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Spectroscopic evidence for a H bond network at Y356 located at the subunit interface of active E. coli ribonucleotide reductase.

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Nick,  T. U.
Research Group of Electron Paramagnetic Resonance, MPI for Biophysical Chemistry, Max Planck Society;

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Kasanmascheff,  M.
Research Group of Electron Paramagnetic Resonance, MPI for Biophysical Chemistry, Max Planck Society;

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Bennati,  M.
Research Group of Electron Paramagnetic Resonance, MPI for Biophysical Chemistry, Max Planck Society;

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Citation

Nick, T. U., Ravichandran, K. R., Stubbe, J., Kasanmascheff, M., & Bennati, M. (2017). Spectroscopic evidence for a H bond network at Y356 located at the subunit interface of active E. coli ribonucleotide reductase. Biochemistry, 56(28), 3647-3656. doi:10.1021/acs.biochem.7b00462.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002D-800E-D
Abstract
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.