Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer

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 ∼35 Å from the stable diferric/tyrosyl-radical (Y₁₂₂^•) cofactor in the β subunit to cysteine 439 (C₄₃₉) in the active site of the α 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 β 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 Y₁₂₂^• by the first residue in the hopping pathway, a water ligand to Fe₁ 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 Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe₁. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe₁-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^•, but also directly in catalysis.