Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

ABSTRACT

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the ß2 subunit (Y122•) necessary for nucleotide reductase activity. Upon binding the cognate α2 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 α2ß2 complex from the Y122• site in ß2 to the substrate activating cysteine residue (C439) in α2 via a pathway of redox active amino acids (Y122[ß] ↔ W48[ß]? ↔ Y356[ß] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]) spanning >35 Å. Ionizable residues at the α2ß2 interface are essential in mediating RT, and therefore control activity. One of these mutations, E350X (where X = A, D, Q) in ß2, obviates all RT, though the mechanism of control by which E350 mediates RT remains unclear. Herein, we utilize an E350Q-photoß2 construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y122• → Y356) is replaced by direct photochemical radical generation of Y356•. These data present compelling evidence that E350 conveys allosteric information between the α2 and ß2 subunits facilitating conformational gating of RT that specifically targets Y122• reduction, while the fidelity of the remainder of the RT pathway is retained.