long-range radical transport (RT) pathway consisting of proton-coupled electron transfer (PCET) events through redox-active aromatic amino acid residues: Y₁₂₂* <--> [W₄₈] <--> Y₃₅₆ in [beta]2 to Y₇₃₁ <--> Y₇₃₀ <--> C₄₃₉ in [alpha]2. Once formed, the transient C₄₃₉* initiates nucleotide reduction. Both the long-range oxidation and the nucleotide reduction chemistries are kinetically masked by rate-limiting protein conformational change(s). To overcome this conformational change, the unnatural amino acid probe 3-aminotyrosine (NH₂Y) has been sitespecifically incorporated at multiple positions (Y₃₅₆, Y₇₃₁, Y₇₃₀) into the RT pathway. Herein, the NH₂Y probe is characterized as pertaining to the previously demonstrated ability for NH₂Y-incorporated RNR (NH₂Y-RNR) to form product. The reduction potential of NH₂Y produces a thermodynamic barrier that RNR cannot overcome. To explain NH₂Y-RNR activity, mass spectrometry was used for relative quantitation of contaminating wt-RNR in the NH₂Y-RNR, lending credence to the fact that the NH₂Y-RNRs are actually inactive. These results provide clarity to the long-standing mystery behind the low activities of the NH₂Y-RNRs. The use of the NH₂Y probe to generate stable radicals on the RT pathway has revealed further remarkable insight, demonstrating a hydrogen bonding network in the [alpha]2 subunit by employing advanced EPR methods on NH₂Y₇₃₀* and NH₂Y₇₃₁*. The evidence for a collinear PCET mechanism is provided with the NH₂Y₇₃₀/Y₇₃₁F and NH₂Y₇₃₁/C₄₃₉A mutants. Mutation of an R₄₁₁ to alanine in [alpha]2 allowed the detection of a \"flipped\" NH₂Y₇₃₁* conformation using advanced EPR techniques. Herein, photo cross-linked RNR is studied by tandem mass spectrometry (MS/MS). The study of a photo cross-linked [alpha]2[beta]2 complex using a 4-N-maleimido-benzophenone covalently attached to the C-terminal tail of [beta]2 yielded no photo cross-linked peptides. These studies taken together provide additional insight at the [alpha][beta] interface and provide additional tools to study this interaction.","abstract_html":"Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2&#x27;- deoxynucleotides. The focus of this thesis is the F coli class la RNR, which is comprised of two homodimeric subunits, [alpha]2 and [beta]2, forming an active [alpha]2[beta]2 complex. The [beta]2 subunit harbors the stable diferric-tyrosyl radical cofactor (Y 122*) that reversibly oxidizes the active site cysteine (C₄₃₉) in [alpha]2. This oxidation requires a long-range radical transport (RT) pathway consisting of proton-coupled electron transfer (PCET) events through redox-active aromatic amino acid residues: Y₁₂₂* &lt;--&gt; [W₄₈] &lt;--&gt; Y₃₅₆ in [beta]2 to Y₇₃₁ &lt;--&gt; Y₇₃₀ &lt;--&gt; C₄₃₉ in [alpha]2. Once formed, the transient C₄₃₉* initiates nucleotide reduction. Both the long-range oxidation and the nucleotide reduction chemistries are kinetically masked by rate-limiting protein conformational change(s). To overcome this conformational change, the unnatural amino acid probe 3-aminotyrosine (NH₂Y) has been sitespecifically incorporated at multiple positions (Y₃₅₆, Y₇₃₁, Y₇₃₀) into the RT pathway. Herein, the NH₂Y probe is characterized as pertaining to the previously demonstrated ability for NH₂Y-incorporated RNR (NH₂Y-RNR) to form product. The reduction potential of NH₂Y produces a thermodynamic barrier that RNR cannot overcome. To explain NH₂Y-RNR activity, mass spectrometry was used for relative quantitation of contaminating wt-RNR in the NH₂Y-RNR, lending credence to the fact that the NH₂Y-RNRs are actually inactive. These results provide clarity to the long-standing mystery behind the low activities of the NH₂Y-RNRs. The use of the NH₂Y probe to generate stable radicals on the RT pathway has revealed further remarkable insight, demonstrating a hydrogen bonding network in the [alpha]2 subunit by employing advanced EPR methods on NH₂Y₇₃₀* and NH₂Y₇₃₁*. The evidence for a collinear PCET mechanism is provided with the NH₂Y₇₃₀/Y₇₃₁F and NH₂Y₇₃₁/C₄₃₉A mutants. Mutation of an R₄₁₁ to