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

• DOI: 10.1021/jacs.2c02906
• Publication Date: 2022-06-02T14:52:43Z
• Authors: Andreas Meyer; Annemarie Kehl; Chang Cui; Fehmke A. K. Reic...; Fabian Hecker; Lisa-Marie Funk; Manas K. Ghosh; Kuan-Ting Pan; Henning Urlaub; Kai Tittmann; JoAnne Stubbe; Marina Bennati
• Keywords: Ribonucleotide reductase; Ribonucleotide; Deoxyribonucleotides; Electron acceptor; Pulsed EPR; Acceptor

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 α2β2 enzyme complex that uses reversible multistep radical transfer (RT) over 32 Å across its two subunits, α β, initiate, using metallo-cofactor β2, nucleotide α2. Each step proposed involve distinct proton-coupled electron-transfer (PCET) process. An unresolved RT involving Y356(β) Y731(α) α/β interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731-α2, GDP (substrate) TTP (allosteric effector), Y356• intermediate was trapped identity verified by 263 GHz electron paramagnetic resonance (EPR) 34 pulse electron–electron double spectroscopies. 94 19F electron-nuclear spectroscopy allowed measuring interspin distances between nuclei 3,5-F2Y731 this mutant. Similar experiments mutant E52Q/F3Y122-β2 were carried out for comparison recently published cryo-EM structure holo complex. For both combinations, distance measurements reveal conformations 3,5-F2Y731. Remarkably, one conformation consistent within H-bond Y356•, whereas second observed structure. The observations unexpectedly suggest possibility colinear PCET, which proton are transferred from same donor acceptor Y356 Y731. results highlight important state-of-the-art EPR decipher mechanism.