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

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. class Ia RNR is an αβ 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, nucleotide reduction in α. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y(β) and Y(α) across the α/β interface. Using 2,3,5-FY-β with 3,5-FY-α, GDP (substrate) and TTP (allosteric effector), a Y 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 F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y and the F nuclei of 3,5-FY in this RNR mutant. Similar experiments with the double mutant EQ/FY-β 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-FY. Remarkably, one conformation is consistent with 3,5-FY within the H-bond distance to Y, 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 and Y. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.