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

The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and β subunits that form an active α2β2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. β2 contains the diferric tyrosyl radical (Y₁₂₂·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y₁₂₂· ⇆ [W₄₈] ⇆ Y₃₅₆ in β2 to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and β2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/β) interface. Here, we report unique EPR spectroscopic features of Y₃₅₆·-β, the pathway intermediate generated by the reaction of 2,3,5-F₃Y₁₂₂·-β2/CDP/ATP with wt-α2, Y₇₃₁F-α2, or Y₇₃₀F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [¹H] and [²H]-ENDOR reveal two exchangeable H bonds to Y₃₅₆·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (r_O-H ∼1.8-1.9 Å) are required to reproduce the g_x value of Y₃₅₆· (wt-α2). 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 Y₃₅₆· 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 Y₃₅₆ oxidation.