Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase β2

Abstract

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2'-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric-tyrosyl radical (Y122O(•)) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O(•) reduction mechanism in Escherichia coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O(•) was studied using reaction-induced FT-IR spectroscopy and [(13)C]aspartate-labeled β2. These Y122O(•) minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated β2.

Figure 1

Possible scenarios for carboxylate reorganization at the diiron cofactor in E. coli class Ia β2. The active cofactor is first assembled by reaction of diferrous β2, O₂, and reductant (A). The stable Y122O• is reduced concomitantly with C439 oxidation by reversible, long-range PCET during catalysis (B). Alternatively, the cofactor may be rendered inactive by small molecule- mediated reduction of Y122O• (C). In this report, we monitor changes to the amino acids in red with HU-mediated inactivation.

Figure 2

Schematic of the FT-IR isotope-edited spectrum, reflecting [4-¹³C]-Asp (40%) labeling and reduction of Y122O• with HU (A). Band assignments to υas of D84 are labeled in red. (B) shows the NA–minus–U-¹³C₄Asp isotope-edited spectrum (top, see also Figs. S3–S4) for the HA reaction. The reaction was 100 μM β2 and 25 mM HA in 5mM Hepes, pD 7.6 (20°C, 50 μm spacer). Band assignments were established by spectral simulation (bottom). The bottom trace is the simulated isotope-edited spectrum, which accounts for the data and and was produced from the NA spectrum in Fig. S3F assuming ¹³C shifts of −43 cm⁻¹. Red and blue labels represent assignments to υas and amide I of D84, respectively. Bands labeled in bold face (solid shading) and italics (dashed shading) represent NA and ¹³C isotope, respectively. Tick marks in (B) are 2×10⁻⁴ AU.

Figure 3

Reaction-induced FT-IR spectra associated with Y122O• reduction by HU, recorded at 20°C (see also Figs. S3 and S4). The NA difference spectrum for the HU reaction is shown in (A). In (B–E), isotope-edited HU spectra were constructed, (B) NA–minus–¹³C global (all carbons), (C) NA–minus–U-¹³C₄Asp (uniform), (D) NA–minus–4-¹³CAsp (side chain), and (E) NA–minus–4-¹³CAsp/1-¹³CTyr (double label). The spectra were offset along the y-axis for comparison. Reactions were collected in a FT-IR sample cell equipped either with a (A–B) ~6 μm or a (C–F) 50 μm spacer. The β2 concentrations were (A–B) 250 μM and (C–F) 100 μM in 5 mM Hepes, pD 7.6. The HU concentration was 50 mM in the same buffer. Red and blue labels represent assignments to υas and amide I of D84, respectively (Table S1). Bands labeled in bold face and italics represent NA and ¹³C isotopologue, respectively. Tick marks are 2.5×10⁻⁴ AU. (F) shows a control double difference spectrum, which provides an estimate of the baseline. (E) and (F) were baseline corrected with a straight-line fit for presentation purposes.

Figure 4

Top: Schematic of D84 hydrogen bond shift, which is linked to Y122O• reduction (PCET) by HU. Bottom: Structure of met (Y122OH, Fe³⁺) β2. Waters and oxygen are red and blue spheres, respectively.^, Y122OH-D84 O-O distance is shown.