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. 2013 Jul 18;117(28):8457-68.
doi: 10.1021/jp404757r. Epub 2013 Jul 3.

Redox-linked conformational control of proton-coupled electron transfer: Y122 in the ribonucleotide reductase β2 subunit

Adam R Offenbacher  1 , Lori A Burns, C David Sherrill, Bridgette A Barry
Affiliations

Redox-linked conformational control of proton-coupled electron transfer: Y122 in the ribonucleotide reductase β2 subunit

Adam R Offenbacher et al. J Phys Chem B. .

Abstract

Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of α2 and β2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(•))-diferric cofactor is located in β2. Although Y122O(•) is extremely stable in free β2, Y122O(•) is highly reactive in the quaternary substrate-α2β2 complex and serves as a radical initiator in catalytic PCET between β2 and α2. In this report, we investigate the structural interactions that control the reactivity of Y122O(•) in a model system, isolated E. coli β2. Y122O(•) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific (13)C-labeling of the tyrosine C1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 Å translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 ± 1.0 at 25 °C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(•) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(•)-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(•) and the iron cluster.

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Figures

Figure 1
Figure 1
Crystal structure of the 1.4 Å met (Y122OH; PDB# 1MXR) β2 structure.14 Distances are in Ångstroms. Iron atoms are orange spheres. (B) shows the schematic diagram of the proposed Y122O• conformational change presented in ref3 (pink) and this work (green). For comparison, the singlet form is in light gray. The arrow illustrates the amide carbonyl translation, which is required in the model in ref 3, but is not predicted by the model described here. (C) Schematic for generating isotope-edited spectra (see Figures 3 and 6), which reflect bands sensitive to Y122O• reduction and to incorporation of the 13C isotope in the Y-T amide bond.
Figure 2
Figure 2
Proton inventory for the Y122O• PCET reaction with hydroxyurea, collected at 25°C and in triplicate. The reactions were carried out in 5 mM Hepes, pL 7.6. The experimental data (Table S1) are shown as black dots. The error bars are one standard deviation. The equations used to model the data are derived from the Gross-Butler expression and given in Table 1. The red line simulates a one proton mechanism with the experimentally observed SIE (16.7). The blue line simulates a two proton, transition state mechanism with equivalent solvent isotope effects (4.09). The green line simulates a many proton mechanism, and the gray line simulates a reactant-state mechanism. The black line fits the data and simulates a two proton, transition-state mechanism with non-equivalent solvent isotope effects (1.4 and 11.9).
Figure 3
Figure 3
Reaction-induced FT-IR difference spectra associated with Y122O• reduction by HU, recorded at 20°C. The difference spectra, generated as Y122O•–minus–Y122OH, represent (A) NA, (B) global 13C (all carbons enriched), and (C) 1-13C tyrosine (YT amide linker only) labeled β2. The β2 concentrations were 250 µM in a buffer containing 5 mM Hepes, pD 7.6. HU was prepared at 50 mM concentration in the same buffer. Spectra are averages of (A) 20, (B) 8, and (C) 10 reactions. Isotope-edited FT-IR spectra, generated as 12C-minus- 13C, represent as (D) 12C– minus–13 C global (A-minus-B) and (E) 12C-minus– 13C tyrosine (A-minus-C). (F) represents a control double difference spectrum, which was generated by subtraction of one half of the data in (A) from the other half and division by . This control double difference spectrum is a negative control, which provides an estimate of the signal-to-noise and baseline fluctuations. Vibrational bands (in cm−1 ) discussed in the text are labeled in bold faced. The spectra are displayed with vertical offsets. The y-axis tick marks represent 1 × 10−4 absorbance units.
Figure 4
Figure 4
Structures of the singlet and radical dipeptide (YT) models employed for electronic structure calculations (A), with tyrosine carbons numbered. The carbonyl carbon, enriched with 13C isotope, is designated by the red asterisks. The Newman projections of the relevant conformers are shown in Table 3. (B–F) shows the simulated DFT B3LYP-D/aug-cc-pVDZ) infrared spectra for the singlet (YOH) and radical (YO) forms of the YT dipeptide model. The data were generated for the (B) YOH conformer A, (C) YOH conformer B, (D) YO• conformer A, (E) YO• conformer B, and (F) YO• conformer C. The black and red spectra are predicted for the 12C and 13C isotopologs, respectively. Gaussian lineshapes were generated from the computed frequencies and amplitudes (sticks).
Figure 5
Figure 5
Simulated reaction-induced FT-IR spectra for the YT dipeptide model. Difference spectra were calculated as radical YO• minus singlet YOH (Figure 5). The data are computed as (A) A–minus–A, (B) B–minus–B, (C) A–minus–B, (D) B–minus–A, and (E) C–minus–A. The italics refer to radical conformational state; plain text refers to the singlet conformational state.
Figure 6
Figure 6
Comparison of experimental and simulated isotope-edited, reaction-induced FT-IR spectra. (A) is from experimental FT-IR data of β2 and reproduced from Figure 3E. Simulated (YT model) isotope-edited spectra in (B–E), calculated as 12C–minus–13C, are generated from 12C (Figure 5) and 13C difference spectra for (B) B minus A, (C) A minus A, (D) C minus A, and (E) B minus B. The blue and red shaded areas (with arrows) denote the isotope shifts for the radical (red) and singlet (blue) states. The 13C isotope shifts, designated by ∆, are in cm−1 and denoted with arrows. In (A), the 13C shift of (–) 1662 cm−1 is not resolved, but is expected to have a ∼40 cm−1 downshift (see dashed line), based on the 13C global label (see Figures 3B and D). The italics refer to radical conformational state; plain text refers to the singlet conformational state.
Figure 7
Figure 7
Comparison of the structures for the singlet and radical states of Y122 in β2 (A) and the YT dipeptide model (B). In (A), structural refinements of the 1.4 Å met β2 (Y122OH) form (top), obtained from X-ray crystal analysis (PDB 1MXR),14and radical (Y122O•) form (bottom). The radical form was modeled as the B conformer, predicted by the model dihedral angles, and is presented in gray. Distances (in Ångstroms) between Y122 and D84 are shown in green. For the singlet (top), the dihedral angles of the A conformer were also structurally refined; deviations from the met crystal structure are highlighted in pink. For Y122O• (bottom), the ring dihedral angles predicted previously from experimental, or calculated hyperfine coupling constants are shown in green and purple, respectively. The structures in (B) are derived from DFT calculations on the model dipeptides.
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