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

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.