Radical Transport at the Subunit Interface of Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the transformation of nucleoside 5´-di(tri)phosphates [(ND(T)Ps, where N is A, U, C, or G] to their corresponding dND(T)Ps. In the prototypical Escherichia coli class Ia enzyme, NDP reduction proceeds via unprecedented, long-range proton-coupled electron transfer (PCET) involving the generation of a transient active site thiyl radical (C439•) in an α subunit from a stable tyrosyl radical (Y122•) in a β subunit within the active α2β2 complex. On each turnover, reversible radical transport (RT) is proposed to occur by hopping along the 32 Å RT pathway via a series of conserved redox-active amino acids (Y122•[β] ⇌ W48?[β] ⇌ Y356[β] :::⇌::: Y731[α] ⇌ Y730[α] ⇌ C439[α]) as a single-step tunneling mechanism between Y122•[β] and C439[α] would be prohibitively slow to account for the turnover number. In this thesis, we scrutinize the radical transport pathway at the subunit interface of E. coli class Ia RNR, aiming to deepen our understanding of the underlying mechanisms governing biological PCET in these essential enzymes.

In Chapter 2, we report the synthesis of an active photochemical RNR complex that allows direct analysis of the photoinjection of a radical into the active site of α in the absence of the interfacial Y356[β]. This intersubunit PCET pathway is investigated by ns laser spectroscopy in the presence of substrate (CDP) and effector (ATP) on a [Re] photosensitizer located in place of Y356[β] and a site-specifically incorporated ionizable reporter in α, yielding the complex, [Re356]-β2:F3Y731-α2. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y731[α], which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y356[β] and Y731[α], at the subunit interface.

In Chapter 3, we examine a polar region formed at the subunit interface that is characterized by a series of ionizable residues proposed to be involved in facilitating proton exchange between the RT pathway and bulk solvent. Differences in the generation and RT behavior of Y356• caused by variants of these residues were targeted using a [Re] photosensitizer covalently attached adjacent to position Y356[β]. Mutagenesis studies, transient absorption spectroscopy, and light-dependent substrate turnover assays collectively indicate that the E52[β], R331[α], E326[α], and E326[α′] network plays the essential role of shuttling protons associated with Y356 oxidation during PCET across the αβ interface.

In Chapter 4, we design an active photochemical chimeric construct of RNR that consolidates the α/β subunit interface region and active site into a unimolecular system based on a unifying evolutionary apparatus common to the natural classes of RNRs. Combination of this photoRNR chimera with CDP, ATP, and light resulted in the generation of the interfacial Y356•, along with the production of dCDP and cytosine, and an active site chemistry consistent with both the consensus mechanism of nucleotide reduction and chemistry observed when RNR is inactivated by mechanism-based inhibitors. The enzymatic activity of the RNR photochimera in the absence of the essential β metallocofactor highlights the adaptability of the 10-stranded αβ barrel catalytic fold to support deoxynucleotide formation and accommodate the design of new RNRs.

In Chapter 5, we explore future research directions, including photoreactive crosslinkers to investigate conformational dynamics at the α/β interface, unnatural tryptophan analogs to probe the RT pathway, and incorporation of fluorinated tryptophan analogs to perturb PCET kinetics and activity. Additionally, we consider the potential for reverse RT experiments to capture W48 on pathway and disentangle the fate of the proton at that position during turnover. Seeking to enhance our understanding of the regulation and activity of RNR, we also assess the utility of fluorescent non-canonical amino acids to report on the structural interconversions between active and inactive complexes.