ENDOR Spectroscopy and DFT Calculations: Evidence for the Hydrogen-Bond Network Within α2 in the PCET of E. coli Ribonucleotide Reductase

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y₁₂₂^•) cofactor required to generate a thiyl radical (C₄₃₉^•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y₁₂₂ ⇆ W₄₈[?] ⇆ Y₃₅₆ in β2, across the subunit interface to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH₂Y) replaced Y₇₃₀ in α2, and this mutant was incubated with β2, cytidine 5′-diphosphate, and adenosine 5′-triphosphate to generate a NH₂Y₇₃₀^• in D₂O. [²H]-Electron–nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C₄₃₉ and Y₇₃₁. A third proton was assigned to a water molecule in close proximity (2.2 Å O–H···O distance) to residue 730. The calculations also suggest that the unusual <i>g</i>-values measured for NH₂Y₇₃₀^• are consistent with the combined effect of the hydrogen bonds to Cys₄₃₉ and Tyr₇₃₁, both nearly perpendicular to the ring plane of NH₂Y_730. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available