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(center dot)) is a transient species generated by radical transfer (RT) from a stable diferrictyrosyl radical cofactor located >35 angstrom away across the alpha(2):beta(2) subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y-122 beta <-> [W-48 beta?] <-> Y-356 beta <-> Y-731 alpha <-> Y-730 alpha <-> C-439 alpha). The mutant R(411)A(alpha) disrupts the H-bonding environment and conformation of Y-731, ostensibly breaking the RT pathway in alpha(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 (NH2Y) by amber codon suppression at positions Y-731 or Y-730 and investigation of the NH2Y center dot trapped state in the active alpha(2):beta(2) complex by HYSCORE spectroscopy validate that the perturbed conformation of Y-731 in R(411)A-alpha(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-mu s 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 x 10(4) s(-1) when comparing wild type to R(411)A-alpha(2), respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.