alanine in [alpha]2 allowed the detection of a &quot;flipped&quot; NH₂Y₇₃₁* conformation using advanced EPR techniques. Herein, photo cross-linked RNR is studied by tandem mass spectrometry (MS/MS). The study of a photo cross-linked [alpha]2[beta]2 complex using a 4-N-maleimido-benzophenone covalently attached to the C-terminal tail of [beta]2 yielded no photo cross-linked peptides. These studies taken together provide additional insight at the [alpha][beta] interface and provide additional tools to study this interaction.","abstract_has_math":false,"creators":["Lee, Wankyu"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Chemistry.","school":null,"contributors":[],"advisors":["JoAnne Stubbe."],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018","date_published":"2018","updated_at":"2026-07-22T22:20:47Z","subjects":["Chemistry."],"languages":["eng"],"rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/115805","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["JoAnne Stubbe."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Chemistry."]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. Department of Chemistry."]},{"key":"dc:creator","label":"Author","values":["Lee, Wankyu"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2018-05-23T16:35:39Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2018-05-23T16:35:39Z"]},{"key":"dc:date.issued","label":"Date","values":["2018"]},{"key":"dc:publisher","label":"Institution","values":["Massachusetts Institute of Technology"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Chemistry."]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/115805"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.","Cataloged from PDF version of thesis.","Includes bibliographical references."]},{"key":"dc:description.abstract","label":"Abstract","values":["Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'- deoxynucleotides. The focus of this thesis is the F coli class la RNR, which is comprised of two homodimeric subunits, [alpha]2 and [beta]2, forming an active [alpha]2[beta]2 complex. The [beta]2 subunit harbors the stable diferric-tyrosyl radical cofactor (Y 122*) that reversibly oxidizes the active site cysteine (C₄₃₉) in [alpha]2. This oxidation requires a long-range radical transport (RT) pathway consisting of proton-coupled electron transfer (PCET) events through redox-active aromatic amino acid residues: Y₁₂₂* <--> [W₄₈] <--> Y₃₅₆ in [beta]2 to Y₇₃₁ <--> Y₇₃₀ <--> C₄₃₉ in [alpha]2. Once formed, the transient C₄₃₉* initiates nucleotide reduction. Both the long-range oxidation and the nucleotide reduction chemistries are kinetically masked by rate-limiting protein conformational change(s). To overcome this conformational change, the unnatural amino acid probe 3-aminotyrosine (NH₂Y) has been sitespecifically incorporated at multiple positions (Y₃₅₆, Y₇₃₁, Y₇₃₀) into the RT pathway. Herein, the NH₂Y probe is characterized as pertaining to the previously demonstrated ability for NH₂Y-incorporated RNR (NH₂Y-RNR) to form product. The reduction potential of NH₂Y produces a thermodynamic barrier that RNR cannot overcome. To explain NH₂Y-RNR activity, mass spectrometry was used for relative quantitation of contaminating wt-RNR in the NH₂Y-RNR, lending credence to the fact that the NH₂Y-RNRs are actually inactive. These results provide clarity to the long-standing mystery behind the low activities of the NH₂Y-RNRs. The use of the NH₂Y probe to generate stable radicals on the RT pathway has revealed further remarkable insight, demonstrating a hydrogen bonding network in the [alpha]2 subunit by employing advanced EPR methods on NH₂Y₇₃₀* and NH₂Y₇₃₁*. The evidence for a collinear PCET mechanism is provided with the NH₂Y₇₃₀/Y₇₃₁F and NH₂Y₇₃₁/C₄₃₉A mutants. Mutation of an R₄₁₁ to alanine in [alpha]2 allowed the detection of a \"flipped\" NH₂Y₇₃₁* conformation using advanced EPR techniques. Herein, photo cross-linked RNR is studied by tandem mass spectrometry (MS/MS). The study of a photo cross-linked [alpha]2[beta]2 complex using a 4-N-maleimido-benzophenone covalently attached to the C-terminal tail of [beta]2 yielded no photo cross-linked peptides. These studies taken together provide additional insight at the [alpha][beta] interface and provide additional tools to study this interaction."