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

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 α₂β₂enzyme 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 β₂, nucleotide reduction in α₂. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y₃₅₆(β) and Y₇₃₁(α) across the α/β interface. Using 2,3,5-F₃Y₁₂₂-β₂with 3,5-F₂Y₇₃₁-α₂, GDP (substrate) and TTP (allosteric effector), a Y₃₅₆^•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 ¹⁹F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y₃₅₆^•and the ¹⁹F nuclei of 3,5-F₂Y₇₃₁in this RNR mutant. Similar experiments with the double mutant E₅₂Q/F₃Y₁₂₂-β₂were 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-F₂Y₇₃₁. Remarkably, one conformation is consistent with 3,5-F₂Y₇₃₁within the H-bond distance to Y₃₅₆^•, 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 Y₃₅₆and Y₇₃₁. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.