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

5 years ago

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

Thomas U. Nick, Marina Bennati, JoAnne Stubbe, Kanchana R. Ravichandran, Müge Kasanmascheff

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.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00462

DOI: 10.1021/acs.biochem.7b00462