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

Abstract

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.