Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and β. The active form is an asymmetric αα'ββ' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C₄₃₉•), and the β subunit houses the diferric-tyrosyl radical (Y₁₂₂•) that is essential for C₄₃₉• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y₁₂₂•[β] ↔ W₄₈?[β] ↔ Y₃₅₆[β] ↔ Y₇₃₁[α] ↔ Y₇₃₀[α] ↔ C₄₃₉[α]. In a recent cryo-EM structure, Y₃₅₆[β] was revealed for the first time and it, along with Y₇₃₁[α], spans the asymmetric α/β interface. An E₅₂[β] residue, which is essential for Y₃₅₆ oxidation, allows access to the interface and resides at the head of a polar region comprising R₃₃₁[α], E₃₂₆[α], and E₃₂₆[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y₃₅₆• was photochemically generated using a photosensitizer covalently attached adjacent to Y₃₅₆[β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation 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 from the interface to bulk solvent.

Figure 1.

Cryo-EM asymmetric structure of RNR (αα′ββ′) trapped using F₃Y₁₂₂•/E₅₂Q-β₂ with α₂, GDP, and TTP and the proposed residues within the α/β subunit interface (PDB ID: 6W4X). (A) The asymmetric αα′ββ′ complex (α in two shades of blue and β in red and orange) showing the proposed long-range (32-Å) PCET radical transport pathway from Y₁₂₂•-β to C₄₃₉-α (vertical axis) and the proposed proton transfer network to enable Y₃₅₆[β] oxidation for radical transport across the asymmetric α/β interface (horizontal axis). The red dashed line indicates the proposed path E₅₂-β, R₃₃₁-α, E₃₂₆-α, and E₃₂₆-α′) for H⁺ release to the bulk solvent. (B) An enlargement of the α/β asymmetric interface highlighting the residues (in red) that form the polar network for proton transfer between the interface and bulk solvent.

Figure 2.

The steady state turnover of α₂ mutants (0.15 μM) assayed with wt-β₂ (0.75 μM): E₃₂₆D (brown circles), E₃₂₆Q (red circles), R₃₃₁K (blue circles), R₃₃₁Q (purple circles).

Figure 3.

Transient absorption spectra of α₂ mutants and photoβ₂. (A) Transient absorption spectra of 50 μM R₃₃₁Q-α₂ in the presence of 20 μM photoβ₂ (blue) or Y₃₅₆F-photoβ₂ (black). (B) Transient absorption spectra of 50 μM E₃₂₆Q-α₂ in the presence of 20 μM photoβ₂ (red) or Y₃₅₆F-photoβ₂ (black). All spectra were measured with 3 mM ATP/1 mM CDP and 10 mM Ru(NH₃)₆Cl₃ in assay buffer pH 7.6. The samples were excited at 355 nm with 1.5 mJ/pulse. The spectra were collected after 1 μs delay. The baseline of TA spectra was normalized to the ΔOD between 350–375 nm.

Figure 4.

Single turnover photochemical assay of α₂ mutants. (A) Photochemical turnover of E₃₂₆Q-α₂ (red circles), wt-α₂ (black squares) and R₃₃₁Q-α₂ (blue triangles) with photoβ₂ at pH 7.6. (B) Photochemical turnover of 2,3,5-F₃Y₇₃₁/R₃₃₁Q-α₂ with photoβ₂ at pH 8.2 (green triangles) and 3,5-F₂Y₇₃₁/R₃₃₁Q-α₂ with photoβ₂ at pH 8.2 (light purple circles), 7.6 (purple triangle) and 6.2 (dark purple squares). The reactions were initiated by photoexcitation at 370 nm and quenched by ice-cold 2% HClO₄ after reacting for 20 s.

Figure 5.

Blue mesh highlights the “empty volume” observed in the cryo-EM structure, beginning at Y₃₅₆ at the interface and leading out to bulk solvent via E₅₂[β], R₃₃₁[α], E₃₂₆[α] and E₃₂₆[α′].

Figure 6.

Proposed model for radical transport across the RNR interface following Y₃₅₆•[β] radical initiation in (A) wt RNR and pathway mutants (B) E₅₂Q, (C) R₃₃₁Q, (D) R₃₃₁Q/F_nY₇₃₁, and (E) E₃₂₆Q. The yellow-orange and blue backgrounds represent β and α subunits, respectively. Systems (A), (D) and (E) support substrate turnover. The H⁺ from Y₃₅₆ (indicated by red arrows) is shown to traverse the empty volume about the residues (dashed region outlined in blue), which we ascribe to be occupied by water. In Figure 6C, the blue pathway indicates that the second release of a proton is impeded upon Y₇₃₁ oxidation.