(19)F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase

[Image: see text] Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α(2)β(2) enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β(2), nucleotide reduction in α(2). Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y(356)(β) and Y(731)(α) across the α/β interface. Using 2,3,5-F(3)Y(122)-β(2) with 3,5-F(2)Y(731)-α(2), GDP (substrate) and TTP (allosteric effector), a Y(356)(•) intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz (19)F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y(356)(•) and the (19)F nuclei of 3,5-F(2)Y(731) in this RNR mutant. Similar experiments with the double mutant E(52)Q/F(3)Y(122)-β(2) were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F(2)Y(731). Remarkably, one conformation is consistent with 3,5-F(2)Y(731) within the H-bond distance to Y(356)(•), whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y(356) and Y(731). The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.