Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase.

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C ₄₃₉ ^• ) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α ₂ :β ₂ subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y ₁₂₂ β ↔ [W ₄₈ β?] ↔ Y ₃₅₆ β ↔ Y ₇₃₁ α ↔ Y ₇₃₀ α ↔ C ₄₃₉ α). The mutant R ₄₁₁ A(α) disrupts the H-bonding environment and conformation of Y ₇₃₁ , ostensibly breaking the RT pathway in α ₂ . However, the R ₄₁₁ A protein retains significant enzymatic activity, suggesting Y ₇₃₁ is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH ₂ Y) by amber codon suppression at positions Y ₇₃₁ or Y ₇₃₀ and investigation of the NH ₂ Y ^• trapped state in the active α ₂ :β ₂ complex by HYSCORE spectroscopy validate that the perturbed conformation of Y ₇₃₁ in R ₄₁₁ A-α ₂ is dynamic, reforming the H-bond between Y ₇₃₁ and Y ₇₃₀ to allow RT to propagate to Y ₇₃₀ . Kinetic studies facilitated by photochemical radical generation reveal that Y ₇₃₁ changes conformation on the ns-μs time scale, significantly faster than the enzymatic k _cat . Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 10 ⁴ s ^-1 when comparing wild type to R ₄₁₁ A-α ₂ , respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.