Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxy-nucleotides and is composed of two subunits: alpha 2 and beta 2. beta 2 contains a stable di-iron tyrosyl radical cofactor required to generate a thiyl radical (C-439(center dot)) in alpha 2 over a distance of 35 angstrom, 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: Y-122 reversible arrow W-48[?] reversible arrow Y-356 in beta 2, across the subunit interface to Y-731 reversible arrow Y-730 reversible arrow C-439 in alpha 2. Within alpha 2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y-730 in alpha 2, and this mutant was incubated with beta 2, cytidine 5'-diphosphate, and adenosine 5'-triphosphate to generate a NH2Y730 center dot in D2O. [H-2]-Electron nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of alpha 2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C-439 and Y-731. A third proton was assigned to a water molecule in close proximity (2.2 angstrom O-H center dot center dot center dot O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730 center dot are consistent with the combined effect of the hydrogen bonds to Cys(439) and Tyr(731), both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in alpha 2 of the active RNR complex, for which no structural data are available.