Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by (17)O Electron–Nuclear Double Resonance Spectroscopy

[Image: see text] The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate (17)O high-frequency electron–nuclear double resonance (ENDOR) in conjunction with H(2)(17)O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α(2)β(2)E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···(17)O distances in the range 2.8–3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.