5 years ago

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

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 (rO–H ∼1.8–1.9 Å) are required to reproduce the gx 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

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