Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the β subunit (Y) necessary for nucleotide reductase activity. Upon binding the cognate α 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 αβ complex from the Y site in β to the substrate activating cysteine residue (C) in α via a pathway of redox active amino acids (Y[β] ↔ W[β]? ↔ Y[β] ↔ Y[α] ↔ Y[α] ↔ C[α]) spanning >35 Å. Ionizable residues at the αβ interface are essential in mediating RT, and therefore control activity. One of these mutations, EX (where X = A, D, Q) in β, obviates all RT, though the mechanism of control by which E mediates RT remains unclear. Herein, we utilize an EQ-photoβ construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y → Y) is replaced by direct photochemical radical generation of Y. These data present compelling evidence that E conveys allosteric information between the α and β subunits facilitating conformational gating of RT that specifically targets Y reduction, while the fidelity of the remainder of the RT pathway is retained.