Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase

Abstract

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 (C₄₃₉^•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α₂:β₂ subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y₁₂₂β ↔ [W₄₈β?] ↔ Y₃₅₆β ↔ Y₇₃₁α ↔ Y₇₃₀α ↔ C₄₃₉α). The mutant R₄₁₁A(α) disrupts the H-bonding environment and conformation of Y₇₃₁, ostensibly breaking the RT pathway in α₂. However, the R₄₁₁A protein retains significant enzymatic activity, suggesting Y₇₃₁ is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH₂Y) by amber codon suppression at positions Y₇₃₁ or Y₇₃₀ and investigation of the NH₂Y^• trapped state in the active α₂:β₂ complex by HYSCORE spectroscopy validate that the perturbed conformation of Y₇₃₁ in R₄₁₁A-α₂ is dynamic, reforming the H-bond between Y₇₃₁ and Y₇₃₀ to allow RT to propagate to Y₇₃₀. Kinetic studies facilitated by photochemical radical generation reveal that Y₇₃₁ 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 × 10⁴ s^-1 when comparing wild type to R₄₁₁A-α₂, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.