Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the beta(2) subunit (Y-122(center dot)) necessary for nucleotide reductase activity. Upon binding the cognate alpha(2) subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active alpha(2)beta(2) complex from the Y-122(center dot) site in beta(2) to the substrate activating cysteine residue (C439) in a2 via a pathway of redox active amino acids (Y-122 [beta] <-> W-48 [beta] ? <-> Y-356[beta] <-> Y-731[alpha] <-> Y-730[alpha] <-> C-439[alpha]) spanning >35 A. Ionizable residues at the alpha(2)beta(2) interface are essential in mediating RT, and therefore control activity. One of these mutations, E350X (where X = A, D, Q) in beta(2), obviates all RT, though the mechanism of control by which E-350 mediates RT remains unclear. Herein, we utilize an E(350)Q-photo beta(2) construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y-122(center dot) -> Y-356) is replaced by direct photochemical radical generation of Y-356(center dot). These data present compelling evidence that E-350 conveys information between the alpha(2) and beta(2) subunits facilitating conformational gating of RT that specifically targets Y-122(center dot) reduction, while the fidelity of the remainder of the RT pathway is retained.