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D. in Biological Chemistry"]},{"key":"dc:title","label":"Title","values":["Mechanistic studies of the radical transport pathway in aminotyrosine-substituted class Ia ribonucleotide reductase"]}]}],"canonical_facts":{"dc:contributor.advisor":["JoAnne Stubbe."],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Chemistry."],"dc:contributor.other":["Massachusetts Institute of Technology. Department of Chemistry."],"dc:creator":["Lee, Wankyu"],"dc:date.accessioned":["2018-05-23T16:35:39Z"],"dc:date.available":["2018-05-23T16:35:39Z"],"dc:date.issued":["2018"],"dc:description":["Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.","Cataloged from PDF version of thesis.","Includes bibliographical references."],"dc:description.abstract":["Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'- deoxynucleotides. The focus of this thesis is the F coli class la RNR, which is comprised of two homodimeric subunits, [alpha]2 and [beta]2, forming an active [alpha]2[beta]2 complex. The [beta]2 subunit harbors the stable diferric-tyrosyl radical cofactor (Y 122*) that reversibly oxidizes the active site cysteine (C₄₃₉) in [alpha]2. This oxidation requires a long-range radical transport (RT) pathway consisting of proton-coupled electron transfer (PCET) events through redox-active aromatic amino acid residues: Y₁₂₂* <--> [W₄₈] <--> Y₃₅₆ in [beta]2 to Y₇₃₁ <--> Y₇₃₀ <--> C₄₃₉ in [alpha]2. Once formed, the transient C₄₃₉* initiates nucleotide reduction. Both the long-range oxidation and the nucleotide reduction chemistries are kinetically masked by rate-limiting protein conformational change(s). To overcome this conformational change, the unnatural amino acid probe 3-aminotyrosine (NH₂Y) has been sitespecifically incorporated at multiple positions (Y₃₅₆, Y₇₃₁, Y₇₃₀) into the RT pathway. Herein, the NH₂Y probe is characterized as pertaining to the previously demonstrated ability for NH₂Y-incorporated RNR (NH₂Y-RNR) to form product. The reduction potential of NH₂Y produces a thermodynamic barrier that RNR cannot overcome. To explain NH₂Y-RNR activity, mass spectrometry was used for relative quantitation of contaminating wt-RNR in the NH₂Y-RNR, lending credence to the fact that the NH₂Y-RNRs are actually inactive. These results provide clarity to the long-standing mystery behind the low activities of the NH₂Y-RNRs. The use of the NH₂Y probe to generate stable radicals on the RT pathway has revealed further remarkable insight, demonstrating a hydrogen bonding network in the [alpha]2 subunit by employing advanced EPR methods on NH₂Y₇₃₀* and NH₂Y₇₃₁*. The evidence for a collinear PCET mechanism is provided with the NH₂Y₇₃₀/Y₇₃₁F and NH₂Y₇₃₁/C₄₃₉A mutants. Mutation of an R₄₁₁ to alanine in [alpha]2 allowed the detection of a \"flipped\" NH₂Y₇₃₁* conformation using advanced EPR techniques. Herein, photo cross-linked RNR is studied by tandem mass spectrometry (MS/MS). The study of a photo cross-linked [alpha]2[beta]2 complex using a 4-N-maleimido-benzophenone covalently attached to the C-terminal tail of [beta]2 yielded no photo cross-linked peptides. These studies taken together provide additional insight at the [alpha][beta] interface and provide additional tools to study this interaction."],"dc:description.degree":["Ph. D. in Biological Chemistry"],"dc:identifier.uri":["http://hdl.handle.net/1721.1/115805"],"dc:language.iso":["eng"],"dc:publisher":["Massachusetts Institute of Technology"],"dc:rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"dc:rights.uri":["http://dspace.mit.edu/handle/1721.1/7582"],"dc:subject":["Chemistry."],"dc:title":["Mechanistic studies of the radical transport pathway in aminotyrosine-substituted class Ia ribonucleotide reductase"],"dc:type":["Thesis"]},"updated_at":"2026-07-22T22:20:47Z"}