Mechanistic investigations of the radical transport pathway in fluorotyrosine-substituted class Ia ribonucleotide reductases

Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'- deoxynucleotides, providing the monomeric precursors for DNA replication and repair. The focus of this thesis is on the E. coli class la RNR that is composed of two homodimeric subunits ([alpha]a2 and [beta]2), which form an active [alpha]2[beta]2 complex. A stable diferric-tyrosyl radical (Y₁₂₂*) in [beta]2 reversibly oxidizes an active site cysteine (C₄₃₉*) in [alpha]2 via multiple proton-coupled electron transfer (PCET) steps through conserved aromatic amino acid residues: Y₁₂₂* <-> [W₄₈] <-> Y₃₅₆ in [beta]2 to Y₇₃₁ <-> Y₇₃₀ <-> C₄₃₉ in [alpha]2. The transient C₄₃₉* is responsible for initiating nucleotide reduction. Long-range radical transport (RT) and nucleotide reduction are kinetically masked by rate-limiting protein conformational changes. Herein, the stable Y₁₂₂₈ is site-specifically replaced with a 2,3,5-trifluorotyrosyl radical (2,3,5-F₃Y*) that modulates the driving force for RT. This 2,3,5-F₃Y-substituted RNR perturbs PCET kinetics such that a radical intermediate (Y₃₅₆*) can be observed and characterized. Rapid kinetic studies demonstrate that Y₃₅₆* is kinetically and chemically competent for nucleotide reduction, and provide the first evidence for a pathway Yo that can complete the RNR catalytic cycle. Temperature and pH dependent studies show equilibration of the stable 2,3,5-F₃Y* with the pathway radical intermediate, Y₃₅₆*. These data are corroborated by similar experiments performed with 3,5-difluorotyrosine (3,5-F₂Y) in place of Y₃₅₆, which demonstrate equilibration of Y₁₂₂*. with 3,5-F₂Y*. These studies together provide insight into the thermodynamic landscape of the RT pathway. A model is proposed in which the RT pathway is thermodynamically uphill and driven forward by rapid irreversible water loss that occurs during nucleotide reduction. The 3,5-F₂Y analog is further utilized to test the ability of E₃₅₀, a conserved [beta]2 C-terminal tail residue, to function as the proton acceptor for Y₃₅₆ or Y₇₃₁ . A model is put forth in which E₃₅₀ does not participate in proton transfer, but is involved in [alpha]2[beta]2 subunit interaction and in controlling radical initiation. Finally, an X-ray crystal structure of the active [alpha]2[beta]2 complex has remained elusive. Herein, Ni-NTA pull-down assays are presented, demonstrating that injection of a single electron into the diferric cluster site generates a stable [alpha]2[beta]2 complex. These studies afford the opportunity to structurally characterize the [alpha]2[beta]2 complex with the goal of understanding PCET across the [beta]a interface.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2016

Author dc:creator

• Ravichandran, Kanchana

Advisor dc:contributor.advisor

• JoAnne Stubbe.

• Chemistry.

Statement dc:rights

• M.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.

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/1721.1/105044

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/105044