Glutamate 52-β at the α/β subunit interface of Escherichia coli 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. Escherichia coli class Ia RNR is composed of alpha and beta subunits that form a transient, active alpha 2 beta 2 complex. The E. coli RNR is rate- limited by S/e- dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 angstrom across the subunit (alpha/beta) 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 alpha 2 beta 2 created from X- ray structures of alpha and beta and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the alpha/beta interface (Glu(350) and Glu(52) in beta 2 and Arg(329) and Arg(639) in alpha 2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-beta 2, weakened subunit affinity. An RNR mutant with 2,3,5- trifluorotyrosine radical (F3Y122 center dot) replacing the stable Tyr122(center dot) in WT-beta 2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His(6)-alpha 2/S/e resulted in anRNRcapable of catalyzing pathway- radical formation (Tyr(356 center dot) -beta 2), 0.5 eq of dCDP/F3Y122 center dot, and formation of an alpha 2 beta 2 complex that is isolable in pulldown assays over 2 h. Negative stainEMimages with S/e (GDP/TTP) revealed the uniformity of the alpha 2 beta 2 complex formed.