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

• Published in: Journal of the American Chemical Society 2022-06, Vol.144 (25), p.11270-11282
• Main Authors: Meyer, Andreas, Kehl, Annemarie, Cui, Chang, Reichardt, Fehmke A K, Hecker, Fabian, Funk, Lisa-Marie, Pan, Kuan-Ting, Urlaub, Henning, Tittmann, Kai, Stubbe, JoAnne, Bennati, Marina
• Format: Article
• Language: English
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.2c02906

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 α β 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-F Y -β with 3,5-F Y -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-F Y in this RNR mutant. Similar experiments with the double mutant E Q/F Y -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-F Y . Remarkably, one conformation is consistent with 3,5-F Y 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.