The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of similar to 35 angstrom from the stable diferric/tyrosyl-radical (Y-122(center dot)) cofactor in the beta subunit to cysteine 439 (C-439) in the active site of the alpha subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton transfer steps and conformational gating within the beta sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided In the key first step, the reduction of Y-122(center dot) by the first residue in the hopping pathway, a water ligand to Fe-1 of the diferric cluster was suggested to donate a proton to yield the neutral Y-122. Here we show that forward radical translocation is associated with perturbation of the Mossbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe-1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe-1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y-122(center dot), but also directly in catalysis.