F-19 Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the alpha/beta 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 alpha(2)beta(2) enzyme complex that uses a reversible multistep radical transfer (RT) over 32 angstrom across its two subunits, alpha and beta, to initiate, using its metallo-cofactor in beta(2), nucleotide reduction in alpha(2). Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(beta) and Y-731(alpha) across the alpha/beta interface. Using 2,3,5-F3Y122-beta(2) with 3,5-F2Y731-alpha(2), GDP (substrate) and TTP (allosteric effector), a Y-356(center dot) 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-19 electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y-356(center dot) and the F-19 nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E(52)Q/F2Y731-beta(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-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y-356(center dot), 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.