Glutamate 52-β at the α/β subunit interface of class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of the Ia RNR (Iag), we identified and investigated four residues at the α/β interface (Glu and Glu in β2 and Arg and Arg in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (FY) replacing the stable Tyr in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr-β2), 0.5 eq of dCDP/FY, and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed.