Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase

Abstract

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122•) that generates a thiyl radical (C439•) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y•-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s–1). Rapid freeze–quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y•-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356• and 0.5 equiv of dCDP, both at 30 s–1. In the absence of an external reducing system, Y356• reduction occurs concomitant with F3Y reoxidation (0.4 s–1) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s–1) is observed prior to steady-state turnover (1.7 s–1). The [Y356•] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122• is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes

“…22 We hypothesized that if we carried out the same experiment with a block in the pathway (Y 731 F-α2 or Y 730 F-α2) 13 then equilibration of F 3 Y 122 • and Y 356 • could be measured by EPR spectroscopy as a function of temperature, allowing determination of Δ E °′(F 3 Y 122 •–Y 356 •). F 3 Y 122 •-β2, CDP, and ATP were incubated with Y 731 F-α2 at varying temperatures from 2 to 37 °C for 20 s or 1 min.…”

“…The amount of F 3 Y 122 • is typically 0.6–0.8 per β2 (instead of the theoretical 2 F 3 Y•/β2), with active β2 containing a F 3 Y 122 • in each β monomer. 22 Furthermore, while the active form of RNR is α2β2, the enzyme exhibits half-sites reactivity where only one of the two Y 122 •’s (one α/β pair) is active at a time. 22 A consequence of these phenomena is the presence of 50% of the total spin as residual F 3 Y 122 • in all reaction mixtures.…”

“…22 Furthermore, while the active form of RNR is α2β2, the enzyme exhibits half-sites reactivity where only one of the two Y 122 •’s (one α/β pair) is active at a time. 22 A consequence of these phenomena is the presence of 50% of the total spin as residual F 3 Y 122 • in all reaction mixtures. Thus, the data shown in Figures 3, S3, and S4 are presented using method A described in the experimental section to allow the small changes in the amounts of Y 356 • as a function of temperature (2, 5, 8, 10, 12, and 15 °C) to be more clearly observable.…”

“…43 In the case of F 3 Y 122 •-β2, we have measured formation of Y 356 • (20–30 s –1 ) and demonstrated that reoxidation of F 3 Y 122 by Y 356 • is slow (0.4–1.7 s –1 ) and rate-limiting for multiple turnovers. 22 In the NO 2 Y 122 •-β2 system, Y 356 • accumulates (100–300 s –1 ) due to the inability of this pathway radical to reoxidize NO 2 Y – subsequent to the first turnover. 17 Taken together, these studies suggest that Y 356 • can be observed during turnover only when reverse RT is slowed down (F 3 Y 122 •-β2) or completely inhibited (NO 2 Y 122 •-β2) and is partly a result of the potential difference between Y 122 and Y 356 .…”

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

“…29, 73, 75 The same reaction with His 6 -NH 2 Y 730(731) -α2, wt-β2, ATP, N 3 CDP formed N• at ~15 % of the total initial Y 122 •. 13 The 6% contaminating wt-α2, assuming this reaction is analogous to the one with the wt RNR, should yield 3% N•.…”