Spectroscopic Evidence for a H Bond Network at Y356 Located at the Subunit Interface of Active E. coli Ribonucleotide Reductase

The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of a and beta subunits that form an active alpha 2 beta 2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. beta 2 contains the diferric tyrosyl radical (Y-122.) cofactor that initiates radical transfer (RT) over 35 A via a specific pathway of amino acids (Y-122. reversible arrow [W-48] reversible arrow Y-356 in beta 2 to Y-731 reversible arrow Y-730 reversible arrow C-439 in alpha 2). Experimental evidence exists for colinear and orthogonal PCET in a2 and beta 2, respectively. No mechanistic model yet exists for the PCET across the subunit (alpha/beta) interface. Here, we report unique EPR spectroscopic features of Y-356.-beta, the pathway intermediate generated by the reaction of 2,3,5-F3Y122.beta 2/CDP/ATP with wt-alpha 2, Y731F-alpha 2, or Y730F-alpha 2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [H-1] and [H-2]-ENDOR reveal two exchangeable H bonds to Y-356: a moderate one almost in-plane with the pi-system and a weak one. DFT calculation on small models of Y. indicates that two in-plane, moderate H bonds (r(O-H) similar to 1.8-1.9 A) are required to reproduce the g(x) value of Y-356. (wt-alpha 2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y-356. that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H+ release to the solvent during Y-356. oxidation.