Mechanistic studies of the radical transport pathway in aminotyrosine-substituted class Ia ribonucleotide reductase

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2018

Author dc:creator

• Lee, Wankyu

Advisor dc:contributor.advisor

• JoAnne Stubbe.

• Chemistry.

Statement 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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/115805

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/115805